| ObjectivePostmenopausal osteoporosis（PMOP）is a common systemic bone meta-bolic disease characterised by the decreased bone mineral density,severe bone loss and destruction of bone tissue structure,which may cause great disturb-ance to the health of middle-aged and elderly women and even the quality of life.Previous studies by our group have shown that the Bushen Huatan （BSHT）Decoction has good clinical efficacy in regulating bone metabolism and improving osteoporosis with multiple signalling pathways and targets.Based on the distribution characteristics of clinical TCM syndromes,this study further discusses the rationale of"kidney deficiency and phlegm"in causing PMOP,and proposed BSHT Decoction according to the therapeutic rules.Furthermore,we conducted studies on the pharmacological mechanisms and toxicology to verify the clinical safety of BSHT Decoction.Additionally,in conjunction with the previous studies of the original formula,we explored whether the mechanism of improving bone metabolism by this formula could be related to the bile acid-parathyroid hormone-related peptide/β-arrestin2 （PTHr P/β-arrestin2）axis,in order to further clarify the mechanism underlying the prevention and treatment of PMOP with BSHT Decoction.Methods（1）By reviewing ancient TCM books,the origin of phlegm and the char-acteristics of its pathogenesis were first discussed,leading to the proposal of a syndrome type‘kidney deficiency and phlegm turbidity’in PMOP.Based on the domestic and international literature review,the traditional and modern medical connotations of‘kidney deficiency and phlegm turbidity’regarding the pathogenesis of PMOP were discussed.According to extensive clinical and pharmacological studies,composition of the drugs in this formula and their ef-fects on bone metabolism were discussed,providing a theoretical basis for the proposal of BSHT Decoction.（2）A network pharmacology study was conducted to clarify the effect of BSHT Decoction in treating PMOP.Based on public databases such as TCMSP and ETCM,the active ingredients of BSHT Decoction were searched and the common targets for treating PMOP were screened and uploaded to the STRING database to build a protein interaction network.GO and KEGG data-bases were utilized to conduct the enrichment analysis on common targets,ex-ploring the potential mechanism of BSHT Decoction in treating PMOP.（3）（1）Acute oral toxicity test:16 Sprague-Dawley（SD）rats were ran-domly divided into 2 groups with 8 rats in each,half male and half female.Rats in the intervention group were administered BSHT decoction （37.6g/kg·bw）by gavage for 2 weeks according to the maximum tolerated dose,observing the growth and toxicity of rats.（2）30-day feeding test:64 SD rats,with half male and half female,were randomly divided into low（9.4g/kg·bw）,medium（18.8g/kg·bw）and high dose（37.6 g/kg·bw）groups as well as blank group（0.9%saline）.BSHT de-coction were administered to the low,medium and high dose groups respec-tively by gavage at 1ml/100g,while the blank group was given an equal vol-ume of 0.9%saline for 30 consecutive days.At the end of the month,rats were anesthetized and executed for sampling.Effects of BSHT Decoction on the blood,liver and kidney functions,morphology and coefficients of organs of rats in each group were compared.（3）Bone marrow micronucleus test:50 Kunming（KM）rats were ran-domly divided into low,medium and high dose groups of BSHT decoction,and the negative control（0.9%saline）as well as the positive control group（40mg/kg·bw cyclophosphamide）,with half male and half female in each.Each group was gavaged at 0.2 ml/10g in five consecutive days.At the end of the5th day,rats were executed and the bone marrow were prepared and examined microscopically to compare the ratio of polymorphic erythrocytes（PCE）to to-tal erythrocytes（PCE+NCE）in each group.（4）Sperm Malformation Test:Another 25 male KM rats were randomly divided into five groups,with 5 in each.Groups and interventions were similar to those in the bone marrow micronucleus test.On the 35th day after the first intervention,rats were executed and the bilateral epididymis were taken to ob-serve the sperm deformation,calculating and comparing the sperm malfor-mation rate of rats in each group.The acute toxicity test in rats,30-day feeding test,bone marrow cell micronucleus test and sperm malformation test in mice were conducted to verify whether BSHT Decoction has delay or accumulation of symptoms caused by hepatic and renal as well as reproductive toxicity,so as to provide a scientific basis for the safety of medication.（4）PMOP model was established by ovariectomy（OVX）in female SD rats.45 SD female rats were randomly divided into the model group（OVX group,n=15）,BSHT group（n=15）,and the Sham group（sham-operated）were set as the negative control（n=15）.The femoral tissues of the rats were scanned with Micro-CT to observe the changes in bone microstructure to con-firm whether the model was successfully operated.（1）Metagenomic sequencing technology was used to observe the differ-ence in the composition of intestinal flora of rats in each group,and the effect of BSHT Decoction on the diversity of intestinal flora.Non-targeted metabo-lomics technology was used to screen out the differential metabolites in the faeces and serum of rats in each group.The correlation and pathway enrich-ment analysis of the metabolites of intestinal flora were performed to clarify the potential mechanism and pathways of BSHT Decoction improving the in-testinal flora.（2）Quantitative analysis of bile acids,which is the differential metabolite identified in the last experiment,was carried out by the targeted metabolomics techniques to identify the bile acids and to figure out those whose changes were the most significant in both serum and faeces samples.The effect of bile acids on the proliferation of osteoblasts were investigated in vitro and whether the serum containing BSHT Decoction could reverse this effect was further discussed.（3）Expression levels of the related genes and protein PTHr P/β-arrestin 2in the serum of rats in each group were measured by reverse transcription-pol-ymerase（RT-PCR）and western blot（WB）to clarify the mechanism of BSHT decoction in promoting the bone reconstruction through PTHr P/β-arrestin2 axis.Results（1）Theoretical research:both TCM and western medicine have a pro-found understanding of phlegm generation and pathogenesis:all five organs can produce phlegm,which then can act as a pathological product and damage the organism back.The basic pathogenesis of osteoporosis（OP）in TCM is kidney deficiency,which generates phlegm and then moves into the bone,re-sulting in bone paralysis and impotence.The proposal of BSHT decoction is in line with the ideology of dialectical treatment in TCM.Moreover,clinical and pharmacological studies have provided some scientific basis for the effects of components and drug pairs in this formula on regulating the bone metabolism.（2）There are 54 bioactive ingredients in BSHT formula after screening,of which there are twenty-three ingredients from Epimedium,four from Rhi-zoma Drynariae,eleven from Cuscuta,eleven from Trichosanthis fructus,four from Hawthorn and one from Red Yeast Rice.Main components include ses-quiterpene,quercetin,isorhamnetin,kaempferol,epimedium glycosides,muxi-fenesin and isoflavones,etc.Moreover,Interleukin-6（IL-6）,matrix metallo-protein 9（MMP9）and Interleukin-1β（IL-1β）are the core genes regarding the treatment of PMOP with BSHT Formula.Biological processes containing re-sponse to stimulus,developmental processes,locomotion,positive regulation of biological processes,regulation of biological processes,etc.,are involved.The KEGG enrichments analysis shows that potential mechanisms are related to MAPK,PI3K/Akt,PPAR and osteoblast differentiation signalling pathways.（3）（1）Acute oral toxicity test showed that rats were in a normal state of growth,with no obvious toxic symptoms or deaths within two weeks.There were no significant abnormalities were observed after the autopsy at the end.Therefore,the decoction has no toxicity.（2）30-day feeding test showed that there was no statistically significant difference in the body weight of rats in each group at the beginning and at the end of each week;and there was no significant difference regarding the blood test,the liver and kidney test（ALT,BUN,Cr,total CHOL and GLU）of rats in each group;the differences in the body weight and organ coefficients of the heart,liver,lung,spleen and kidney were not significant（P>0.05）,suggesting that BSHT decoction would not adversely affect the rats.（3）Results of the micronucleus test showed that the PCE/NCE and micro-nucleus rate of the low,medium and high dose groups had no statistic differ-ence compared with those of saline group（P>0.05）,while the PCE/NCE and micronucleus rate of the cyclophosphamide group were changed significantly（P<0.01）,suggesting that cyclophosphamide is toxic to rats,while the BSHT Decoction is in safe for the bone marrow of rats.（4）The sperm malformation rate was significantly higher in the cyclo-phosphamide group than that in the saline group（P<0.01）,while there was no significant difference in the sperm aberration rate of the low,middle and high dose groups when comparing to that in the negative control（P>0.05）,suggesting that BSHT Decoction would not induce sperm malformation in rats.（4）（1）Micro-CT was used to scan the femur from each group,showing that the femur of Sham group was morphologically intact,with closely ar-ranged and uniformed bone trabeculae,while the trabeculae of the OVX group were broken,shortened and thinned.BMD,Tb.N,Tb.Th and BV/TV of the fe-mur in OVX group were significantly lower（P<0.05）,while Tb.Sp and BS/BV were significantly higher comparing to Sham group（P<0.05）,sug-gesting that PMOP model was operated successfully.The bone microstructure of the femur of rats in BSHT group was improved significantly（P<0.05）,suggesting that the bone loss owing to the OVX and estrogen deficiency could be alleviated to a certain extent BSHT Decoction.（2）Sequencing analysis of intestinal flora showed that the number of OTUs of rats in the OVX group was significantly decreased compared with that in the Sham group,while the number of Operational Taxonomic Units（OTUs）was significantly increased compared with that in the OVX group af-ter the intervention of BSHT Decoction,suggesting that rats with PMOP could reduce the diversity of intestinal flora,while BSHT Decoction could increase the diversity of intestinal flora.The results of non-targeted metabolomics showed that there were 22 up-regulated metabolites and 5 down-regulated me-tabolites in the OVX group,however,15 of which could be reversed by BSHT Decoction.KEGG enrichment analysis of the intestinal flora showed that its metabolic pathways were mainly focused on metabolism,where the top four pathways were bile acid（KO3453）,butyrate kinase（K00929）,propionate ki-nase（K00932）and acetate kinase（K00925）metabolic pathways.Moreover,the intestinal flora involved in intestinal flora-bile acid metabolism mainly in-cluded Bacteroidetes,Clostridium,Lactobacillus,Bifidobacterium,Braun-schweiger and Ruminococcaceae.（3）Targeted metabolomics results showed that secondary bile acids with the same trend of changes and have statistically significant differences in both serum and faecal samples were taurocholic deoxycholic acid（THDCA）,tauro-deoxycholic acid（TDCA）and taurodeoxycholic acid（TCDCA）,etc.In con-trast,BSHT Decoction could regulate these secondary bile acids to some ex-tent（P<0.05）.THDCA was the most significant,which was selected with dif-ferent concentrations to conduct the in vitro experiment.Results showed that THDCA could inhibit the proliferation of OB which was dose-dependent,while BSHT Decoction could alleviate the inhibition of OB proliferation.（P<0.05）.（4）RT-PCR showed that levels of PTHr P andβ-arrestin2 m RNA of rats were significantly decreased in the OVX group compared with that in Sham group,however,expressions of two genes were significantly increased after the BSHT intervention.（P<0.05）.WB showed that levels ofβ-arrestin2 and PTHr P were significantly reduced in the OVX group compared with the that in Sham group,whilst expressions ofβ-arrestin2 and PTHr P in BSHT group were significantly increased after the BSHT intervention（P<0.05）.Conclusion:（1）The identification of PMOP syndrome type as kidney deficiency and phlegm turbidity has a certain theoretical basis,and the application of BSHT decoction is reasonable in terms of etiology and pathogenesis,clinical sympto-matology and pharmacological research.（2）BSHT can effectively improve the bone microstructure and the disor-der of intestinal flora by inhibiting the production of secondary bile acids and alleviating the negative regulation of bile acids on osteoblasts,the mechanism of which is related to the PTHr P/β-arrestin2 pathway. |
PDF Full Text Request