Objective: To study the effects of Synaptotagmin1 gene knockout (Syt1^+/-) on emotional behavior in mice and explore its possible mechanisms. Methods: Five 8-week-old male Syt1^+/-mice and five wild-type (WT) mice in the same litter were selected. The expressions of Syt1 in 6 mice brain regions of prelimbic cortex (PL), hippocampus (HIP), amygdala (AMY), accumbens nucleus (ACB), caudoputamen (CP) and ventral tegmental area (VTA) were detected by Immunofluorescence staining. Nine 8-week-old male Syt1^+/-mice and ten WT mice were selected as controls. The anxiety-like behaviors of adult Syt1^+/- mice and WT mice were detected by open field test, elevated plus maze test and forced swim test. In addition, five 8-week-old male Syt1^+/-mice and five WT mice were selected to detect the glutamate content in prelimbic cortex, hippocampus and amygdala. Results: Compared with WT mice, the number of Syt1 positive cells in PL, HIP, AMY, ACB, CP and VTA were decreased significantly in Syt1^+/- mice (P＜0.01); Syt1^+/- mice had less total movement distance in open field test (P＜0.01), more preference for peripheral area (P＜0.01) and less desire to explore the central platform (P＜0.01), while Syt1^+/- mice preferred to stay in a closed and safe environment (P＜0.01); the number (P＜0.05) and the time spent in open-arm explorations (P＜0.01) were reduced significantly; the immobile time of Syt1^+/- mice was increased in the forced swim test (P＜0.01). Meanwhile, the concentration of glutamate in the amygdala of Syt1^+/- mice was increased significantly (P＜0.01). Conclusion: Syt1 gene knockout leads to significant anxiety-like behavior in mice, which is deduced that related to the increase of glutamate content in the amygdala.

Objective: To investigate the liver injury induced by lung ischemia / reperfusion(LI/R) and the role of autophagy in its prevention and treatment. Methods: The lung ischemia/reperfusion injury(LI/RI) model was prepared by anesthetizing the rats, cutting the trachea for mechanical ventilation, and using an arterial clamp to close the pulmonary hilum to simulate the ischemic process, and releasing the arterial clamp after 30 min to resume perfusion for 3 h. SD rats(n=24)were randomly divided into sham operation(sham)group,ischemia/reperfusion(I/R)group,solvent(DMSO)group and autophagy inhibitor (3-MA) group, 6 rats in each group. The rats were intraperitoneally injected with medicine before operation. After the rat LI/RI model was established,the rats were killed, and the lung wet/dry weight ratio was used to evaluate the success of modeling, the venous blood was collected to measure the contents of ALT and AST, and the liver tissues were collected. Light and electron microscopes were used to observed the liver tissues and cell shapes. The protein and mRNA expression levels of autophagy related proteins were determined by Western blot and RT-qPCR to suggest autophagy levels. Results: Compared with sham group, the lung wet/dry weight ratios in other groups were elevated, and the liver tissues of other groups were damaged significantly. Serum levels of AST and ALT were increased significantly and liver tissue damage was obvious, especially in I/R group. The light microscopy showed that the arrangement of hepatic cords was disordered or broken, hepatic sinuses were dilated, and edema of liver cells were observed; transmission electron microscopy showed varying degrees of mitochondria swelling up in liver cells in the other groups. At the same time, the expressions of AMPK, Beclin 1 and LC3 mRNA were increased, but the expressions of mTOR and p62 mRNA were decreased; the protein expressions of p-AMPK, Beclin 1, LC3-B were increased significantly, but those of p-mTOR and p62 were decreased (all P＜0.05). Compared with DMSO group, the injury of liver tissue in 3-MA group was alleviated, the damage degree of mitochondrial ultrastructure was lower, the levels of AST and ALT were decreased, the transcription and protein expression levels of autophagy related protein in liver tissue were decreased (P＜0.05). However, the injury degree of IR and DMSO groups were similar, and there was no significant differences in each index (P＞0.05). Conclusion: Lung ischemia/reperfusion can cause liver injury in rats. Autophagy can mediate liver injury induced by lung ischemia / reperfusion in rats and inhibiting autophagy can effectively reduce liver injury induced by LI/R in rats.

Objective: To construct the lentivirus overexpression vector with two label genes fused with CopGFP and Puro^R and to detect the emission of green fluorescence as well as resistance to puromycin in liver cancer cells infected with lentivirus packaged with the above vector. Methods: Firstly, two fragments containing copGFP and Puro^R coding sequences were amplified from pCDH-CMV-MCS-copGFP and pLKO.1 respectively; secondly, the two amplified regions were fused with each other by recombinant PCR; thirdly, the fusion DNA fragment was cut and inserted into pCDH-CMV-MCS-copGFP vector, which was linearized with the same restriction endonuclease as used to digest fusion DNA fragment: BamH Ⅰ and Sal Ⅰ. The fusion region in the constructed vector was confirmed by DNA sequencing. The checked vector was co-transfected with package assistant plasmids, namely PLP1, PLP2 and VSVG into in 293T cells and the culture supernatant was subjected to centrifuge and infect liver cancer MHCC97H cells, which were then used to detect their resistance to puromycin (infected cells were treated with 1 mg/ml puromycin for 7 days after infection) and to observe green fluorescence emission in microscope. To determine its efficiency in expressing foreign target protein, the Sp1 coding region was inserted into the MCS sites of the vector, and Sp1 mRNA and protein expression levels were compared with the vehicle vector by RT-qPCR and Western blot. Results: The lentivirus overexpression vector with two label genes fused with CopGFP and Puro^R was successfully constructed, and the liver cancer cells infected with lentivirus packaged with the vector expressing two labeling genes fused with CopGFP and Puro^Rshowed both emission of green fluorescence and resistance to puromycin simultaneously, while cells containing with the vector inserted with Sp1 coding region improved Sp1 mRNA level with 3.3 fold and protein level with 2.2 fold higher in comparison with cells containing the vehicle vector (P＜0.01). Conclusion: The fused label genes consisting of copGFP and Puro^R are correctly cloned into the lentivirus vector and confer cells with the ability to emission of green fluorescence and resistance to puromycin, besides, the vector may promote the expression of the target gene with long coding sequence.

Objective: To investigate the effects of intrathecal injection of IRF8 SiRNA on the pain threshold and activation of spinal cord microglia in rats with postoperative persistent pain. Methods: One hundred and twenty male Sprague-Dawley rats were randomly divided into sham group (SH, n=12), SMIR group (SM, n=48), SMIR + DEPC group (SD, n=12) and SMIR + irf8 SiRNA group (SS, n=48). In the SM group, the persistent postsurgical pain(PPsP) model was established according to the skin/muscle incision and retraction (SMIR), and the SH group was only incised without retracted. The SD group and SS group received intrathecal catheterization one week before SMIR, the SS group was injected with 20 μl of IRF8 SiRNA solution (dissolved in DEPC-treated water, 150 pmol) intrathecally on the 5^th and 6^th day after SMIR, and the SD group was injected with the same amount of DEPC-treated water. The paw withdrawal threshold (PWT) of each group was measured and recorded before SMIR and on the 1^st, 3^rd, 7^th, 12^th, 22^nd and 33^rd days after SMIR. Western blot was used to detect the expression of Iba-1 in the dorsal horn of spinal cord on the 12^th days after SMIR, and the saphenous nerves in the SH group and SM group were collected to observe their ultrastructural changes under electron microscope. The flow cytometry was used to detect the activation of microglia in spinal cord dorsal horn before SMIR and on the 1^st, 3^rd, 7^th, 12^th, 22^nd and 33^rd days after SMIR in the SM group and SS group. Results: Compared with D0, the PWT of SM group was decreased on the 1^th to 22^nd day after SMIR (P＜0.05 or P＜0.01), and returned to normal level on the 33^rd day after SMIR (P＞ 0.05). Compared with the SH group, the PWT of the SM group was decreased on the 1^th to 22^nd day after SMIR (P＜0.05 or P＜ 0.01). However, compared with the SD group, the PWT of the SS group was increased on the 7^th to 22^nd day after SMIR (P＜0.05 or P＜0.01). Compared with SH group, the PWT of SS group was decreased on the 7^th to 22^nd day after SMIR (P＜0.05 or P＜0.01). The average thickness of saphenous nerve myelin was (377.0 3±69.60) nm in the SH group and (369.50±73.26) nm in the SM group, and there was no significant difference between the two groups (P＞0.05). Compared with the SH group, the expression level of Iba-1 was increased significantly (P＜0.01) in the SM group. Compared with the SD group, the expression of Iba-1 was inhibited (P＜0.05) in the SS group, and compared with the SH group, the expression of Iba-1 was also statistically different (P＜0.05) in the SS group, while the expression of Iba-1 was not statistically significant between the SM group and the SD group (P＞0.05). Compared with D0, the activation ratio of microglia was increased significantly on the 3^rd to 22^nd day after SMIR (P＜0.01) in the SM group , while the activation of microglia reached a peak on 3rd day after SMIR (P＜0.01) in the SS group. After intrathecal administration, the activation rate of microglia in the spinal dorsal horn of the SS group was decreased significantly, and compared with the SM group, it was decreased significantly on the 7^th to 12^th day after SMIR (P＜0.01). Conclusion: The significant and persistent mechanical hyperalgesia in PPsP induced by SMIR was caused non-obvious peripheral nerve injury, which may be mediated by the activation of microglia in the dorsal horn of the spinal cord. IRF8 SiRNA administrated by intrathecal injection could inhibit the activation of microglia and reverse SMIR-induced hyperalgesia.

Objective: To investigate the effects of the peroxisome proliferator-activated receptor δ (PPARδ) agonist GW501516 on the proliferation of primary rat proliferation of pulmonary artery smooth muscle cells ( PASMCs ) induced by hypoxia, in order to discover new drugs for the treatment and prevention of pulmonary vascular remodeling. Methods: The PASMCs in the control group were cultured with 21% oxygen, while the PASMCs in the hypoxia group were cultured with 3% oxygen to induce cell proliferation. PASMCs were incubated with GW501516 at the concentrations of 10, 30 and 100 nmol/L under hypoxic conditions for different time points (12, 24, and 48 h) to find out the appropriate concentrations of GW501516 for inhibition the proliferation. PASMCs were incubated with 100 nmol/L GW501516 and ( or ) protein kinase B (AKT) agonist SC79 for 24 h to explore related mechanisms of GW501516 in regulating the proliferation. The proliferation and DNA synthesis were determined by CCK-8 and BrdU kit. The cell cycle progression was analyzed by flow cytometry. The mRNA expressions of Cyclin D1 and the cyclin kinase inhibitor p27(p27) were measured by quantitative real-time PCR (RT-PCR). The expressions of PPARδ, total and phosphorylated forms AKT and glycogen synthase kinase 3β (GSK3β) were detected by Western blot. Results: Compared with the hypoxia group, PASMCs incubated with different concentrations of GW501516 (10, 30, 100 nmol/L) for 12, 24, 48 h under hypoxic conditions could inhibit the proliferation and DNA synthesis, and the greatest level of suppression of proliferation was induced by GW501516 at the concentration of 100 nmol/L(P＜0.05 or P＜0.01). Compared with the control group, the expression of PPARδ was upregulated markedly in PASMCs incubated with 100 nmol/L GW501516 for 24 h,while hypoxia could downregulate the expression of PPARδ significantly(P＜0.01). Compared with the hypoxia group, 100 nmol/L GW501516 blocked the proliferation and DNA synthesis of PASMCs significantly(P＜0.01), increased the proportion of PASMCs in G0 /G1 phase while decreased the proportion of PASMCs in S phase and G2 /M phase(P＜0.05 or P＜0.01), markedly downregulated the mRNA expression of cyclin D1 and upregulated the mRNA expression of p27(P＜0.01), significantly inhibited the protein expressions of phosphorylated AKT and GSK3β(P＜0.01). Compared with the 100 nmol/L GW501516 hypoxia group, AKT agonist SC79 reversed all the above effects of 100 nmol/L GW501516 on hypoxia stimulated PASMCs(P＜0.05 or P＜0.01). Conclusion: GW501516 inhibits hypoxia induced proliferation in PASMCs via inactivating AKT/GSK3β signaling pathway.

Objective: To investigate the expression level of podocyte slit diaphragm protein in rats after one-time exhaustive exercise, to explore the effect of PKC inhibitor on the protein expression level, and to reveal the mechanism of PKC in the formation of exercise-induced proteinuria. Methods: Thirty male SD rats were randomly divided into control group (C), exercise group (E) and exercise combining with PKC inhibitor group (EPI), with 10 rats in each group. Rats in group E and EPI performed one single bout of exhaustive exercise (25 m/min), rats in group EPI were intraperitoneally injected with a PKC inhibitor (chelerythrine, 5 mg/kg) 1 day and 1 hour before exercise respectively, while rats in group C and E were injected with the same volume of saline. Results: ①Compared with group C, the levels of urine protein, uric acid, urine sugar, blood urea, and blood uric acid of rats in group E were increased significantly (P＜0.05), the level of blood glucose was reduced significantly (P＜0.01), and renal ROS production was increased significantly (P＜0.01). The expressions of nephrin and podocin protein in renal tissue were decreased significantly (P＜ 0.05), while the expressions of PKC, Nox2, and Nox4 protein were increased significantly (P＜0.05). ②Compared with group E, the levels of urinary protein,urine glucose and blood urea in EPI group were decreased significantly (P＜0.05), the level of blood glucose was increased significantly (P＜0.01), renal ROS production was reduced significantly (P＜0.01). the expressions of nephrin and podocin protein in renal tissues of the EPI group were increased significantly (P＜0.05), while the expressions of PKC and Nox2 protein was reduced significantly (P＜0.05, P＜0.01). Conclusion: One-time exhaustive exercise can down-regulate the expressions of nephrin and podocin through PKC/Nox/ROS pathways in the kidney of rats; PKC inhibitor alleviates the decrease in the expression of podocyte slit diaphragm protein caused by exhaustive exercise, and prevents the occurrence of exercise-induced proteinuria.

Objective: To investigate the effects of 6 weeks of aerobic exercise on the sarcoplasmic reticulum calcium regulatory proteins in skeletal muscle of apolipoprotein E (ApoE) knockout mice fed by high-fat diet. Methods: There were a total of twenty five 9-week-old ApoE knockout mice (ApoE knockout mice, ApoE KO), five of which were selected randomly for the maximum running speed test. The running speed would be increased by 1.2 m/min every 3 min after a 5-min duration of initial speed of 4.8 m/min without slope until exhaustion, then the final speed was set as maximal speed, and the test result of the maximum running speed was (27.0±2.4)m/min. The remaining 20 ApoE KO mice were randomly divided into ApoE KO mouse high-fat diet group (KO) and ApoE KO mouse high-fat diet + aerobic exercise group (KE), 10 mice per group. Ten 9-week-old wild-type C57BL/6J mice were used as a blank control group (wild-type, WT). High fat diet composition: fat content was 21% (w/w) and cholesterol content was 1.5% (w/w). Exercise intervention was initiated 1 week after adaptive training in the KE group with an exercise protocol consisting of 40% maximal running speed (10.8 m/min), 40 min/d and a frequency of 3 d/w for a total of 6 weeks. All mice were anesthetized and sacrificed by cardiac puncture then bilateral gastrocnemius muscles were isolated immediately 48 h after the final exercise. Skeletal muscle Ca²⁺ concentration was detected by visible light colorimetry; the expression levels of RyR, CaM, CaMK Ⅱ, SERCA1 and SERCA2, which are calcium regulated proteins of sarcoplasmic reticulum, in mouse skeletal muscle, was detected by Western blotting. Results: Compared with WT mice, the Ca²⁺ concentration, the expression levels of the sarcoplasmic reticulum calcium releasing proteins RyR, CaMKⅡ, and the calcium recycling proteins SERCA1 and SERCA2 were decreased significantly in skeletal muscle of the KO mice (P＜0.05, P＜0.01), while the expression of CaM protein did not change. Skeletal muscle Ca²⁺ concentration and the levels of sarcoplasmic reticulum calcium recycling proteins SERCA1 and SERCA2 were increased significantly (P＜0.05) in KE mice compared with KO mice, but there were no significant differences in the expressions of the sarcoplasmic reticulum calcium release proteins RyR, CaM and CaMK II. Conclusion: High fat diet can reduce the concentration of Ca²⁺ in skeletal muscle and weaken the release and recovery of sarcoplasmic reticulum calcium in ApoE knockout mice. 6-week aerobic exercise training can significantly increase its Ca²⁺concentration and promote the recovery of sarcoplasmic reticulum calcium.

Objective: To illuminate the protective effects of pathway in inhibiting ferroptosis by glutathione peroxidase 4 (GPX4) activated by nuclear factor erythroid 2-related factor 2 (Nrf2) during aerobic exercise against myocardial injury in high-fat diet mice. Methods: Forty 5-week-old SPF C57BL/6 male mice were randomly divided into the control group (NC), the exercise group (NE), the high fat group (HC) and the high fat diet with exercise group (HE, began at the same time). There were 10 mice in each group. The mice in the high fat diet group were fed with 60% Kcal SPF high fat model diet. Aerobic exercise was performed using increasing load platform exercise, 5 days /week, 60 min/d, the speed started from 13m/min, and increased by 1m/min every two weeks. Myocardium and blood samples were collected after 14 weeks. Structural changes of myocardial tissues were observed by HE staining. Western blot was used to detect the expressions of Nrf2/GPX4/Ferroptosis related proteins in myocardium. Myocardial peroxide concentration and antioxidant enzyme activity were measured by spectrophotometry. Myocardial mitochondrial 8-hydroxy-2 deoxyguanosine (8-OHdG) and serum insulin were measured by ELISA. Results: Compared with the NC group, there was more lipid accumulation in the myocardial fiber space in the HC group, and the levels of FBG and FINS were increased significantly, while ISI was decreased significantly (P＜0.01). Compared with the HC group, the lipid concentration was decreased in the HE group, and the activities of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) and glutathione (GSH) were increased significantly, while the levels of mitochondrial 8-OHdG and myocardial iron content were decreased (P＜0.01). The expression levels of Ferroportin1 (FPN1), ferritin heavy chain 1 (FTH1), GPX4, glucose transporter (GLUT1) and Nrf2 in the HE group were significantly higher than those in the HC group (P＜0.01). Conclusion: The expression of GPX4 was enhanced by more Nrf2 transposition into the nuclear during aerobic exercise, which inhibited the occurrence of myocardial ferroptosis. The activities of antioxidant enzymes were promoted and inhibited the peroxidation damage of myocardial mitochondria.

Objective: To investigate the possible protective effects of combined dexamethasone and valsartan against cigarette induced chronic obstructive pulmonary disease (COPD) in mice. Methods: Forty C57BL/6 mice were randomly divided into control group, COPD group, dexamethasone treated group, valsartan treated group and dexamethasone + valsartan combined treatment group, with 8 mice in each group. Mice in COPD group were exposed to cigarette for 8 weeks. On the basis of cigarette exposure, mice in dexamethasone treated group were intraperitoneally injected with dexamethasone (2 mg / kg) before cigarette exposure for 5-8 weeks. Mice in valsartan treated group were intraperitoneally injected with valsartan (30 mg/kg) before cigarette exposure for 1-8 weeks. Dexamethasone (2 mg/kg) and valsartan (30 mg/kg) were injected intraperitoneally into mice in the dexamethasone + valsartan combined treatment group. After 8 weeks, the lung tissues and bronchoalveolar lavage fluid (BALF) of mice in each group were collected. The pathological score of lung tissue was evaluated. The activities of superoxide dismutase(SOD) and matrix metalloproteinase-9 (MMP-9), and the contents of malondialdehyde (MDA), intracellular adhesion molecule-1 (ICAM-1), C-reactive protein (CRP) and nitric oxide (NO) in BALF were determined. Results: Compared with the control group, COPD mice had emphysema and alveolar congestion, the levels of MDA, ICAM-1, MMP-9, CRP and lymphocytes in BALF were increased, while the levels of SOD, macrophages and NO were decreased (all P＜0.05). Compared with COPD group, there was no significant improvement in emphysema and alveolar congestion, the levels of SOD and NO in BALF were increased, and the levels of MDA, lymphocytes and macrophages were decreased in dexamethasone or valsartan group (all P＜0.05). Compared with dexamethasone or valsartan group, the dexamethasone + valsartan combined treatment was more effective in preventing pulmonary emphysema and alveolar congestion caused by cigarette smoke. The levels of MDA, ICAM-1, MMP-9, CRP and lymphocyte in BALF were decreased, while the levels of SOD, macrophage and NO were increased (all P＜0.05). Conclusion: Compared with dexamethasone or valsartan, dexamethasone combined with valsartan has a more effective protective effect in COPD mice by inhibiting oxidative stress and inflammation.

Objective: To investigate the effects and mechanisms of Astragalus polysaccharide on improving imiquimod-induced psoriasiform dermatitis in mice. Methods: Forty healthy female C57BL/6 mice were randomly divided into 5 groups, including blank control group, model group, astragalus polysaccharide high-dose group (200 mg/kg), medium-dose group (100 mg/kg) and low-dose group (50 mg/kg), with 8 mice in each group. The mice in model group and astragalus polysaccharide treatment group were treated with 5% imiquimod cream on the back to induce psoriasiform dermatitis. PASI score was monitored, and the secretion of inflammatory factors was determined by ELISA. The secretion of inflammatory factors was closely related to the infiltration of macrophages. The infiltration of macrophages in skin was detected by flow cytometry to further explore the effect of different concentrations of APS on psoriasis. Results: Compared with control group, the PASI score and the serum levels of TNF-α, IL-1β and IL-6 were increased significantly (P＜0.05), and the infiltration of macrophages in skin tissue was increased significantly in model group (P＜0.05). Compared with model group, the PASI score was decreased significantly (P＜0.05), and the serum levels of TNF-α, IL-1β and IL-6 were down-regulated significantly in astragalus polysaccharide high-dose and medium-dose groups (P＜0.05). The infiltrating macrophages in skin tissue were decreased significantly in Astragalus polysaccharide high-dose group (P＜0.05). Conclusion: Astragalus polysaccharide improve psoriasiform dermatitis in mice by inhibiting the infiltration of macrophages in skin tissue and decreasing the secretion of TNF-α, IL-1β and IL-6 in serum.

Objective: To investigate the protective effect of edaravone on chlorpyrifos-induced neuronal apoptosis and its mitochondrial mechanism. Methods: Under the principle of randomization and double-blindness, the rats were divided into control group, chlorpyrifos group, and edaravone group (n=6). The rats in edaravone group were treated with edaravone (10 mg/1.6 ml/kg, ip.) 1 h after chlorpyrifos injection. After continuous injection of chlorpyrifos and edaravone for 28 days, the learning and memory abilities of the rats were tested by open field and water maze tests. The rat brain tissue was collected after cardiac perfusion, and the neuronal damage in the hippocampus of the brain was detected by HE staining and the mitochondrial and nuclear damage were observed by transmission electron microscopy. The contents of Na⁺-K⁺-ATPase and ATP were measured to evaluate mitochondrial damage. The expression of mitochondrial fission protein DRP1 and phosphorylation at Ser 637 of DRP1 were determined by immunohistochemistry and immunoblotting. Results: Compared with the control group, the total movement distance and average speed of the rats in the chlorpyrifos group were decreased significantly within 3 minutes of the open field test (P＜0.01), and the escape latency within 1 minute of the water maze test was prolonged significantly. The number of platform crossings was reduced significantly (P＜0.01), the activity of ATPase in brain tissue was decreased significantly (P＜0.01) , the content of ATP and the phosphorylation level of Ser637 of mitochondrial DRP1 were decreased significantly (P＜0.05, P＜0.01). After edaravone treatment, the total movement distance and average speed of rats in the open field test were increased (P＜0.05), the latency in the water maze test was decreased, and the number of crossing platforms was increased (P＜0.01), brain pathological sections showed that nerve cells were arranged neatly, nucleus and mitochondrial damage was significantly improved, the activity of ATPase in brain tissue was increased (P＜0.01), the levels of ATP and mitochondrial DRP1 Ser637 phosphorylation increased (P＜0.05, P＜0.01).Conclusion: Edaravone alleviates chlorpyrifos-induced brain injury in rats by promoting the phosphorylation of DRP1 at Ser637.

Objective: To investigate the inhibitory effects of thalidomide on the expressions of VEGF and bFGF in human lung adenocarcinoma A549 cells and human hepatocellular carcinomas HepG2 cells mediated by cereblon (CRBN). Methods: shRNA technology was used to construct the A549 cell line (A549_CRBN) and HepG2 cell line (HepG2_CRBN) with stable knockdown of CRBN, which was verified by real-time PCR and Western blot. A549 cells were divided into negative control group (A549_luciferase) and CRBN down-regulation group (A549_CRBN); HepG2 cells were divided into negative control group (HepG2_luciferase) and CRBN down-regulation group (HepG2_CRBN). The above cells were seeded into 6-well plates at 3×10⁵ cells/well, and cultured in a 37℃, 5% CO₂ incubator for 24 h. Then, 1 ml medium containing 100 μmol/L thalidomide (thalidomide group) and 1 ml medium containing 1‰ DMSO (control group) were added respectively, and the culture was continued for 24 hours before subsequent experiments. Each group was designed with three replicate wells. The effect of thalidomide on the activity of A549 cell line was detected by MTS assay. Real-time PCR was performed to detect mRNA expression levels of VEGF, bFGF and c-jun. ELISA assay was performed to detect protein expressions of VEGF and bFGF. Results: Compared with the control group, thalidomide at the concentrations of 1, 10, 50 and 100 μmol/L had no significant effects on the proliferation of A549 and HepG2 cells (P＞0.05). VEGF and bFGF levels in the A549_CRBN or HepG2_luciferase groups were significantly lower than those in the A549_CRBN or HepG2_CRBN groups (P＜0.05). Compared with the control group of the A549_luciferase or HepG2_luciferase, thalidomide inhibited the expressions of VEGF and bFGF in A549_luciferase and HepG2_luciferase cells (P＜ 0.05), but did not inhibit the expressions of VEGF and bFGF in A549_CRBN and HepG2_CRBN cells. Compared with the control group of the HepG2_luciferase, thalidomide inhibited c-Jun expression in HepG2_luciferase cells (P＜0.01), but did not significantly inhibit c-Jun expression in HepG2_CRBN cells. Conclusion: The inhibitory effects of thalidomide on VEGF and bFGF expressions may be mediated by CRBN in A549 and HepG2 cells, and c-Jun may be one of the key transcription factors responsible for this inhibition.

Objective: To investigate the effects of SI-4650, a novel small molecule inhibitor of spermine oxidase (SMO), on the proliferation and epithelial mesenchymal transformation (EMT) of human ovarian cancer SKVO-3 cells as well as its underlying molecular mechanisms. Methods: SKVO-3 cells treated with 0 μmol/L SI-4650 were used as control group, SKVO-3 cells treated with 30, 60 μmol/L SI-4650 were used as experimental group. The effects of SI-4650 on the activity of SMO, the polyamine contents and the cellular reactive oxygen species (ROS) were detected. Cell proliferation, cell cycle and mitochondrial membrane potential change of SKVO-3 cells were tested. The effects of SI-4650 on apoptosis, migration and invasion were investigated. The effects of SI-4650 on Bax, Bcl-2, Caspase3, E-cadherin, N-cadherin, Vimentin, matrix metalloproteinase 2 ( MMP2) and MMP 9 expression levels in SKVO-3 cells were detected. Results: Comparison between blank control group and experimental groups,SI-4650 could improve the content of SI-4650 in SKVO-3 cells. SI-4650 could inhibit the activity of SMO (P＜0.01), reduce the ROS (P＜0.01)and polyamine content in SKVO-3 cells (P＜0.01). Treatment of SKVO-3 cells with SI-4650 inhibited the proliferation (the inhibition rate was 32.27% and 47.31% in experimental groups), caused S-phase cell cycle arrest (P＜0.01) and induced apoptosis (P＜0.01). The expressions of Bax and c-Caspase3 in SKVO-3 cells were increased (P＜0.01),the content of Bcl-2 was decreased (P＜0.01), and the mitochondrial membrane potential was decreased (P＜0.01), and the number of apoptotic cells was increased(31.41% and 43.51% in experimental groups). At the same time, SI-4650 could change the expression levels of EMT-related factors, increased the expression level of E-cad , decreased the expression levels of N-cad, Vimentin, MMP-2 and MMP-9, and inhibited the migration and invasion of SKVO-3 cells. Conclusion: SI-4650 can effectively inhibit proliferation, invasion and metastasis of human ovarian cancer SKVO-3 cells, and the mechanism may be related to its ability to depress the activity of SMO, interfere polyamine metabolism and induce cell cycle arrest, mitochondrial apoptosis and inhibit EMT. This study reveals potential application of SI-4650 in the treatment of ovarian cancer.

Objective: To investigate the effects of berberine on glucose and lipid metabolism, sex hormone binding protein, adiponectin (LPS), NF-κB and MAPK signaling pathways in polycystic ovary syndrome (PCOS) model rats. Methods: Female SD rats were randomly divided into control group, PCOS model group, berberine (0.216 g/kg) group, metformin (0.135 g/kg) group and Dyne-35 (0.18 mg/kg) group, 10 rats in each group. The rats in PCOS model group were treated with letrozole 1 mg.kg^-1 by ig for 3 weeks. After 28 days of drug intervention, the body constitution, ovarian and uterine indexes of the rats were detected, and the changes in the number of ovarian follicles were observed by HE staining. The levels of serum sex hormone, glucose and insulin, triglyceride and cholesterol, sex hormone-binding protein and adiponectin were determined by ELISA, and the protein expressions of p38-MAPK, C-Jun and NF-κB in ovarian tissues were determined by Western blot. Results: Compared with control group, body weight of model group was increased (P＜0.05), and uterine index was decreased (P＜0.05); The number of follicles was increased (P＜0.05). Serum levels of luteinizing hormone (LH), testosterone (T) and LH/FSH ratio were increased (P＜0.05), follicular estrogen (FSH) level was decreased (P＜0.05), total cholesterol (TC), triglyceride (TG), fasting insulin and insulin index (HOMA) were increased (P＜ 0.05). The content of sex hormone binding protein (SHBG) was decreased and the content of adiponectin (LPS) was increased (P＜0.05). The protein expressions of p38-MAPK, c-Jun and NF-κB in ovarian tissue were up-regulated (P＜0.05). Compared with model group, in berberine group, the uterine index and the number of secondary follicles were increased(P＜0.05), the serum levels of luteinizing hormone (LH) , testosterone (T) and the ratio of LH/FSH were decreased (P＜0.05), and the protein expression levels of p38-MAPK and NF-κB in ovarian tissue were down-regulated (P＜0.05), which were similar to those of Dyne-35 group. Berberine significantly decreased serum triglyceride (TG), insulin level and insulin index (P＜0.05), increased serum SHBG level and decreased serum LPS level (P＜0.05), which were similar to those of metformin. Conclusion: Berberine can regulate sex hormone disorder and insulin resistance (IR) in PCOS rats by down-regulating the expressions of p38-MAPK and NF-κB protein in ovarian tissues and decreasing the serum content of LPS.

Objective: By isolating and purifying primary hepatocytes and primary Kupffer cells from rats with nonalcoholic steatohepatitis (NASH), and establishing the primary cell model of NASH in vitro, to provide reliable technical support for cell experiment in the study of NASH. Methods: Forty SD rats were selected and randomly divided into the control group and the NASH group. The rats in the control group were fed with common feed, and the rats in the NASH group were fed with a high-fat diet (88% basal feed + 10% lard + 2% cholesterol). After 6-8 weeks, using the NASH score table, the liver tissue section steatosis + intralobular inflammation + ballooning degeneration score ≥ 4 points under pathological observation, indicating that the rat NASH model was successfully established. And the primary hepatocytes of NASH rats were isolated and purified by collagenase in situ perfusion. Cells were identified by CK-18 and CD68 immunofluorescence and ink swallowing test. The lipid accumulation was tested by Oil red O staining, and the contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined to evaluate the liver function in primary hepatocytes of NASH rats. The expressions of inflammatory factors of primary Kupffer cells were detected by Western blot. Finally, primary hepatocytes and primary Kupffer cells were co cultured at the ratio of 6:1 and observed under microscope. Results: NASH primary hepatocytes and primary Kupffer cells were successfully isolated and purified. Compared with the control group, Oil red O staining showed that the primary hepatocytes of the NASH group had obvious fat deposition, and the AST and ALT levels in the primary hepatocytes of the NASH group were significantly higher than those of the control group, indicating obvious liver damage (P＜ 0.05). The Western blot result showed that the levels of TNF-α, IL-1β and MCP-1 in primary Kupffer cells was significantly higher than the control group (P＜0.05). Conclusion: The primary hepatocytes and primary Kupffer cells of NASH rats were isolated successfully by collagenase in situ perfusion. At the same time, a proportional co-culture rat in vitro primary cell NASH model was successfully established.