Experimental Study On The Intervention Of Bushen Qiangdu Prescription On Pathological New Bone Formation In Ankylosing Spondyliti

OBJECTIVE:To study the effects of Bushenqiangdu recipe on the expression of Wnt/β-catenin pathway-related factors,microRNA miR-29a and long-chain non-coding RNAlnkRNA H19 during osteogenesis of ankylosing spondylitis based on animal experiments and cell experiments.To explore the specific target mechanism of Bushenqiangdu recipe in the intervention of pathological new bone formation in ankylosing spondylitis.METHODS:Bone marrow mesenchymal stem cells(BMSCs)were selected as cell models in the cell experiments.BMSCs cells were divided into blank control group,western medicine control group,and Bushenqiangdu recipe low-medium-high dose group for osteogenic differentiation and alizarin red staining.The expression of GSK3β,wnt5a and SOST mRNA was detected by real-time fluorescent quantitative PCR,and the expression of GSK3 β,wnt5a and SOST proteins in Wnt/β-catenin pathway were detected by Western blot.The expression of miR-29a in each group was detected by real-time fluorescent quantitative PCR.In the animal experiment,male DBA/1 mice were used as animal models of ankylosing spondylitis.They were randomly divided into model group,Bushenqiangdu recipe high,low dose group and western medicine control group.C57BL/6 mice were also used as blank group.After 20-week-old DBA/1 mice developed joint inflammation,the rats in the high-dose and low-dose groups were given the high and low concentrations of Bushenqiangdu recipe,and the western medicine control group was administered with celecoxib capsules.And the blank group was orally administered with normal saline.Continuous feeding and gavage for 12 weeks,and the symptoms of arthritis in mice were evaluated once every two weeks.After sacrifice,the mice were treated with HE staining.The expressions of SOST,Wnt3a,β-catenin mRNA and lnkRNA H19 and miR-29a were analyzed by real-time fluorescent quantitative PCR.The SOST,Wnt3a and β-catenin protein expressionwere detected by immunohistochemistry and Western blotting.RESULTS:In the cell experiment section,obvious mineralized nodules were formed after osteogenic differentiation of BMSCs in the blank group.No obvious mineralized nodules were observed in the intervention group of Bushenqiangdu recipe.Western Blot assay showed that the expression of SOST and GSK3β protein in BMSCs of Bushenqiangdu recipe serum group was significantly increased compared with the blank control group,and the expression of Wnt5a protein was significantly decreased(P<0.05).The expression of SOST and GSK3βmRNA in BMSCs of Bushenqiangdu recipe serum group was significantly increased compared with the blank control group,and the expression of Wnt5a mRNA was significantly decreased(P<0.05).Compared with the undifferentiated group,the expression of miR-29a in the blank serum control group was increased.Compared with the blank serum control group,the expression level of miR-29a in the high-dose group was significantly lower(P<0.05).In the animal experiment section,compared with the blank control group,the arthritis physical sign score of the model group was significantly higher,while the scores of the Bushenqiangdu recipe and the western medicine control group were lower than the model group(P<0.05).Histopathological observation of Achilles tendon showed that compared with the blank group,the ossification score of the model group was higher,while the score of the high-dose group of Bushenqiangdu recipe was lower than that of the model group,the difference was statistically significant(P<0.05).Compared with the blank group,the expression of SOST mRNA and protein in the model group was increased.Compared with the model group,the expression level of SOST mRNA and protein in the high-dose group of Bushenqiangdu recipe was significantly increased.Compared with the blank group,the expression of Wnt3a mRNA and protein in the model group was increased.Compared with the model group,the expression of Wnt3a mRNA and protein was decreased in the high-dose group.Compared with the model group,the expression levels of β-catenin mRNA and protein in the high and low dose groups of Bushenqiangdu recipe and western medicine control groups were significantly lower.Compared with the blank group,there was no significant difference in the expression of lnkRNA H19 in the model group(P>0.05).Compared with the model group,the expression level of lnkRNA H19 in the high-dose group of Bushenqiangdu recipe was significantly increased.Compared with the blank group,there was no significant difference in the expression of miR-29a in the model group(P>0.05).Compared with the model group,the expression level of miR-29a in the high-and low-dose groups of Bushenqiangdu recipe and western medicine groups was significantly lower.Conclusion:Bushenqiangdu recipe can inhibit Wnt/β-catenin signaling pathway in DBA/1 mice of ankylosing spondylitis and inhibit osteogenesis.Bushenqiangdu drug-containing serum has the effect of inhibiting ossification of ankylosing spondylitis,and its mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway in BMSCs osteogenesis.This side can also up-regulate lnkRNA H19 level and down-regulate miR-29a level,which may have a suggestive effect on the pathological osteogenesis target of ankylosing spondylitis in the regulation of Wnt/β-catenin signaling pathway by Bushenqiangdu recipe.