基于Wnt-β连环蛋白信号通路探讨补肾壮骨汤对骨质疏松性骨折大鼠骨折愈合的影响及作用机制OA

Objective:To investigate the effects of Bushen Zhuanggu Tang(补肾壮骨汤,BSZGT)on fracture healing in osteoporotic fracture(OPF)rats,and to explore its underlying mechanism.Methods:Forty 12-week-old SPF-grade female Sprague-Dawley(SD)rats were randomly divided into sham-operated group,model group,BSZGT group,and inhibitor group,with 10 rats in each group.All rats but the ones in sham-operated group were subjected to bilateral ovariectomy for inducing osteoporosis followed by midshaft femoral osteotomy to build OPF model.On the day after the end of modeling procedure,the rats in BSZGT group were intervened by intragastric administration with BSZGT concentrate at the doses of 0.01 mL/g/day,the ones in inhibitor group with BSZGT concentrate at the doses of 0.01 mL/g/day,fol-lowed by subcutaneous injection of XAV-939 at the doses of 1.0 × 10-4 mL/g/day,with an interval of no more than 1 hour between the two interventions;while the ones in the model group and sham-operated group were intervened by intragastric administration and subcutaneous injection with the same dose of sterile saline.All rats in the 4 groups were intervened once a day for consecutive 8 weeks.Following the end of intervention,the serum levels of alkaline phosphatase(ALP),collagen type Ⅰ cross-linked C-telopeptide(CTX),and osteocalcin(OC)were quantified using ELISA kits;the femoral fracture healing was assessed in each group by taking anteroposterior X-ray films of the right hind femur using a high-resolution digital X-ray imaging system;the bone mineral density(BMD)at the fracture site of the right hind femur was measured using dual-energy X-ray absorptiometry;the biomechanical properties of the right hind femur,including maximum load and maximum stress,were evaluated using a mechanical testing apparatus;moreover,the bone formation of the right hind femur was assessed by fluorescence microscopy,and the mineral apposition rate(MAR)was quantitatively analyzed.Apart from that,the morphology of callus tissue was observed via HE staining and tartrate-resistant acid phosphatase(TRAP)staining,and the number of osteoclasts was calculated,be-sides,the protein expression levels of Wnt3a,β-catenin,Cyclin D1,and c-Myc in the callus tissue were detected by Western Blot.In addi-tion,the bone samples from the distal femoral metaphysis of the right hindlimb were scanned using Micro-CT to analyze trabecular thickness(Tb.Th),trabecular number(Tb.N),and bone volume/tissue volume(BV/TV)in the distal femoral metaphysis.Results:① Detection re-sults of serum ALP,CTX,and OC levels.The serum ALP and OC levels were lower,while the serum CTX level was higher in the model group compared to the sham-operated group(P=0.000,P=0.000,P=0.000).Conversely,the BSZGT group showed increased serum ALP and OC levels along with a decreased serum CTX level compared to the model group(P=0.000,P=0.000,P=0.000).Whereas,the serum ALP and OC levels were lower and the serum CTX level was higher in the inhibitor group compared to the BSZGT group(P=0.000,P=0.000,P=0.000).②Evaluation results of femoral fracture healing.The femoral fracture healing score was lower in model group com-pared to the sham-operated group(P=0.000),while it increased in the BSZGT group compared to the model group(P=0.000),but de-creased in the inhibitor group compared to the BSZGT group(P=0.000).③Measurement results of BMD at the femoral fracture site.The BMD at the femoral fracture site was lower in the model group compared to the sham-operated group(P=0.000),while it increased in the BSZGT group compared to the model group(P=0.000),but decreased in the inhibitor group compared to the BSZGT group(P=0.000).④Results of femoral biomechanical testing.Both the maximum load and maximum stress of the femur were lower in the model group com-pared to the sham-operated group(P=0.000,P=0.000),while these two parameters increased in the BSZGT group compared to the model group(P=0.000,P=0.000),but decreased in the inhibitor group compared to the BSZGT group(P=0.000,P=0.000).⑤Results of bone formation assay.The MAR was lower in the model group compared to the sham-operated group(P=0.000),while it was higher in the BSZGT group compared to the model group(P=0.000),but lower in the inhibitor group compared to the BSZGT group(P=0.000).⑥Results of histopathological observation of callus tissue.Histological analysis showed that the osteoclasts was more in the model group compared to the sham-operated group(P=0.000),while it was less in the BSZGT group compared to the model group(P=0.000),but more in the inhibitor group compared to the BSZGT group(P=0.000).⑦Detection results of protein expression levels of Wnt3a,β-cate-nin,Cyclin D1,and c-Myc in callus tissue.Western Blot analysis showed that the protein expression levels of Wnt3a,β-catenin,Cyclin D1,and c-Myc in callus tissue were significantly lower in the model group compared to the sham-operated group(P=0.000,P=0.000,P=0.000,P=0.000),while these parameters increased in the BSZGT group compared to the model group(P=0.000,P=0.000,P=0.000,P=0.000),but decreased in the inhibitor group compared to the BSZGT group(P=0.000,P=0.000,P=0.000,P=0.000).⑧Detec-tion results of bone microstructure in the distal femoral metaphysis.Micro-CT analysis revealed that the Tb.Th,Tb.N,and BV/TV were re-duced in the model group compared to the sham-operated group(P=0.000,P=0.000,P=0.000);while these parameters were signifi-cantly improved in the BSZGT group compared to the model group(P=0.000,P=0.000,P=0.000).However,the Th.Th,Tb.N,and BV/TV were reduced in the inhibitor group compared to the BSZGT group(P=0.004,P=0.000,P=0.000).Conclusion:BSZGT can promote the fracture healing in OPF rats,it may work by activating the Wnt-β-catenin signaling pathway.