Intervention Effect And Mechanism Of Erzhi Wan On Postmenopausal Osteoporosis

Objective To clarify the intervention effect of EZW on bone formation and bone resorption in PMOP rats.From the perspective of OB and OC,the efficacy of EZW was confirmed.At the same time,the mechanism of action of chemerin on PMOP was explored in vivo and in vitro.Methods 1.In vivo experiments: 70 female SD rats of SPF grade were randomly divided into sham operation group,model group,alendronate group,sodium fluoride group,EZW group,NZZ group,MHL group,10 rats in each group.Rat ovariectomy was used to prepare PMOP animal models.After 8 weeks,each group was given by intragastric administration,The doses administered were alendronate sodium group(1 mg/kg),sodium fluoride group(5 mg/kg),EZW group(2 g/kg),NZZ group(1 g/kg),MHL group(1 g/kg),The sham operation group and the model group were given the same amount of normal saline(5 ml/kg),8 consecutive weeks.Micro-CT was used to scan the bone microstructure of the femur of each group.The bone quality of each group of rats was detected by dual energy X-ray absorption method and vertebral body compression method.The concentration changes of BALP,TRACP-5b,?-catenin,RANKL,OPG,IL-1,IL-6 and chemerin were detected by ELISA.The gene expression of RANKL,OPG,?-catenin and chemerin in bone tissue was detected by qPCR.2.In vitro experiments: The optimal concentration of EZW,NZZ and MHL for growth of MC3T3-E1 and RAW 264.7 was screened by MTT colorimetry.An osteogenic differentiation medium was established to induce MC3T3-E1 cells to differentiate into OB.LPS induced RAW 264.7 cells to establish a model of differentiation to OC.After the differentiation culture,qPCR was used to detect the gene expression levels of osteogenic differentiation markers OCN,RUNX2,ALP and the expression levels of osteoclast differentiation markers CTSK,NFATc1,TRACP.BMSCs were extracted and cultured in the IL-1? environment,The chemerin secretion level and the effect of drugs on the chemerin level in the inflammatory environment were detected by ELISA,and the chemerin mRNA expression level was detected by qPCR.Results 1.PMOP rat model modeling results show that: DXA results show that the BMD of the model group rats is significantly lower than the sham operation group(P<0.05),Micro-CT two-dimensional femur and DXA results are consistent,proving the PMOP rat model modeling success.2.The bone strength test results showed: In terms of bone microstructure,Micro-CT shows that compared with the model group,the number of bone trabeculae in each administration group increased,the arrangement was dense and ordered,and the bone microstructure repair was obvious.In terms of bone mass,the BMD of the model group was significantly lower than that of the sham operation group(P<0.01),and the other groups were significantly increased(P<0.05,P<0.01).The maximum load of bone in the model group was significantly reduced(P<0.01),and the other groups were significantly increased(P<0.05).Micro-CT showed that compared with the model group,the number of bone trabeculae increased in each administration group,and the arrangement was dense and orderly,and the repair of bone microstructure was obvious.3.ELISA detection of bone metabolism markers in serum showed: Compared with the sham operation group,the serum levels of BALP,OPG and ?-catenin in the model group were significantly reduced(P<0.01),TRACP-5b and RANKL secretion levels were significantly increased(P<0.05).Compared with the model group,the serum BALP,OPG and ?-catenin secretion levels of EZW group,NZZ group and MHL group were significantly increased(P<0.05,P<0.01),TRACP-5b and RANKL secretion levels Significantly reduced(P<0.01).4.ELISA detection of serum inflammation-related factors showed: Compared with the sham operation group,the serum IL-1 and IL-6 secretion levels of the model group were significantly increased(P<0.05).Compared with the model group,the serum IL-1 and IL-6 secretion levels of EZW group,NZZ group and MHL group were significantly reduced(P<0.05,P<0.01).5.The results of qPCR detection of bone metabolism markers in bone tissue showed: Compared with the sham operation group,the expression of OPG mRNA and ?-catenin mRNA in the model group was significantly reduced(P<0.05),and the expression of RANKL mRNA was significantly increased(P<0.01).Compared with the model group,the expressions of OPG mRNA and ?-catenin mRNA in the EZW group,NZZ group and MHL group increased significantly(P<0.05,P<0.01),and the expression of RANKL mRNA decreased significantly(P<0.05,P<0.01).6.In vivo chemerin experimental research results show: The level of chemerin secretion and expression in the model group was significantly higher than that in the sham operation group(P<0.01).Compared with the model group,chemerin secretion and expression levels in the EZW group,NZZ group and MHL group were significantly reduced(P<0.05,P<0.01).7.MTT test cell viability results show: MC3T3-E1 and RAW 264.7 were administered at a concentration of NZZ 50 ?g/mL,MHL 50 ?g/mL,EZW 100?g/mL.8.qPCR detection of MC3T3-E1 osteogenic differentiation-related indicators show: Compared with the control group,the expressions of OCN,RUNX2,and ALP mRNA in the EZW group,NZZ group,and MHL group increased significantly(P<0.01,P<0.05).9.Osteoclast differentiation index results show: The expression of osteoclast differentiation indexes CTSK,NFATc1 and TRACP in model group increased significantly(P<0.01).The expressions of CTSK,NFATc1 and TRACP mRNA in EZW group,NZZ group and MHL group were significantly reduced(P<0.01).10.The effect of EZW on chemerin level in inflammatory environment show: The level of chemerin secretion and expression in the model group was significantly higher than that in the control group(P<0.01).Compared with the model group,the chemerin secretion level in EZW group and MHL group was significantly reduced(P<0.01).Chemerin mRNA expression decreased most significantly in Erzhiwan group(P<0.01).Conclusion EZW and its single medicine can effectively enhance bone formation and inhibit bone resorption,thereby improving the abnormal bone metabolism state of PMOP.Its mechanism may be related to chemerin regulating inflammatory factors affecting bone reconstruction.