Possible Mechanism Of Static Progressive Stretching Combined With Extracorporeal Shock Wave Therapy In Reducing Extended Knee Joint Contracture In Rats Based On MAPK/ERK Pathway

Objective: The study aimed to observe the therapeutic effect of static progressive stretching(SPS)combined with extracorporeal shock wave therapy(ESWT)on extension knee joint contracture in rats and the effect on mitogen-activated protein kinase(MAPK)/ extracellular signal-regulated kinase(ERK)pathway in joint capsule.Methods: Thirty-six male SD rats with mature bones were randomly divided into control group(C),immobilization model group(IM),natural recovery group(NR),extracorporeal shock wave intervention group(EI),static progressive stretching intervention group(SI),extracorporeal shock wave combined with static progressive stretching intervention group(CI),with 6 rats in each group.The left-knee joint of rats were fixed with an external fixation brace for 4 weeks at full extension to form joint contracture,except group C.The joint range of motion(ROM)of the left knee of rats before and after myotomy was measured at different time points,to calculate the total contracture and arthrogenic contracture of the knee.The changes of the number of total cell,cell morphology and collagen deposition in the anterior joint capsule of the left knee joint of rats were observed by H&E and Masson staining.The protein expression levels of transforming growth factor-β1(TGF-β1),fibroblast growth factor-2(FGF-2)and extracellular signal-regulated kinase 2(ERK2)in the anterior joint capsule,as well as the mean optical density(MOD)of upstream rat sarcoma(RAS)protein and downstream extracellular signal-regulated kinase 2(ERK2)protein positive expression in the MAPK/ERK pathway were detected by Western blot and immunohistochemistry.The therapeutic effects of each intervention group were evaluated after quantitative comparison of each test index.Results: Static progressive stretching combined with extracorporeal shock wave therapy was more effective in relieving total contracture and arthrogenic contracture,promoting the recovery of knee joint ROM more than single therapy,improving the histopathological changes of the anterior joint capsule,and suppressing the high expression of target proteins and the over-activated MAPK/ERK pathway.In the results of total contracture and arthrogenic contracture,Compared with the IM group,the total contracture of the NR group was improved(P <0.05),but there was no statistical significance in the comparison of arthrogenic contracture(P >0.05),the total contracture and arthrogenic contracture of the CI group were significantly smaller than those of the EI group(P <0.05)and SI group(P <0.05).The results of H&E and Masson staining showed that the number of total cells and collagen deposition in the CI group were significantly less than that in the EI group(P <0.05)and SI group(P<0.05),and there was no statistical significance between the EI and the SI groups(P>0.05).In the comparison of protein expression levels of Western blot.The expression levels of TGF-β1,FGF-2 and ERK2 in the three intervention groups was significantly lower than that in the NR group(P <0.05),and the expression levels of TGF-β1,FGF-2 and ERK2 in the CI group was lower than that in the EI group(P <0.05)and SI group(P <0.05);the expression level of TGF-β1 in the CI group was statistically different from that in the C group(P <0.05),but the expression level of FGF-2 and ERK2 in the CI group was not different from that in the C group(P >0.05).The results of Immunohistochemical staining showed that compared with the NR group,the MOD of the RAS and ERK2 positive expression in each group was significantly lower than that in the NR group after different interventions(P <0.05),and the MOD in the CI group was lower than that in the EI group(P <0.05)and SI group(P <0.05);the MOD of the RAS positive expression in the CI group was significantly different from that in the C group(P <0.05),but the MOD of the ERK2 positive expression in the CI group was not significantly different from that in the C group(P >0.05).Conclusion: The over-activated MAPK/ERK pathway is involved in the formation of extension knee joint contracture in rats.SPS combined with ESWT was effective in relieving joint contracture and fibrosis of joint capsular.Moreover,the inhibition of the over-activated MAPK/ERK pathway may be one of the potential molecular mechanism for its therapeutic effect.