| ObjectiveTo explore the mechanism of Huashi Dingtong decoction on knee osteoarthritis(KOA),the model of KOA was established to observe the effects of Huashidingtong decoction on synovium and Ras/Raf/Mek/Erk signaling pathway on synovium of KOA rats.Methods1.The establishment of KOA model rats and groupingⅡ collagenase was injected in knee joint of rats to estabilsh KOA model,then 38 SD rats were randomly divided into control group and model group.The rats in model group were injected in Ⅱ collagenase in the first and fourth day and the rats in control group were injected in saline in the same day.MRI was used to evaluate whether the model were established successfully.The rats of model group were randomly divided into model group,Huashidingtong decoction group and MEK inhibitor group after MRI evaluation.2.Model identification:On the 7th day,3 rats were selected from the blank group and the modeling group respectively for knee MRI scan,and MOAKS score was used to evaluate whether the model were successfully established.3.Drug intervention:Rats in blank group and model group were given normal saline intragastrically;The rats in Huashi Dingtong Decoction group were given Huashi Dingtong Decoction by gavage.Rats in MEK inhibitor group were intraperitoneally injected with MEK inhibitor PD98059.4.Measurement of the degree of knee swelling in rats:Knee swelling on the affected side of rats were measured with an electronic vernier caliper on the 0,7th,14 th,21th,28 th,35th day.5.The measurement of T2 values of the cartilage in each group:T2 mapping of MRI was used to scan the knee joint of rats in each group and measured the T2 value of the cartilage of rats.6.Morphological observation of rat synovial tissue:H&E staining was used to observe the inflammatory infiltration of rat synovial tissue7.Observation and detection of synovial cell indicators:the m RNA expressions of Ras,Raf,Mek and Erk in synovial tissue of rats in each group were detected by RT-q PCR,and the protein expressions of Ras,Raf,p-Mek and p-Erk in synovial tissue of rats in each group were detected by Western-blot.8.Serum ELISA of rats:ELISA was used to detect the contents of IL-1 and IL-18 in serum of rats in each group.Results1.Identification of KOA rat modelIn the control group,the surface of knee cartilage on the affected side of rats was smooth and complete,the joint space was the same width,the rectus femoris tendon was normal without edema,there was a small amount of effusion in the joint cavity,and there was no soft tissue swelling and fascia effusion around the joint.In the model group,the surface of the knee cartilage was rough,the joint space was relatively equal,the rectus femoris tendon edema was obvious,there was a large amount of fluid in the joint cavity,and the soft tissue around the joint was swollen and there was fascia effusion.Compared with the control group,the knee MRI semi-quantitative score in the model group was significantly higher,and the difference was statistically significant(P<0.05).2.Effect of Huashi Dingtong Decoction on knee joint diameter in KOA ratsThe affected limbs of rats in control group showed no significant changes and no ecchymosis.On the 7th day after modeling,the affected knee joint of rats in the model group had different degrees of swelling and ecchymoses compared with the control group,and the joint diameter was significantly increased compared with the control group,with statistical significance(P < 0.05).On the 28 th day after administration,compared with model group,knee joint diameter of the Huashi Dingtong Decoction group and MEK inhibitor group was significantly decreased,with statistical significance(P<0.05).3.Effect of Huashi Dingtong Decoction on T2 value of cartilage in KOA ratsCompared with the blank group,the T2 value of the cartilage of rats in the model group was significantly lower,which indicating that the cartilage of the model rats was seriously damaged.The T2 value of cartilage in Huashi Dingtong decoction was lower than that in model group,which indicating that the drug can promote cartilage repairment in rats.4.Effect of Huashi Dingtong Decoction on the pathological morphology of synovium in the joint of KOA ratsIn control group,the synovial lining cell layer was thinner and arranged regularly,and there were abundant adipocytes under the synovial lining cells,and less vascular hyperplasia and inflammatory cell infiltration.In the model group,the synovial lining cell layer was significantly proliferated,and the adipocytes under the lining cells were significantly reduced.The infiltration of inflammatory cells was obvious,and the cell arrangement was disorderly,with obvious vascular hyperplasia.Compared with the model group,the synovial lining cell layer in the Huashidingtong decoction group and the MEK inhibitor group was thinner;inflammatory cells in both groups showed diffuse infiltration,which was better than that in the model group;there were more adipocytes under the lining cells than that in the model group;and vascular hyperplasia was improved.5.Effect of Huashidingtong Decoction on m RNA expressions of Ras,Raf,Mek and Erk in synovium of joint of KOA ratsThe m RNA expression levels of Ras,Raf,Mek and Erk in synovium of model group were significantly increased compared with blank group(P<0.01).Compared with model group,m RNA expressions of Ras,Raf,Mek and Erk in Huashi Dingtong decoction group and Mek inhibitor group were significantly decreased,with statistical significance(P<0.01).6.Effect of Huashijingtong Decoction on expression of Ras,Raf,p-Mek,p-Erk protein in synovium of joint of KOA ratsCompared with blank group,the expressions of Ras,Raf,p-Mek and p-Erk in synovium of model group were significantly increased(P<0.01).Compared with model group,the expressions of Ras,Raf,p-MEK and p-ERK in synovium of rats in Huashi Dingtong decoction group and MEK inhibitor group were significantly decreased(P <0.01).7.Effect of Huashijingtong Decoction on expression of IL-1β,IL-18 in serumCompared with blank group,the expressions of IL-1β,IL-18 in serum of model group were significantly increased(P < 0.01).Compared with model group the expressions of IL-1β,IL-18 were decreased significantly(P<0.01).Conclusion1.Huashi Dingtong Decoction can improve the inflammation of synovial tissue in KOA rats;2.The mechanism of Huashi Dingtong Decoction in the treatment of KOA is related to its regulation of the expression of factors of Ras/Raf/Mek/Erk pathway. |
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