Study On Clinical Effect And Mechanism Of Bushen Huatan Recipe In Treating Postmenopausal Osteoporosis

Background:Postmenopausal osteoporosis（PMOP）is a common systemic metabolic disease characterized by decreasing bone mineral density,severe bone mass loss and destruction of bone tissue structure.Patients with PMOP can cause fracture under slight external force,which is a brittle fracture.The fracture of PMOP patients is difficult to heal,accompanied by a high rate of disability and mortality,which will seriously affect the daily life quality and cause serve economic burden to patients.With the change of population structure and population aging,the prevalence rate of PMOP in Asia is47.3%,which has already exceeded the total incidence of heart disease,stroke and breast cancer.In China,the prevalence rate of PMOP among women over 50 years old is 35.8%which will become a serious public health problem in the near future,thus the prevention and treatment of this disease has gradually became an important research hotspot.Estrogen replacement therapy is effective in the treatment of patients with PMOP and further reduces the risk of fracture.However,long-term high doses of estrogen can not only lead to breast pain,uterine bleeding but also increase the risk of breast and endometrial cancer.Clinically,western medicine bisphosphonates,denitrifying amines,teripapeptide and other drugs have been proved to be effective in the prevention and treatment of osteoporosis,but their long-term adverse reactions would have a great impact on the body as well.In general,PMOP is a systematic and complex systemic disease,which is the result of the comprehensive action of a variety of pathogenic factors.Based on the holistic concept and dialectical treatment,traditional Chinese medicine has certain advantages in the use of multi-components and multi-targets in the treatment of PMOP.Bushen Huatan recipe is a combination of Bushen Huatan pill with Danxixin method cucumber lotus pill.Though early clinical basis shows that Bushen Huatan recipe can effectively reduce the loss of bone mineral density,regulating bone metabolism,relieving pain and improve the level of active vitamin D in serum of patients with PMOP effectively,its main mechanism is not clear up to now.Thus,the purpose of this paper is to clarify the specific mechanism of Bushen Huatan recipe and determine its related signal pathway.Methods:The clinical experimental study of Bushen Huatan recipe was carried out.The bone mineral density（BMD）,serum vitamin D level,pain score,TCM syndrome score and SF-36 quality of life score were used to evaluate the therapeutic effect of Bushen Huatan recipe.The rat model of osteoporosisinduced by bilateral ovariectomy（Ovariotomy,OVX）was established,and Bushen Huatan recipe was put forward.The effects of Bushen Huatan recipe on bone and kidney of OVX rats were compared by histopathology and molecular biology.Differential renal metabolites were screened by metabonomics and bioinformatics analysis.The action targets and related signal pathways of Bushen Huatan recipe on PMOP were identified as well.Results:In the clinical trial,Bushen Huatan recipe increased the level of serum active vitamin D in patients with PMOP and decreased the pain score and syndrome score.Moreover,the T value of bone mineral density of lumbar L1-L4 and femoral neck have been increased as well.Compared with those before treatment,the total scores of body pain,general health,social function and mental health were significantly increased.The evidence from animal studies showed that Bushen Huatan recipe could increase bone volume,bone mineral density,total collagen positive ratio,alkaline phosphatase positive area and osteocalcin positive area,while decrease tartrate resistant acid phosphatase positive area,RANKEL gene and protein expression level in OVX rats.Nevertheless,it also increases the total antioxidant capacity of bone tissue,decreasing the content of malondialdehyde,hydrogen peroxide and the expression level of apoptosis-related protein in bone tissue of OVX rats.Bushen Huatan recipe increased the expression of Bmi1 protein in bone tissue of OVX rats,decreasing the expression of aging-related proteins p16,p19,p53,p27,p21,the ratio of positive cells of 8-OHd G andβ-gal by immunohistochemical staining.Bushen Huatan recipe could also decrease the positive area of Masson staining,type I collagen（Col-I）andα-smooth muscle actin（α-SMA）in kidney of OVX rats,down-regulated the expression level ofα-SMA gene and protein in kidney,up-regulated the expression level of cadherin（E-cadherin）gene and protein in epithelial cells.What’s more,the recipe could down-regulate the expression of TGF-βsignal pathway related proteins and genes in kidney of OVX rats,reducing the infiltration of inflammatory factors and the expression of apoptotic protein in kidney tissue as well as improving DNA damage and increasing the number of mitochondria.Last but not least,Bushen Huatan recipe can increase the blood flow velocity of renal artery and the level of serum creatinine clearance as well as the protein expression level of 1αhydroxylase（1α（OH）ase）coding gene CYP27B1.The results of metabonomics showed that the metabolic profile of Bushen Huatan recipe changed significantly after intervention in the kidney of OVX rats.The results of principal component（PCA）analysis exhibited that Bushen Huatan group was close to the sham operation group while far away from the OVX group,and returned to the sham operation group.Among the 42 differential metabolites in the kidney of OVX rats,Bushen Huatan recipe can regulate 35 of them,of which 22 have statistical significance.The metabolites of Bushen Huatan recipe are mainly related to glycine,serine and threonine metabolic pathway,glycerophospholipid metabolic pathway,purine metabolic pathway,arachidonic acid metabolic pathway and taurine metabolic pathway.Conclusion:Clinical trials show that Bushen Huatan recipe can effectively relieve pain,reduce bone mass loss and improve patients’quality of life in patients with PMOP.The results of animal model and metabonomic experiments exhibit that Bushen Huatan recipe can delay bone aging,inhibiting bone oxidative stress and apoptosis,promoting bone formation and inhibiting bone resorption.The recipe can delay kidney aging,inhibiting kidney cells secreting cell aging-related phenotypes,reducing oxidative stress level and inflammatory reaction,inhibiting kidney cell apoptosis and kidney tissue TGF-β1 activation by regulating the disorders of glycine,serine and threonine metabolic pathways,glycerophospholipid metabolic pathways,purine metabolic pathways,arachidonic acid metabolic pathways and taurine metabolic pathways.Furthermore,it can inhibit the phosphorylation of Smad2,blocking the process of EMT,delaying renal tubular atrophy and interstitial fibrosis,enhancing the expression activity of 1α-hydroxylase in renal tubular epithelial cells,increasing the expression level of1,25（OH）₂D₃,regulating the homeostasis of calcium and phosphorus and delays the loss of bone mass in postmenopausal osteoporosis through the antioxidation of1,25（OH）₂D₃.