四物汤对卵巢功能低下模型大鼠肠道菌群的影响

Effect of Siwutang on Diminished Ovarian Reserve in Rats Based on 16S rRNA Sequencing of Intestinal Flora

目的:采用16S rRNA技术研究四物汤对雷公藤多苷致卵巢功能低下模型大鼠肠道菌群的影响,探讨四物汤改善卵巢功能低下的作用机制。方法:20只8周龄雌性SD大鼠随机分为4组,分别为正常组、模型组、四物汤高、低剂量组。除正常组外,其他3组均按50 mg·kg^(-1)灌胃雷公藤多苷片造模,连续给药14 d,第15天停止造模,高剂量组按人临床日用量(40 g)的3倍,低剂量组按人临床日用量的1.5倍给药,连续灌胃18 d,模型组和正常组灌胃等体积生理盐水。第18天,收集粪便用16S rRNA进行基因测序,给药后1 h,麻醉后腹主动脉取血,采用放射免疫法检测激素水平,取双侧卵巢,包埋切片后苏木素-伊红(HE)染色进行病理学观察。结果:与正常组比较,模型组黄体生成素(LH)水平明显升高(P<0.05),雌二醇(E2)水平明显降低(P<0.05);与模型组比较,四物汤高、低剂量组的LH水平均明显降低(P<0.05),E2水平均明显升高(P<0.05),四物汤给药后,能不同程度扭转雷公藤多苷造成的血清促卵泡激素(FSH)、LH水平明显升高,E2、孕酮(P)水平明显降低;正常组有大量处于不同发育阶段的各级卵泡,模型组中闭锁卵泡明显增多,四物汤高剂量组可见大量成熟卵泡、次级卵泡及初级卵泡,低剂量组黄体增加,可见原始卵泡、次级卵泡;与正常组和给药组比较,模型组疣微菌门和Epsilonbacteraeota的丰度明显减少;与正常组比较,模型组在门、纲、目、科、属分类层面上均存在差异菌群,其中模型组的厚壁菌门和拟杆菌门比例增加;雷公藤多苷造模后,毛螺菌科丰度明显降低,瘤胃菌属UCG-005丰度明显增加;四物汤给药组与正常组、模型组能明显区分开,且给药组具有向正常组靠近的趋势。结论:四物汤对雷公藤多苷致卵巢功能低下模型大鼠有改善作用,其机制可能与调节肠道菌群多样性有关。

Objective:To study the effect of Siwutang(SWT) on intestinal flora in rats with diminished ovarian reserve(DOR)induced by Tripterygium wilfordii polyglycoside(TWP)based on 16S rRNA sequencing.Method:Twenty 8-week-old female SD rats were randomly assigned into four groups:blank group,model group,SWT high-dose group,and SWT low-dose group.Except the blank group,the other three groups were underwent intragastric administration of TWP tablets at a dose of 50 mg·kg^(-1)for 14 days.On day15,the high-dose group was administrated at 3 times of the human dosage(40 g/person/day),the low-dose group at 1.5 times of the human dosage,and the model group and the blank group with the same volume of normal saline for 18 days.Then,feces were collected for 16S rRNA sequencing.One hour after administration,blood was sampled from abdominal aorta after anesthesia for the measurement of hormone levels by radioimmunoassay,and ovaries were sampled,embedded,sliced,and stained with haematoxylin-eosin(HE)for pathological observation.Result:The model group had higher level of luteinizing hormone(LH,P<0.05)and lower level of estradiol(E2,P<0.05)than the blank group.The SWT high-dose group and low-dose group had lower LH levels(P<0.05)and higher E2 levels than the model group(P<0.05).SWT reversed the elevation in follicle-stimulating hormone(FSH)and LH levels and the decline in E2 and progesterone(P)levels caused by TWP to some extent.There were a large number of follicles at different developmental stages in the blank group,while atretic follicles increased significantly in the model group.A large number of mature follicles,secondary follicles,and primary follicles were observed in the high-dose SWT group,and primordial follicles,secondary follicles,and increased corpus luteum in the low-dose SWT group.Compared with that in the blank group and the administration group,the abundance of Verrucomicrobia and Epsilonbacteraeota in the model group significantly reduced.Compared with the blank group,the model group had different intestinal flora in phylum,class,order,family,and genus levels.Specifically,the model group had increased proportions of Firmicutes and Bacteroidetes.After TWP modeling,the abundance of Lachnospiraceae decreased significantly while that of Ruminococcaceae UCG-005 increased significantly.SWT groups,blank group,and model group can be clearly distinguished,and SWT groups had a tendency to approach the blank group.Conclusion:SWT may improve the ovarian function of rats with TWP-induced DOR by regulating intestinal flora diversity.