Objective: Increasing activities in anterior cingulate cortex (ACC) can enhance the aversion reactions associated with noxious stimuli. It has been known that opioid receptors activation can trigger endogenous analgesic effect. This study tried to explore whether opioid receptors activation in the ACC region could reduce the aversion associated with noxious stimuli. Methods: The experimental rats were randomly divided into seven groups, Complete Freund's adjuvant (CFA) + normal saline (NS) group, normal saline (NS) + normal saline (NS) group, normal saline (NS) +DAMGO ((DAla², NMe-Phe⁴, Gly-ol⁵)enkephinlin, μ-opioid receptor agonist) group, complete Freund's adjuvant (CFA)+ 0.01/0.04/0.2/1 μg/μl DAMGO group(n=6). The experimental period was three days. The basal value was measured on the first day. The second day, 1 μl was administered through the ACC area, and then 0.08 ml of complete Freund's adjuvant (CFA) was injected into the left hind paw of the rat. CPA response, paw withdrawal reflex latency (PWL) and electrical activity in the ACC brain region of rats were observed on the third day. Results: ①PWL was significantly decreased in rats after CFA was injected into left hind paw compared with post-injection(P＜0.05).② In the pain side of the apparatus, it took rats less residence time than that in the non-pain side. ③ 0.04/0.2/1 μg/μl DAMGO was given before CFA-injection, C-CPA reactions could be revised significantly. ④ Given 0.04/0.2/1 μg/μl DAMGO in the ACC region could decrease the increasing discharge frequency induced by CFA in ACC neurons. Conclusion: The activation of the mu-opioid receptor in the ACC region alleviates the aversion induced by noxious stimulation.

Objective: To observe the effects of the neurogenesis in the dentate gyrus (DG) of the hippocampus on depression-like behaviors in adult Wistar Kyoto (WKY) rats. Methods: There were three groups in total (n = 10): ① the control (Wistar) group: 9-week-old Wistar rats were treated with saline for 3 weeks (10 mg/kg, intragastric administration); ② the depression model (WKY) group: WKY rats of the same age, tested for depression-like behaviors, were as a rat model of depression, and were treated with saline for 3 weeks (10 mg/kg, intragastric administration); ③ the positive control (AMI+WKY) group: WKY rats of the same age were treated with amitriptyline for 3 weeks (10 mg/kg, intragastric administration). The neurogenesis in hippocampus was detected by immunofluorescence staining for Ki67 (a neuronal proliferation marker) and DCX (an immature neuronal marker). The depression-like behaviors were assessed by sucrose preference test (SPT), open field test (OFT) and forced swimming test (FST). Results: ① When compared with Wistar rats, the number of Ki67⁺ cells and DCX⁺ cells of the DG in WKY rats were decreased by 33.0% (P＜0.01) and 39.2% (P＜0.01), respectively; amitriptyline treatment significantly increased the number of Ki67⁺cells and DCX⁺ cells in the DG by 43.8% (P＜0.01) and 46.7% (P＜0.01), respectively, as compared with WKY rats. ② When compared with control group, WKY 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, the immobility time in the FST was increased significantly (P＜0.01). Amitriptyline treatment significantly improved the depression-like behaviors in WKY rats. Conclusion: ① The proliferation and differentiation of hippocampal neural stem cells in adult WKY rats are significantly lower than those of Wistar rats, suggesting that the neurogenesis in adult WKY rats is impaired. ②Amelioration of impaired hippocampal neurogenesis can partially reverse the depression-like behaviors in adult WKY rats.

Objective: To investigated the effects of dihydromyricetin on cognitive and affective disorders induced by chronic social defeat stress and its possible mechanism in mice. Methods: C57BL/6J mice were randomly divided into control group (Control), chronic social defeat stress group (CSDS) and chronic social defeat stress + DHM group (CSDS+DHM) (14 mice in each group). The mice received chronic social defeat stress and were injected with DHM or vehicle intraperitoneally. A part of mice were subjected to (10 mice of each group) novel object recognition test (NOR), Y maze test, open field test (OFT), social interaction test (SIT), forced swimming test (FST) and tail suspension test (TST). The other mice (4 mice of each group) were decapitated and the expression levels of SIRT1 in hippocampus were detected by Western blot. Results: Compared with the control group, the learning and memory of the CSDS group were reduced significantly, the anxiety level was increased significantly, the immobility time in TST and FST was increased significantly, and the SIRT1 protein level in hippocampus was reduced significantly (P＜ 0.05 or P＜ 0.01); Compared with the CSDS group, the learning and memory of the CSDS + DHM group were improved significantly, the anxiety level of the mice was reduced significantly, and the immobility time in TST and FST was reduced significantly. The protein level of SIRT1 in hippocampus was increased significantly (P＜ 0.05 or P＜ 0.01). Conclusion: DHM ameliorates the cognitive impairment, anxiety like behavior and depression like behavior of mice induced by CSDS and up-regulates the expression of SIRT1 protein.

Objective: To study the mechanisms of curcumin alleviating oxidative stress and spleen apoptosis induced by overtraining in rats by regulating Kelch-like ECH-associated protein-1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway. Methods: Male Wistar rats of 7 weeks old were divided into control group (C group, 12), overtraining group (OM group, 11), curcumin + overtraining group (COM group, 14). The C Group did not undergo any exercise intervention. The OM and COM group underwent 8-week incremental load swimming training. During the training, rats in the COM group were treated with curcumin at the dose of 200 mg/(kg·d) in the volume of 5 ml/kg by gavage, and rats in the other groups were given an equal volume of solvent, 0.5% sodium carboxymethylcellulose. Twenty-four hours after the last training, the spleen index was calculated by weighing, the pathological changes of the spleen were observed by light microscopy, and the biochemical indicators of blood and spleen were detected. Results: The spleen structure of C group was normal under light microscope; the spleen index of OM group was significantly lower than that of C group (P＜0.01) and pathological changes were obvious; the spleen index of COM group was significantly higher than that of OM group (P＜0.05) and histomorphological changes were relieved. Compared with C group, in OM group, serum corticosterone (Cor) level, spleen apoptosis level, malondialdehyde (MDA) concentration and the expression of proapoptotic Bcl-2 associated X protein (Bax) in spleen were increased (P＜0.05 or P＜0.01); the body weight, serum testosterone (T), superoxide dismutase (SOD) activity, the expressions of heme oxygenase-1 (HO-1) and anti-apoptotic B cell lymphoma-2 protein (Bcl-2) in spleen were decreased (P＜0.05 or P＜0.01); the expression of Nrf2 was not changed significantly (P＞ 0.05). Compared with OM group, in COM group, there were no significant changes in body weight (P＞0.05), serum T level, SOD activity, the expressions of Bcl-2, Nrf2 and HO-1 in spleen were increased (P＜0.05 or P＜0.01); serum Cor level, spleen apoptosis level, MDA concentration and the expression of Bax were decreased (P＜0.05 or P＜0.01). The change trend of T/Cor ratio between groups was consistent with the change of testosterone, and the change trend of Bcl-2/Bax ratio was consistent with the change of Bcl-2. Conclusion: The 8-week incremental load excessive swimming training aggravated spleen apoptosis, led to pathological changes and dysfunction of spleen. Curcumin can up-regulate expression of Nrf2 and HO-1, alleviate oxidative stress induced by overtraining, enhance Bcl-2 expression and attenuate Bax expression, thereby inhibiting excessive spleen apoptosis of rats, protecting the structure and function of spleen.

Objective: To study the effects of Salvia mil on Th17 cells and related cytokines in non-alcoholic fatty liver disease (NAFLD) rats. Methods: SD rats were randomly divided into control group, model group, Pueraria control group and Salvia mil treated group (8 rats in each group). Rats in Salvia mil treated group and Pueraria control group were treated with Salvia mil or Pueraria at the dose of 6.25g·kg^-1·d^-1 by gavage once a day for 4 weeks. Except for control group, rats in other groups were fed with high-fat diet for 4 weeks. The levels of serum lipids (TC, TG, LDL-C, HDL-C), liver function indexes (AST, ALT) and liver index were measured, and the pathological changes of liver tissues were observed. Th17 and Treg cells in peripheral blood were detected by flow cytometry. Serum levels of IL-6, IL-17 and TNF-alpha were detected by ELISA. The expression level of RORγt gene in liver tissue was detected by RT-PCR. Results: NAFLD rat model was successfully established by feeding high-fat diet for 4 weeks. The liver of model group had steatosis and a large number of inflammatory cells infiltrated. Compared with model group, the levels of TC, TG, LDL-C, ALT, AST and liver index in Salvia mil treated group and Pueraria control group were significantly lower than those of model group (P＜0.05), and the level of HDL-C was significantly higher than that of model group (P＜0.05). The content of Th17 cells in peripheral blood of rats in Salvia mil treated group was 1.58%±0.18%, significantly lower than that of model group 3.50%±0.16% and Pueraria control group 1.69%±0.20% (P＜0.05), Treg cell content was 4.58%±0.18%, higher than that of model group 3.71%±0.22% (P＜0.05) and Pueraria control group 4.52%±0.19%(P＞0.05), Treg/Th17 was 2.91±0.25, significantly higher than that of model group 1.06±0.24 and Pueraria control group 2.67±0.26 (P＜0.05). The levels of IL-6, IL-17 and TNF-α in Salvia mil treated group were lower than those of model group and Pueraria control group, and the levels of IL-6 and IL-17 were decreased significantly (P＜ 0.05). The expression level of RORγt gene in Salvia mil treated group was 0.72±0.09, significantly lower than that of model group 2.16±0.14 and Pueraria control group 1.32±0.08 (P＜0.05). Conclusion: Salvia mil can decrease the levels of IL-6, IL-17 and TNF-α, inhibit the expression of RORγt gene, decrease the content of Th17 cells, increase the content of Treg cells, adjust the balance of Th17/Treg, then inhibit the development of NAFLD.

Objective: To investigate the protective effects of nitidine chloride (NC) on dextran sodium sulfate (DSS) - induced ulcerative colitis (UC) in mice by targeting miR-31 and its underlying mechanisms. Methods: DSS at the concentration of 1% was used to induce UC in mice. Thirty C57BL/6 male mice were randomly divided into four groups: normal control group (n=7), DSS group (n=8), DSS + NC group (7.27 mg/kg) (n=8) and NC group (n=7). DSS was added in drinking water, and NC was administrated by gavage. The period of modeling lasted for 3 weeks. The control group and NC group drank sterile water every day, DSS group and DSS + NC group drank 1% DSS water in the first week, normal water in the second week and 1% DSS water in the third week. In the last week of modeling, mice in control group and DSS group were given 0.5% CMC-Na by gavage, while mice in DSS + NC group and NC group were given NC by gavage. After the establishment of the model, the disease activity index (DAI) related to colitis was observed, the pathological score of colon tissue was evaluated by HE staining, the expression level of miR-31 in colon tissue was detected by qPCR, and the protein expressions of NF -κ B and COX-2 in colon tissue were detected by Western blot. Results: ① Compared with DSS group, the DAI in the DSS + NC group was decreased (P＜0.01). The colonic pathological injury was obviously ameliorated after treated by NC. ② Compared with normal control group, the expression of miR-31 in colonic tissue of DSS group was increased significantly(P＜0.01), compared with DSS group, the expression of miR-31 was decreased after treatment with NC(P＜ 0.05). ③ Compared with DSS group, the levels of inflammatory protein NF-κB and COX-2 in DSS + NC group was decreased significantly (P＜0.05). Conclusion: Nitidine chloride has obvious therapeutic effects on DSS induced mouse colitis, and its anti-inflammatory mechanism is related to the down-regulation of miR-31 expression.

Objective: To investigate the interventive effects of Salvia przewalskii Maxim.(SPM)on high-altitude pulmonary hypertension(HAPH)in rats and possible mechanism. Methods: The male SD rats were randomly divided into the control group, the hypoxia group and SPM(0.5 g/kg,1 g/kg and 2 g/kg) group. There were 14 rats in each group. The rats in control group were feed in Xining(with an altitude about 2 260 m), and the other group rats were all feed in Maduo county people’s hospital(with an altitude about 4 260 m). The rats in SPM groups were treated with SPM at the doses of 0.5 g/kg,1 g/kg and 2 g/kg by gavage respectively (100 g/ml). The rats in control and the hypoxia groups were received equal volume of distilled water, once a day. After 4 weeks, the mean pulmonary artery pressure (mPAP) of rats was measured and the same part of lung tissue of each rat was collected and stored in liquid nitrogen. Then the relative mRNA expression levels of the proliferation cell nuclear antigen(PCNA), the cell cycle dependent kinase 4(CDK4), CyclinD1, RhoA, ROCK1, ROCK2 in lung tissues of each group rats were all tested by RT-PCR. Results: Compared with the control group, the mPAP and the relative mRNA expression levels of PCNA, CDK4, CyclinD1, RhoA, ROCK1 and ROCK2 were increased significantly in the hypoxia group(P＜0.01). Compared with the hypoxia group, the mPAP and the relative mRNA expression levels of PCNA, CDK4, CyclinD1, RhoA, ROCK1 and ROCK2 in the lung tissues of the SPM group rats were all decreased significantly(P＜ 0.05 or P＜0.01). Conclusion: SPM can prevent the HAPH in rats, and the mechanisms may be related to the inhibition of the excessive proliferation of smooth muscle cells in pulmonary artery and the excessive activation of the RhoA/Rho kinase(ROCK) signaling pathway.

Objective: To investigate the effects of psychological stress on xanthine oxidase (XO) expression, activity and related markers in adipose tissue of mice. Methods: Twenty male Kunming mice were randomly divided into two groups (10 in each group), stress group and control group (10 in each group). Stress group were restrained in self-made restraint device for 2 hours per day for 14 days, then blood samples and white adipose tissues(WAT) were collected. The expression levels of XO and NADPH oxidase-4 (Nox-4) in WAT were detected by immunohistochemistry. The expression of XO, Nox-4, antioxidant proteins (manganese superoxide dismutase (Mn SOD), glutathione peroxidase (GSH-Px), and catalase (CAT)), adipocytokines (adiponectin (ADPN), monocyte chemotactic protein 1 (MCP-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)) in WAT were further detected by quantitative PCR. Relative expressions of glucose metabolism (insulin receptor substrate-1(IRS-1) and glucose transporter type 4(GLUT-4)) and thrombin(tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1))were measured. XO activity and serum concentrations of (triglyceride (TG), total cholesterol (T-Cho), free fatty acid, (FFA), and uric acid (UA)) were detected by ELISA. Results: XO expressed in stress mice inguinal WAT was deeper and more abundant than that of control group, mainly expressed in adipocytes. RT-PCR and ELISA results showed that the levels of XO mRNA, serum XO concentration and the activities of XO enzyme in WAT of stress group were significantly higher than those of control group(P ＜0.01). Compared with control group the concentrations of free fatty acid (FFA) and uric acid (UA) in stress group were increased significantly than in control group (P＜ 0.01). Nox-4 positive cells mainly expressed in adipocytes. The expression of Nox-4 in WAT of stress group was significantly higher(P ＜0.01); The levels of antioxidant proteins (Mn-SOD, GSH-Px, Catalase) in WAT of stress group were significantly lower (P＜ 0.01). WAT in stress group showed a large number of infiltration reactions and inflammatory changes of monocytes, neutrophils, eosinophils and plasma cells. Stress significantly decreased the expression of adiponectin in WAT, and significantly increased the expressions of MCP-1, IL-6 and TNF-α (P＜0.01). The levels of IRS-1 and GLUT-4 in WAT of stress mice were increased significantly (P＜ 0.01). The expressions of TF and PAI-1 in WAT of stress mice and blood concentrations were significantly higher (P＜0.01). Conclusion: Stress can induce excessive expressions of XO in adipose tissue, which eventaully can lead to adipose inflammation, glycometabolism and abnormal prothrombin.

Objective: To investigate the effects of fatty acid synthase (FASN) on proliferation, migration and invasion of bladder cancer UMUC3 cell lines and possible mechanism. Methods: The expression levels of FASN protein in 30 cases of bladder cancer and 15 cases of normal bladder tissues were detected by Immunohistochemistry. FASN siRNA and nonsense siRNA were transfected into UMUC3 cell lines by lipofectamine 2000 respectively, and the stable siFASN and siControl cell lines were successfully obtained after screening and identification for several times. The siFASN cell lines were set as the experimental group, while the siControl cell lines were set as the control group. The expressions of FASN protein and mRNA in the experimental group and the control group were detected by Western blot and real-time quantitative PCR (RT-PCR) respectively. Cell proliferation activities in two groups were detected by MTT assay and cell invasion and migration in two groups were detected by cell scratch test and Transwell invasive assays respectively. Results: FASN protein was overexpressed in bladder cancer tissues, and it was closely correlated with pathological stage and grade (P＜0.05). Compared with the siControl group, the expressions of FASN mRNA and protein in the siFASN group cell lines were decreased significantly (P＜0.05). The cell proliferation ability, the migration ability and the number of transmembrane cells of siFASN group cell lines were reduced significantly (P＜0.05). Conclusion: The FASN overexpression may play an essential role in the development and progression of bladder cancer. Down-regulation of FASN expression can inhibit the proliferation, migration and invasion of bladder cancer cells, and inhibition of FASN expression is expected to be a new treatment for bladder cancer.

Objective: To study the protective effects of azithromycin on renal damage induced by doxorubicin and albumin in mice. Methods: Forty male BALB/c mice were randomly divided into blank control group (Ctrl group), renal damage model group (ADR+BSA group), azithromycin treated group (Azm group) and prednisone acetate positive control group (Pdn group) in accordance with random number table method. Mice in ADR+BSA, AZM and Pdn group were injected intravenously with 9.8 mg·kg^-1 doxorubicin five days a week, 10 mg·kg^-1 serum albumin was injected intraperitoneally, and normal saline was administered to the control group for 4 weeks to establish renal damage model. After that, AZM group was given daily. 62.5 mg·kg^-1 azithromycin was intragastrically administered. The Pdn group was given 12.5 mg·kg^-1 prednisone acetate daily, the other two groups were given the same amount of normal saline. After 6 weeks, the urine volume was collected and recorded for 24 hours to detected urine protein amount and endogenous creatinine clearance rate (Ccr). Serum biochemical indicators and serum immune factors were detected. Results: Compared with the Ctrl group, the 24 h urine protein level of the ADR+BSA group was increased significantly (P＜0.05), and the Ccr was decreased significantly (P＜0.05). After the azithromycin treatment, the 24 h urine protein was decreased significantly (P＜0.05), while the Ccr was increased significantly (P＜0.05) compared with ADR+BSA group. Conclusion: Azithromycin has a protective effects on the renal damage induced by doxorubicin and albumin in mice.

Objective: To investigate the effects of oral exposure of zinc oxide nanoparticles on multiple peripheral organs of C57BL/6J mice. Methods: Twenty male C57BL/6J mice were randomly divided into control group and experimental group, with 10 mice in each group. The experimental group was treated with continuous gavage administration of zinc oxide nanoparticle solution at a dose of 20 mg/kg body weight for 60 days, and the control group was given the corresponding amount of normal saline; the mice were weighed once a week. After the end of the exposure, blood samples was collected from the eyeballs, and the levels of blood sugar and lipids, liver and kidney function, and inflammatory factors such as platelet activating factor (PAF), interleukin-6 (IL-6) and tumor septicemia (TNF-α) were detected. Then, tissues sections of the heart, liver, spleen, lung, kidney and small intestine were prepared and their morphological changes were observed after hematoxylin-eosin staining. Results: There was no significant difference in body weight between control group and the experimental group. Compared with control group, the serum levels of albumin (ALB), albumin/globulin ratio(A/G), alkaline phosphatase (ALP) activity, aspartate aminotransferase/alanine aminotransferase ratio(AST/ALT), uric acid (UA) and blood urea in the experimental group were increased significantly (P＜0.05 or P＜0.01). There was no significant change in serum inflammatory factors. Pathological examination showed myocardial turbidity, mild inflammatory lesions (focal or small necrosis) in liver, decreased pigmentation in spleen, mild or moderate interstitial inflammation in lungs, and no obvious pathological changes in the kidneys or small intestine. Conclusion: Sixty days of oral exposure to nanometer zinc oxide did not cause inflammation in the blood system of C57BL / 6J mice, but it could induce mild pathological changes in the heart, liver, spleen and lungs, and lead to abnormal liver and kidney function.

Objective: To study the effects of bergapten (BP) on damages of osteocytes MLO-Y4 induced by tricalcium phosphate (TCP) wear particles and its mechanism. Methods: MLO-Y4 cells were treated with TCP wear particles for 48 h to establish the model of osteocytes injuries in vitro. The MLO-Y4 cells were divided into the following five groups: control group, TCP wear particles treated (0.1 mg/ml) group, bergapten (1, 5 and 20 μmol/L) treated groups. MTT assay and Calcein-AM staining were used to determine the viability of MLO-Y4 cells; Hoechst 33342 staining and the flow cytometry were applied to detect the apoptosis of MLO-Y4; real-time PCR was performed to examine the mRNA levels of dentin matrix protein1 (DMP-1), sclerostin (SOST) and fibroblast growth factor23 (FGF23); Western blot was performed to examine protein expressions of glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK) phospho-PERK (p-PERK), eukaryotic initiation factor 2α (eIF2α), phospho-eIF2α (p-eIF2α), activating transcription factor 4 (AFT4), C/EBP homologous protein (CHOP) and caspase-3 in MLO-Y4 cells. Results: Compared with control group, the MLO-Y4 viability and DMP-1 mRNA level in TCP group were decreased significantly (P＜0.05), while the percentage of apoptosis and mRNA levels of SOST and FGF23 were obviously increased (P＜0.05), and protein expressions of GRP78, AFT4, CHOP, p-PERK/PERK and p-eIF2α/eIF2α were up-regulated significantly in MLO-Y4 cells (P＜0.05). Compared with TCP group, the damages of MLO-Y4 and cell apoptosis in bergapten treated groups were decrease obviously (P＜0.05), the expressions of GRP78, AFT4, CHOP, p-PERK/PERK and p-eIF2α/eIF2α were down-regulated remarkably (P＜0.05). Conclusion: Bergapten can inhibit osteocytes damages induced by TCP wear particles, which may be related to reducing ER stress and PERK pathway activation.

Objective: To construct pcDNA3.1(+) eukaryotic expression plasmid of connective tissue growth factor(CTGF), and detected its expression in human osteoblast-like cells SaOS-2, which provides a technical support for further research on the mechanism of CTGF gene in bone development and bone repair process. Methods: The whole sequence of CTGF gene was cloned in vitro by polymerase chain reaction(PCR) method and connected to the linear pcDNA3.1(+) vector for constructing pcDNA3.1(+)-CTGF eukaryotic expression plasmid by homologous recombination technology. The plasmid was identified by sequencing. After identification, it was transfected into SaOS-2 cells and its expression was detected at 48 h. Results: pcDNA3.1(+)-CTGF eukaryotic expression recombinant plasmid was successfully constructed, which was confirmed by sequencing. Compared with the control group, CTGF expression level was significantly up-regulated after transfection of SaOS-2 cells for 48 h, up to five times as much as the control group. Conclusion: pcDNA3.1(+)-CTGF eukaryotic expression plasmid was successfully constructed and could be stably expressed in human osteoblasts-like cell SaOS-2, which laid a foundation for further study on the regulatory mechanism of CTGF gene on bone formation.