摘要
目的探讨粪菌移植对骨质疏松症的改善作用,以及大鼠肠道菌群对青娥丸生、盐品中主要化学成分的代谢作用。方法将36只SD大鼠随机分为6组,假手术组、模型组、仙灵骨葆胶囊治疗组(阳性药组)、青娥丸生品粪菌移植治疗组(青娥丸生品移植组)、青娥丸盐品粪菌移植治疗组(青娥丸盐品移植组)以及益生菌组。采用去除双侧卵巢法制备大鼠骨质疏松症模型。青娥丸生、盐品移植组自造模6周后开始分别给予相应的粪菌液(每100 g体质量给予2 mL),对照组和模型组给予等体积生理盐水,益生菌组给予乳酸杆菌菌液1 mL。给药8周后,采用Micro-CT检测大鼠骨密度(BMD);通过ELISA法检测大鼠血清雌二醇(E2)、骨钙素(BGP)、碱性磷酸酶(BAP)和抗酒石酸酸性磷酸酶(TRAP)的含量以及炎症因子IL-17、IL-6和TNF-α的水平;采用三点弯曲试验法检测大鼠股骨力学指标的变化;采用GC-MS技术测定大鼠肠道内容物中乙酸、丙酸和丁酸的含量。体外实验将青娥丸生、盐品分别与大鼠肠道菌群共孵育,采用UHPLC-Q-TOF/MS技术对共孵育样品中青娥丸主要成分的原型和代谢产物进行检测与分析,并推测其代谢途径。结果与假手术组相比,骨质疏松模型组大鼠表现为BMD降低(P<0.001),血清中E2水平降低(P<0.001),BGP、BAP、TRAP水平升高(P<0.001),骨力学参数中最大载荷、最大挠度和刚度降低(P<0.05,P<0.01,P<0.001),血清中IL-17、IL-6和TNF-α水平升高(P<0.001),肠道内容物中乙酸、丙酸和丁酸含量降低(P<0.01,P<0.001)。与模型组相比,青娥丸生、盐品移植组上述指标均有不同程度的改变,趋于假手术组水平,但青娥丸生、盐品移植组之间各指标无显著性差异。体外共孵育实验结果表明,青娥丸方中4种主要原型成分在经肠道菌群代谢后共产生14种代谢产物,其中主要代谢产物为肠二醇、肠内酯、补骨脂素和异补骨脂素等,而青娥丸生、盐品共孵育样品的代谢产物之间无显著差异。结论青娥丸可通过肠道菌群减少大鼠血清中炎症因子的产生,增加大鼠肠道中短链脂肪酸的含量,改善大鼠骨质疏松;大鼠肠道菌群能够影响青娥丸方中主要成分的代谢。肠道菌群是青娥丸治疗骨质疏松症的靶点,青娥丸和肠道菌群之间相互作用可能是青娥丸改善骨质疏松症的机制之一。
OBJECTIVE To explore the effect of the intestinal flora transplantation in rats on osteoporosis,and to explore the metabolization of the main chemical components in Qing'e pills raw and salt products by rats intestinal flora.METHODS 36 SD rats were randomly divided into sham operation group,model group,Xianling Gubao capsule treatment group(positive drug group),Qing'e pills raw product fecal bacteria transplantation treatment group(Qing'e pills raw product transplantation group),Qing'e pills salt fecal bacteria transplantation treatment group(Qing'e pills salt transplantation group)and probiotics group.A rat model of osteoporosis was prepared by removing both ovaries.The Qing'e pills raw product transplantation group and the Qing'e pills salt transplantation group were given 2 mL per 100 g(body weigh)of fecal bacteria solution 6 weeks after the model was established.The control group and the model group were given equal volumes of normal saline,and the probiotic group was given 1 mL of lactobacillus liquid prepared.After 8 weeks of administration,the bone mineral density(BMD)of rats was measured by Micro-CT;the serum estradiol(E2),osteocalcin(BGP),alkaline phosphatase(BAP)and anti-tartaric acid were measured by ELISA method.The content of acid phosphatase(TRAP);the three-point bending test method was used to detect the changes in rat femur mechanics;ELISA method was used to detect the levels of IL-17,IL-6 and TNF-αin the rat serum;GC-MS was used The technique measures the contents of acetic acid,propionic acid and butyric acid in the intestinal contents of the rat ileum.In vitro,Qing'e pills and salt products were incubated with rat intestinal flora.UHPLC-Q-TOF/MS was used to detect and analyze the prototypes and metabolites of the main components of Qing'e pills in the co-incubated samples.Speculate its metabolic pathway.RESULTS Compared with the sham operation group,rats in the osteoporosis model group showed decreased BMD(P<0.001),decreased E2 level(P<0.001),increased BGP,BAP,and TRAP levels(P<0.001),decreased maximum load,maximum deflection and stiffness in bone mechanical parameters(P<0.05,P<0.01,P<0.001),and increased IL-17,IL-6 and TNF-α(P<0.001),and decreased the contents of acetic acid,propionic acid and butyric acid in the ileum and intestinal contents(P<0.01,P<0.001).After intervention treatment of fecal bacteria transplantation group,compared with model group,the above indicators all changed to different degrees,tending to the level of sham operation group,but there was no significant difference between Qing'e pills raw product transplantation group and salt transplantation group.In vitro Qing'e pills raw materials,salt products and rat intestinal flora co-incubation samples were tested and 14 metabolites were produced from the four main prototype components found in Qing'e pills prescription produced by the intestinal flora,of which the main metabolites were intestinal diol,intestinal fat,psoralen and isopsoralen.There was no difference between the metabolites of Qing'e pills raw and salt products.CONCLUSION Qing'e pills can reduce the production of inflammatory factors in the rat serum through the intestinal flora,increase the content of short-chain fatty acids in the rat's intestine,and improve the osteoporosis of rats.The rat's intestinal flora can affect the metabolism of certain main ingredients in Qing'e pills prescription.Intestinal flora is the target of Qing'e pills in the treatment of osteoporosis.The interaction between Qing'e pills and intestinal flora may be one of the mechanisms by which Qing'e pills can improve osteoporosis.
作者
谢辉
林上阳
华政颖
郭梦雨
于垠廷
李伟东
XIE Hui;LIN Shang-yang;HUA Zheng-ying;GUO Meng-yu;YU Yin-ting;LI Wei-dong(School of Pharmacy,Engineering Center of State Ministry of Education for Standardization of Chinese Medicine Processing,Nanjing University of Chinese Medicine,Nanjing,210023,China)
出处
《南京中医药大学学报》
CAS
CSCD
北大核心
2021年第4期521-528,共8页
Journal of Nanjing University of Traditional Chinese Medicine
基金
国家自然科学基金(81773902,81973484)。
关键词
青娥丸
粪菌移植
骨质疏松
药物代谢
作用机制
Qing'e pills
fecal microbiota transplantation
osteoporosis
drug metabolism
mechanism of action
