Objective: Primary cultured cochlear stria vascularis endothelial cells (ECs) of guinea pig were used to investigate the expression changes of TMEM16A and its effect on apoptosis and senescence of ECs in the cochlear stria vascularis. Methods: Primary cultured ECs in the cochlear stria vascularis were used to establish aging models according to CCK-8 and SA-β-galactosidase. Senescent cells were randomly divided into senescent group (P12), DMSO group (P12+DMSO), T16Ainh-A01 group (P12+T16Ainh-A01). Immunofluorescence and Western blot were used to detect the expression of TMEM16A in ECs. Flow cytometry was used to detect the apoptotic rate. Western blot was used to detect the protein expressions of Bax, Bcl-2 and cleaved casepase-3 in each group. Results: The positive rate of primary cultured cochlear stria vascularis ECs was above 95%, and the 12th generation cochlear stria vascularis ECs were determined as the senescence group, and the expression of TMEM16A in protein and fluorescence was increased (P＜0.05). After intervention with T16Ainh-A01 for 24 h, the protein expressions of Bax and cleaved casepase-3 were down-regulated (P＜0.01), the protein expression of Bcl-2 was increased (P＜0.05), the apoptotic rate and the positive rate of SA-β-gal were down-regulated (P＜0.01). Conclusion: It was found that apoptosis and TMEM16A expression were increased in cochlear stria vascularis senescent ECs, TMEM16A specific blocker T16Ainh-A01 could reduce the apoptosis and senescence in ECs of the cochlear stria vascularis. These results suggest that TMEM16A may participate in apoptosis and senescence of ECs in the cochlear stria vascularis.

Objective: To investigate the effects of Apelin-13 on barrier function injury of human umbilical vein endothelial cells (HUVECs) induced by LPS. Methods: The HUVECs cultured in vitro were divided into 4 groups: Control group, LPS group, Apelin-13+LPS group, Apelin-13 group. HUVECs were treated by 5 μg/ml LPS for 24 h to replicate the model with endothelial barrier impaired. Apelin-13 at the concentration of 1 μmol/L was given 30 min before LPS treatment. The cell viabillity of HUVECs was measured by CCK-8 assay. Protein expressions of VE-cadherin and F-actin were measured by Western blot and immunofluorescence. Nuclear factor κB p65(NF-κB p65) was detected by immunofluorescence. Results: Compared with the control group, the cell viabillity of HUVECs and protein expression of VE-cadherin were decreased by LPS, but the protein expression of F-actin and activation of NF-κB p65 were increased by LPS. These effects were attenuated by Apelin-13 administration. Conclusion: Apelin-13 ameliorates LPS-induced barrier function injury of HUVECs, which may be related to the inhibition of inflammation.

Objective: To investigate the effects of nano-SiO₂ and cold on the cytotoxicity and secretion of inflammatory factors in human lung adenocarcinoma epithelial cell line A549. Methods: A549 was used as experimental subject, a single factor multilevel experiment was designed, A549 cells were exposed to 10, 50, 100, 200 μg/ml nano-SiO₂ particles and/or cultured at 31℃, 33℃, 35℃ for 48 h. After that, cell morphology was observed and relative cell survival rate was detected. According to the results of single factor analysis and based on the selection of nano-SiO₂ dose and temperature that significantly reduced the relative survival rate of A549 cells, the experiment was designed according to 2×2 factor analysis , they were divided into 4 groups: control group(37℃), Nano-SiO₂ exposure group, low temperature exposure group, Nano-SiO₂ and low temperature composite group. After exposure for 48 h, the supernatant of cell culture medium was collected for detecting the LDH activity by colorimetric method and the levels of cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) were determined by ELISA. The mRNA levels of cellular IL-6 and IL-8 were detected by qRT-PCR. Results: The activities of A549 cells in 100 μg/ml Nano-SiO₂ group and 31℃ low temperature group were decreased significantly. Under the combined conditions, the activity of A549 cells was most inhibited (P＜0.01), and the levels of inflammatory factors IL-6 and IL-8 and mRNA were significantly increased (P＜0.01). Conclusion: 100 μg/ml Nano-SiO₂ combined with 31℃ cold exposure can synergistically reduce the activity of A549 cells and increase the expression level of inflammatory factors IL-6 and IL-8 .

Objective: To investigate the protective effects of gliclazide on myocardium of diabetic rats and its possible mechanisms. Methods: Sixty healthy SD rats were randomly divided into two groups: normal group (NC, n=10) and model group (n=50). Rats in model group were fed with high glucose and high fat diet for 4 weeks and then intraperitoneally injected with STZ (45 mg/kg) to establish a diabetic model and randomly selected FBG ≥ 16.7 mmol / L as a successful diabetes model. Thirty-eight diabetic rats were randomly divided into model group (MC, n=9), gliclazide group (Glic, 80 mg/kg, n=10), glibenclamide group (Glib, 2.5 mg/kg, n=10) and fasudil group (Fas, 10 mg/kg, n=9). NC group and MC group were given equal volume distilled water by gavage, Glic group and Glib group were treated with gliclazide or glibenclamide by gavage, and the Fas group was treated with fasudil by intraperitoneal injection. Rats in each group were given once a day and recorded body mass and fasting blood glucose (FBG) weekly for 8 weeks. At the end of the experiment, the heart weight was measured, and the heart weight index (HWI) was calculated; the contents of glycosylated hemoglobin (HbA1c), total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL-C), low density lipoprotein (LDL-C), the level of serum malondialdehyde MDA) and the activity of superoxide dismutase (SOD) were measured; the pathological changes of myocardial tissue were observed by HE and Masson staining. The expressions of RhoA, ROCK1, eNOS, Bcl-2 and Bax protein were detected by Western blot. Results: Compared with NC group, in MC group, the levels of FBG, HWI, HbA1c, TC, TG, LDL-C, MDA, myocardial collagen deposition and cardiomyocyte apoptosis rate and RhoA, ROCK1, Bax protein in myocardial tissue were increased significantly, while the SOD activity, the levels of HDL-C, eNOS, Bcl-2 and body weight were decreased significantly (P＜0.01). Compared with MC group, Glic treatment decreased the levels of FBG, HWI, HbA1c, LDL-C, TG, TC and MDA, increased the levels of SOD activity and HDL-C (P＜0.01 or P＜0.05); decreased myocardial collagen deposition, inhibited cardiomyocyte apoptosis (P ＜ 0.01); decreased the expression levels of RhoA, ROCK1 and Bax protein; increased the levels of eNOS and Bcl-2 protein (P＜0.01 or P＜0.05). Compared with Glic group, in Glib group, the levels of blood lipids, BM, FBG, HWI, MDA, myocardial fibrosis and cardiomyocyte apoptosis rate were increased, the levels of SOD and Bcl-2 were decreased, and the expressions of RhoA, ROCK1 and Bax in myocardial tissue were upregulated (P＜0.01 or P＜0.05). Conclusion: Gliclazide significantly alleviates myocardial injury and reduces myocardial apoptosis in diabetic rats, and its mechanism may be related to lowering blood glucose, improving oxidative stress and regulating RhoA / ROCK1 / eNOS signaling pathway.

Objective: To explore the positive inotropic effect of atractylodin which is major active component of Rhzoma Atractylodis Lanceae and its underlying mechanism. Methods: For in vivo study, six male SD rats were randomly selected for the heart pressure-volume loop (P-V loop) experiment. The effects of atractylodin (3 mg/kg, intraperitoneal injection) on hemodynamic parameters such as LVDP (left ventricular developed pressure), SW (stroke work), HR (heart rate), CO (cardiac output), SBP (systolic blood pressure) and DBP (diastolic blood pressure) were analyzed. For in vitro study, left ventricular developed pressure (LVDP) from the Langendroff-perfused isolated rat heart was analyzed before as the control and after atractylodin perfusion. For in vitro study, the effects of atractylodin and atractylodin with H89 (PKA inhibitor) or KN-93 (CaMKII inhibitor or Calyculin A (PP1, PP2A inhibitor) on LVDP were analyzed. The experiments were separated into four parts with six isolated hearts for each as follows: (1) Control→0.1→1→10 μmol/L atractylodin; (2) Control→200 nmol/L H89 (PKA inhibitor)→200 nmol/L H89+10 μmol/L atractylodin; (3) Control→500 nmol/L KN-93 (CaMKII inhibitor)→500 nmol/L KN-93+10 μmol/L atractylodin; (4) Control→10 nmol/L Calyculin A (PP1, PP2A inhibitor)→10 nmol/L Calyculin A+10 μmol/L atractylodin. For the study of rat left ventricular myocyte Ca²⁺ transient induced by field stimulation, the experiment design was the same as in vitro study. The six cells from the different rats were used for each part experiment. Results: ① Atractylodin (3 mg/kg) significantly increased the heart rate, cardiac output and stroke work (P＜0.05) and decreased the diastolic blood pressure (P＜0.05). ② Atractylodin (0.1, 1, 10 μmol/L) significantly increased the LVDP in a concentration dependent manner (P＜0.05). The positive inotropic effect of atractylodin could be blocked by PKA inhibitor H89. ③ Atractylodin (10 μmol/L) significantly increased the amplitude of SR Ca²⁺ transient amplitude mediated by facilitating sarcoplasmic reticulum SERCA2a. The enhanced amplitude of SR Ca²⁺ transient could be blocked by PKA inhibitor H89. Conclusion: Atractylodin had positive inotropic effect in rat heart both in vivo and in vitro with decreased diastolic blood pressure and its underlying mechanism was mediated by PKA. Based on the fact that the atractylodin exerted its positive inotropic effect was mediated by PKA, the PKA-SERCA2a signaling pathway might be the mechanism of the atractylodin's positive inotropy.

Objective: To explore the role of transforming growth factor-β (TGF-β) signaling pathway in xiaotan huayu liqiao traditional Chinese medicine compound (XC)'s anti-myocardial fibrosis in chronic intermittent hypoxia (CIH) rats. Methods: Forty SD rats were randomly divided into normoxia group, oxygen + traditional Chinese medicine compound group ( TCMC), Chronic intermittent hypoxia model group (CIH), TCMC + CIH, 10 in each group. CIH cabin was built by filling with nitrogen and oxygen. Firstly, the volume fraction of oxygen in the cabin reduced from 21% to 9% in 90 s by filling the cabin with nitrogen. And then it gradually rose to 21% by reoxygenating in 90s, as a cycle. CIH and TCMC+CIH group rats were placed in the CIH device, while normoxia and TCMC group rats were placed in the normal oxygen chamber. In addition, rats in TCMC +CIH group and TCMC group were treated with XC crude drug (24 g/kg) daily by gavage, while rats in CIH group and normoxia group were given equal volume normal saline. Using sirius red staining, the collagen in myocardial interstitium was visualized. The protein expressions of collagen I, collagen III and fibronectin were detected by Western blot, p-Smad3, p-Smad2 and TGF-β protein in the TGF-β/Smads signaling pathway were also analyzed by Western blot. The mRNA expressions of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinase -2(TIMP-2) were measured by real-time quantitative polymerase chain reaction (PCR). Results: Compared with the rats exposed to normoxia, the CIH rats showed obvious collagen deposition, protein expressions of collagen I, collagen III and fibronectin were significantly increased in the myocardial tissue (P＜0.01). The protein expression levels of TGF-β, p-smad2 and p-smad3 in the myocardial tissue of the CIH rats were also significantly increased (P＜0.01). The up-regulation of TIMP-2 mRNA in the myocardial tissues resulted in the decrease of MMP-2 mRNA(P＜0.01). XC reduced myocardial fibrosis of CIH rats and inhibited the expressions of collagen I and collagen III and fibronectin protein (P＜0.05,P＜0.01,P＜0.05, respectively). The further mechanism study showed that XC inhibited the expression of TGF-β (P＜0.01), which down-regulated the expressions of p-smad2, p-smad3 and TIMP-2 (P＜0.05). Conclusion: XC could reduce the expression of TGF-β and smad2/3 phosphorylation, down-regulate the expression of TIMP-2, which would inhibit the formation of myocardial fibrosis in CIH rats, and improve the myocardial function of CIH rats.

Objective: To explore the different effects of rapid and progressive ascent to Tibet plateau on cardiovascular function and stress factors in pre-selected expeditioners for the 31^st, 32^nd and 33^rd Chinese Antarctic expedition for inland station, to provide a scientific basis for the selection of qualified expeditioners. Methods: A total of 85 pre-selected expeditioners for the 31^st, 32^nd and 33^rd Chinese Antarctic expedition for Kunlun station were enrolled in this study. According to the different manners of entering the plateau, they were divided into the rapid ascent group by aircraft (RAG, n=55) and the progressive ascent group by train (PAG, n=30). Hemodynamics and electrocardiogram were examined at 4 m (Shanghai), areas at altitude of 3 658 m (Lhasa) and 4 300 m(Yangbajain), respectively. Saliva levels of stress factors, including testosterone (T), cortisol (COR) and immunoglobulin A (IgA), were tested by ELISA. Results: The heart rates (HR) were increased significantly, while the SpO₂ was decreased significantly in the two groups within 24 hours at altitudes of 3 658 m and 4 300 m (P＜0.05), while there was no significant difference between the two groups at the same altitude. Compared with 4 m, the blood pressure (BP) of the two groups at 3 658 m and 4 300 m was significantly increased (P＜0.05), and some indexes of myocardial contraction and pumping function were significantly reduced (P＜0.05). However, due to the increase of HR, there was no significant change in Cardiac Output (CO). At 4 300 m, the Thoracic Fluid Content (TFC) of the rapid ascent group was significantly higher than that of the progressive ascent group (P＜0.05). Compared with 4m, there was no significant difference in salivary testosterone change between the two groups at 3 658 m above sea level (P＞0.05), while COR and IgA changes in the rapid ascent group were significantly higher than those in another group (P＜0.05). Conclusion: Compared with the progressive ascent by train,expeditioners that rapid ascent to high altitude have significant effects on cardiovascular function and the stress hormones and immunoglobulin levels in saliva. It's suggested that hypoxia adaptation before Antarctic expediting for Kunlun Station could ensure the selected expeditioners' physical and psychological health, so that the mission could be finished smoothly.

Objective: To investigate the sensitivity of carotid body to hypoxia and the effect of dopamine on the sensitivity of carotid body to hypoxia after acute intermittent hypoxia stimulation in rats. Methods: The isolated carotid body-sinus nerve in rat was transferred to incubator, and then the isolated sinus nerve was inhaled into the recorded glass electrode for recording electrical signals. The baseline buffer was bubbled with 95% O ₂ + 5% CO ₂ mixture gas, and the hypoxic stress was treated with 5% O ₂ + 5% CO ₂ + 90% N₂ mixture gas, hypoxic stimulation was given for 30 seconds, 95% O ₂ + 5% CO ₂ for 90 seconds, a total of 10 cycles. No less than 5 rats in each group. Results: In this experiment, the electrical activity of sinus nerve isolated from rats was enhanced by hypoxia stimulation after acute intermittent hypoxia, but the response of sinus nerve to hypoxia was inhibited by dopamine. Before acute intermittent hypoxic stress, dopamine also inhibited the firing activity of sinus nerve, but after acute intermittent hypoxic cycle, the inhibition of dopamine on the firing activity of sinus nerve was strengthened. Conclusion: Acute intermittent hypoxia enhances the response of sinus nerve isolated from rats to hypoxia, dopamine inhibits the enhancement of carotid body sensitivity to hypoxia induced by acute intermittent hypoxic.

Objective: To investigate the effects of simulated hypoxia environment at an altitude of 5 500 meters on hypothalamic-pituitary-thyroid (HPT) axis and intestinal flora of rats and the correlation between them. Methods: The hypoxia model of adult male SD rats was established by hypobaric chamber with simulated altitude of 5 500 m. The hypoxia groups were set for 1, 3, 7, 14, 21 and 28 days, and the normoxic recovery group were set for 1 and 3 days after hypoxia (8 rats per group, hypoxia time 24h per day). Daily body weight and food intake of rats were recorded. The serum levels of HPT axis hormones were detected by enzyme-linked immunosorbent assay (ELISA). Intestinal flora was analyzed by 16s rDNA sequencing. The correlation between intestinal flora and serum HPT axis hormone was analyzed by Spearman correlation analysis. Results: Compared with the normoxic group, the body weight and food intake were significantly reduced (P＜0.01). In the 1-day and 3-day groups, the levels of thyrotropin releasing hormone (TRH) and thyroid stimulating hormone (TSH) were decreased significantly (P＜0.05), the levels of total thyroxine (TT4), total triiodothyronine (TT3), free thyroxine (FT4) and free triiodothyronine (FT3) were increased significantly (P＜0.05), and the abundance of Prevotella was decreased significantly (P＜0.05), while those of Parabacteroides, Lactobacillus, Butyricimonas, Bacteroides, Odoribacter and RC4-4 were increased significantly (P＜0.05). In the 14-day, 21-day and 28-day hypoxia groups, the levels of TRH and TSH were decreased significantly (P＜0.05), and the levels of TT4 and TT3 were increased significantly (P＜0.05), and there was no significant difference in other indexes, the abundance of Parabacteroides, Sphaerochaeta, Akkermansia, Elusimicrobium was increased significantly, but that of Lactobacillus and Sutterella was decreased significantly (P＜0.05). Correlation analysis of intestinal flora and HPT axis hormones showed that Butyricimonas, Elusimicrobium and Sutterella were significantly negatively correlated with TRH and TSH (P＜ 0.05), Prevotella, Bacteroides, Odoribacter and Parabacteroides were significantly correlated with TSH, TT4, TT3 and FT4 (P＜ 0.05), respectively. Lactobacillus was significantly correlated with TRH, TSH and FT4 (P＜0.05). Akkermansia was significantly correlated with TRH and FT4 (P＜0.05). RC4-4 was significantly associated with TSH and TT3 (P＜0.05). Conclusion: Hypoxia stress at an altitude simulating 5500 meters significantly changed the composition of the intestinal flora of SD rats. This may be a change in thyroid function adapted to the hypoxia environment, and the degree of change is related to the time of hypoxia stress. The change of intestinal microflora is significantly correlated with the hormone level of HPT axis.

Objective: To investigate the effects of fatty acid binding protein 5(FABP5)- peroxisome proliferator-activated receptor gamma(PPARγ) signaling pathway on learning-memory ability and lipid metabolism in rats with vascular dementia(VD) and its mechanisms. Methods: ①VD model rats were established by ligating bilateral common carotid artery. These rats were divided into three groups: normal group (WT group), sham-operated group (sham group) and VD model group; ②WT group and FABP5 inhibitor group were set up. After four weeks, Morris water maze test was used to detect spatial learning and memory ability in rats. RT-qPCR and Western blot methods were used to detect the expressions of FABP5, PPARγ, p-PPARγ and lipoprotein lipase (LPL) in the brain at the mRNA and protein levels. The levels of TC, TG and FFA in the brain were detected by assay kits. Results: Compared with the WT group and sham group, the learning-memory ability of the VD model and the FABP5 inhibitor group were significantly decreased, and the expressions of FABP5, PPARγ, p-PPARγ and LPL were significantly decreased at mRNA level and protein level in the brain; and the levels of TC, TG and FFA were increased significantly in the brain. Conclusion: FABP5 can affect the learning-memory ability and lipid metabolism in VD rats through PPARγ and LPL.

Objective: To investigate the effects of Ganmai Dazao Tang on behavior and monoamine neurotransmitters in rats with depression, and to explore its potential mechanism from synaptic structure. Methods: Sixty SD rats were randomly divided into 5 groups: normal control group, model group, fluoxetine group (10.8 mg/kg), Ganmai Dazao Tang high and low dose group (9.72, 4.86 g/kg), 12 rats in each group. Except the control group, the rats in the other groups were all chronically unpredictable mild stress (CUMS) to establish a depression model, and were treated by intragastric administration for 21 days. The depression-like behaviors of rats were evaluated by sucrose consumption test and open field test. The contents of serotonin (5-HT) and norepinephrine (NE) in hippocampus were detected by ELISA. The synaptic damage of neurons was observed by Golgi staining. The synaptic structure protein expression levels of MAP-2 and GAP-43 of hippocampus were detected by Immunohistochemistry and Western blot. Results: Compared with control group, the sucrose preference and autonomic activity scores of the depression model rats were decreased significantly (P＜0.01), the levels of 5-HT and NE in hippocampus were decreased significantly (P＜0.01), and the dendritic spines were absent, and the expressions of MAP-2 and GAP-43 were down-regulated significantly (P＜0.01). After treated with Ganmai Dazao Tang, the depression-like behavior of the model rats was significantly relieved (P＜0.01), and the levels of 5-HT and NE were increased (P＜ 0.05). Dendritic spine density, length and branching were increased, the expressions of MAP-2 and GAP-43 were increased (P＜ 0.01). Conclusion: Ganmai Dazao Tang can improve the depression-like behavior of depression model rats and increase the monoamine neurotransmitter content in hippocampus, which may be related to up-regulation of synaptic structural proteins and relief of synaptic damage in neurons.

Objective: To evaluate the neuroprotective effects of linagliptin, a dipeptidyl peptidase-4(DPP-4) inhibitor, on cerebral ischemia/reperfusion(I/R) injury in mice. Methods: BALB/c mice were randomly divided into Sham group, I/R group and linagliptin (2.5, 5 and 10 mg/kg) +I/R group, 8 mice in each group. The mice in the linagliptin group were administrated by gavage 3 weeks before I/R. I/R injury model was induced by MCAO, neurological deficit scores(n=8) and infarct volume(n=4) were assessed 24 h following reperfusion. Forty-eight hours following reperfusion, mice were euthanized, the contents of glutathione (GSH), malondialdehyde (MDA), phosphoinositide 3 kinase (PI3K), phosphoprotein kinase B (p-Akt) and rapamycin target protein (mTOR) in brain tissue were measured (n=4). Results: Compared with the I/R group, the neurological deficit score and infarct volume were significantly decreased in the linagliptin pretreatment group after 24 h reperfusion (P＜0.05); the MDA content in the brain was significantly decreased (P＜0.05), while the GSH, PI3K, p-Akt and mTOR levels were significantly increased (P＜0.05). Conclusion: This study proves that linagliptin exerted a neuroprotective effect in I/R mice, which may be mediated by activation of the PI3K/AKT/mTOR pathway.

Objective: To investigate the changes of pyroptosis-related proteins in the hippocampus of insulin-resistant mice and the regulation of resistance training on pyroptosis-related proteins. Methods: Six-week-old male C57BL/6J mice were randomly divided into control group (C, n=12) and high-fat diet group (HFD, n=26) for normal or high-fat diet for 12 weeks. Subsequently, according to the results of glucose tolerance test (GTT) and insulin tolerance test (ITT), the rats fed with high-fat diet were divided into insulin resistance group (IR, n=10) and resistance exercise group (RT, n=10) as well as to maintain high-fat diet. At the same time, mice in the RT group were subjected to resistance training. After 12 weeks, all mice were sacrificed after anesthesia, brain was removed and hippocampus was exfoliated, and the expressions of pyroptosis-related proteins were detected by Western blot. Results: Compared with the C group, NF-κB, the NLRP3 inflammasome proteins, their downstream pyroptosis-related proteins GSDMD-N and GSDMD as well as inflammation factors IL-1β and IL-18 in hippocampus of IR group were significantly increased (P＜0.05), and the expression levels of SIRT1 and p-AMPK protein were significantly decreased (P＜0.05). Compared with the IR group, NF-κB, the NLRP3 inflammasome proteins, their downstream pyroptosis-related proteins GSDMD-N and GSDMD as well as inflammation factors IL-1β and IL-18 in hippocampus of RT group were significantly decreased (P＜0.05), and the expression levels of SIRT1 and p-AMPK protein were significantly increased (P＜0.01). Conclusion: NLRP3 inflammasome in the hippocampus of insulin-resistant mice is activated, which mediates pyroptosis in the hippocampus. Twelve weeks of resistance training can effectively inhibit the activation of NLRP3 inflammasome and decrease pyroptosis and improve inflammation in the hippocampus.

Objective: To investigate the changes of metabolites of teenage football players after exercise-induced fatigue. Methods: Twelve male teenage football players (14～16 yrs) were selected as experimental subjects in this study. And an exercise model including aerobic and anaerobic exercise as one group exercise was established by using power bicycle: completion 6 min 150 W load, 60～65 r/min of riding exercise and 30 s of riding exercise which load was the maximum speed set by the tester's weight. The rest took 1 min in the middle of one group exercise, and repeat 3 times of one group exercise, then rest for 3 min after one group exercise. The maximum oxygen uptake (VO₂ max) and average anaerobic power were measured after each group exercise. Their urine samples were collected before and after the whole exercise model, and gas chromatograph-mass spectrometer (GC-MS) was used to detect the differential metabolites. Results: The teenage football players had a significant decrease in anaerobic capacity after fatigue. Compared with pre-exercise, a total of 25 differential metabolites were screened out, of which 3 metabolites were significantly higher and 22 metabolites were markedly lower. The related metabolic pathways of above differential metabolites were classified as glycine-serine-threonine metabolism, tricarboxylic acid cycle, tyrosine metabolism, nitrogen metabolism and glycerophospholipid metabolism, respectively. Conclusion: After exercise-induced fatigue occurs in teenage football players, the body's metabolites: sarcosine, L-allothreonine, creatine, serine, succinic acid, citric acid, 4-hydroxyphenylacetic acid, hydroxylamine, and ethanolamine produce significant changes. The above-mentioned differential metabolites can be used as indicators for teenage football players' exercise-induced fatigue evaluation.

Objective: To investigate the effects of 4-week electroacupuncture intervention on “Browning” of white fat in rats, and to explore its molecular mechanisms. Methods: Twenty-four 8-week-old male SD rats were randomly divided into sedentary group (Sed), aerobic exercise group (Exe) and electroacupuncture group (ElA), 8 rats in each group. Exe group used 65% Max oxygen uptake intensity treadmill exercise, 1 h/d,6 d/w, while the ElA group used electric acupuncture to stimulate “zusanli” and “tianshu” points, 20 min/d,6 d/w, and the weight of rats was recorded every week. After 4 weeks of intervention, blood samples were collected from the apex and abdominal aorta. The wet weight of scapular fat and perirenal fat of rats was detected and the body fat and the serum levels of Irisin were determined. What's more, the expressions of adenosine 5'-monophosphate activated protein kinase-α (AMPKα), phosphorylation of adenosine 5'-monophosphate activated protein kinase-α (p-AMPKα), peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α), fibronectin type III domain containing 5 (FNDC5) and uncoupling protein (UCP1) and brown adipose tissue lipid droplets in rats were detected. Results: Compared with the Sed group, the weight gain of rats in the Exe and ElA groups was decreased significantly from the second week. After 4 weeks, the body weight and body fat of the Exe and ElA groups were decreased significantly (P＜0.01), while there was no significant difference between the Exe and ElA groups (P＞0.05). ②After 4 weeks of intervention, the white fat wet weight of ElA group and Exe group was significantly was reduced (P＜0.05 or P＜0.01). ③The serum levels of Irisin in Exe and ElA groups were significantly increased (P＜0.05), and the expressions of p-AMPKα/AMPKα, PGC-1α, FNDC5 in the gastrocnemius and the expressions of UCP1 in white fat and brown adipose tissue of Exe and ElA groups were increased (P＜0.05). ④The area of lipid droplets in white and brown adipose tissue of Exe and ElA groups was significantly reduced (P＜ 0.01). Conclusion: 4 weeks of electroacupuncture intervention can effectively control the weight of rats and induce “browning” of white fat, and its effect was similar to aerobic exercise, which may be release Irisin through the AMPKα-PGC-1α-FNDC5-Irisin signaling pathway, and then “crosstalk” with adipose tissue, up-regulate the expression of UCP1, and induce “browning” of white adipose tissue.

Objective: To investigate the changes of nuclear factor erythroid 2-related factor 2 (Nrf2) and superoxide dismutase (SOD) in the prevention of insulin resistance in skeletal muscle of rats by aerobic exercise. Methods: Twenty-four 12-month old Sprague-Dawley (SD) rats were randomly divided into control (C), insulin resistance model (IR) and insulin resistance model and exercise (IRE) groups. IR was induced by high- glucose and high-fat diet. Rats of IRE received the increasing load running on the treadmill for 6 W. The activity of total superoxide dismutase (T-SOD), catalase (CAT), Malonaldehyde (MDA) and glutathione / L-glutathione oxidized (GSH/GSSG) were measured by experiment kits. The content of 8-hydroxy-2 deoxyguanosine (8-OHdG) was measured by ELISA Kits. The expressions of Nrf2 and Glucose transporters 4 (GLUT4) in gastrocnemius were detected by Western Blot. Results: ①Compared with IR, the content of HOMA-IR was decreased in IRE (P＜0.05), while the content of muscle glycogen was increased in IRE (P＜0.01). ② The activities of T-SOD, CAT, and GSH/GSSG in IRE were significantly increased compared with those in IR (P＜0.01). Compared with IR, the contents of 8-OHdG and MDA in IRE were significantly decreased (P＜0.01). ③ The expressions of Nrf2 and GLUT4 in gastrocnemius of IRE rats were increased than those in IR (P＜0.01). Conclusion: The Nrf2 pathway was activated in gastrocnemius of rats after aerobic exercise, which promoted the activity of antioxidant enzymes, and prevented IR induced by high- glucose and high-fat diet.

Objective: To investigate the effects of Sini San prescription(SNS) on the proliferation and apoptosis of HepG2 cells and its molecular mechanism. Methods: The morphological changes of hepatocellular carcinoma HepG2 cells treated by SNS were observed by inverted microscope. MTT assay was used to detect the inhibitory effect of SNS on cell proliferation. Fluorescence staining and flow cytometry were employed to analyze the effect of SNS on apoptosis of HepG2 cells. Rho123 (Rhodamine 123) staining method was performed to detect the changes of mitochondrial membrane potential, and Western blot was used to evaluate the expression of proteins related to apoptosis. Results: The number of hepatocellular carcinoma HepG2 cells were significantly decreased (P＜0.01) and cells showed typical apoptotic cell morphology after treated with serum contained SNS. The inhibition rate of HepG2 cells was increased with the increase of concentration of serum contained SNS. The number of cells in G1 phase was significantly increased, while G2 phase was decreased after treated with serum contained SNS(P＜0.05).The apoptosis rate and mitochondrial membrane potential of HepG2 cells were significantly increased and decreased after treated with serum contained SNS(P＜0.05). The expression levels of Bax, caspase-3,-9 and cyt-c were significantly increased, while the expression of bcl-2 was decreased in HepG2 cells treated with serum contained SNS(P＜0.05).Conclusion: Sini San prescription can inhibit the proliferation of HepG2 cells and induce apoptosis by mitochondrial pathway.

Objective: To investigate the effect and mechanism of lipoxygen on acute liver injury. Methods: Twenty-four SD rats were randomly divided into 4 groups (n=6): normal control group: subcutaneous injection of olive oil at a dose of 1.8 ml/kg; model group: subcutaneous injection of 40% carbon tetrachloride(CCL4)oil (olive oil as a solvent) at a dose of 3 ml/kg; BML-111 treatment group: Lipoxin receptor agonist BML-111 was injected subcutaneously at a dose of 1 mg/kg, and treated in the same model group after 30 minutes; BOC-2 blocker group: Lipoxin receptor blocker BOC-2 was injected subcutaneously at a dose of 50 μg/kg. After 30 minutes, the treatment was the same as BML-111 group. HE staining was used to observe the pathological changes of liver tissues to judge the liver injury. Serological detection was used to determine the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST); The myeloperoxidase (MPO) activity in rat liver tissue was detected by kit method; the contents of of angiotensin converting enzyme (ACE) , Angiotensin converting enzyme 2 (ACE2), angiotensin II (AngII) and angiotensin 1-7 (Ang- (1-7)) were detected by ELISA. Western Blot was used to detect the protein contents of Ang II and Ang- (1-7) in liver tissue. Results: The treatment group had less liver damage than the model group and the blocker group; BML-111 decreased the levels of ACE and AngII in serum of rats with CCL4 injury (P＜0.01) and increased the levels of ACE2 and Ang- (1 in serum -7) content (P＜0.01). BML-111 increased the content of Ang- (1-7) in liver tissue and decreased the content of AngII in CCL4 injured rat tissue. Conclusion: The results showed that the intervention effect and mechanism of lipoxygen receptor agonist BML-111 on acute liver injury in rats may be related to the regulation of AngII and Ang-(1-7).

Objective: To investigate the preventive and therapeutic effects of Qiwei Yugan Granule (QYG) on hepatic fibrosis in rats based on MMP-13/TIMP-1 imbalance. Methods: The rats were randomly divided into control group, model group, colchicine group (1.0×10^-4 g/kg) and QYG treated groups (3.7, 7.4, 14.8 g/kg) (n=8). The rat model of hepatic fibrosis was established by injected with carbon tetrachloride subcutaneously for 4 weeks and treated with ethanol by gavage for 6 weeks. The effects of QYG on liver function, histopathology of liver, related indexes of serum liver fibrosis, and MMP-13, TIMP-1 in hepatic tissue were observed. Results: QYG at the doses of 14.8、7.4、3.7 g/kg could significantly decrease the serum levels of ALT, AST, HA, PCⅢ and C-Ⅳ, relieve the pathological changes of hepatic fibrosis, increase the activity of MMP-13, decrease the activity of TIMP-1 and alleviate the imbalance of MMP-13/TIMP-1. Among them, QYG had a certain trend of dose-effect relationship with TIMP-1 and MMP-13/TIMP-1 (P＜0.05, 0.01). Conclusion: QYG has the effect of preventing and treating liver fibrosis and one of mechanisms is to promote MMP-13/TIMP-1 to restore balance.

Objective: To investigate the potential effects of microRNA-106b (miR-106b) on gluconeogenesis in normal human liver cell line L02 and its underlying mechanisms. Methods: Normal human liver L02 cells were cultured in DMEM containing 10% FBS and transfected with 20 nmol/L of miR-106b mimic or antagomiR-106b, respectively. Twenty-four hours later after transfection, Western blot was performed to detect the levels of proteins or phosphorylated proteins. Quantitative RT-PCR was carried out to measure the mRNA expressions of gluconeogenesis-related genes. Glucose Assay Kit was used to detect the glucose contents in the medium. Results: MiR-106b mimic significantly increased the protein abundances of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase)(P＜0.01,P＜0.01, respectively), enhanced the mRNA expression of phosphoenolpyruvate carboxykinase 1 (PCK1) (P＜0.01), and decreased the mRNA level of glucokinase (GCK) (P＜0.01). AntagomiR-106b dramatically reduced the protein levels of PEPCK and G6Pase (P＜0.01,P＜0.01, respectively), suppressed the mRNA level of PCK1 (P＜0.01), and increased the mRNA level of GCK(P＜0.01). In addition,miR-106b mimic or antagomiR-106b significantly reduced or enhanced the protein levels of signal transducer and activator of transcription 3 (STAT3)(P＜0.01,P＜0.01, respectively).The inhibition of STAT3 by its specific inhibitor abolished the inhibitory effects of antagomiR-106b on hepatic gluconeogenesis. Conclusion: miR-106b increases hepatic gluconeogenesis by inhibiting the STAT3 signaling pathway.

Objective: To study the effects of Youguiwan on the osteoglycin (OGN), osteonectin (ON), fibrinogen 2 (FBN2) of articular cartilage tissue in the model of knee osteoarthritis (KOA). Methods: Sixty SD rats were randomly divided into six groups: sham control group, model group, glucosamine sulfate group, Youguiwan (high-dose, middle-dose and low-dose )group. The modified Hulth method was used to establish KOA models for 6 weeks. The sham control group and the model group were treated with normal saline. The rats in Youguiwan high-dose, middle-dose, low-dose groups were treated with Youguiwan at the doses of 4.8, 2.4, 1.2 g/kg by gavage respectively, and the glucosamine sulfate group was treated with glucosamine sulfate 0.17 g/kg. The rats were administrated for 8 weeks according to the dose. After intervening, articular cartilage of rats were obtained, the pathological changes were observed by using HE staining method, and Mankin score was evaluated. The expressions of OGN, ON and FBN2 in articular cartilage were detected by immunohistochemistry. The expression of GSK-3β in articular cartilage was detected by Western blot. Results: Compared with the sham control group, the Mankin score was obviously increased in the model group, the protein expression of FBN2 was increased significantly, yet the protein expressions of OGN, ON and GSK-3β were decreased significantly (P＜0.01), articular cartilage was seriously damaged, and chondrocytes were arranged in disorder. Compared with the model group, the Mankin score was declined obviously in the high-dose Youguiwan group, the protein expression of FBN2 was significantly decreased, but the protein expression of GSK-3β was significantly increased, the protein expressions of OGN and ON were significantly increased in the middle-dose and high-dose Youguiwan group (P＜0.05 or P＜0.01), cartilage structure was tended to be normal, the chondrocytes distribution was uneven, and articular cartilage surface was not smooth. Conclusion: Youguiwan can significantly improve the articular cartilage degeneration of KOA rats, its mechanism maybe raise OGN and ON protein expression level to promote the ossification and reconstruction of articular cartilage.

Objective: To identify the compulsive drug-seeking behavior of the individual in the heroin-addicted rat, a novel analysis method of telemetering electroencephalogram (EEG) in the frontal association cortex (FrA) induced by heroin-dependent position preference in rats. Methods: Thirty clean-grade Wistar rats after implantation of prefrontal cortex electrodes, were randomly divided into the surgical control group (n=10) and heroin-inducing group (n=20). The heroin-induced group was subcutaneously injected with heroin 0.5 mg/(kg.d), and then increased daily by 0.25 mg/kg for seven days. The control group was injected with the same amount of normal saline at the same time. Using the CPP video system combined with electroencephalogram (EEG) wireless telemetry technology, EEG signals in FrA areas of the addicted rats were recorded simultaneously in four behaviors: white-black shuttle, black-white shuttle, black-chamber stay and white-chamber stay. The areas with EMG and other noisy signals in the original EEG were identified, and wavelet decomposition and amplitude threshold denoising pre-processing were used. The sample entropy values of EEG data and wavelet coefficients corresponding to 4 rhythm frequencies under different behavioral states standard deviation were extracted, and support vector machine algorithm (SVM) was used to achieve real-time identification of different behavioral states of heroin-addicted rats. Results: SVM real-time classification recognition rate of 20 heroin abstinence rats, which are staying in black or white chamber of video box, shuttling between black-white chambers or between white - black chambers, was about 80%. Among them, the real-time recognition rate of black-white shuttle, which is closely related to drug-seeking behavior, reached 83.88%. Conclusion: In this paper, the real-time identification method of heroin-induced obsessive-compulsive drug-seeking behavior in rats can be used as an effective method to detect the initiation and occurrence of heroin-seeking drug-seeking behavior in rats. It can be used for the clinical observation of heroin-dependent patients and the prevention of drug-seeking behavior.

Objective: To establish an efficient method for extracting exosomes from large-volume cell perfusate. Methods: EA.HY926, an immortalized cell line produced by the hybridization of human umbilical vein endothelial cells and human lung adenocarcinoma cell line A549, was cultured with M199 culture medium containing 10% fetal bovine serum. Flexcell STR-4000 parallel plate flow chamber system was employed to apply shear stress to EA.HY926. And then the perfusate was collected. The cell debris was removed by centrifugation. The supernatant was freeze-dried into the dry powder and was resuspended by small-volume medium. The dialysis was used to desalt and purify the suspension. The exoEasy Maxi Kit was used to extract the exosomes. The morphology of exosomes was observed by electron microscopy. The size of exosomes was detected by nanometer particle size analyzer. The activity of exosomes was detected by PKH26 staining. BCA protein quantification method was used to detect the protein concentration of exosomes. The expressions of exosomal specific proteins CD9 and CD81 were detected by Western blot. The quantitative RT-PCR was used to detect the expression of related genes in the exosomes. Results: The exosomes extracted by this method were uniform in size, showing a typical and complete vesicle-like structure. The particle size was concentrated at 30～150 nm, and the peak value was at 97.63 nm, indicating that the size was appropriate and the purity was high. Moreover, exosomes-specific protein CD9 and CD81 were expressed. PKH26 could bind to the membranous structure of exosomes and exosomes could be efficiently taken up by cells. Endothelial cells-associated CD31, vWF mRNA, and microRNA molecules such as miR-126, miR-21, miR-155 were expressed in exosomes secreted by EA.HY926. Conclusion: This method can effectively extract structurally intact, high-concentration, high-quality exosomes from large-volume cell perfusate.