To Explore the Effect of Qing'e Pills on Anti-Osteoporosis Based on Fecal Microbiota Transplantation

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-α in 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.