心理应激对小鼠脂肪组织黄嘌呤氧化酶表达、活性及相关指标的影响

Effects of psychological stress on xanthine oxidase expression,activity and related markers in adipose tissue of mice

目的:探讨心理应激对小鼠脂肪组织黄嘌呤氧化酶表达、活性及相关指标的作用。方法:雄性无特定病原体(SPF)级20只昆明小鼠随机分2组(每组10只),即慢性束缚应激(Stress)组和正常对照(Control)组。Stress组小鼠每天在自制式束缚器中限制活动2 h,其余时间两组小鼠在相同环境中自由饮水摄食,实验持续14 d,取血和白色脂肪组织(WAT);观察脂肪组织病理学改变,检测WAT中黄嘌呤氧化酶(XO)和烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(Nox-4)的蛋白水平,检测WAT组织中XO、Nox-4、超氧化物歧化酶(Mn SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、脂联素(ADPN)、单核细胞趋化蛋白1(MCP-1)、白介素6(IL-6)、肿瘤坏死因子α(TNF-α)、胰岛素受体底物1(IRS-1)、葡萄糖转运蛋白4(GLUT-4)、组织因子(TF)、纤溶酶原激活物抑制物1(PAI-1)的mRNA表达,检测血清和WAT组织中XO酶活性以及血清甘油三酯(TG)、总胆固醇(T-Cho)、游离脂肪酸(FFA)、尿酸(UA)的含量。结果:与control组比较,stress小鼠腹股沟WAT组织中XO免疫染色阳性着色细胞黄褐色沉淀深且丰富,WAT中出现大量的单核细胞、中性粒细胞、嗜酸性粒细胞及浆细胞浸润反应和炎症性的改变;血清XO浓度、WAT组织中XO mRNA水平和XO的酶活性显著升高(P<0.01),血清游离脂肪酸(FFA)和尿酸(UA)的含量显著增高(P<0.01),WAT组织中Nox-4蛋白、MCP-1、IL-6、TNF-α、TF、PAI-1mRNA的表达水平显著增高(P<0.01),而Mn-SOD、GSH-Px、CAT、ADPN、IRS-1和GLUT-4的mRNA水平则显著降低(P<0.01)。结论:心理应激可诱发脂肪XO过量表达及其活性增高,进而引起脂肪炎症、糖代谢及凝血酶原异常等反应。

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.