The Research Of "Layers Adjusting External Application" Treating Knee Osteoarthritis Pain Based On The Mechanical Hyperalgesia Mediators TRPV1 And TPRV4 Calcium Channels

Objective:Explore the relationship between transient potential receptor channels(TRPV1,TRPV4)and Knee Osteoarthritis(KOA),elucidate the therapeutic mechanism of "Layers Adjusting External Application" treating KOA pain through the TRPV1 and TRPV4,search for the possible connections between the external application and its function of alleviating the pain,reducing the inflammation.Method:1.Compare the WOMAC knee pain index and single knee support test results between KOA patients and healthy volunteers;compare the protein and mRNA expression of TRPV1 and TRPV4 in synovial tissue of healthy population and KOA patients;2.KOA model was established in rats in order to analyze the pain behavior,protein and mRNA expression of TRPV1 and TRPV4 in synovium,the effect of selective TRPV1 and TRPV4 inhibitors on mechanical pain threshold;3.Observe the changes of calcium influx in rat synovial cells after using selective TRPV1 and TRPV4 inhibitors and agonists respectively;4.KOA model was established in rats to analyze the effect of "Layers Adjusting External Application" on the pain behavior.the inhibition effect of the TRPV1 and TRPV4 protein and mRNA expression in synovial tissue,measure the effect of the application on the alleviation of mechanical hyperalgesia;5.KOA model was established in rats to analyze the effect of "Layers Adjusting External Application" on the inflammation-related cytokines in the progress of KOA by HE staining and ELISA.Result:1.The scores of WOMAC index and the positive rates of single knee support test in KOA patients were significantly higher than those in normal volunteers(P<0.05).Within the synovial tissue,both the TRPV1 and TRPV4 protein and mRNA expressions of KOA patients were significantly increased comparing with the healthy people(P<0.05);2.The results of pain behavior test showed that the mechanical stimulus pain threshold and limb force of the rats in KOA group were decreased significantly comparing with those in control group(P<0.05).The protein and mRNA expressions of TRPV1 and TRPV4 in synovial tissue of KOA rats were significantly up-regulated(P<0.05).Injection of selective inhibitors of TRPV1 and TRPV4 could significantly increase the pain threshold of mechanical stimulation in KOA rats(P<0.05).3.In rat synovial cells,the intervention of TRPV1 and TRPV4 selective inhibitors could reduce the channel capacity,the calcium influx was significantly reduced.The intervention of TRPV1 and TRPV4 selective agonists,can increase the channel capacity,the calcium influx was significantly increased.4.The "Layers Adjusting External Application" can effectively alleviate the mechanical pain threshold in KOA rats(P<0.05),meanwhile,the protein expression of TRPV1 and TRPV4 were inhibited(P<0.05),but it had no effect on the mRNA expression of both.5.Local application of The "Layers Adjusting External Application" may partially down-regulate the expression or concentration of some proinflammatory cytokines produced in the progression of KOA rats.At the same time,the expression or concentration of some cytokines that suppress inflammation may be up-regulated by the application(P<0.05).Conclusion:1.In the clinical trials,KOA patients showed mechanical pain-sensitive features,TRP channel members--TRPV1,TRPV4 may have correlation with this phenomenon;2.TRPV1 and TRPV4 may mediate the mechanical pain sensitivity in the basic experiments.Suppressing and activating TRPV1 and TRPV4 respectively could result in rapid changes of mechanical hyperalgesia both in vitro and in vivo;3.External treatment of traditional Chinese medicine--The "Layers Adjusting External Application" can relieve the mechanical pain sensitivity mediated by TRPV1 and TRPV4.Meanwhile,the application may effect varies of cytokines in the progression of KOA,reverse the inflammatory environment.4.The "warming channels and activating blood circulation,truncating and reversing" method represented by The "Layers Adjusting External Application" is a good therapy of KOA and may have broad research prospect in the field of Chinese medicine.