黄芪甲苷减轻环磷酰胺诱导的早发性卵巢功能不全模型大鼠卵巢炎症反应

Astragaloside IV alleviates ovarian inflammation in a rat model of pre⁃mature ovarian insufficiency induced by cyclophosphamide

目的:研究黄芪甲苷(astragaloside Ⅳ,AS-IV)是否通过抑制核因子κB(nuclear factor-κB,NF-κB)信号通路降低环磷酰胺(cyclophosphamide,CTX)诱导的早发性卵巢功能不全(premature ovarian insufficiency,POI)模型大鼠炎症反应。方法:动情周期正常的雌性SD大鼠80只,选取12只为正常对照组,其余68只大鼠用CTX腹腔注射诱导建立POI模型。将模型制备成功的60只大鼠随机分为5组,每组12只,分别为POI模型组、芬吗通组、低剂量AS-IV组、中剂量AS-IV组和高剂量AS-IV组,干预28 d后取样。采用ELISA法测定血清中17β-雌二醇(17β-estradiol,17β-E2)、抗缪勒管激素(anti-Müllerian hormone,AMH)、促卵泡生成素(follicle-stimulating hormone,FSH)和黄体生成素(luteinizing hormone,LH)水平;转录组测序筛选差异表达基因及功能富集;HE染色法观察卵巢组织病理变化并进行卵泡计数;免疫组织化学染色法和Western blot法分别进行p65(胞核)、p-p65和p-IκBα及下游促炎因子IL-6、IL-1β、TNF-α及IL-8的定位表达和相对表达水平检测。结果:用CTX诱导15 d建立的POI模型大鼠动情周期紊乱;与正常对照组相比,POI模型大鼠血清17β-E2和AMH显著降低、FSH和LH显著升高(P<0.01)。经各组相应干预28 d后,与POI模型组相比,中剂量AS-IV组大鼠血清17β-E2和AMH水平升高、FSH和LH水平降低,差异显著(P<0.01)。基于转录组测序结果发现,POI模型组与中剂量AS-IV组有274个差异表达基因,富集在炎症相关功能基因簇。HE染色显示,与正常对照组相比,POI模型组大鼠卵巢组织结构紊乱,初级卵泡和成熟卵泡数目减少,闭锁卵泡数目增多(P<0.01);与POI模型组相比,中剂量AS-IV组大鼠卵巢组织初级卵泡和成熟卵泡数目增多(P<0.01)。芬吗通治疗后初级卵泡数量较模型组没有差异,可见的有腔卵泡数量较模型组增多,但多为闭锁卵泡。与正常对照组相比,POI模型组p65(胞核)、p-p65、p-IκBα、IL-6、IL-1β、TNF-α及IL-8的蛋白表达量显著升高(P<0.05);与POI模型组相比,芬吗通组和中剂量AS-IV组p65(胞核)、p-p65/p65、p-IκBα、IL-6、IL-1β、TNF-α及IL-8的蛋白水平显著降低(P<0.05)。结论:AS-IV可能通过介导NF-κB信号通路抑制卵巢炎症反应,从而改善CTX诱导的POI模型大鼠的卵巢形态结构和功能。

AIM:To investigate the effect of astragaloside IV(AS-IV)on the inflammatory response in a rat model of premature ovarian insufficiency(POI)induced by cyclophosphamide(CTX).METHODS:Twelve female SD rats with normal estrous cycle were selected as the control group,and 68 rats were intraperitoneally injected with CTX to establish the POI model.Sixty POI rats were randomly divided into 5 groups(12 rats per group):POI model group,femoston group,low-dose AS-IV group,middle-dose AS-IV group and high-dose AS-IV group.After 28 d of treatment,the serum levels of 17β-estradiol(17β-E2),anti-Müllerian hormone(AMH),follicle-stimulating hormone(FSH)and luteinizing hormone(LH)of the rats were measured by ELISA.The differentially expressed genes and functional enrichment were screened by transcriptome sequencing.The pathological changes of ovarian tissues were observed by HE staining,and follicles were counted.The protein levels of nuclear p65,p-p65,p-IκBα,IL-6,IL-1β,TNF-αand IL-8 were quantitated by immunohistochemistry and Western blot.RESULTS:The POI estrous cycle disorder in female rats was induced by CTX injection over 15 d.Compared with control group,the serum levels of 17β-E2 and AMH in POI rats were significantly decreased,while the levels of FSH and LH were significantly increased(P<0.01).After 28 d of AS-IV treatment,the serum levels of 17β-E2 and AMH were increased and the levels of FSH and LH were decreased in middle-dose AS-IV group(P<0.01),compared with POI model group.Based on RNA-Seq analysis,274 differentially expressed genes were found between POI model group and middle-dose AS-IV group,enriched in inflammation-related functional gene clusters.HE staining showed that compared with control group,the ovarian tissue structure in POI model group was disordered,the number of primary and mature follicles was decreased(P<0.01),and the number of atretic follicles was increased(P<0.01).Compared with POI model group,the number of primary and mature follicles was increased in middle-dose AS-IV group(P<0.01).There was no difference in the number of primary follicles between femoston group and POI model group.The number of antral follicles in femoston group was higher than that in POI model group,but most of them were atretic follicles.The protein levels of nuclear p65,p-p65,p-IκBα,IL-6,IL-1β,TNF-αand IL-8 in POI model group were significantly increased compared with control group(P<0.05),while those in middle-dose AS-IV group and femoston group were significantly decreased compared with POI model group(P<0.05).CONCLUSION:Astragaloside IV may inhibit the ovarian inflammatory response by modulating the NF-κB pathway,thus improving the ovarian structure and function in the CTX-induced POI model rats.