Mechanism Research Of Electro-acupuncture Treatment On Knee Osteoarthritis Cartilage Degeneration By Ras-Raf-MEK1/2-ERK1/2 Signaling Pathway

Objective:To explore the mechanism of electro-acupuncture in the treatment of osteoarthritis cartilage degeneration based on the Ras-Raf-MEK1/2-ERK1/2 signal pathway.Methods:1.40 Two-month-old male SPF rats were randomly divided into 4 groups,namely sham operated control group:normal feeding after sham operation,without any intervention;model group:a normal feeding fter modeling,without any intervention;Experiment 1 group:EA 15 min/day after modeling;group 2:EA 30 min/day after modeling.Weight was recorded every two weeks,preparation of arterial serum,synovial membrane of knee joint,femoral condyle cartilage,femur and tibia.To observe the changes of femoral condylar cartilage surface morphology by HE and toluidine blue staining;expression of IL-1 beta detection in the synovium by Elisa;Western-blot to detect changes expression of ADAMTS-4,ADAMTS-5,MMP-3,MMP-13,Ras,Raf,MEK1/2,ERK1/2,p-ERK1/2,c-fos,c-jun and c-myc protein in articular cartilage.2.The mechanical enzyme digestion method was used to obtain rat chondrocytes,cultured and passaged in vitro,Inverted phase contrast microscope was used to observe the internal and external morphology of chondrocytes,toluidine blue staining and immunohistochemical staining for type II collagen were identified on chondrocytes in vitro.Different concentrations of LPS were used to interfere with the chondrocytes in vitro,and Elisa assay was used to detect the optimal concentration and time of LPS.LPS and each group containing serum culture medium(normal group,model group,U0126 group,electro acupuncture 15min group,electro acupuncture 30min group)were respectively used to interfere with chondrocytes,the expression of Collagen II and ADAMTS-4 protein were observed by confocal laser scanning;Western-blot was used to detect the expression changes of Ras、Raf、MEK1/2、ERK1/2、p-ERK 1/2、c-fos、c-jun and c-myc protein in chondrocytes.Q-PCR was used to detect the expression changes of miR-34a,miR-146a and miR-27b in chondrocytes.Results:1.The average body weight of model group was significantly higher than that of blank group(P<0.05),the experimental 1 group and the experimental 2 group were significantly lower than those of model group(P<0.01);The expression of IL-1β in synovial tissue of knee joint of model group was significantly higher than that of blank group(P<0.01),while the experimental 1 group and the experimental 2 group were significantly lower than those of the model group(P<0.01);The expression of ADAMTS-4,ADAMTS-5,MMP-3,MMP-13,Ras,Raf,MEK,p-ERK1/2,c-fos,c-jun and c-myc in cartilage tissue of model group was significantly higher than that of blank group(P<0.01),while the experimental 1 group and the experimental 2 group were significantly lower than those of the model group(P<0.01,P<0.05).2.The suitable concentration of LPS induced inflammatory model of chondrocytes in vitro was 10 ng/ml and the intervention time was 8 h;Expression of MMP-3,MMP-9 and MMP-13 in the supernatant of chondrocytes of model group were significantly higher than that of normal group(P<0.01,P<0.05),while EA 15 min group,EA 30 min group and U0126 group were significantly lower than those of model group(P<0.01,P<0.05);Expression of Collagen Ⅱ protein in the model group was lower than that in the normal group while EA 15 min group,EA 30 min group and U0126 group were higher than that in the model group;Expression of miR-34a and miR-146a gene in chondrocytes of model group were significantly higher than that of normal group(P<0.05),while EA 15 min group,EA 30 min group and U0126 group were significantly lower than those of model group(P<0.05);Expression of miR-27b gene in chondrocytes of model group was significantly lower than that of normal group(P<0.05),while EA 15 min group,EA 30 min group and U0126 group were significantly higher than those of model group(P<0.05).Conclusions:1.Electro-acupuncture significantly improve the morphological structure of articular cartilage of osteoarthritis,protect articular cartilage,inhibit the activation of signal transduction pathway of synovitis and articular cartilage,and delay the degeneration of cartilage of osteoarthritis.2.Electro acupuncture serum(EAS)decrease the expression of Pro-apoptotic factors,inhibit the activation of inflammatory ERK1/2 signaling pathway and its downstream factors,inhance the expression of chondrocytes collagen Ⅱ,and by regulating the expression of miRNAs,the initial regulatory factor mRNA,inhibition of inflammation mediated apoptosis of cartilage cells.