The Changes Of Bone Metabolism In Er? And ? Knockout Mice And Intervention Of Quercetin

Objective: To observe the change of ER? and ER? knockdown mice in bone metabolism.Evaluating the impact of ER? and ER? on bone metabolism,and further research that the effects and potential mechanism of quercetin(QUE)on bone metabolism of ER? knockdown mice.Method: 1.ER? and ER? knockout mice were identified by Northern blot,Western blot and qPCR.2.Micro-CT was used to detect the bone microstructure of femur in three-month-old female WT,ER?-/-and ER?-/-mice.Morphological change of bone tissue was observed by HE staining.The expression levels of bone formation related factors(RUNX2 and ALP)and bone resorption related factors(TRAP and RANKL) were detected by Western blot and qPCR.3.RNA-Seq was used to screen the differentially expressed genes of mouse femur after ER? and ER? knockout,and some genes were randomly selected for qPCR verification to evaluate the consistency with sequencing results.The function of differentially expressed genes was analysed using KEGG.4.Three-month-old ER?-/-female mice were selected as a model of bone metabolism disorder to observe the effect of QUE.The experiments were divided into: WT group(0.4 mL saline),ER?-/-(0.4 mL saline)group and ER?-/-+QUE (100 mg/kg·d)group,and each group has five mice.Collecting bone tissue samples after ten weeks of gavage.5.The morphological changes of the femoral head in each group after QUE intervention were observed by HE staining.The bone formation related factors (RUNX2 and ALP)and osteoclast differentiation-related factors(TRAP and RANKL)expression of each group were observed by Western blot and qPCR.Result: 1.Northern-blot results showed that WT,ER?-/-and ER?-/-mice's DNA expressed at 110 bp,189 bp and 550 bp,respectively.Western blot and qPCR results showed that compared with WT group,the expression of ER? protein and mRNA in ER?-/-mice decreased(P<0.05),and the expression of ER? protein and mRNA in ER?-/-mice decreased(P<0.05).2.Compared with WT mice,MircoCT results in the ER?-/-group showed an increase in BMD,Tb.N and BV/TV,and no difference in Tb.Sp(P<0.05);there was no change in BMD,Tb.N,BV/TV and Tb.Sp in the ER?-/-group(P>0.05).HE staining of the femoral head showed that compared with the WT group,the number of trabecular bone were decreased,the arrangement was sparse,part of the fracture occurred,and the medullary cavity was increased in the ER?-/-group,while in the ER?-/-group the trabecular bone was thicker and fuller,with better connectivity and integrity,good mesh continuity,tight and regular arrangement,and small trabecular space.Western blot and qPCR results showed that compared with WT group,the expression levels of protein and mRNA of RUNX2 and ALP were significantly decreased(P<0.05),and the expressions of protein and mRNA of bone resorption related factors RANKL and TRAP were significantly increased(P<0.05)in ER?-/-group,while the expression levels of protein and mRNA of RUNX2 and ALP and bone resorption related factors TRAP and RANKL had no significantly change in ER?-/-group(P> 0.05).3.RNA-Seq results showed that compared with WT group,there were 261 differential genes in the ER?-/-group,and 126 differential genes related to bone metabolism mainly interacting with nuclear membrane receptors,complement and coagulation cascades and adhesions,which was related to the plaque and P13K Akt signaling pathway.There were 252 differential genes in the ER?-/- group,and 58 differential genes related to bone metabolism,which were involved in nuclear membrane receptor interaction,protein digestion and absorption,Wnt signaling pathway and PI3 K Akt signaling pathway.The three differentially expressed genes,Ddit4,Scd1 and Cfd were randomly selected for verification and the mRNA expression of which were consistent with the sequencing results.4.After ten weeks of QUE intervention,compared with the ER?-/-group,HE staining of the femoral head showed that the number of trabecular bone increased,the area of the bone marrow cavity decreased,and the free end of the bone decreased in the ER?-/-(QUE)group;Western blot and qPCR results showed that the expression levels of RUNX2 and ALP protein and mRNA up-regulated in the ER?-/-(QUE)group,while the absorption-related factors TRAP and RANKL protein and mRNA down-regulated in the ER?-/-(QUE)group(P<0.05).Conclusion: 1.Successfully obtained gene knockout homozygous mice by expansion and identification.2.After the ER? gene knockout,the microstructure of the femur bone of the mouse was destroyed,which might be related to the decrease of bone formation and the increase of bone resorption.3.After the ER? gene knockout,the microstructure of femur bone in mice was improved.4.There were dysregulated 261 and 252 genes caused by ER? and ER? gene knockout.Besides,there were 126 and 58 genes which were involved in bone metabolism and enriched in nuclear membrane receptor interaction and P13 K Akt signaling pathway.5.QUE has an improved effect on bone metabolism in ER? knockout mice,and its mechanism is related to promoting bone formation and inhibiting bone resorption.