补肾宁心汤通过microRNA144/AKT/mTOR轴抑制过度自噬改善早发性卵巢功能不全大鼠卵巢功能的研究

Bushen Ningxin Decoction(补肾宁心汤)Inhibiting Excessive Autophagy through microRNA144/AKT/mTOR Axis to Improve Ovarian Function of Rats with POI

目的观察补肾宁心汤对早发性卵巢功能不全(premature ovarian insufficiency,POI)大鼠的疗效,并探讨作用机制。方法21日龄大鼠随机分为对照组,模型组,补肾宁心汤低、中、高剂量组。对照组给予腹腔注射芝麻油,其余各组给予腹腔注射VCD溶液造模,造模同时,中药组分别给予低、中、高剂量的中药,对照组及模型组给予等体积的0.9%氯化钠溶液,持续45 d后,大鼠处死取材,测定相应指标。采用高通量测序结合公共数据库挖掘POI中miRNA和mRNA表达谱的变化,并预测microRNA-144的靶基因和潜在功能。结果随时间延长,模型组大鼠动情周期逐渐紊乱,表现为动情期缩短,动情间期延长,中药治疗后有改善。卵巢HE染色发现,模型组大鼠卵泡及黄体数均明显减少,中药干预后,卵泡和黄体数增加,中剂量组和高剂量组较为明显。子宫HE染色发现,模型组子宫内膜变薄,管壁子宫腺体数量明显减少,中药治疗后,内膜增厚,腺体数量明显增加。数据库分析提示microRNA144在POI中显著下调,与该实验结果一致。同时测序发现Beclin-1、LC3Ⅰ、ULK1在POI大鼠中显著上调。靶基因预测和功能富集分析显示:microRNA144与Beclin-1、LC3Ⅰ、ULK1存在相互作用且其靶基因显著富集在自噬、AKT/mTOR通路中。PCR测定大鼠卵巢中microRNA-144表达,发现模型组中该表达显著下降,中药治疗后显著升高。WB测定AKT/mTOR、自噬相关蛋白,结果发现以上指标在模型组中的表达分别受到抑制和促进作用,中药治疗后,均有改善。结论补肾宁心汤对POI大鼠的卵巢功能有保护作用,可改善大鼠内分泌和减缓卵泡闭锁,起效的机制可能与上调microRNA144/AKT/mTOR通路抑制过度自噬有关。

Objective To observe the effect of Bushen Ningxin Decoction(补肾宁心汤,BSNXD)on rats with premature ovarian insufficiency(POI)and explore its mechanism.Methods The 21-day-old rats were randomly divided into control group,model group,BSNXD low,medium and high dose groups.The control group was given intraperitoneal injection of sesame oil,and the other groups were given intraperitoneal injection of 4-vinylcyclohexene diepoxide(VCD)solution for 15 days.And then the treatment groups were given low,medium and high dose of BSNXD respectively.The control group and the model group were given the same volume of normal saline.Forty-five days later,the rats were sacrificed for sampling.Then,high-throughput sequencing technology combined with public database mining was used to explore the changes of miRNA and mRNA expression profiles and predict the target genes and potential functions of microRNA-144.Results As time went on,the estrous cycle of rats in the model group was gradually becoming disorganized,which was manifested as shortening of estrous period and prolongation of estrous interval.The improvement was found after BSNXD treatment.HE staining of ovary showed that the follicular and luteal counts of the model group were significantly reduced.After BSNXD intervention,the follicular and luteal counts were significantly increased,especially in the medium and high dose groups.HE staining of uterus showed the endometrium of the model group was thinned and the number of uterine glands in the tube wall was significantly reduced.After BSNXD intervention,the endometrium was thickened and the number of glands was significantly increased.The results of public database analysis suggested that microRNA144 was significantly down-regulated in POI rats,which was consistent with the results of high-throughput sequencing.Meanwhile,the expressions of Beclin-1,LC3Ⅰand ULK1 were significantly up-regulated in POI rats.MiRNA target gene prediction and functional enrichment analysis showed that microRNA144 interacted with Beclin-1,LC3Ⅰand ULK1 and microRNA144 target genes were significantly enriched in autophagy and AKT/mTOR pathways.PCR results showed that microRNA-144 expression significantly decreased in the model group,and significantly increased after BSNXD treatment.Western blot results showed that the expressions of AKT/mTOR pathway and autophagy pathway-related proteins,including Beclin-1,LC3Ⅰand ULK1 in the model group were inhibited and promoted,respectively.After BSNXD treatment,the expression of AKT/mTOR protein was significantly increased,while the expression of autophagy related proteins was significantly decreased.Conclusion BSNXD can protect the ovarian reserve function of rats with premature ovarian insufficiency and improve the endocrine state of rats and slow down follicular atresia.The mechanism may be related to the up-regulation of microRNA144/AKT/mTOR pathway to inhibit excessive autophagy.