心理应激对雌性小鼠胃黏膜炎症和卵巢功能早衰的影响

Effects of stress on gastric mucosal inflammation and premature ovarian failure in female mice

目的探讨心理应激对雌性小鼠胃黏膜炎症和卵巢功能的影响,以及芬吗通对其改善的作用。方法8周龄,雌性SPF级昆明小鼠随机分为正常对照(Control)组,心理应激(Stress)组和芬吗通干预(Femoston)组(每组6只);Stress组和Femoston干预组雌鼠每天在自制式束缚器中限制活动2 h,持续14 d。通过H&E染色在光镜下观察胃黏膜和卵巢组织病理学改变,采用免疫组化方法观察NADPH氧化酶-4(Nox-4)在雌鼠胃黏膜和卵巢组织中的表达,进一步通过实时定量RT-PCR和酶联免疫吸附法分别检测雌鼠卵巢组织及血液中Nox-4、抗氧化酶[锰超氧化物歧化酶(Mn-SOD)、胱甘肽过氧化物酶(GSH-Px)、Catalase]及炎性因子[白细胞介素-1β(IL-1β)、IL-6、肿瘤坏死因子-α(TNF-α)]等指标的mRNA表达及其在血液释放浓度。结果Stress雌鼠胃黏膜出现中性粒细胞和淋巴细胞等免疫细胞浸润现象和炎症改变;stress组胃黏膜炎症评分显著高于Control组(P<0.01)。Stress雌鼠胃黏膜组织中Nox-4大量表达,同时其mRNA表达水平显著上升(P<0.01)。Stress显著上升胃黏膜炎性因子(IL1-β、IL-6、TNF-α)的mRNA表达水平(P<0.01)。Stress组雌鼠卵巢组织出现原始卵泡(pr)、初级卵泡(prm)、次级卵泡(sf)、炎症(ii)及卵泡结构的变形(fd)等卵泡微结构明显的改变,而芬吗通干预显著改善此卵泡结构的异常。Stress雌鼠卵巢组织中Nox-4主要表达于卵泡细胞中;其mRNA表达水平显著上升(P<0.001);同时,stress雌鼠血液中Nox-4的含量显著上升(P<0.001)。芬吗通干预能够有效降低心理应激雌鼠卵巢组织和血液中Nox-4的高表达(P<0.01)。Stress雌鼠显著增高血液中的促卵泡激素(FSH)及黄体生成素(LH)水平;并降低雌二醇(E2)和孕酮(Prog)的水平(P<0.001)。芬吗通干预显著改善E2的水平(P<0.01)。Stress雌鼠显著降低卵巢组织抗氧化酶(Mn-SOD、GSH-Px、Catalase)的mRNA水平(P<0.001);芬吗通干预显著增高抗氧化酶的表达水平(P<0.01)。Stress雌鼠显著增高血液中以及卵巢组织中IL-1β、IL-6及TNF-α的含量及其mRNA表达水平(P<0.001),芬吗通有效抑制雌鼠卵巢组织和血液中炎性因子的高表达(P<0.01)。Pearson相关性分析结果显示,Stress组Nox-4血清含量与性激素(FSH、LH、E2、Prog)的含量之间存在正相关关系(P<0.01),相关系数分别为r=0.94、0.97、0.93、0.96。同时,血清Nox-4与炎症因子IL-1β、IL-6、TNF-α释放浓度之间存在正相关关系(P<0.01),相关系数分别为r=0.95、0.97、0.98。结论心理应激诱导雌鼠胃黏膜炎症和卵巢功能异常是局部组织和血液中Nox-4高表达有关,芬吗通有效改善卵巢组织结构及功能的异常,其深入的机制有待进一步探索。

Objective To investigate the effects of stress on gastric mucosal inflammation and ovarian function in female mice,and the inhibitory effect of Femoston.Methods 8-week old female SPF Kunming mice were randomly divided into control,stress and Femoston intervention group(6 in each group);Female mice in the stress group and Femoston group were restricted in the self-made restraint apparatus for 2 hours every day for 14 days.The histopathological changes of gastric mucosa and ovary were observed under light microscope by H&E staining.The expression of Nox-4 in gastric mucosa and ovary was observed by immunohistochemistry.Further,the levels of Nox-4,antioxidant enzymes(Mn SOD,GSH Px,Catalase)and cytokines(IL1-β,IL-6,TNF-α)in mice ovarian tissue and blood were detected by real-time quantitative RT-PCR and ELISA.Results H&E staining showed that neutrophils,lymphocytes infiltration and inflammatory changes in gastric mucosa of stress mice;The score of gastric mucositis in stress group was higher(P<0.01).Nox-4 was highly expressed in the gastric mucosa,and its mRNA expression level was increased(P<0.01).Stress increased the mRNA level of gastric cytokines(IL1-β,IL-6,TNF-α).The ovarian tissue of stress showed obvious changes in follicle microstructure,such as primordial follicle(pr),primary follicle(prm),secondary follicle(sf),inflammation(ii)and follicle structure deformation(fd).Femoston improved the abnormality of this follicle structure.The Nox-4 in ovarian mainly expressed in follicular cells;Its mRNA expression level increased(P<0.001);The content of Nox-4 in the blood of stress mice increased(P<0.001).Femoston can effectively reduce the high expression of Nox-4 in ovarian tissue and blood of stressed mice(P<0.01).Stress mice increased the levels of follicle stimulating hormone(FSH)and luteinizing hormone(LH)in blood;The levels of estradiol(E2)and progesterone(Prog)were decreased(P<0.001).Femoston improved the level of E2(P<0.01).Stress mice decreased the mRNA level of antioxidant enzymes(Mn SOD,GSH Px,Catalase)in ovarian tissue(P<0.001);Femoston increased the expression levels of antioxidant enzymes(P<0.01).Stress increased in blood and ovarian tissue IL1-β,IL-6,TNF-αmRNA levlels,and Femoston effectively inhibited the high expressions of those cytokines(P<0.01).Pearson correlation analysis showed that there was a positive correlation between the serum level of Nox-4 and sex hormones(FSH,LH,E2,Prog)in the stress group(P<0.01).Serum Nox-4 and cytokines(IL1-β,IL-6,TNF-α)were a positive correlation between concentrations(P<0.01).Conclusion The high expression of Nox-4 in local tissues and blood is related to the gastric mucositis and abnormal ovarian function induced by stress.Femoston can effectively improve the abnormality of ovarian structure and function,deeper mechanism needs further exploration.