基于自噬研究麒麟丸对环磷酰胺诱导的卵巢储备功能减退模型小鼠卵巢储备功能的影响

Effects of Qilin Pills on ovarian reserve function in cyclophosphamide-induced decreased orarian reserve mice model based on autophagy

目的:探讨麒麟丸对环磷酰胺诱导的卵巢储备功能减退(DOR)模型小鼠卵巢功能及自噬水平的影响。方法:18只SPF级雌性C57BL/6小鼠随机分为3组:正常对照组,模型对照组,麒麟丸组。麒麟丸组予麒麟丸溶液灌胃(5.46g·kg^(-1)·d^(-1)),正常对照组及模型对照组予等体积0.5%CMC-Na溶液灌胃,连续5周;自第2周起,模型对照组及麒麟丸组予环磷酰胺腹腔注射(75mg/kg),正常对照组予等体积0.9%氯化钠注射液腹腔注射,每周1次,共4周。ELISA法检测血清抗米勒管激素(AMH)水平,HE染色统计各级卵泡数及比例,RT-qPCR检测LC3B、P62、Becn1、Atg5、Atg7 mRNA表达,免疫组化法检测卵巢LC3B蛋白表达,Western Blot法检测分析卵巢LC3B、P62蛋白表达。结果:与正常对照组比较,模型对照组小鼠卵巢系数、AMH水平显著降低(P<0.01),始基卵泡、初级卵泡及次级卵泡数量显著减少(P<0.05,P<0.01),闭锁卵泡显著增加(P<0.01);卵巢内Becn1、LC3B、Atg7 mRNA水平显著升高,P62 mRNA水平显著降低(P<0.01);LC3B-Ⅱ蛋白水平显著升高,P62蛋白水平显著下降(P<0.01)。与模型对照组比较,麒麟丸组小鼠卵巢系数及AMH水平显著升高(P<0.05),始基卵泡数显著增加,发育中卵泡及闭锁卵泡比例显著降低(P<0.01,P<0.05)。Becn1、LC3B、Atg7 mRNA水平显著降低,P62 mRNA表达显著升高(P<0.05,P<0.01)。LC3B-Ⅱ蛋白表达显著降低,P62蛋白表达显著增加(P<0.01)。结论:麒麟丸可抑制环磷酰胺诱导的C57BL/6小鼠始基卵泡过度激活,减少卵泡闭锁,保护卵巢储备功能,其作用机制可能与抑制卵巢内过度自噬有关。

Objective:To investigate the effects of Qilin Pills on ovarian reserve function and autophagy level in cyclophosphamide induced decreased ovarian reserve(DOR)model mice.Methods:Eighteen SPF female C57 BL/6 mice were selected and randomly divided into 3 groups:normal control group,model control group and Qilin Pills group.Qilin Pills group was gavaged with Qilin Pills solution(5.46g·kg ^(-1)·d^(-1))for 5 consecutive weeks,while normal group and model group were gavaged with 0.5%CMC-NA solution at the same volume.From the second week,the model group and Qilin Pills group were given intraperitoneal injection of cyclophosphamide(75 mg/kg)once a week for4 weeks,while the normal group was given intraperitoneal injection of isovolumetric sodium chloride.The serum AMH levels were detected by ELISA,the ovarian structure was observed by HE staining,and the number and proportion of follicles at all levels were counted.LC3 B,P62,Becn1,Atg5 and Atg7 mRNA were detected by RT-qPCR.The expression of LC3 B protein in the ovaries was detected by immunohistochemistry,and that of LC3 B and P62 were detected by Western blot.Results:Compared with the normal control group,the ovarian coefficient and AMH level in the model control group were significantly decreased(P<0.01),the number of primary follicles,primary follicles and secondary follicles were significantly decreased(P<0.05,P<0.01),and the number of atresia follicles was significantly increased(P<0.01).Becn1,LC3 B and Atg7 mRNA levels significantly increased,while P62 mRNA levels significantly decreased(P<0.01).LC3 B-I protein level significantly increased,P62 protein were significantly decreased(P<0.01).Compared with the model control group,the ovarian coefficient and AMH level of the Qilin pills group were significantly increased(P<0.05),the number of primary follicles was increased,and the proportion of developing follicles and atresia follicles was significantly decreased(P<0.01,P<0.05).Becn1,LC3 B and Atg7 mRNA levels significantly decreased(P<0.05),while P62 mRNA expression significantly increased(P<0.05,P<0.01).LC3 B-II protein expression significantly reduced(P<0.01),P62 protein expression significantly increased(P<0.01).Conclusion:Qilin pills can inhibit the overactivation of primary follicles in C57 BL/6 mice induced by cyclophosphamide,reduce follicular atresia,and protect ovarian reserve function.The mechanism of action may be related to the inhibition of excessive autophagy in the ovary.