The Mechanism Of Bugu Shengshui Decoction On Postmenopausal Osteoporosis Based On Different Metabonomics Techniques

BackgroundOsteoporosis(OP)is a systemic metabolic disease characterized by decreased bone mineral density(BMD),damaged bone microarchitecture,and increased bone fragility.As a common type of primary osteoporosis,the incidence of PMOP is also increasing year by year.According to the research,the prevalence of OP in people over 50 years old and over 65 years old is 32.1%and 51.6%,respectively.The treatment cost of PMOP is long-term,which has also become the economic burden of the family and society.At present,the treatment of PMOP in China is mainly inclined to western medicine,which has achieved significant clinical efficacy.However,western medicine has certain limitations,which have become an important problem to limit long-term clinical medication.Therefore,Chinese medicine has more advantages because of multicomponents,multi-targets,and small adverse reactions,which have come into the view of clinicians.Traditional Chinese Medicine(TCM)mentions that PMOP is mainly associated with bone and related to Liver,Spleen and Kidney.Liver and Kidney deficiency and Spleen Qi deficiency are the main TCM pathogenesis of PMOP,so clinical treatment of PMOP is mainly based on tonifying Liver,Kidney,and invigorating Spleen,The Bugu Shengsui decoction is mainly composed of Rhizoma Drynariae,Fructus Psoraleae,and Rhizoma Cibotii,etc.Meanwhile,it has the effects of tonifying Kidney,strengthening bone,and relieving pain.At present,this decoction has been used in clinic for more than 30 years,and its clinical effective rate and total effective rate are 46%and 82%,which can improve patients’ bone density,reduce bone resorption and bone loss,and improve related clinical symptoms.In addition,it also has the advantages of high safety and low adverse reactions.Meanwhile,previous vivo experimental studies have confirmed that Bugu Shengsui decoction can improve microcirculation,regulate oxidative stress to control ferroptosis,and mediate ERK phosphorylation to regulate Smad/ERK pathway.Intestinal is an important metabolic organ,which is related to the occurrence of many metabolic diseases.Studies have confirmed that metabolites of intestinal microbiota and hormone-promoting hormone secreted by intestinal cells can regulate target cells in the blood,affecting bone metabolism.Therefore,this experiment speculated whether Bugu Shengsui decoction could affect PMOP by regulating intestinal microbiota and metabolites in serum.ObjectTo explore the possible related pathways and potential targets of PMOP treated by Bugu Shengsui decoction through targeted analysis of short-chain fatty acids metabolites of intestinal flora and non-targeted metabolomics screening of serum.To provide reliable scientific evidence for clinical use,new ideas for new drug research,and new strategies for PMOP treatment.The possible related pathways and potential targets of Bugu Shengsui decoction in the treatment of PMOP were explored through targeted analysis of intestinal flora metabolites of short-chain fatty acids and serum non-targeted metabolomic screening.The study can provide reliable scientific evidence for clinical use,new ideas for new drug development,and new strategies for PMOP treatment.Content(1)Bone mineral density analysis and histopathological examination were used to investigate the effect of Bugu Shengsui decoction on postmenopausal osteoporosis rats and to determine the optimal dosage.(2)Short-chain fatty acid analysis and serum non-targeted metabolomics were used to explore the potential pathways and targets of Bugu Shengsui decoction in the treatment of postmenopausal osteoporosis in rats.Methods1.Eighty rats were randomly divided into SHAM group(3 months),MODEL group(3 months),SHAM group(6 months),MODEL group(6 months),Alendronate sodium group(6 months),Bugu Shengsui decoction(BGSSF)high dose group(6 months),Bugu Shengsui decoction(BGSSF)medium dose group(6 months),Bugu Shengsui decoction(BGSSF)low dose group(6 months).There were 10 rats in each group and modeling was performed by bilateral oophorectomy.Chinese herbs were soaked with distilled water in the container,and immersed for 1 h.Then we added herbs into the vacuum concentration tisanes machine,decocted,filtered,and concentrated in a water-bath water.Finally,we saved decoction in 4 ℃ refrigerator.After feeding for 3 months,the rats were successfully modeled by BMD test and histopathological test,and the drug treatment started.Alendronate sodium group,BGSSF high dose,BGSSF medium dose and BGSSF low dose were administered at a volume of 10 mL/kg,and concentration were 0.84 mg/kg,11.21 g/kg,5.61 g/kg and 2.80 g/kg,respectively.The SHAM group was given the same amount of normal saline.The continuous intragastric administration lasted for 3 months,6 days a week.After intragastric administration,the left and right tibia of rats in each group were taken.BMD test was performed on the left tibia and histopathological test were performed on the right tibia to confirm the success of modeling and optimal effective dosage.2.After confirming the efficacy,30 SPF SD rats were randomly divided into 3 groups,SHAM group(6 months),MODEL group(6 months)and BGSSF high-dose group(6 months),with 10 rats in each group.Bilateral ovariectomy was also used for modeling.After modeling,we also fed for 3 months.Chinese herbs were soaked with distilled water in the container,and immersed after for 1 h.Then,we added herbs into the vacuum concentration tisanes machine,decocted,filtered,and concentrated in a water-bath water enrichment.Finally,we saved decoction in 4℃ refrigerator.The concentration of BGSSF high-dose group was given to 11.21 g/kg with each rat at a volume of 10 mL/kg.The SHAM group was given the same amount of normal saline.The continuous intragastric administration lasted for 3 months,6 days a week.After intragastric administration,feces,and serum of rats in each group were collected for untargeted metabonomics analysis of short-chain fatty acids metabolites and serum,respectively.In conclusion,we tried to explore the possible related pathways and targets of PMOP treatment,and to provide a new direction for future experiments.ResultsExperiment 1:Modeling evaluation of SHAM group(3 months)and MODEL group(3 months):First,BMD of the MODEL group was significantly lower than that of the SHAM group,which difference was statistically significant.Compared with the SHAM group,histopathological examination showed that the adipocytes in SHAM group(3 months)were significantly increased,the medullary cavity was enlarged,and the trabecular bone was thinned,distributed unevenly,arranged sparsely,and mostly fractured.Combined with the BMD values and histologic examination,we confirmed the rats model building has been completed with 3 months.Pharmacodynamics evaluation of Bugu Shengsui decoction for the treatment of postmenopausal osteoporosis in rats:Compared with SHAM group(6 months),the BMD of MODEL group(6 months)was significantly lower.Compared with MODEL group(6 months),the BMD of Alendronate sodium group and BGSSF highdose,medium-dose and low-dose groups increased in varying degrees,and the improvement degree of BGSSF high-dose group was more significant than that of medium-dose and low-dose groups.Compared with SHAM group,the adipose cells in MODEL group were significantly increased,and the trabecular bone was thinner,less evenly distributed,sparsely arranged,and there were multiple trabecular fractures.Compared with MODEL group,the rest of the treatment groups were improved in varying degrees,mainly in increased trabecular bone status,increased thickness,evenly aligned and few fracture.Besides Alendronate sodium group,these performances were most significantly improved in BGSSF high-dose group.Based on the above analysis of BMD and histopathological examination results,we confirmed that Bugu Shengsui decoction has efficacy in the treatment of PMOP,and the high dose is the optimal dose for this experiment.In the follow-up experiments,we will use the high dose as the intervention dose to explore the potential mechanism.Experiment 2:(1)Compared with SHAM group,MODEL group and BGSSF group,the level of acetic acid and propionic acid in MODEL group was lower than those in SHAM group,and the level of BGSSF group was significantly higher than those in MODEL group.The contents of other fatty acids in MODEL group were significantly higher than those in SHAM group,and BGSSF group was significantly lower than MODEL group,among which acetic acid and caproic acid had statistically significant differences.(2)Serum untargeted metabolomics analysis was performed on the serum of SHAM group,MODEL group and BGSSF group.We found 7 metabolic pathways with high correlation:Basal cell carcinoma,mTOR signaling Pathway,Intestinal immune network for IgA production,Protein Digestion and absorption,the Central carbon metabolism in cancer,Aminoacyl tRNA biosynthesis,and Mineral absorption.Therefore,we speculated that the effective mechanism of Bugu Shengsui decoction on PMOP might be related to cancer-related pathways,protein digestion and absorption,mTOR signaling pathway,intestinal related immune network,mineral absorption,and amino-Aminoacyl tRNA biosynthesis.Then,it was shown that the Intestinal immune network for IgA production was closely related to the content of this study by reviewing related research at home and abroad.Conclusion(1)The Bugu Shengsui decoction has a significant effect in the treatment of PMOP,and the high dose is the best dosage.(2)Through targeted analysis of short-chain fatty acids in feces metabolites,the content of acetic acid increased and the content of caproic acid decreased.(3)It was found that the Intestinal immune network for IgA production and the cytokine TGF-β may be the potential metabolic pathway and target of PMOP treatment by the serum untargeted metabolomics.InnovationThe relationship between Bugu Shengsui decoction and the intestinal immune pathway(Intestinal immune network for IgA production)and cytokine TGF-β were discovered by serum metabolomics.The short-chain fatty acid metabolites of intestinal flora were used to analyze the differential changes in the content of acetic acid and caproic acid in intestinal flora metabolites short-chain fatty acids in the treatment of postmenopausal osteoporosis,which provided a new strategy and a new idea for clinical treatment of PMOP.