活骨方对酒精性骨质疏松大鼠骨髓微环境相关炎症-氧化失衡及衰老信号的影响OA

Objective:To investigate the effects of Huogu formula on bone marrow microenvironment-related inflammatory response,distal femoral trabecular microarchitecture,bone antioxidant defense,and p16/p21/p53-related protein changes in rats with alcoholic osteoporosis(AOP),and to evaluate its intervention effects in combination with bone metabolism indices and bone marrow fat deposition.Methods:Fifty SPF male Sprague-Dawley rats aged 6-8 weeks and weighing(200±20)g were randomly divided into normal group(N group),AOP model group(AOP group),and low-,medium-,and high-dose Huogu formula groups(L,M,and H groups),with 10 rats in each group.Except for the N group,rats in all groups received intragastric administration of 20%(vol/vol)ethanol at 10 mL/kg,6 times per week for 20 consecutive weeks to establish the AOP model.At week 16,distal femoral Micro-CT was performed for stage validation of the model.From week 16,on the basis of continued ethanol intervention,rats in the L,M,and H groups were given Huogu formula by gavage at doses of 0.98,1.96,and 3.92 g/kg,respectively,6 times per week for 4 consecutive weeks.At week 20,distal femoral trabecular microarchitecture was re-evaluated by Micro-CT.Serum levels of IL-1β,IL-6,CXCL10,MCP-1,Ca,P,PINP,and CTX-1 were measured.Total antioxidant capacity(T-AOC),superoxide dismutase(SOD),and catalase(CAT)levels in bone tissue were determined.Protein expression of p16,p21,and p53 in femoral tissue was detected by Western Blot.Bone mineralization and bone marrow fat deposition were observed by Alizarin Red staining and Oil Red O staining,respectively.Results:At week 16,Micro-CT showed that,compared with the N group,rats in the AOP group exhibited markedly sparse and thinned trabeculae in the distal femur,accompanied by decreased trabecular tissue mean BMD(P<0.01),reduced trabecular thickness(Tb.Th,P<0.01),trabecular number(Tb.N,P<0.001),and bone volume fraction(BV/TV,P<0.000 1),as well as increased trabecular separation(Tb.Sp,P<0.01),indicating successful model estab-lishment.At week 20,these trabecular microarchitectural abnormalities remained in the AOP group.Compared with the AOP group,Huogu formula improved distal femoral trabecular microarchitecture to varying degrees,with more marked improvement in the high-dose group,as evidenced by increased trabecular tissue mean BMD,Tb.Th,Tb.N,and BV/TV and decreased Tb.Sp(all P<0.05).Meanwhile,compared with the N group,the AOP group showed increased serum IL-1β,IL-6,CXCL10,and MCP-1 levels(all P<0.05),accompanied by decreased serum Ca and P leves,decreased bone tis-sue T-AOC,SOD,and CAT levels(all P<0.05),upregulated p16,p21,and p53 protein expression in femoral tissue(all P<0.05),increased bone marrow fat deposition,and disordered bone metabolism.Huogu formula ameliorated these ab-normalities to different extents,with relatively greater improvement in the high-dose group(all P<0.05).Conclusion:Huogu formula can ameliorate distal femoral trabecular microarchitectural damage,abnormal elevation of inflammatory factors,decreased bone antioxidant capacity,abnormal expression of p16/p21/p53-related proteins,increased bone marrow fat deposition,and bone metabolic disorder in AOP rats.Its protective effect may be related to the improvement of abnor-malities associated with the bone marrow microenvironment.