补骨生髓方调控EphrinB2/EphB4/ERK通路改善去势大鼠骨丢失的作用机制OA

Objective To investigate the mechanism by which Bugu Shengsui Formula ameliorates bone loss in orchidectomized rats based on the EphrinB2/Eph receptor B4(EphB4)/extracellular signal-regulated kinase(ERK)pathway.Methods Male SD rats were randomly divided into sham operation group,model group,alendronate sodium group(0.88 mg·kg-1),and low-,medium-,and high-dose Bugu Shengsui Formula groups(2.94,5.87,11.74 g·kg-1),with 15 rats per group.Except for the sham operation group,all rats underwent bilateral orchidectomy to establish a castration-induced osteoporosis model.Drug intervention began on day 2 post-surgery.The treatment groups received the corresponding doses by intragastric gavage,while the sham operation group and model group received an equal volume of distilled water.Gavage was performed once daily for 6 consecutive days followed by 1 day of rest,for a total of 12 weeks.Micro-CT was employed to evaluate the three-dimensional trabecular microarchitecture parameters and bone mineral density(BMD)of the distal femur,including bone volume fraction(BV/TV),trabecular thickness(Tb.Th),trabecular number(Tb.N),trabecular separation(Tb.Sp),structure model index(SMI),and BMD.Histopathological changes in bone tissue were observed by HE staining.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood urea nitrogen(BUN),and creatinine(CREA)were measured using an automatic biochemical analyzer.Serum bone Gla protein(BGP)and bone alkaline phosphatase(BALP)levels were determined by ELISA.RT-qPCR was performed to detect the mRNA expression levels of ALP and osteocalcin(OCN)in bone tissue.Immunohistochemistry was used to detect the protein expression of Runt-related transcription factor 2(RUNX2).Western Blot was used to measure the protein expression levels of EphrinB2,EphB4,p-EphB4,and ERK in bone tissue.Results Compared with the sham operation group,the model group exhibited significantly decreased BMD,BV/TV,and Tb.N of the distal femur(P＜0.01),significantly increased Tb.Sp and SMI(P＜0.01),significantly reduced serum BALP and BGP levels(P＜0.01),significantly downregulated mRNA expression of ALP and OCN(P＜0.01),and significantly downregulated protein expression of EphrinB2,EphB4,p-EphB4,ERK,and RUNX2,as well as the p-EphB4/EphB4 ratio(P＜0.01),accompanied by a significantly decreased mean optical density of RUNX2(P＜0.01).Compared with the model group,the alendronate sodium group and the medium-and high-dose Bugu Shengsui Formula groups showed significantly increased BMD,BV/TV,and Tb.N(P＜0.01),significantly decreased Tb.Sp and SMI(P＜0.05,P＜0.01),and significantly elevated serum BALP and BGP levels(P＜0.05,P＜0.01).In addition,the medium-dose Bugu Shengsui Formula group demonstrated a significant increase in Tb.Th(P＜0.05),significantly upregulated ALP and OCN mRNA expression(P＜0.01),and significantly upregulated protein expression of EphrinB2,EphB4,p-EphB4,ERK,and RUNX2,as well as the p-EphB4/EphB4 ratio(P＜0.05,P＜0.01),with a significantly increased mean optical density of RUNX2(P＜0.01).No significant differences were found in serum ALT,AST,BUN,or CREA levels among all groups(P＞0.05).Conclusion Bugu Shengsui Formula can improve the three-dimensional microarchitecture of trabecular bone in the distal femur,increase bone mineral density,promote bone formation,and exert osteoprotective effects in orchidectomized rats.The mechanism may be associated with activation of the EphrinB2/EphB4/ERK signaling pathway and enhanced expression of osteogenic factors.