壮骨止痛胶囊对去卵巢致骨质疏松大鼠肠道微生态的影响

Effects of Zhuanggu Zhitong Capsule on intestinal microecology of ovariectomised rats with osteoporosis

目的探讨壮骨止痛胶囊(Zhuanggu Zhitong Capsule,ZGZTC)对去卵巢(ovariectomised,OVX)所致骨质疏松症(osteoporosis,OP)大鼠肠道菌群的影响。方法选用雌性SD大鼠,随机分为假手术(SHAM)组10只、造模组30只。造模组切除双侧卵巢,SHAM组仅切除卵巢周围等质量脂肪组织。1周后,将造模组随机分为OVX组、戊酸雌二醇(estradiol valerate,EV)组和ZGZTC组,每组10只。EV组灌服EV混悬液[0.09 mg/(kg·d)],ZGZTC组灌服ZGZTC混悬液[1.944 g/(kg·d)],SHAM组、OVX组灌服等体积生理盐水,给药12周后取材。HE染色观察股骨、结肠结构变化,RT-PCR法分析结肠闭合小环蛋白-1(zonula occludens-1,ZO-1)、咬合蛋白(Occludin)mRNA的表达,ELISA法检测血清白细胞介素-17(interleukin-17,IL-17)、转化生长因子-β(transforming growth factor-β,TGF-β)、脂多糖(lipopolysaccharide,LPS)的表达,收集大鼠粪便行肠道菌群16S rRNA测序,分析肠道微生物群的差异。结果与SHAM组比较,OVX组大鼠股骨骨小梁稀疏、断裂,结肠上皮细胞形态破坏,炎性细胞浸润;与OVX组比较,ZGZTC组、EV组大鼠股骨骨小梁数目增加,结肠中炎症细胞浸润减少。与SHAM组比较,OVX组大鼠结肠组织Occludin、ZO-1 mRNA相对表达量明显降低(P<0.01),血清IL-17、LPS值升高(P<0.01),TGF-β值降低(P<0.01);与OVX组比较,ZGZTC组和EV组大鼠结肠组织Occludin、ZO-1 mRNA相对表达量明显升高(P<0.01),血清IL-17、LPS值均降低(P<0.01),TGF-β值升高(P<0.01)。与OVX组比较,ZGZTC组、EV组厚壁菌门丰度均增加,拟杆菌门丰度均减少。结论ZGZTC能改善OVX大鼠骨微结构,其作用机制可能同增加肠道菌群的多样性、调节肠道菌群结构有关。

Objective To investigate the effects of Zhuanggu Zhitong Capsule(ZGZTC)on the intestinal flora of rats with ovariectomized(OVX)-induced osteoporosis(OP).Methods Female SD rats were selected and randomized into ten in the sham-operated(SHAM)group and 30 in the modeling group.In the modeling group,both ovaries were excised,and in the SHAM group,only an equal mass of adipose tissue around the ovaries was excised.One week later,the modeling group was randomized into the OVX group,the estradiol valerate(EV)group,and the ZGZTC group,with ten rats in each.The EV group was administered EV suspension 0.09 mg/(kg·d),the ZGZTC group was administered ZGZTC suspension 1.944 g/(kg·d),and the SHAM group and OVX group were administered equal volume of saline.The material was taken after 12 weeks of drug administration.The structural changes of femur and colon were observed by HE staining,the expression of zonula occludens-1(ZO-1)and Occludin mRNA in colon was analyzed by RT-PCR,the expression of serum interleukin-17(IL-17),transforming growth factor-β(TGF-β)and lipopolysaccharide(LPS)was determined by ELISA.The feces of rats were collected for 16S rRNA sequencing of intestinal flora,to analyze the differences of intestinal microbiota.Results Compared with the SHAM group,in the OVX group the femoral trabeculae of rats were sparse and broken,and the morphology of colonic epithelial cells was disrupted and infiltrated by inflammatory cells;compared with the OVX group,in the ZGZTC and EV groups the number of trabeculae increased and the infiltration of inflammatory cells in the colon was reduced.Compared with the SHAM group,in the OVX group the relative expression of Occludin and ZO-1 mRNA in the colon tissues of rats was significantly lower(P<0.01),the values of serum IL-17 and LPS were higher(P<0.01),and the value of TGF-βwas lower(P<0.01).Compared with the OVX group,in the ZGZTC and EV groups the colon tissues of rats showed a significant increase in the expression of Occludin and ZO-1 mRNA relative expression(P<0.01),serum IL-17 and LPS values were reduced(P<0.01),and TGF-βvalues increased(P<0.01).Compared with the OVX group,in the ZGZTC and EV groups the abundance of thick-walled bacillus phylum was increased,and the abundance of anaplastic bacillus phylum was decreased.Conclusion ZGZTC can improve the bone microstructure of OVX rats,and its mechanism may be related to increasing the diversity of intestinal flora and regulating the structure of intestinal flora.