| Objective:This study confirmed the effect of anti-oxidative stress on preventing and treating osteoporosis;clarified the mechanism of anti-oxidative stress regulation of Mongolian medicine Mongolian echinops through FoxO/Wnt/?-catenin signaling pathway to mediate osteoblast differentiation and bone formation;and clarified the related regulatory mechanism and target of anti-oxidative stress of Mongolian medicine Mongolian echinops.To provide experimental basis for Mongolian medicine based on the classical theory of "Supplement warm and essence?Restrain Heyi?Strength Bone",and to prevent and treat postmenopausal osteoporosis through anti-oxidative stress,and enrich the connotation of modernization of Mongolian medicine basic theory.Method:Grouping:Six-month-old SD healthy female sterile rats of SPF grade were randomly divided into 7 groups:sham group,ovariectomized osteoporosis model group(OVX),Echinops-H group,Echinops-M group,Echinops-L group,Herba Epimedii group(HEP)and CoQ10 group of Western medicine control group.Modeling:Postmenopausal osteoporosis model was established by bilateral ovariectomy(castration).Vaginal epithelial keratosis test,bone mineral density test and uterine pathology were used to verify the success of the model.Drug administration:90 days after operation,except the model group and sham operation group,the other groups were given corresponding drug intragastric intervention according to the plan.Sample collection:After the last gastric perfusion,samples were collected.Abdominal aorta puncture was used to collect blood and centrifuge serum;complete femur and tibia were removed and labeled,right femur and tibia were fixed with 4%polyformaldehyde solution,left femur and tibia were preserved in liquid nitrogen storage tank at-196 C.Indicators detection:ELLISA method was used to determine the changes of serum osteocalcin(BGP),osteoprotegerin(OPG)and other bone metabolism related indicators,as well as the changes of serum SOD,glutathione peroxidase and reduced glutathione isooxidative stress indicators after different drug intervention in rats.RT-PCR was used to determine the gene expression of endonuclear receptor transcription factors regulating target gene expression:peroxisome proliferator-activated receptor-gamma(PPAR-?),osteoblast differentiation transcription factor protein(Runx2),osteoclast inhibitor osteoprotegerin(OPG).Bone mineral density(BMD)in the proximal femur and total femoral BMD)were measured by bone mineral density instrument in the right femur.Bone microstructure was measured by micro-CT.The expression of p-p66(S36),p66,beta-catenin,Wnt2 and FoxO3a in the left femur was detected by Western blot.Result:1.Measurement results of bone mineral density and bone morphology in rats:Compared with Sham group.BMD of proximal femur and total BMD of femur in OVX group decreased significantly(P<0.05).Morphometric expression of bone microstructures in OVX group showed abnormal stereoreticular structure of bone microstructures,bone volume fraction(BV/TV),structural model index(SMI),trabecular volume(TBV),trabecular width(Tb.W,tracular width)and bone.The number of trabeculae(Tb.N)and the thickness of bone trabeculae(Tb.th)decreased significantly(P<0.05),and the trabecular spacing(Tb.Sp)increased(P<0.05).Compared with OVX group,BMD,bone microstructures and bone morphometry of Mongolian medicine Echinops-H,medium and low dose group,Chinese medicine control group,Epimedium group,western medicine control group,coenzyme Q10 group and sham operation group were significantly improved.2.Histological measurements of rat bone(HE staining):Histological results showed that compared with sham-operated group,the density and diameter of adipocytes in model group were significantly increased(P<0.05);compared with sham-operated group,the density and diameter of adipocytes in middle dose group and epimedium group had no statistical difference(P>0.05),and there was statistical difference compared with model group(P<0.05).05),suggesting that the anti-bone destruction effect is remarkable.3.Changes of serum bone metabolism indicators:Compared with sham group,the serum osteoprotegerin(OPG)content of model group(OVX)rats was significantly decreased,and the content of osteocalcin(BGP)was significantly increased(P<0.05),with statistical significance.Compared with OVX group,Echinops-H,Echinops-M,Echinops-H,Herba Epimedii,Co Q10 and Sham groups decreased significantly,and the content of osteocalcin(BGP)increased significantly(P<0.05).4.Changes of serum oxidative stress related indicators:Compared with Sham group,the levels of serum antioxidant stress related indicators in OVX group were significantly lower(P<0.05),with statistical significance.Compared with OVX group,the antioxidant stress index of each dose group of Mongolian medicine,Chinese medicine control group and sham operation group increased significantly(P<0.05),with statistical significance.5.Western blot was used to detect the expression of related proteins in bone tissue:Compared with Sham group,the expression of ?-catenin and Wnt protein in OVX rats was significantly decreased(P<0.05),and the expression of FoxO3a was significantly increased(P<0.05),with statistical significance.Compared with OVX group,the contents of ?-catenin and Wnt2 protein in Echinops-H,Echinops-M,Echinops-L,HEP,Co-Q10 group and Sham group increased significantly(P<0.05),while the contents of FoxO3a protein decreased significantly(P<0.05).6.RT-PCR was used to determine the expression of related factors regulating the expression of target genes:Compared with Sham group,the expression of Runx2 and OPG in bone tissue of rats in OVX group decreased significantly(P<0.05),and the expression of PPAR-gamma in OVX group increased significantly(P<0.05).Compared with OVX group,the contents of Echinops-H,Echinops-M,Echinops-L,HEP,Co-Q10 group,Sham group,?-Runx2 and OPG were significantly increased,and PPAR-gamma was significantly decreased(P<0.05).Conclusion:This study showed that the Mongolian medicine Echinops has significant anti-osteoporosis effect on postmenopausal osteoporosis model rats;Echinops can significantly improve the oxidative stress state of postmenopausal osteoporosis model rats;the Echinops can cross-regulate the FoxO/Wnt/?-catenin pathway,inhibit the transcription of FoxO in oxidative stress,up-regulate the expression of Wnt,and promote the bone of model rats.Tissue osteogenesis differentiation and bone formation;Echinops can reduce the related indicators of bone metabolism and bone resorption in osteoporosis model rats.This study shows that the biological effects of Mongolian medicine based on the theory of "Supplement Warm and essence-Strength Bone" on preventing and treating postmenopausal osteoporosis may be achieved by promoting osteogenic differentiation and bone formation of bone tissue,and the biological effects of Mongolian medicine "Restrain Heyi-Strength Bone" may be achieved by anti-oxidative stress mechanism.This study enriches the scientific connotation of Mongolian medicine's theory of"Supplement Warm and essence-"Restrain Heyi-Strength Bone",and reveals the effective ways of this theory and treatment from the perspective of modern biology. |
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