海巴戟天对PCOS作用及机制的初步研究

A preliminary study on the effect and mechanism of PCOS in Morinda citrifolia

目的利用超高效液相色谱-串联质谱技术(ultra-performanceliquidchromatography-tandemmassspectrometry,UPLC-MS/MS)鉴定海巴戟天化学成分,进一步实验验证海巴戟天调控miR-223-3p/NLRP3信号通路抑制多囊卵巢综合征(polycysticovarysyndrome,PCOS)大鼠炎症反应。方法采用UPLC-MS/MS分析海巴戟天化学成分。动物实验:SD雌性大鼠采用随机数字表分为3组:正常对照组(n=5)、PCOS模型组(n=5)和海巴戟天灌胃组(n=5)。HE染色观察大鼠卵巢组织学形态,ELISA方法测定大鼠睾酮(testosterone,T)、黄体生成素(luteinizinghormone,LH)、卵泡刺激素(follicle stimulating hormone,FSH)、白细胞介素(interleukin,IL)表达水平,RT-qPCR法检测大鼠miR-223-3p、NLRP3的m RNA表达,Westernblot法检测大鼠NLRP3的蛋白表达。结果UPLC-MS/MS鉴定出海巴戟天化学成分有641种,包括木犀草素、芹菜素、大黄素等多种成分。与正常对照组比较,PCOS模型组卵巢颗粒细胞层数减少,卵泡囊性扩张,闭锁卵泡增多,T、LH水平升高(P<0.05),FSH水平降低(P<0.05),IL-18、IL-1β水平升高(P<0.05),卵巢组织miR-223-3p mRNA表达降低(P<0.01),NLRP3 mRNA和蛋白表达升高(P<0.01);与PCOS模型组相比,海巴戟天灌胃组各级卵泡比例正常,颗粒细胞层数增多,T、LH水平降低(P<0.05),FSH水平升高(P<0.05),IL-18、IL-1β水平降低(P<0.05),卵巢组织miR-223-3p mRNA表达升高(P<0.05),NLRP3 mRNA和蛋白表达降低(P<0.05)。结论海巴戟天能够改善PCOS大鼠卵巢病理状态,改变异常性激素及降低IL-18、IL-1β水平,其机制可能是通过调控miR-223-3p/NLRP3信号通路抑制PCOS大鼠炎症反应。

Objective To use the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)to authenticate the chemical composition of Morinda citrifolia,and further experiments verify that Morinda citrifolia regulate miR-223-3p/NLRP3 signaling pathway to inhibit the inflammatory response in rats with polycystic ovary syndrome(PCOS).Methods Use UPLC-MS/MS analyze the chemical composition of Morinda citrifolia.SD female rats were randomly divided into three groups:normal control group(n=5),PCOS model group(n=5)and Morinda citrifolia gavage group(n=5).HE staining observe the morphology of ovarian histology,ELISA method detect the expression levels of testosterone(T),luteinizing hormone(LH),follicle stimulating hormone(FSH),interleukin(IL)-1β and IL-18,RT-qPCR detect the mRNA expression of miR-223-3p and NLRP3,and Western blot detect the protein expression of NLRP3.Results UPLC-MS/MS identify 641 chemical composition of Morinda citrifolia,including luteolin,Apigenin,emodin and other composition.Compared with the normal control group,the number of cell layers of ovarian granule in the PCOS model group is reduced,and follicular cystic dilation,atresia follicles increased,T and LH levels increased(P<0.05),FSH levels decreased(P<0.05),IL-18 and IL-1β levels increased(P<0.05),and ovarian tissue miR-223-3p mRNA expression decreased(P<0.01),NLRP3 mRNA and protein expression increased(P<0.01)in PCOS model group.Compared with the PCOS model group,the proportion of follicles at all levels is normal,the number of granule cell layers increased,T and LH levels decreased(P<0.05),FSH levels increased(P<0.05),IL-18 and IL-1β levels decreased(P<0.05),miR-223-3p mRNA expression in ovarian tissue increased(P<0.05),NLRP3 mRNA and protein expression decreased(P<0.05)in Morinda citrifolia gavage group.Conclusion Morinda citrifolia can improve the ovary pathological state of PCOS rats,change the abnormal sex hormones and reduce the levels of inflammatory factors IL-18 and IL-1β.The mechanism maybe regulate the miR-223-3p/NLRP3 signaling pathway to inhibit the inflammatory response in PCOS rats.