Objective: The aim of this study was to investigate the effects of chronic intermittent hypoxia (CIH) on atrial electrical remodeling in Sprague-Dawley (SD) rats, which provide the explication for the mechanisms of CIH promoting atrial fibrillation (AF). Methods: Eighty SD rats were randomly divided into 2 groups: control group and CIH group (n=40). CIH rats were subjected to CIH 8 h/d for 30 days. After the echocardiography and hemodynamics examination, cardiac electrophysiological experiments, histological experiments, and molecular biological experiments were executed. AF susceptibility was measured by isolated heart electrophysiological experiments. Masson's trichrome stain was used to assess the degree of atrial fibrosis. The protein expression levels of sodium voltage-gated channel alpha subunit 5 (SCN5A/Na_v1.5), calcium voltage-gated channel subunit alpha1 C (CACNA1C/Ca_v1.2) and potassium voltage-gated channel subfamily D member 3 (KCND3/K_v4.3) were measured by Western blot. In whole-cell patch clamp experiments, current clamp mode was used to record AP, and APD₉₀ and APD₅₀ were analyzed and compared between the two groups. In voltage clamp mode, I_Na, I_Ca-L, I_to and their kinetic parameters were recorded and compared between the two groups. Results: Compared to the control rats, atrial interstitial collagen deposition (P＜0.01) and AF inducibility (P＜0.05) were increased in CIH rats, whereas the expression levels of Na_v1.5, Ca_v1.2 and K_v4.3 were decreased (P＜0.05). APD₉₀ and APD₅₀ in CIH rats' atrial myocytes were longer than those of control rats, and CIH rats showed decreased current density of I_Na, I_Ca-L(P＜0.01) and I_to(P＜0.01). Conclusion: CIH-induced changes in the protein expression levels of ion channel subunits, current intensity, APD, and AF susceptibility, which may be the mechanisms of CIH promoting AF.

Objective: To investigate the protective effects of three Polyphenolic compounds on intestinal microbial communities in mice exposed intermittent plateau hypoxia. Methods: In this study, 60 healthy male Balb/c mice were randomly divided into plain control group, plateau control group, primary anthocyanin intervention group, quercetin intervention group and resveratrol intervention group, 12 mice in each group. Primary anthocyanin, quercetin and resveratrol were administrated by gavage at the doses of 50, 100 and 20 mg/kg in pharmacological intervention group, respectively. After exposure of the mice to simulation plateau-condition for 30 days, the serum samples were collected for DAO testing, sterile feces were collected in mice, and the diversity and genus level of the mouse gut bacteria were detected by using 16S rRNA technology. Ileum tissue was fixed and stained with HE. Results: HE staining showed that the plateau control group had significant damage to the intestinal tissue structure compared to the plain control group, and the serum DAO concentration was increased (P＜0.05), but there was no statistical difference in the abundance and diversity of intestinal flora species. Contrast to simulated intermittent plateau hypoxia group, the structure of the intestine tissue and the level of DAO in the quercetin intervention group and resveratrol intervention group were improved(P＜0.05), the abundance and α diversity of the intestinal flora were decreased, the relative abundance of Bacteroidetes was reduced(P＜0.05), and the Firmicutes was increased. Concomitantly, significant decreases in relative abundance were observed for Corynebacterium glutamicum and Lactobacillus reuteri(P＜ 0.05). Conclusion: Quercetin and resveratrol showed some degree of protection to mice intestinal microbial communities, and increased the diversity and the abundance of the dominant flora and inhibited the growth of conditional pathogenic bacteria.

Objective: To investigate the effects of blocking lactate synthesis on the HT22 cell injuries caused by hypoxia. Methods: 2-deoxy-D-glucose (2-DG) is a non-metabolized glucose analogue that can inhibit lactate synthesis by blocking glycolysis. HT22 cells were divided into 4 groups: Control group, 2-DG group, Hypoxia group and 2-DG+Hypoxia group. The cells in control group and 2-DG treatment group were cultured in a 37℃, 5% CO₂ incubator, and thecells in hypoxia group and 2-DG + Hypoxia group were cultured in a hypoxia incubator. The concentrations of 2-DG were 2.5 and 5 mmol/L, the concentration of oxygen was 0.3%, and the treatment time was 24 h. Cell activity was detected by CCK-8 assay, the levels of lactate in cell culture medium were detected by spectrophotometry, cell morphology was observed by fluorescence staining, the level of reactive oxygen species (ROS) was detected by flow cytometry, and the activities of superoxide dismutase (SOD) and catalase (CAT) were determined by enzyme activity kits. The protein expression levels of p-p38, t-p38 and β-actin were detected by Western blot. Results: Compared with that in control group, the lactate level in culture medium and cell activity were decreased significantly (P＜0.01), the number of adherent cells was decreased, the level of ROS was increased (P＜0.01), and the enzyme activity of CAT was decreased (P＜0.05) in the 2-DG group. In the hypoxia group, the level of lactate in the culture medium was increased significantly (P＜0.01), the cell activity was decreased (P＜0.01), the number of adherent cells was decreased, the ROS levels were increased (P＜0.01), and the enzyme activities of CAT and SOD were decreased (P＜0.01 or P＜0.05). In 2-DG+Hypoxia group, the level of lactate was decreased significantly (P＜0.05), the cell viability was decreased significantly (P＜0.01), the number of cells was decreased significantly, and the ability of adhere to the wall was weakened significantly. The level of ROS was increased significantly (P＜0.01), the enzyme activities of CAT and SOD were decreased significantly (P＜0.01), the protein expression level of p-p38 was increased significantly (P＜0.05), and there was no change in t-p38. Compared with hypoxia groups, in 2-DG combined with hypoxia group, the level of lactate induced by hypoxia, the cell activity, and the enzyme activity level of CAT were decreased significantly (all P＜0.01), while the level of ROS was increased significantly (P＜ 0.01). Conclusion: Blocking lactate can reduce the cell activity level under hypoxia and aggravate the oxidative stress injury of HT22 cells. The mechanisms may be related to increasing ROS level and activating p38 signal pathway.

Objective: In the present study, we determined whether the glycogen phosphorylase(GP)inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) ameliorates pentylenetetrazole (PTZ)-induced acute seizure, neuroinflammation and memory impairment in rats. Methods: In experiment 1, rats were randomly divided into the Vehicle (n=5) and PTZ (n=5) groups, and received intraperitoneal injection of saline or PTZ (70 mg/kg), respectively. Hippocampal tissues were collected 30 min after drug injection. Western blot was used to examine the levels of GP expression. Colorimetric assay was used to determine the levels of lactate. In experiment 2, rats were randomly divided into the Vehicle+Vehicle (n=18), DAB+Vehicle (n=18), Vehicle+PTZ (n=19) and DAB+PTZ (n=18) groups. Rats received intracerebroventricular injection of PBS or DAB (50 μg/2 μl) 15 min before receiving intraperitoneal injection of saline or PTZ (70 mg/kg). Behavioural assays and the Racine scale were used to evaluate seizure severity. Western blot was used to examine the levels of targeted protein of hippocampal tissues. Novel object recognition test was used to assess memory performance. Results: ① Compared with the Vehicle group, the levels of GP and lactate in the hippocampal tissues of the PTZ group were increased significantly (both P＜0.01). ② Compared with the Vehicle+PTZ group, in the DAB+PTZ group, the levels of myoclonic body jerk latency, forelimb clonus latency and tonic-clonic seizure latency were increased significantly (all P＜0.01), while the duration of seizure and seizure scores were decreased significantly (both P＜0.01). ③ Compared with the Vehicle+Vehicle group, in the Vehicle +PTZ group, the levels of IL-1β, IL-6, TNF-α, IBA-1 and GFAP in the hippocampal tissues were increased significantly (all P＜0.01), and the discrimination index in the novel object recognition test was decreased significantly (P＜0.01). Compared with the Vehicle+PTZ group, in the DAB+PTZ group, the levels of IL-1β, TNF-α, IBA-1 and GFAP in the hippocampal tissues were decreased significantly (all, P＜0.01), while the discrimination index in the novel object recognition test was increased significantly (P＜0.01). Conclusion: DAB ameliorates PTZ-induced seizure, neuroinflammation and memory impairment in rats, suggesting that DAB may serve as a novel agent for potential clinical treatment of epilepsy.

Objective: To investigate the effects of Cathepsin K(CatK) on spatial learning and memory in rat hippocampus and its mechanisms. Methods: Twenty male SD rats were randomly divided into Control group and CatK inhibitor group(CatKⅡ group), which were microinjected with Cathepsin K specific inhibitor(0.5 μg/μl) and artificial cerebrospinal fluid in hippocampal DG area respectively with 5 days. The cultured hippocampal neuron cells were divided into control group (CON group), negative control group(NC group), siRNA interference group(siCatK group). Three re-wells were set for each group, and samples were collected 18～20 h after siRNA transfection. Morris water maze was used to evaluate spatial learning and memory function of rats. Meanwhile, dynamic changes of glutamate(Glu) content in extracellular fluid of DG region during learning and memory were observed by microdialysis and high performance liquid chromatography in conscious rats. Western blot was used to detect CatK-mediated Notch1 activation and other signal molecules. Results: Animal experiments showed that compared with the control group, the spatial learning and memory ability were decreased significantly in CatKII group, and the hippocampus protein expressions of c-Notch1, p-Akt, p-CREB and BDNF were also decreased significantly(P＜0.05); the levels of Glu in DG area of control group and CatK II group were increased significantly with Morris water maze training days, but the increase of CatK II group was significantly weaker than that of control group(P＜ 0.05). The results of cell experiment showed that the expressions of CatK, c-Notch1, p-CREB and BDNF in siCatK group were significantly lower than other groups (P＜0.05). Conclusion: CatK can affect the spatial learning and memory function of rats by activating Notch1 and its memory related signal protein in hippocampus.

Objective: To investigate the effects of 40 Hz acousto-optical stimulation on anxiety like symptoms of post-traumatic stress disorder (PTSD), with emphasis on the possible molecular mechanism stimulation. Methods: Thirty SD rats were randomly divided into three groups: Control group, PTSD group and PTSD+40 Hz group,ten rats in each group. The SPS&S model was established in the rats of the PTSD group and PTSD+40 Hz group and, then PTSD+40 Hz group rats were stimulated with 40 Hz acousto-optical stimulation for 7 days. The behavior of anxiety was tested by elevated plus maze (EPM) and open field test (OFT). The expressions of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB), synapsinⅠand postsynaptic density protein 95 (PSD95) in the rat prefrontal cortex (PFC) and hippocampus (HIP) were detected by Western blot. The mRNA transcription level of BDNF genes in the PFC and HIP was verified by real-time quantitative PCR (RT-PCR) and the distribution of BDNF in the PFC and HIP was determined by immunofluorescence. Results: Compared with the Control group, in the OFT the total distance and the time spending in the center, and in the EPM the total distance were decreased significantly (P＜0.05), the number of entering into the open arm as a percentage of the total number of entering in two arms was decreased,and the expression levels of BDNF, TrkB, PSD95, Synapsin I protein in HIP and PFC, and the mRNA expression level of BDNF were reduced significantly (P＜0.01), the immunofluorescence expression of BDNF was reduced in CA1, DG and PFC in the PTSD group rats; Compared with the PTSD group, the total distance and the time spending in the center in OFT (P＜0.05), the total distance and the number of entering into the open arm as a percentage of the total number were increased significantly (P＜0.05), the protein expression levels of BDNF, TrkB, PSD95, SynapsinⅠin the PFC and HIP, the mRNA expression level of BDNF were increased significantly (P＜0.05), and the immunofluorescence expression of BDNF was increased significantly in CA1, DG and PFC in the PTSD+40 Hz group rats. Conclusion: 40 Hz acousto-optical stimulation improves the formation of anxiety-like symptoms in rats with PTSD, which may be related to the synaptic plasticity influenced by BDNF-TrkB signaling pathway.

Objective: To investigate the effects and molecular mechanisms of miR-125b-5p on cognitive dysfunction caused by traumatic brain injury (TBI). Methods: The rats were randomly divided into control group, TBI group (model group), NC Agomir group (false negative group) and miR-125b-5p agomir group (high expression group), with 5 rats in each group. The false negative group and the high expression group were injected with NC agomir and miR-125b-5p agomir, respectively. The brain injury model was established by modified Feeney method except control group. Animal behavioral experiments were utilized for evaluation of the motor coordination, learning and memory and the degree of nerve damage in rats; and enzyme-linked immunosorbent assays (ELISA) and Western blot (WB) were used for determination of the expression levels of inflammatory factors and nerve-related factors in the hippocampus of rats in each group respectively. Finally, combined with bioinformatics, downstream target genes of miR-125b-5p were predicted and verified by reverse transcription polymerase chain reaction (RT-PCR) and WB. Results: Compared with control group, mir-125b-5p expression level, motor coordination ability, learning and memory ability, brain-derived neurotrophic factor(BDNF) and nerve growth factor(NGF) expression levels of rats in model group and false negative group were decreased significantly, the MNSS score, the expressions of interleukins (IL-1β, IL 6), tumor necrosis factor-α(TNF-α) and glial fibrillary acid protein(GFAF) were increased significantly (P＜0.01);However, compared with model group and false negative group, the above situation of rats in high expression group was opposite (P＜0.01). Bioassay showed that MMP-15 was the downstream target gene of miR-125b-5p. Compared with the control group, the expression of MMP-15 in model group and false negative group was increased significantly (P＜0.01);Compared with model group and false negative group, the expression of MMP-15 in high expression group was decreased significantly (P＜0.01) . Conclusion: miR-125b-5p can improve cognitive dysfunction induced by TBI in rats, which may be related to regulating the expression level of MMP-15, thereby inhibiting the neuroinflammatory response after TBI and promoting neuronal regeneration.

Objective: To study the effects of octadecadienoic acid (ODA) on the proliferation and apoptosis of glioma cells and its mechanisms. Methods: Cultured human glioma cells (cell density 2×10⁶ cells/L) were divided into solvent control group (DMSO, 30 μl/L), 5-FU group (10 mg/L) and octadecadienic acid groups (0.3, 0.6 and 1.2 mg/L groups). The toxicity of ODA on glioma cells was detected by trypan blue and thiazolium blue (MTT). The expression levels of P53, PI3K, P21, PKB/Akt and Caspase-9 in glioma cells were determined by enzyme-linked immunosorbent assay (ELISA). Results: ① Cell count under optical microscope showed that the inhibition rate of cell proliferation in ODA low, medium and high dose groups and 5-FU group was significantly higher than that in the solvent control group (P＜0.01), but there was no statistical significance compared with the 5-FU group (P＞0.05). ② MTT assay showed that the inhibition rate of cell proliferation was increased significantly in ODA low, medium and high dose groups and 5-FU groups (P＜0.01), compared with the solvent control group. Compared with 5-FU group, the inhibition rate of cell proliferation was increased significantly only in ODA high dose group (P＜0.01). ③ The number of G₀/G₁ phase cells in ODA low, medium and high dose groups and 5-FU group were increased significantly (P＜0.05, P＜0.01), the number of G₂/M phase cells were decreased significantly (P＜0.01), and the apoptosis rate was increased significantly (P＜0.01),compared with the solvent control group. Compared with the 5-FU group, the number of cells in G₂/M phase was decreased significantly (P＜0.01) and the apoptosis rate was increased significantly (P＜0.01) in ODA high dose group. ④ ELISA test results showed that the protein expression levels of P53, PI3K and PKB/Akt in ODA low , medium and high dose groups and 5-FU group were significantly lower than those in solvent control group (all P＜0.01), but the protein expression levels in ODA high dose group were significantly lower than those in 5-FU group (P＜0.01). The protein expression levels of P21 and caspase-9 in ODA low , medium and high dose groups and 5-FU group were significantly higher than those in solvent control group (P＜0.05, P＜0.01), but the protein expression levels in ODA high dose group were significantly higher than those in 5-Fu group (P＜0.01). Conclusion: ODA can significantly inhibit the proliferation and promote apoptosis of glioma cells. The mechanisms are related to up-regulating the levels of P21 and caspase-9 to promote apoptosis, down-regulating the levels of P53, PI3K and PKB/Akt to inhibit the cell division cycle, and reducing the activity of PI3K-Akt signal transduction pathway.

Objective: To study the effects of miR-99b-5p (non-coding RNA) in alleviating pathological neuropathic pain after paclitaxel chemotherapy by inhibiting NLRP3 inflammatory vesicle activation and the effects on neuronal cells pyrosis and apoptosis. Methods: SD rats were randomly divided into blank group, model group, agomiR-99b-5P treatment group, and agomiR-NC group, 6 rats in each group. The blank group received saline treatment as a control, the model group established a pain model induced by paclitaxel, and the rats in agomiR-99b-5p treatment group and agomiR-NC group were treated with agomiR-99b-5p and agomiR-NC injections, respectively. The expressions of miR-99b-5p in the blank group, model group, and treatment group were detected by RT-qPCR. The mechanical foot retraction threshold (MWT) of the blank group, model group, and treatment group were detected. TUNEL was used to detect the apoptosis of spinal dorsal horn cells. The levels of ROS, MDA, and SOD were detected by ELISA kits. The protein expressions of NLRP3, caspase-1, and IL-1β were detected by immunofluorescence staining. Results: Compared with the model group, the expression level of miR-99b-5p and the MWT were increased significantly in agomiR-99b-5p treatment group (P＜0.05), the apoptosis of dorsal horn cells was inhibited (P＜0.05), the level of antioxidant stress was increased in rats, the levels of ROS and MDA were decreased (P＜0.05), while the level of SOD was increased (P＜0.05). Immunofluorescence showed that the expressions of NLRP3, caspase-1, and IL-1β were inhibited by miR-99b-5p. Conclusion: miR-99b-5p can alleviate the apoptosis and pyroptosis of neurons after paclitaxel chemotherapy by inhibiting the activation of NLRP3 and improving oxidative stress in vivo.

Objective: To investigate the effects of ZnO nanoparticles (ZnO NPs) on proliferation and apoptosis of human lung epithelial cells BEAS-2B and its molecular mechanisms. Methods: BEAS-2B cells were treated with ZnO NPs at concentrations of 3, 6 and 12 μg/ml for 12 h and 24 h, the control group was not treated with ZnO NPs, each with 3 replicate wells. Cell viability was detected by CCK-8 method, and the half lethal concentration (IC₅₀) was analyzed. Then, the BEAS-2B cells were treated with ZnO NPs at selected concentrations of 3 and 6 μg/ml for 24 h respectively,each group was set with 3 replicate. Cell morphology was observed under inverted microscope. The morphology of cell nuclei was observed by Hochest33342 staining. The morphology of apoptosis was observed by AO staining and scanning electron microscopy. Cell cycle progression, cell apoptosis rate and the level of reactive oxygen species(ROS)were detected by flow cytometry. Western blot was used to detect the expression levels of Bcl-2 and Bax protein. Results: Compared with the control group, the cell viability of cells treated with ZnO NPs were decreased significantly(P＜0.01), and the IC₅₀ was 6.13 μg/ml at 24 h of drug treatment. After the cells were treated with ZnO NPs for 24 h, the levels of ROS were increased significantly(P＜0.05, P＜0.01)in 3 μg/ml, 6 μg/ml groups. The cell cycle was arrested at G2/M phase, chromatin condensation and apoptotic bodies were induced, apoptosis rate was increased significantly(P＜0.01) in 6 μg/ml group. The expression of Bcl-2 was decreased(P＜0.05), and the expression of Bax was increased (P＜0.05) in cells treated with 6 μg/ml ZnO NPs for 24 h. Conclusion: ZnO NPs induced ROS accumulation, blocked progress of cell cycle and induced cell apoptosis in BEAS-2B cells.

Objective: To investigate the mechanisms of Astragaloside Ⅳ on inhibiting apoptosis and delaying kidney aging in rats by regulating SIRT1/p53 signaling pathway. Methods: The aging model was established by subcutaneous injection of D-galactose 200 mg/(kg·d). SPF-grade healthy male SD rats were randomly divided into 4 groups: normal control group (intragastric infusion of 5 ml/(kg·d) normal saline), aging model group (intragastric infusion of 5 ml/(kg·d) normal saline), Astragaloside IV group (intragastric infusion of 40 mg/(kg·d) Astragaloside IV),and SRT1720 group( intragastric infusion of 20 mg/(kg·d) SRT1720), with 10 rats in each group. After 8 weeks, the serum samples of rats were collected to detect the levels of renal function (creatinine and urea nitrogen) and senescent associated secretory phenotype (TGF-β and IL-6) by ELISA. The renal tissues of rats were obtained for HE and Masson staining. The protein and mRNA expressions of SIRT1, p53, Bcl-2, Bax, p21 and pRb were detected by Western blot and RT-PCR. Results: Serum creatinine and urea nitrogen levels in the aging model group were higher than those in the normal group, but there was no significant difference in each group (P＞0.05). The serum levels of TGF-β and IL-6 in the aging model group were higher than those in the normal group (P＜0.05), and which in the Astragaloside IV group and SRT1720 group were lower than those in the model group (P＜0.05). There was no significant differences between Astragaloside IV group and SRT1720 group (P＞0.05). The results of pathological staining of renal tissues showed that, compared with the normal group, the renal tubules dilated, local atrophy, infiltration of inflammatory cells and proliferation of collagen fibers were observed in the aging model group. Compared with the aging model group, the pathological changes were alleviated in Astragaloside IV group and SRT1720 group. The results of Western blot and RT-PCR showed that, compared with the normal group, the protein and mRNA expressions of SIRT1 and pRb in the renal tissue of the aging group were decreased, the protein expression of Bcl-2 was decreased(P＜0.05), and the protein and mRNA expressions of p53 and p21 were increased, the protein expression of Bax was increased(P＜0.05). Compared with the aging group, Astragaloside IV and SRT1720 improved the above-mentioned indexes (P＜0.05). Conclusion: Astragaloside IV can delay kidney aging by regulating the SIRT1/p53 signaling pathway.

Objective: To investigate the protective effects and possible mechanisms of ferulic acid on diabetic nephropathy by observing the effects of ferulic acid on the level of inflammation and autophagy in glomerular mesangial cells induced by high glucose. Methods: SV40 MES 13 cells were cultured and randomly divided into the following groups: normal group (Control, 5.6 mmol/L glucose), mannitol group (Man, 30 mmol/L mannitol), high glucose group (HG, 30 mmol/L glucose), ferulic acid group (FA, 30 mmol/L glucose + 12.5, 25, 50, 100, 200 μmol/L ferulic acid), and the proliferation of SV40 MES 13 cells in each group was observed by MTT method. The levels of tumour necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1) and interleukin 1β(IL-1β)in cell supernatant were determined by enzyme-linked immunosorbent assay (ELISA). The expressions of NLRP3, IL-1β, LC3-II/I and p62 proteins in SV40 MES 13 cells were detected by Western blot. Results: ①The proliferative activity of SV40 MES 13 cells was significantly higher in the HG group compared to the control group (P＜0.01), while the proliferative activity of SV40 MES 13 cells was decreased to different degrees in the FA group compared to the HG group (P＜0.05～0.01). ②Compared to the control group, the levels of TNF-α, MCP-1 and IL-1β were increased significantly in the cell supernatant of HG group (P＜0.01). Compared with the HG group, the levels of TNF-α, MCP-1 and IL-1β were decreased significantly in the FA group (P＜0.01). ③Compared with the control group, LC3-II/Ⅰ protein expression was decreased in the HG group, while the levels of p62, NLRP3 and IL-1β protein were increased significantly (P＜0.01). Compared with the HG group, the expression of LC3-II/Ⅰ protein was elevated significantly (P＜0.05) in the FA group, while the levels of p62, NLRP3 and IL-1β protein in the FA group were decreased significantly (P＜ 0.01). Conclusion: FA can inhibit the abnormal proliferation of SV40 MES 13 cells induced by high glucose. FA can protect glomerular mesangial cells by inhibiting inflammation and increasing the level of autophagy.

Objective: To investigate the effects of silent information regulator 1 (SIRT1) in amygdala on depression-like behaviors in rats using chronic restraint stress (CRS) as a model of depression. Methods: Sixty male SD rats were randomly divided into six groups (n=10 per group): control group (Control), chronic restraint stress group (CRS), CRS + fluoxetine-treated group (CRS + FLU), CRS + saline-treated group (CRS + NaCl), CRS + SIRT1-overexpression group (CRS + AAV-SIRT1), and CRS + empty vector group (CRS + AAV-EGFP). Except for the control group, rats from the other groups were exposed to chronic restraint stress for 21 days. After the modeling, rats in fluoxetine-treated group and saline-treated group were, respectively, treated with fluoxetine (10 mg/kg) or saline (10 mg/kg) by gavage every day for 3 weeks; AAV-SIRT1 or AAV-EGFP was, respectively, stereotaxically injected into the amygdala of rats in SIRT1-overexpression group and empty vector group, and the virus was expressed for 3 weeks. Rats in normal control group and CRS model group were not given any drug treatment. The depression-like behaviors of rats in each group were evaluated by sugar preference test (SPT), open field test (OFT) and forced swimming test (FST). SIRT1 expression in amygdala of rats was assessed by using immunoblot blotting. The number of SIRT1-positive cells in amygdala of rats was detected by immunofluorescence technique. Results: Compared with the normal control group, the level of SIRT1 protein and the number of SIRT1⁺ cells in amygdala of the CRS-exposed rats were decreased significantly (P＜0.01), and CRS-exposed rats showed a significant decrease in sucrose preference (P＜0.01), less total horizontal distance (P＜0.01) and less time entered the center field (P＜0.01) in the OFT, a significant increase in the immobility time of the FST (P＜0.01). Fluoxetine treatment (P＜0.05, P＜0.01) or SIRT1 overexpression (P＜0.01) partially reversed the down-regulation of SIRT1 protein and SIRT1⁺ cells in amygdala of CRS-exposed rats and significantly improved the depression-like behaviors of CRS rats. Conclusion: Fluoxetine treatment partially reversed the down-regulation of SIRT1 level and the number of SIRT1⁺ in CRS rats, and significantly improved the depression-like behaviors. The antidepressant effect of fluoxetine treatment may be related to the up-regulation of SIRT1 in the amygdala of CRS-exposed rats.

Objective: Through aerobic exercise and diet intervention on obese mice, the effects of exercise and diet intervention on testicular oxidative stress and p38MAPK-NF-κB pathway were investigated in obese mice. Methods: Seventeen C57BL/6J mice were randomly divided into a normal diet group (ND), and 37 mice were divided into a high-fat diet group (HFD), the high-fat diet accounted for 40% of fat. After 12 weeks of feeding, 3 obesity-resistant mice were excluded from the HFD group, and the remaining 34 were successfully modeled. The mice in ND group were then divided into normal diet control group (NC, n=8) and normal diet and exercise group (NE, n=9). The mice in HFD group were divided into obese high-fat diet control group (OC, n=8), obese high-fat diet and exercise group (OE, n=9), obese normal diet group (ONC, n=8), and obese normal diet and exercise group (ONE, n=9). Each group continued to feed for 8 weeks, and the NE, OE and ONE groups performed treadmill exercise for 8 weeks at a speed of 20 m/min, 60 min/d, 6 d/week. Blood and testicular tissue samples were collected 36～40 h after the last exercise. Serum testosterone and testicular oxidative stress (MDA, T-SOD, T-AOC) levels were detected by ELISA, and testicular p38MAPK-NF-κB levels were detected by RT-PCR and Western blot. Results: Compared with the NC group, the body fat parameters, testicular MDA and testicular p38MAPK-NF-κB mRNA and protein levels in the OC group were increased significantly (P＜0.01), while the levels of testicular SOD, testis coefficient and blood testosterone were decreased significantly (P＜0.01); the body fat parameters of the mice in the NE group were decreased significantly (P＜0.05), and the serum level of testosterone was increased significantly (P＜0.01). Compared with the OC group, the body fat parameters, testicular MDA and testicular p38MAPK-NF-κB mRNA and protein levels were decreased significantly in the OE group (P＜0.05 or 0.01), and the testicular SOD and blood testosterone levels were increased significantly (P＜0.01); Body fat parameters, testicular MDA and testicular p38MAPK-NF-κB mRNA and protein levels were decreased significantly in ONC group (P＜0.01), while testicular SOD level and testis coefficient were increased significantly (P＜0.05); Body fat parameters, testicular MDA and testicular p38MAPK-NF-κB mRNA and protein levels of mice in ONE group were decreased significantly (P＜0.01), while testicular SOD, testis coefficient and blood testosterone levels were increased significantly (P＜0.01). Conclusion: Obesity induces oxidative stress in the testis of mice, up-regulates the level of p38MAPK-NF-κB, and reduces the level of blood testosterone; exercise, diet and exercise*diet interventions can reduce testicular oxidative stress and down-regulate testicular p38MAPK-NF-κB levels by reducing body fat.

Objective: To investigate the effects of glucocorticoid receptor agonists on hyperalgesia in rats with neuropathic pain (NPP) by regulating nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/interleukin-1β (IL-1β) pathway and its mechanisms. Methods: Forty SD rats were divided into control group, NPP model group, NPP treated with NLRP3 inhibitor group and dexamethasone treatment group with 10 rats in each group. The NPP rat model was induced by vincristine. The model group was established according to the above method, the NLRP3 inhibitor group was treated with NLRP3 inhibitor (MCC950) after the NPP model was established, and the treatment group was treated with glucocorticoid receptor agonist (dexamethasone) after the model was established according to the design. The rats of the control group were given the same amount of normal saline. After 7 days of intervention, the mechanical pain threshold, thermal pain threshold, morphological changes of spinal dorsal horn, pain factors (prostaglandin E2 (PGE2), substance P (SP), 5-hydroxytryptamine (5-HT)), inflammatory factors (interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6)), and NLRP3/IL-1β protein expressions were determined and compared among the four groups. Results: Compared with the model group, the pathological changes of spinal dorsal horn neurons in NLRP3 inhibitor group and treatment group were alleviated significantly, the arrangement of neurons was tended to be close, the number of neurons was gradually returned to normal, and the pyknosis of neurons was decreased. Compared with the control group, the mechanical pain threshold and thermal pain threshold of the model group were decreased significantly (P＜0.05), and the expressions of inflammatory factors, pain factors and NLRP3, IL-1β protein were increased significantly (P＜0.05); compared with the model group, the mechanical pain threshold and thermal pain threshold of the NLRP3 inhibitor group and the dexamethasone treatment group were increased significantly (P＜0.05), and the expressions of inflammatory factors, pain factors and NLRP3, IL-1β protein were decreased significantly (P＜ 0.05). The difference between NLRP3 inhibitor group and treatment group was not statistically significant (P＞0.05). Conclusion: Glucocorticoid receptor agonists may reduce the hyperalgesia of neuropathic pain rat model by down regulating NLRP3/IL-1β pathway, which may be the mechanism of dexamethasone on antiinflammatory of analgesia in early stage of NPP.

Objective: To investigate the effects of Angelicae Sinensis Radix (ASR) on cyclic adenosine monophosphate (cAMP) /exchange protein activated by cAMP (Epac) signaling pathway in the treatment of chronically infected cough mice with Yin deficiency syndrome. Methods: Mice were randomly divided into blank control group, model control group, positive control group and ASR group (n=8). The chronic cough mouse model of hyperreactive and infected airway with Yin deficiency syndrome was established with fumigation (once a day, 30 days in total), lipopolysaccharide nasal drip (every 3 days 10 μl, 10 times in total), intragastric administration of thyroid gland (120 mg/kg, once a day, a total of 15 days) and inhalation of ammonia (3 min / time × 10 times). On the basis of observing eating and drinking water, body weight and autonomic activities, the effects of ASR on metabolic level, autonomous activities, antitussive effect, cell factor in bronchoalveolar lavage fluid (BALF) brain tissue 5-HT and lung tissue related active factors(SP, PGP9.5, cAMP, Epac1) were detected. Results: ASR could significantly restrain cough, alleviate the pathological changes of bronchioles, reduce the contents of IL-4, IL-13, TNF-α in BALF and the levels of SP, PGP9.5, cAMP and Epac1 in lung tissues, increase the content of 5-HT in brain tissue (P＜0.05, 0.01). Conclusion: ASR has some effects on restraining cough and one of its mechanisms is to down-regulate cAMP/Epac signaling pathway, to alleviate airway neurogenic inflammation and reduce sensitivity of cough neural pathway.

Objective: To investigate the effects of Butylphthalide on the expressions of HMGB1 and RAGE in frontal lobe of rats after chronic sleep deprivation. Methods: Chronic sleep deprivation and butylphthalide treatment was performed in Sprague Dawley(SD)rats and the rats were divided into three groups (n＝6): platform control group, chronic sleep deprivation group and chronic sleep deprivation + butylphthalide intervention group. Rats suffering chronic sleep deprivation were put in multiple platforms box for 18 h per day and sleep deprivation lasted for 28 days. Rats in butylphthalide intervention group were intraperitoneally injected with butylphthalide 100 mg/(kg·d) for 14 days after sleep deprivation. After collecting brains, high-mobility group box (HMGB1) and nuclear transcription factor kappB (NF-κB)p65 were detected by immunohistochemistry. The expression of HMGB1, silent information regulator of transcription 1 (SIRT1), receptor for advanced glycation end-products (RAGE) and NF-κB in frontal lobe were determinated by Western blot. Results: Compared with platform control group, the expression levels of HMGB1, RAGE and nuclear NF-κB p65 were increased significantly, while the expression of SIRT1 was decreased siginificantly in frontal lobe of chronic sleep deprivation group (all P＜0.05). Compared with chronic sleep deprivation group, the expression levels of of HMGB1, RAGE and nuclear NF-κB p65 were decreased significantly, while the expression of SIRT1 was increased significantly in chronic sleep deprivation + butylphthalide intervention group (all P＜0.05). Conclusion: Butylphthalide can inhibit HMGB1/RAGE/NF-κB pathway in frontal lobe of rats after chronic sleep deprivation by changing the expression of HMGB1 and RAGE, and reducing the nuclear translocation of NF-κBp65.

Objective: To investigate the electrophysiological properties of pyramidal neurons in mouse motor cortex during the early postnatal development. Methods: Thirty-six mice were randomly divided into postnatal 1-, 2-, 3-Week and 1-, 2-,3-Month groups (n=6). Membrane properties, action potentials (AP) and spontaneous excitatory postsynaptic currents (sEPSCs) of motor cortex pyramidal neurons were recorded to evaluate the changes in the intrinsic electrophysilogical characteristics by using whole cell patch clamp. Pyramidal neurons and interneurons were distinguished according to the AP firing patterns. Results: Comparing with interneurons, pyramidal neurons exhibited regular spiking (RS) with smaller frequency. During the period of postnatal 1 Week-3 Months, some of the intrinsic membrane properties of motor cortex pyramidal neurons changed. Compared to the 1-Week mice, the resting membrane potential (RMP) of 2-Week decreased significantly (P＜0.01), and the membrane input resistance (R_in) of 1-Month got a hyperpolarization (P＜0.01), and they showed no significant change in the next period, while the membrane capacitance (Cm) showed no significant changes during the whole postnatal development. The AP dynamic properties changed significantly during this period. Compared to the 1-Week mice, the absolute value of the AP threshold and the AP amplitude of the 3-Week increased significantly (P＜0.01), while the spike half width of the 2-Week decreased substantially (P＜0.05), and they showed no significant change in the next period. The sEPSCs frequency and amplitude of 1- Month increased significantly compared to the 1-Week mice(P＜0.01), while during the period of next 1 Month-3 Months, the amplitude and frequency showed no significant change. Conclusion: These results suggest that the motor cortex pyramidal neurons have time-specific eletrophysilogical properties during the postnatal development. The electrophysiological properties can be used as a functional index to detect the degree of neurons maturity, and as a marker to distinguish the pyramidal neurons and interneurons.

Objective: To investigate the effects of glutamate aspartate transporter (GLAST)deletion on the normal auditory function of mice. Methods: We hybridized GLAST^+/- mice with C57BL/6J background and identified the genotypes of their offspring by agarose gel electrophoresis. 9-10-week-old mice were selected to detect the expression of GLAST protein in the cochlea by immunofluorescence staining and to verify the knockout results(n=3). The changes in weight from 7 days to 30 days after birth and the 30-day body length of male and female mice were compared(n=8). The auditory brainstem response(ABR) was used to detect the auditory threshold and the amplitude of wave I in 9-10-week-old male and female mice(n=5). Results: Male GLAST^-/- mice had shown significantly lower weight and body length compared to male GLAST^+/+ and GLAST^+/- mice(P＜0.01), and male GLAST^-/- mice showed significant differences compared to GLAST^+/+ from P7 to P30 statistical time. Male GLAST^-/- mice exhibited a significant reduction in weight after P15 compared to male GLAST^+/- mice. In contrast, no significant differences in weight and body length were observed in female GLAST^-/- mice compared with female GLAST^+/+ and GLAST^+/- mice. There was no difference in the hearing threshold detected by ABR between the three genotypes in both male and female mice, but the amplitude of wave I in GLAST^-/- mice was significantly lower than that in male GLAST^+/+ mice(P＜0.01). In contrast, the amplitude of wave I in females was reduced throughout the stimulus intensity but was most significant only at high-intensity stimulation (e.g.80 dB, 90 dB) (P＜0.05). Conclusion: GLAST knockout affects the normal growth and development of male mice, and decreases the amplitude of wave I, but do not change the threshold, suggesting that GLAST knockout may lead to synaptic pathological changes, and there are gender differences in this effect.

Objective: To investigate the effects of mitochondrion-targeted cyanine fluorescent small molecule IR-61 on cardiac injury induced by exhaustive exercise in rats. Methods: Thirty-six adult male SD rats were randomly divided into 3 groups(n=12),control group (Ctrl), exhaustive exercise group (EE) and IR-61+ exhaustive exercise group (IR-61+EE). IR-61+EE group were intraperitoneally injected with 2 mg/kg IR-61 at the same time on day 1, 4 and 7. One hour after the end of the last drug administration, the two exhaustive exercise groups were subjected to exhaustive exercise modeling. The rats were placed on an animal treadmill with a slope of 0° at a speed of 10~15 m/min to coordinate their limbs running posture, and then ran at a speed of 25~30 m/min until exhaustion about 15 minutes later. After the animal models established, ECG was recorded by physiological recorder, myocardial injury was observed by light microscope, mitochondrial injury was observed by transmission electron microscope, myocardial cell apoptosis was detected by TUNEL method, markers of myocardial injury were detected by ELISA, and myocardial mitochondrial respiration rate was measured by high-resolution Oxygraph-2K mitochondrial instrument. Results: ① Compared with Ctrl group, heart rate was increased, PR interval was shortened, QRS interval was prolonged, QTc was prolonged and ST segment was depressed significantly in EE group (P＜0.05). In EE group, myocardial fiber fracture and mitochondrial inner chamber swelling were obvious, mitochondrial crest was fuzzy, mitochondrial outer membrane was incomplete, and a large number of mitochondrial rupture and fusion were visible. In EE group, TUNEL staining cells were abundant, chromatin concentration and marginalization, nuclear membrane lysis, chromatin fragmentation into massive apoptotic bodies, apoptosis score increased (P＜0.05). The levels of creatine kinase isoenzyme-MB (CK-MB), cardiac troponin I(cTn-I) and N-terminal B-type natriuretic peptide (NT-proBNP) were increased in EE group (P＜0.05). Basal respiration rate, oxidative respiration rate of fatty acids and respiration rate of complex Ⅰ, Ⅱ and Ⅳ were all decreased (P＜ 0.05). ② Compared with EE group, the heart rate in IR-61+EE group was increased, PR interval was prolonged, QRS interval was shortened, QTc was shortened, ST segment was not significantly depressed (P＜0.05). In IR-61+EE group, myocardial fiber arrangement was loose, no obvious fracture was observed, mitochondrial inner ventricle was swelling, mitochondrial outer membrane was intact, TUNEL stained cells and unstained cells were observed, the overall morphology was more similar to Ctrl group. Apoptosis index was decreased (P＜0.05), the levels of CK-MB and cTn-I were decreased in IR-61+EE group (P＜0.05). The oxidative respiration rate of fatty acids and the respiration rate of complex Ⅱ and Ⅳ were increased (P＜0.05). Conclusion: Mitochondrion-targeted cyanine fluorescent small molecule IR-61 can improve cardiac electrical activity, reduce myocardial cell injury and mitochondrial injury, reduce myocardial cell apoptosis, and improve the myocardial mitochondrial energy metabolism condition in exhausted rats.

Objective: To investigate the effects of p53 upregulated modulator of apoptosis (PUMA) on the apoptosis of H9C2 cardiomyocytes induced by high glucose and its mechanisms. Methods: H9C2 cardiomyocytes were treated with 5.5mmol/L (control group) or 35 mmol/L glucose (HG group) for 6 h, 12 h, 24 h or 48 h respectively to induce apoptosis, each group sets 5 multiple wells. Apoptosis was tested by TUNEL assay. PUMA mRNA was measured by RT-PCR and protein expression was measured by Western blot assay. The mitochondrial membrane potential was detected by JC-1 method. The expressions of cleaved caspase-3 and cytochrome C (Cyt C) protein in mitochondria and cytoplasm were determined by Western blot assay. H9C2 cardiomyocytes were randomly divided into four groups, control group (5.5 mmol/L), HG (35 mmol/L) group, HG+si-scramble group(si-scramble treatment for 24 h, then 35 mmol/L high glucose treatment for 24 h) and HG-si-PUMA group (si-PUMA treatment for 24 h, then 35mmol/L high glucose treatment for 24 h). Si-PUMA was transfected into cardiomyocytes and the effects of PUMA on high glucose-induced apoptosis were studied. Results: Compared with the control group, high glucose increased cardiomyocyte apoptosis and enhanced PUMA mRNA and protein expressions significantly (P＜0.05 or P＜0.01). Cell injury and increased PUMA expression were time-dependent and there was no significant difference between the high glucose 24 h group and the high glucose 48h group. The following experiment used high glucose 24 h as the stimulation time. The cardiomyocytes transfected with si-PUMA to inhibit PUMA expression had decreased apoptotic rate and cleaved caspase-3, increased mitochondria membrane potential and decreased Cyt C release (P＜0.05 or P＜0.01). There were no significant differences between the HG+si-scramble group and the high glucose group (P＞0.05). Conclusion: PUMA mediates high glucose-induced cardiomyocyte apoptosis suggesting PUMA may be an important target gene of diabetic cardiomyopathy.

Objective: To investigate the effects of sphingosine-1-phosphate (S1P) on cardiac hypertrophic response in H9c2 cells. Methods: H9c2 cells were randomly divided into four groups: normal control group, S1P (1 μmol/L) treated group, Phenylephrine (PE) (100 μmol/L) treated group, PE (100 μmol/L) treated group combined with S1P (1 μmol/L) treatment. Each group has 3 duplicated wells. After 24 hours, the size of H9c2 cells in each group was detected by Actin-Trakcer Green immunofluorescence staining. Transcriptional levels of hypertrophic markers ( ANP, BNP and β-MHC) in H9c2 cells were determined by real-time PCR. Western blot was performed to examine the expression level of ANP in each group. Then H9c2 cells were randomly divided into five groups: normal control group, PE (100 μmol/L) treated group, PE (100 μmol/L) with S1P low-dose (0.1 μmol/L) treated group, PE (100 μmol/L) with S1P middle-dose (1 μmol/L) treated group and PE (100 μmol/L) with S1P high-dose (10 μmol/L) treated group. Each group has 3 duplicated wells. After 24 hours, Western blot was performed to examine the expressions of phosphorylated Janus kinase 2 (JAK2) and signal transducers and activators of transcription 3 (STAT3) under low, medium and high concentrations of S1P. Each experiment was repeated three times. Results: Compared with normal control group, the surface area of H9c2 cells in PE group was increased significantly (P＜0.05), meanwhile, the transcription levels of ANP, BNP and β-MHC were increased significantly (all P＜0.05), and the expression of ANP was also increased significantly (P＜0.05) in PE group. While compared with PE group, the surface area of H9c2 cells in PE + S1P group was decreased significantly (P＜0.05), the transcription levels of ANP, BNP and β-MHC and the expression of ANP were also decreased significantly (all P＜0.05) in PE + S1P group. After treated with PE and different concentrations of S1P, the expressions of p-JAK2 and p-STAT3 were increased significantly compared with the normal control group and PE group (P＜0.05), in a dose-dependent manner. Conclusion: S1P could protect H9c2 cells against hypertrophic response induced by PE, which may be achieved by activating JAK2/STAT3 signal pathway.

Objective: To investigate the effects of ferrostatin-1 (Fer-1) on cardiomyocyte hypoxia/reoxygenation injury and its mechanisms. Methods: The original generation of myocardial cells were extracted from 1～3 d newborn SD rats, which were randomly divided into normal control group (control), hypoxia reoxygenation (H/R) group and hypoxia reoxygenation + iron death inhibitors group (H/R + Fer-1). After 52 h of culture, cells in H/R group were added with 4 mmol/L Na₂S₂O₄ solution. After 1 h of hypoxia, cells were reoxygenated with DMEM medium containing 10% calf serum for 3 h.The H/R+ Fer-1 group was pretreated with Fer-1 (2 μmol/L) for 24 h and then subjected to hypoxia and reoxygenation. The release rate of lactate dehydrogenase (LDH) was measured by UV spectrophotometry, the cell survival rate was measured by CCK-8 method, SOD was measured by xanthine oxidase method, MDA was measured by chemical coloration, and the changes of mitochondrial membrane potential and reactive oxygen species (ROS) were observed by immunofluorescence. Western blot was used to detect the expressions of ACSL4 and GPX4. Results: Compared with the control group, the cell activity, SOD release and MMP level were decreased (P＜0.05), the levels of LDH, MDA and ROS were increased (P＜0.05), the protein expression of ACSL4 was increased (P＜0.05), and the protein expression of GPX4 was decreased (P＜0.05) in H/R group. Compared with the H/R group, the cell activity, SOD release and MMP level were increased (P＜0.05), the level of LDH, MDA and ROS were decreased (P＜0.05), the protein expression of ACSL4 was decreased (P＜0.05), and the protein expression of GPX4 was increased (P＜0.05) in H/R+Fer-1 group. Conclusion: Fer-1 can inhibit the production of intracellular reactive oxygen species by regulating ACSL4 and GPX4, thereby alleviating the hypoxia and reoxygenation injury of primary cardiomyocytes caused by iron death.

Objective: To investigate the effects and related molecular mechanisms of Astragalin on undifferentiated gastric cancer cell HGC-27. Methods: Astragalin was used to treat HGC-27 cells, the cell proliferation activity was detected by CCK-8 method, the cell morphology was observed under inverted microscope, hoechst 33342 and JC-1 staining were used to observe the changes of nucleus formation and mitochondrial membrane potential, the cell cycle and apoptosis rate were detected by flow cytometry, the reverse transcription level of the gene was analyzed by the second-generation sequencer. Results: Astragalin inhibited the proliferation of HGC-27 significantly (P＜0.01), down-regulated mitochondrial membrane potential, induced cell apoptosis, blocked the cell cycle in G1 prophase. At the same time, Astragalin up-regulated the transcription levels of genes bax and bad, down-regulated the transcription levels of genes egf, egfr, pik3cb, pdk1, akt3 and bcl-2. Western blot analysis also showed that the expressions of PI3K and Akt protein were decreased, and the proportion of Bax and BCL-2 protein was increased significantly (P＜0.01). Conclusion: The apoptosis of undifferentiated gastric cancer cell line HGC-27 can be induced by Astragalin through inhibition of EGFR/PDK/Akt signaling pathway, and the cell cycle can be blocked in G1 phase, which has a certain therapeutic effect on undifferentiated gastric cancer.

Objective: To investigate the effect of α-lipoic acid in ameliorating liver injury in rats with type 2 diabetes mellitus via activating adenosine 5'-monophosphate-activate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway. Methods: The T2DM rat models were established by feeding with high-fat, high-sucrose diet and intraperitoneal injection of 27.5 mg/(kg·d) streptozotocin. The 32 rats with T2DM were randomly divided into 4 groups: T2DM group, α-lipoic acid group (LA), Compound C group (Comp C, an inhibitor of AMPK) and LA+Comp C group, with 8 rats in each group. Additionally, 8 Sprague-Dawlay (SD) rats without diabetes were set as normal control. The rats received α-lipoic acid at a dosage of 100 mg/(kg·d) or Compound C at a dosage of 20 mg/(kg·d) by intraperitoneal injection for 8 weeks as needed. The levels of relevant biochemical indexes were detected. The weight of liver was recorded to calculate liver weight index (LWI), and the pathological changes of liver tissues were detected by light and electron microscopy. The levels of AMPK, p-AMPK, mTOR, p-mTOR in rat liver were detected by Western blot. Results: Compared with control group, the levels of LWI, homeostasis model assessment of insulin resistance, fasting blood glucose, alanine transaminase, aspartate transaminase, gamma glutamyl transferase and triglyceride in T2DM group were increased significantly (all P＜0.05). The liver tissue lesions were more serious and hepatic steatosis grade was higher. The expression of p-AMPK was decreased (P＜0.05) and the expression of p-mTOR was increased significantly(P＜0.05). α-lipoic acid could reverse the above-mentioned changes, ameliorate insulin resistance (all P＜0.05), protect the structure and function of liver, and activate the AMPK/mTOR pathway (P＜0.05). The protection of α-lipoic acid was weakened by the inhibition of AMPK with Compound C (P＜0.05). Conclusion: α-lipoic acid could protect the liver of rats with T2DM by activating AMPK/mTOR pathway.

Objective: To study the effects of exogenous calcium-load on promoting muscle-derived IL-6 secretion, and regulating AMPK and p38MAPK signal pathway to improve insulin resistance. Methods: C2C12 cell lines and palmitic acid-induced insulin resistance C2C12 cell lines were selected as the experimental objects. Preliminary experiment was aimed to determinate the glucose concentrations of culture solutions and observe contraction status of cells under microscope following different calcium concentrations culture 24 h. In the first official experiment, cells were divided into four groups: control group (A group, normal culture solution), IR group(B group, 0.6 mmol/L palmitic acid culture cells 24 h), 1 000 ng/ml IL-6 culture IR B group cells 48 h(IL-6+IR group) and IL-6 shRNA culture A group cells (IL-6shRNA group). In the second official experiment, cells were divided into three groups: IR group(A group), 100 μmol/L CaCl₂ culture IR group cells 48 h(CaCl₂+IR group) and 100 μmol/L CaCl₂ and IL-6shRNA co- culture IR group cells 48 h(CaCl₂+IL-6shRNA+IR group). The expression levels of GLUT4 mRNA and IL-6 mRNA were measured by real-time PCR, the protein expression levels of p-AMPK, p-p38MAPK, p-IRS-1 and p-PI-3K were measured by Western blot. Results: Preliminary experiment results showed that compared with 0 μmol/L CaCl₂ group, the glucose concentrations were decreased significantly after cells treated with CaCl₂, at different concentrations. The cell contractions were observed under microscope and the cell contraction was most obvious treated with 100 μmol/L CaCl₂. The first official experiment results showed that compared with IR group, the contents of p-AMP-activated protein kinase(p-AMPK), p-insulin receptor substrate 1(p-IRS-1), p-phosphoinositide-3 kinase(p-PI-3K), the expression level of glucose transporter 4(GLUT4) mRNA and the glucose uptake of IL-6+IR group were increased significantly(P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was decreased significantly (P＜0.01) ; Compared with control group, the expression levels of p-AMPK, P-IRS-1, p-PI-3K, the expression level of GLUT4 mRNA and the glucose uptake of IL-6shRNA group were decreased significantly (P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was increased significantly (P＜0.01). The second official experiment results showed that compared with IR group, the expression levels of p-AMPK, P-IRS-1, p-PI-3K, the level of GLUT4 mRNA of CaCl₂+IR group were increased significantly (P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was decreased significantly (P＜0.01); Compared with CaCl₂+IR group, the contents of p-AMPK, P-IRS-1, p-PI-3K, the expression level of GLUT4 mRNA and the glucose uptake of CaCl₂+IL-6 shRNA+IR group were decreased significantly (P＜0.05 or P＜0.01), the p-p38MAPK protein expression level was increased significantly (P＜0.01). Conclusion: Exogenous Ca-load can stimulate muscle cells contraction, and exercise-induced IL-6 improves insulin resistance by activating AMPK, PI-3Kand inhibiting p38MAPK signal pathway.

Objective: To investigate the effects of paeonol on low-density lipoprotein-induced human vascular endothelial cell injury and its molecular mechanisms. Methods: Human umbilical vein endothelial cells (HUVECs) were divided into 9 groups, normal control (NC) group, ox-LDL group (100 ng/L ox-LDL), low, medium, and high-dose paeonol groups (60 μmol/L, 120 μmol/L, 240 μmol/L paeonol+100 ng/L ox-LDL), ox-LDL+small interfering RNA negative control (si-NC) group, ox-LDL+circ_0003204 small interfering RNA (si-circ_0003204) group, middle dose group+ox-LDL+circ_0003204 overexpression negative control (pcDNA-NC) group, middle dose group+ox-LDL+circ_0003204 overexpression (pcDNA-circ_0003204) group, three replicate wells in each group. MTT flow cytometry, and Western blot were used to detect cell proliferation, apoptosis and protein (CDK2, Bcl2, p27, Bax) expressions, respectively. Malondialdehyde (MDA) and superoxide dismutase (SOD) kit were used to detect MDA content and SOD activity; real-time quantitative PCR (RT-qPCR) was used to detect the expression of circ_0003204. Results: Compared with the NC group, the proliferation activity, protein expressions of CDK2 and Bcl2, and SOD activity of HUVECs in the ox-LDL group were decreased significantly (P＜0.05), and the apoptosis rate, protein expressions of p27 and Bax, MDA content, and circ_0003204 expression were increased significantly (P＜ 0.05). Compared with the ox-LDL group, the proliferation activity, protein (CDK2, Bcl2) expressions and SOD activity of HUVECs in the low, medium and high dose paeonol groups were increased significantly (P＜0.05), and the apoptosis rate, protein (p27, Bax) expressions, MDA content And circ_0003204 expression were decreased significantly (P＜ 0.05). Compared with ox-LDL+si-NC group, the proliferation activity, protein (CDK2, Bcl2) expressions, SOD activity of HUVECs in ox-LDL+si-circ_0003204 group were increased significantly (P＜0.05), the apoptosis rate, protein (p27, Bax) expressions, and the content of MDA were decreased significantly (P＜0.05). Compared with the middle-dose+ox-LDL+pcDNA-NC group, the HUVECs proliferation activity, protein (CDK2, Bcl2) expressions, and SOD activity in the middle-dose+ox-LDL+pcDNA-circ_0003204 group were decreased significantly (P＜0.05), and the levels of circ_0003204, apoptosis rate, protein (p27, Bax) expressions and MDA content were increased significantly (P＜0.05). Conclusion: Paeonol can inhibit ox-LDL-induced apoptosis and oxidative stress of human umbilical vein endothelial cells, and alleviate human umbilical vein endothelial cell injury. The mechanism of action may be related to the down-regulation of circ_0003204 expression.

Objective: To investigate whether probenecid (PROB) could improve the proliferation and migration ability of rats' pulmonary artery smooth muscle cells induced by platelet-derived growth factor-BB (PDGF-BB). Methods: Primary pulmonary artery smooth muscle cells (PASMCs) of SD rats were cultured in vitro, and were randomly divided into control group (CON group), PDGF-BB group (10 ng/ml PDGF-BB treatment for 24 h) and PDGF-BB+PROB group (10 ng/ml PDGF-BB and 200 μmol/L PROB treatment for 24 h, PROB is a specific blocker of pannexin-1). CCK-8 method was used to select the suitable intervention concentrations of PROB and PDGF-BB, and to detect the proliferation of PASMCs in each group. The migration ability of PASMCs was detected by Transwell^TM assay and cell scratch test. Immunofluorescence cytochemistry and Western blot were used to detect the protein expressions and distribution of osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) in PASMCs. Results: Compared with CON group, the migration and proliferation ability of PASMCs in PDGF-BB group were enhanced (P＜0.05). After treated with PROB, the migration and proliferation ability of PASMCs in PDGF-BB+PROB group were decreased significantly (P＜0.05). Compared with CON group, the expression and protein levels of OPN and PCNA in PDGF-BB group were increased significantly (P＜0.05), while the expression and protein levels of OPN and PCNA in PDGF-BB+PROB were decreased significantly (P＜0.05). Conclusion: Probenecid inhibits the migration and proliferation of PDGF-BB-induced PASMCs by blocking Pannexin-1.

Objective: To investigate the injury of cyanate on the pulmonary function and morphology of C57/BL6N mice. Methods: Forty male C57/BL6N mice were randomly divided into two groups: normal control group (20 mice) and cyanate group (20 mice). Mice were exposed to 100 mmol/L cyanate feeding for 4 weeks, and pulmonary Raw (Resistance in Air Way) was measured at the beginning and end of the experiment. The mice were sacrificed at the end of the fourth week of the experiment, and the lung tissues were collected for pathological observation and molecular detection of E-Cadherin and Fibronectin. Well-growing A549 cells in logarithmic growth phase were treated with cyanate at the concentrations of 0, 0.25, 0.5 and 1 mmol/L for 24 h, and the cell viability was detected by CCK8 method; reactive oxygen species ROS fluorescent probe (DCFH-DA) was used to detect the changes of ROS levels, and expressions of E-Cadherin and Fibronectin in cells and pulmonary tissues were detected by Western blot. Results: At the beginning of the experiment, the pulmonary airway resistance values of the mice in the normal control group and the cyanate group were (1.82±0.76)cmH₂O/(L·s) and (1.85±0.78)cmH₂O/(L·s), respectively, with no significant difference. Four weeks later, the pulmonary airway resistance value of mice in the cyanate group was increased to (4.86±0.87)cmH₂O/(L·s) (P＜0.01). The HE staining showed that, compared with the normal control group, the injured alveolar structure, the thickened tracheal wall and the significantly proliferated pulmonary interstitial tissue were observed in the cyanate group. The Masson staining showed that elastic fibers were deposited around the trachea of mice in the cyanate group. The results of CCK8 assay for the viability of A549 cells showed that 0.5 mmol/L cyanate exposure could reduce the viability (P＜0.01). The immunofluorescence staining showed that cyanate could increase ROS level in A549 cells by producing green fluorescence in a concentration-dependent manner. The results of Western blotting showed that 0.5 mmol/L of cyanate treatment on A549 cells could reduce the expression of E-Cadherin (P＜0.01) with increasing concentration of cyanate. The expression level of Fibronectin in A549 cells was increased with the increasing cyanate concentration, and there was a significant difference (P＜0.01) on 1 mmol/L cyanate. Western blot results of lung showed the decreasing expression of E-Cadherin (P＜0.01) and increasing expression of Fibronectin (P＜0.01) in cyanate mice. Conclusion: Pathological concentrations of cyanate can induce the proliferation of pulmonary interstitial tissue, fibrous deposition, and increased pulmonary airway resistance in mice, which may be related to damaged pulmonary epithelial cell viability, enhanced ROS production, and induced pathologic changes of extracellular matrix by cyanate.

Objective: To investigate the effect of hydroxysafflower yellow A (HSYA) on pulmonary fibrosis induced by bleomycin in mice and transforming growth factor β 1(TGF-β1) /Smad signal transduction pathway regulation. Methods: The pulmonary fibrosis model was prepared by intranasal injection of bleomycin 50 μl (15 mg/kg). ICR mice were randomly divided into control group, model group, HSYA group(6 mg/kg) and dexamethasone (Dex) group(3 mg/kg), with 15 mice in each group. From the next day of modeling, HSYA and Dex groups were intraperitoneally injected with corresponding drugs, while the control group and model group were intraperitoneally injected with the same volume of normal saline, once a day, for 28 consecutive days. After 4 weeks, the mice were sacrificed and the lungs were collected. HE and Masson staining were used to observe the pathological damage of lung tissue; Immunohistochemistry, RT-qPCR and Western blot were used to detect the expressions of TGF-β1/Smad signaling pathway in lung tissues. Results: Compared with the control group, the model group showed severe alveolitis and pulmonary fibrosis. The mRNA and protein expressions of TGF-β1 and Smad3 in lung tissues were increased significantly (P＜0.01), while the mRNA and protein expressions of Smad7 were decreased significantly (P＜0.01). Compared with the model group, the degree of alveolitis and pulmonary fibrosis in the HSYA and Dex groups was reduced significantly. The mRNA and protein expressions of TGF-β1 and Smad3 in lung tissues of HSYA and Dex groups were decreased significantly (P＜0.01), while the mRNA and protein expressions of Smad7 were increased significantly(P＜0.01). Conclusion: HSYA can alleviate the pathogenesis of pulmonary fibrosis, and its mechanism may be related to the regulation of TGF-β1/Smad signaling pathway.

Objective: To investigate the effects of chronic intermittent hypoxia (CIH) on the expression of transforming growth factor-β (TGF-β), P-samd3, serum laminin (LN) and hyaluronidase (HA) in mouse lung tissues and the protective effects of Bu Zhong Yi Qi decoction on lung interstitial deposition damage in CIH mice. Methods: Fifty SPF-grade C57BL mice were randomly divided into five groups (n=10): blank control group, CIH model group, and CIH+ low, medium and high doses of Bu Zhong Yi Qi decoction group. Mice were placed under normoxia or CIH conditions, respectively. The Chinese medicine group was given the corresponding doses of drugs. HE staining was performed to assess pathological changes and Masson staining was performed to assess collagen deposition. Western blot was performed to detect the expressions of channel proteins such as TGF-β1, P-smad3 and down stream α-SMA and Collagen I. ELISA was performed to detect the serum levels of TGF-β1, LN and HA. Results: HE staining showed alveolar collapse, septal thickening and epithelial cell necrosis in CIH mice, Masson showed massive collagen fiber proliferation and deposition in lung interstitium, while the above changes in lung tissues were significantly improved in the CIH + Bu Zhong Yi Qi decoction groups compared with the CIH group. TGF-β1, P-smad3 and Collagen I, Collagen Ⅲ, and α-SMA expression levels were increased compared with the blank control group (P＜0.05), and the expressions of TGF-β1 and LN in serum were upregulated (P＜0.05). The expressions of TGF-β1, P-smad3, Collagen I protein and SMA-α in the lung tissues of the CIH+ Bu Zhong Yi Qi decoction groups were downregulated significantly compared with those of the CIH group (P＜0.05), and the improvement of multiple indexes in the CIH+high-dose CIH intervention group was better than those of the low-dose group (P＜0.05). Conclusion: Bu Zhong Yi Qi decoction can inhibit alveolar structural changes and excessive collagen deposition in the interstitium of CIH mice, and then improve lung function in CIH mice. The mechanism may be related to the down-regulation of protein expression related to TGF-β/smads signaling pathway by Bu Zhong Yi Qi decoction.

Objective: To analyze the molecular mechanisms of skeletal muscle cells apoptosis induced by heavy-load exercise with Omi as the entry point. Methods: One hundred and twenty-six adult SD rats were randomly divided into five groups: control group(C), eccentric exercise group (E), simple blocking group (U), DMSO group (D) and exercise block group (EU). In addition to the C group, the other four groups were randomly divided into 0 h after experiment, 12 h after experiment, 24 h after experiment, 48 h after experiment and 72 h after experiment with 6 rats in each group. E and EU group were submitted to a heavy-load exercise on a treadmill down a 16° decline, 16 m/min for 90 minutes. U, D and EU group were one-time intervened with drugs. U and EU groups were intraperitoneally injected with 1.5 μmol/kg ucf-101, D group were intraperitoneally injected with 1.5 μmoL/kg 0.5% DMSO. The rats were sacrificed in batches at different time points after experiment, then the soleus were saved to detect the Caspase-3,-8,-9,-12 activities and protein expressions of Omi and XIAP. Results: Compared with group C, the mitochondrial distribution and morphology appeared the typical ultrastructure pathological changes, the opening degree of MPTP was increased significantly (P＜0.01) or (P＜0.05), protein expressions of Omi and XIAP were increased significantly (P＜0.01 or P＜0.05), the activities of Caspase-9 and Caspase-3 were increased significantly (P＜0.01 or P＜0.05) in group E. Compared with group C, there was no significant difference in XIAP protein and caspase-9, - 3 activities in group U and Group D. The change trend of XIAP protein and Caspase-9, - 3 activities was the same as those between EU group and E group, but the change range of XIAP protein in EU group was significantly higher than that in E group (P＜0.01), and the change ranges of caspase-9, - 3 activities in EU group were significantly lower than those in E group (P＜0.01). Conclusion: A single heavy-load exercise can induce changes in the mitochondria morphology and structure in rats, open the high permeability of MPTP, and improve the expression of Omi protein, then through its downstream XIAP-Caspase pathway, start the mitochondrial apoptosis pathway mediated by caspase-9, and finally lead to myocyte apoptosis. The inhibition of Omi can reduce the cell apoptosis level of motor induced skeletal muscle cells.

Objective: The effects of Alternate-day modified fasting combined exercise on fat mass, muscle mass, and serum Irisin, FNDC5 and UCP1 proteins were investigated in rats with 4 weeks of aerobic exercise and modified alternate-day fasting intervention. Methods: Thirty-two healthy 8-week-old SPF male SD rats were randomly divided into control group, exercise group, alternate-day modified fasting and alternate-day modified fasting combined with exercise group, 8 rats in each group. The exercise group performed treadmill exercise with moderate exercise intensity(60 min/d,5 d/w), the alternate-day modified fasting group alternated between fasting and free feeding every other day, and fed 25% basal energy feed on fasting days, and the alternate-day modified fasting combined exercise group received two combined interventions. After 4 weeks of intervention, the body fat rate of rats was measured by apical blood sampling and abdominal aortic blood sampling, and the serum was preserved and centrifuged, and the wet weights of bilateral gastrocnemius, bilateral perirenal fat and brown fat at the scapula were weighed, and samples were collected for paraffin sectioning and HE staining, and the cell areas were counted; serum Irisin levels were measured by ELISA, and FNDC5 protein expression in gastrocnemius and UCP1 protein expression in adipose tissue were detected by Western blot. Results: After 4 weeks of intervention, compared with the Con group, energy intake, body weight and body fat were decreased significantly in the Exer, ADMF and ADMF-Exer groups (P＜0.05), the wet weight/body weight and adipocyte area of white fat were reduced significantly (P＜0.01), and there was no significant difference in scapular fat wet weight/body weight (P＞0.05). Compared with the Con group, the gastrocnemius wet weight/body weight in the ADMF group was reduced significantly (P＜0.05), while that in the ADMF-Exer group was increased significantly (P＜0.05), the muscle cross-sectional areas in the Exer group and the ADMF-Exer group were increased (P＜0.05), and the content of gastrocnemius FNDC5 protein, serum Irisin level and expression of adipose UCP1 protein in the ADMF-Exer group were increased significantly (P＜0.05). After 4 weeks of intervention, energy intake was reduced significantly in both ADMF and ADMF-Exer groups (P＜0.01) and body weight of ADMF-Exer group was decreased (P＜0.05) compared with the Exer group. Compared with the Exer group, there were no significant differences in body fat content, white fat wet weight/body weight and scapular fat wet weight/body weight between ADMF group and ADMF-Exer group (P＞0.05), and adipocyte area in ADMF-Exer group was reduced significantly (P＜0.05). Compared with the Exer group, the gastrocnemius muscle wet weight/body weight was reduced significantly in the ADMF group (P＜0.05), and the expression of FNDC5 protein, serum Irisin and adipose UCP1 protein in the ADMF-Exer group were increased significantly compared with the Exer group (P＜0.05). Conclusion: Alternate-day modified fasting combined with exercise intervention can effectively control body weight and reduce body fat in rats, and the mechanism may be through the FNDC5/Irisin-UCP1 pathway to induce browning of white adipose tissue and increase thermogenesis of brown fat.

Objective: To investigate the effects of rhein on proliferation and apoptosis of gastric cancer cell line HGC-27 and its related mechanisms. Methods: Human gastric cancer cells HGC-27 were treated with 0, 5, 10 or 20 mg/L rhein respectively for 24, 48 and 72 h in vitro, three duplicate wells were set in each group. The proliferation activity of HGC-27 cells was detected with CCK-8 method, the growth status of HGC-27 cells was observed by small high-content microscope, hoechst staining was used to analyze the karyotype of HGC-27 cells. Mitochondrial membrane potential was detected by JC-1 staining and flow cytometry, cell cycle was analyzed with flow cytometry, the levels of mRNA transcribing of bcl-2, bax, caspase-3, jak1, jak2, stat3 and notch genes were investigated with RT-qPCR method. Protein expressions were determined by Western blot. Results: Compared with HGC-27 cells treated with 0 mg/L rhein, HGC-27 cells treated with 5, 10 and 20 mg/L rhein for 24 h showed decreased mitochondrial membrane potential ( P＜0.01), the cell proliferation activity was inhibited and apoptosis was induced. The effects were enhanced with the increase of rhein concentration and the extension of treatment time, but the cell cycle did not change significantly, and the expressions of bcl-2, jak1, jak2, stat3 and notch genes were down-regulated. The expression levels of bax and caspase-3 genes were increased significantly ( P＜0.01). Conclusion: Rhein can induce apoptosis of HGC-27 cells by influencing NOTCH/JAK/STAT signaling pathway, and has anti-gastric cancer effect.

Objective: To investigate the effects of down-regulating MDR1 gene expression by CRISPRi on enhancing the sensitivity of lung adenocarcinoma A549/DDP cells to cisplatin. Methods: The potential CRISPRi interference sites on the MDR1 gene promoter were predicted by bioinformatics software, and the interference fragments were designed and constructed. The mRNA and protein expression levels of MDR1 gene in each group of cells were detected by qRT-PCR and Western blot methods, and the recombinant vectors with high interference efficiency were screened. Human lung cancer A549/DDP cells were divided into three groups: A549/DDP, Scrambed and sgRNA-MDR1-1, with three multiple holes in each group. After each vector was transfected into the cells for 48 h, the efflux of cells in each group was detected by flow cytometry, the IC₅₀ value of cells in each group was detected by MTT method, and the cell morphology of cells treated with cisplatin was observed under laser confocal microscope. Results: After sequencing and comparison, two kinds of CRISPRi recombinant vectors interfering with MDR1 gene transcription were constructed successfully. After transfection of A549/DDP cells, the mRNA and protein levels of MDR1 gene in all transfection groups were decreased significantly (P＜ 0.01). Among them, the interference efficiency of sgRNA-MDR1-1 was the highest, and the interference efficiency of mRNA and protein was 60% and 51%, respectively. After transfection of sgRNA-MDR1-1 vector, compared with the control group, the efflux ability of cells was decreased (P＜0.01), the IC₅₀ value of cells to cisplatin was decreased significantly (P＜0.01), and the intracellular chromatin gathered and marginalized, and apoptotic bodies appeared. Conclusion: CRISPRi interference with MDR1 gene in drug-resistant A549/DDP cells can significantly enhance the sensitivity to cisplatin.