独活寄生汤基于miR-22-3p靶向RhoA/ROCK通路调节骨重建平衡改善骨质疏松症大鼠骨密度OA

Objective:To investigate the effects of Duhuo Jisheng decoction(DHJSD)on bone mineral density and bone reconstitution balance in osteoporotic(OP)rats by targeting and regulating the Ras homolog gene family member A(RhoA)/Rho-associated coiled-coil-containing kinase(ROCK)pathway through miR-22-3p.Methods:Female SD rats were randomly divided into normal control group(sham group),os-teoporosis model group(OP group),low-dose DHJSD and high-dose DHJSD groups(L-DHJSD group,H-DHJSD group),high-dose DHJSD+antiagomir-NC group(H-DHJSD+antiagomir-NC group),and high-dose DHJSD+miR-22-3p antiagomir group(H-DHJSD+miR-22-3p antiagomir group).The levels of bone gla protein(BGP),osteoprotegerin(OPG),and alkaline phosphatase(AKP)in the serum of rats in each group were detected by ELISA.The bone mineral density of the femur of rats in each group was detected by dual-energy X-ray absorptiometry.Micro-CT was used to detect the changes of bone microstructure of rats in each group.HE staining was used to observe the pathological morphological changes in the metaphyseal region of the femoral tissue in rats of each group.The changes in the number of osteoclasts in femoral tissue were ob-served by tartrate-resistant acid phosphatase staining.The relationship between miR-22-3p and RhoA was examined by dual-luciferase reporter assay.Immunohistochemical staining was used to detect the positive ex-pression of BMP2 and Runt-related transcription factor 2(Runx2)in bone tissue;qRT-PCR was employed to measure the expression levels of miR-22-3p in rat femoral tissue;Western blot was performed to assess the expression levels of proteins related to the RhoA/ROCK pathway in the femoral tissue of rats of each group.Results:Compared with the sham group,the integrity of the trabecular bone structure in the femoral tissue of rats in the OP group was disrupted,with trabeculae markedly thinned and fractured,and the bone marrow cavity markedly dilated.The levels of serum BGP,OPG,AKP,femoral bone mineral density,trabecular bone number(Tb.N),trabecular bone thickness(Tb.Th),bone volume fraction(BV/TV),positive rate of BMP2,positive rate of Runx2,and expression of miR-22-3p were decreased,while the trabecular spacing(Tb.Sp),the number of osteoclasts,the expressions of RhoA and ROCK were increased(P<0.05).Com-pared with the OP group,the trabecular bone structure of the femoral tissue in the L-DHJSD group and the H-DHJSD group was improved,and the bone marrow cavities were reduced.The levels of serum BGP,OPG,AKP,femoral bone mineral density,Tb.N,Tb.Th,BV/TV,positive rate of BMP2,positive rate of Runx2,and expression of miR-22-3p were increased,while the Tb.Sp,number of osteoclasts,and the expressions of RhoA and ROCK were decreased(P<0.05).Compared with the H-DHJSD group and the H-DHJSD+anti-agomir-NC group,the trabecular bone structure of the femoral tissue in the H-DHJSD+miR-22-3p anti-agomir group was severely damaged,and the bone marrow cavity was dilated.The levels of serum BGP,OPG,AKP,femoral bone mineral density,Tb.N,Tb.Th,BV/TV,positive rate of BMP2,positive rate of Runx2,and expression of miR-22-3p were lower,while the Tb.Sp,number of osteoclasts,and the expres-sions of RhoA and ROCK were higher(P<0.05).The results of the dual-luciferase reporter gene showed that the fluorescence activity of the miR-22-3p mimic group was markedly lower than that of the miR-NC group(P<0.05).Conclusions:DHJSD may increase bone mineral density in OP rats and improve the balance of bone remodeling by up-regulating the expression of miR-22-3p,and targeting to inhibit the activation of the RhoA/ROCK pathway.