| ObjectivePrevious studies have shown that Bushen Huatan Decoction can regulate bone metabolism and correct gut flora disturbance in PMOP model rats.However,its underlying mechanism has not yet been elucidated.Therefore,this study intends to further clarify the potential mechanism of Bushen Huatan Decoction in preventing and treating PMOP,focusing on the gut flora metabolite SCFAs butyrate,autophagy activity,and AMPK/m TOR/ULK1 signaling pathway.Furthermore,the preliminary exploration of Bushen Huatan Decoction to correct gut bacteria and improve bone metabolism is related to the regulation of autophagy by SCFAs butyrate.MethodBy reviewing ancient books and the latest research progress widely,based on the basic theory of traditional Chinese medicine,the potential relationship between gut flora and autophagy and PMOP was comprehensively discussed,and the theoretical origin and basis of treating PMOP from "kidney phlegm" were discussed from a theoretical level.The PMOP model rats were established by bilateral oophorectomy as the model group(OVX group,n=15).The rats were treated with LKB1-AMPK activator Metformin as the positive control group(Met group,n=15),and the rats in the sham-operated group(Sham group,n=15)were used as the negative control.The curative effect of Bushen Hutan Decoction on bone metabolism in PMOP model rats.Hematoxylin-eosin staining was used to observe the effect of Bushen Huatan Decoction on bone formation in PMOP model rats.Tartaric acid phosphatase histochemical staining and immunohistochemical staining were used to observe the effect of Bushen Huatan Decoction on bone resorption in PMOP model rats.The expression of ROS in bone tissue was detected by flow cytometry,and the effect of the Bushen Huatan Decoction on the level of oxidative stress in bone tissue of PMOP model rats was observed.Combined with 16 s r RNA amplicon sequencing and metagenomic sequencing,the differences in the composition and structure of the intestinal flora of PMOP model rats in each group were observed,and the effect of Bushen Huatan Decoction intervention on the differences in the structure of intestinal flora in the model rats,and the effect on the function of the flora.Annotation was performed to obtain the functional diversity of differential flora in PMOP model rats.Targeted metabolomics technology,the content of short-chain fatty acids in the feces and serum of rats in each group,was screened.The short-chain fatty acids were screened,including acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,valeric acid,and caproic acid.They were combined with bioinformatics methods to analyze the metabolic pathways involved in differential metabolites to clarify the effect of Bushen Huatan Decoction on the metabolites of intestinal flora in PMOP model rats.Reverse transcription-polymerase(RT-PCR)and Western blotting(WB)were used to detect the expression levels of autophagy-related genes and proteins and AMPK/m TOR/ULK1 signaling pathway-related indicators in rat bone tissue.Immunofluorescence cell imaging and immunohistochemical staining The changes of autophagosome and autophagolysosome in bone tissue were observed,the level of autophagy in PMOP model rats,and the effect of Bushen Huatan Decoction on autophagy activity in model rats through AMPK/m TOR/ULK1 signaling pathway.Results(1)"Kidney phlegm" is a pathological product of abnormal water metabolism caused by kidney deficiency."Kidney phlegm" affects the bones;the muscles and bones are unfavorable,the back is strong and painful,and the feet and knees are sore and weak,which is precisely the pathogenesis and clinical manifestations of PMOP.Therefore,kidney deficiency produces phlegm,and phlegm enters the bones,one of the critical factors that cannot be ignored in PMOP.The two influence each other and participate in diseases which have been confirmed in diseases such as osteoarthritis and inflammatory bowel disease.In vitro and in vivo studies have shown that gut microbiota and autophagy are involved in the pathogenesis of PMOP,but whether the m TOR target mediated by intestinal microbiota metabolites SCFAs to regulate autophagy is involved in the regulation of bone metabolism remains to be further elucidated.The homeostasis of gut microbes and a moderate level of autophagy are crucial to maintaining the orderly life activities of the body,which are in line with the philosophical viewpoints of Chinese medicine,such as the holistic view of life,the balance of yin and yang,and the view of constant movement.Therefore,there is a theoretical basis for traditional Chinese medicine to balance intestinal flora and regulate autophagy in multiple ways.(2)Micro-CT results showed that compared with the Sham group,the BMD,trabecular number,trabecular thickness,and trabecular volume/bone tissue volume percentage of the tibia were significantly lower in the model group(P <0.05).The degree of trabecular bone separation and the percentage of trabecular bone surface area/bone volume increased significantly(P <0.05).Compared with the OVX group,the bone mineral density,trabecular bone number,trabecular bone thickness,and trabecular bone volume/bone tissue volume percentage of the tibia of the rats in the BH group and Met group were significantly improved(P <0.05),and the degree of trabecular bone separation was significantly improved(P <0.05).The trabecular bone surface area/bone volume percentage decreased significantly(P <0.05).The number of tibial osteoclasts in the OVX group was significantly higher than that in the Sham group(P < 0.05);the number of osteoclasts in the BH group and Met group was significantly lower than that in the OVX group(P <0.05).Flow cytometry showed that the level of ROS in the bone tissue of the OVX group was significantly higher than that of the Sham group(P <0.05),and the expression of ROS in the bone tissue of the BH group and Met group was significantly lower than that of the OVX group(P <0.05).(3)The diversity of intestinal flora of rats in the Sham group was better than that of the OVX group,and the diversity of intestinal flora of rats in the BH and Met groups was better than that of the OVX group.Species taxonomy showed that at the phylum level,compared with the Sham group,the abundance of Firmicutes in the OVX group decreased,while the proportions of Bacteroidetes,Proteobacteria,and Moleiobacterium increased.Compared with the OVX group,Bacteroides and proteobacteria decreased,while the abundance of Firmicutes increased in the BH and Met groups.At the genus level,compared with the Sham group,the abundance of Bacteroides and Ruminococcaceae?UCG-005 in the OVX group increased,while Clostridium,Lactobacillus,Lachnospira,Ruminobacter,and Odribactor decreased;Compared with the OVX group,the abundances of Ruminobacteria,Clostridium,Lactobacillus,Lachnospira,and Odribactor increased in BH and Met groups,while Bacteroides and Ruminococcaceae?UCG-014 decreased.At the species level,compared with the rats in the Sham group,the abundance of Marvinbryantia?formatexigens in the OVX group increased,while the abundance of Firmicutes,Clostridium,and Firmicutes decreased.The abundance of Clostridium was relatively elevated.Based on the KEGG database,the intestinal flora function of each group of rats was predicted,and it was found that it was related to bile acid metabolism,short-chain fatty acid transport,and branched-chain amino acid transport system permease protein,acetate kinase,propionate kinase,and butyrate kinase.(4)Targeted metabolomics showed metabolites in feces and serum of the OVX group were significantly separated from those of the Sham group,and the metabolites in the feces and serum of the BH and Met groups were slightly close to those of the Sham group.The quantitative results of SCFAs showed that compared with the Sham group,the content of total SCFAs in the fecal tissue and serum samples of the rats in the OVX group was significantly decreased(P <0.05).The contents of total SCFAs in the feces and serum of rats in the BH and Met groups were slightly increased(P<0.05).Acetic acid,propionic acid,and butyric acid accounted for 90% of the total SCFA content.Compared with the Sham group,the contents of acetate,propionate,and butyrate in the fecal tissue and serum samples of the rats in the OVX group were significantly decreased(P <0.05).The contents of acetic acid,propionic acid,and butyric acid in fecal tissue and serum samples of rats in the BH and Met groups increased(P <0.05).(5)Compared with the Sham group,there were no significant differences in the protein and gene expressions of AMPK,p-AMPK,m TOR,p-m TOR,ULK1,and ATG13 in the OVX group(P>0.05),after drug intervention,the expression levels of AMPK,p-AMPK,ULK1,and ATG13 in bone tissue of rats in BH group and Met group were significantly higher than those in Sham group and OVX group(P<0.05),and m TOR,p-m TOR The expression level was down-regulated(P<0.05).The protein and gene expression levels of Beclin1,LC3?,LC3?,and p62 were not significantly different in the Sham group and the OVX group.The protein and gene expressions of Beclin1,LC3?,and LC3? in the BH group and the Met group were significantly higher than those in the OVX group and the Sham group(P<0.05),the p62 level decreased significantly(P<0.05).Conclusion1.Kidney deficiency produces phlegm,and kidney phlegm in the bone is one of the critical factors that cannot be ignored in PMOP.The treatment of PMOP from "kidney phlegm" and the elimination of phlegm based on invigorating the kidney is in line with the treatment principle of TCM syndrome differentiation and treatment.Traditional Chinese medicine has a solid theoretical and experimental basis for regulating autophagy and intestinal flora from multiple levels and angles,which is a concrete manifestation of the modernization of traditional Chinese medicine.2.Bushen Huatan Decoction can significantly correct intestinal flora disturbance in PMOP model rats,enrich the diversity of intestinal flora,and increase the abundance of short-chain fatty acid-producing bacteria in the intestinal tract of model rats.The abundance and content of producing bacteria are related to promoting the content of propionic acid,butyric acid and acetate in feces and serum,and promoting the autophagy level in PMOP model rats through the AMPK/m TOR/ULK1 signaling pathway. |
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