| ObjestiveTo explore the relationship between the activation of Transient Receptor Potential Ankyrin 1(TRPA1)and the pathological lesion of Knee Osteoarithritis(KOA),so as to elucidate the molecular mechanism of "cold evil" as a pathogenic factor in the KOA,as well as the therapeutic mechanism of "Wenjing HUoxue External Application”treating KOA.Methods1.Some data base were used to enroll the clinical trials which discuss the pain sensitivity in knee joint with OA.RevMan 5.3 was used to analyze the differences of pressure pain threshold(PPT),cold pain threshold(CPT),and heat pain threshold(HPT)among KOA patients and control group.2.The KOA inflammatory model was established in synoviocytes in vitro,and then the inflammatory supernatant was collected in synoviocytes to culture the dorsal root ganglion(DRG)neurons.The intracellular calcium influx was measured by using imaging technique in the supernatant treated DRG neurons.3.The KOA inflammatory model was established in synoviocytes in vitro,and then the expression of TRPA1 in synoviocytes was assessed by polymerase chain reaction and western blot,and the functionality of TRPA1 channel by Ca2+ influx measurements.Meanwhile,production of interleukin(IL)-6,matrix metalloproteinase(MMP)-1,and MMP-3 after inhibition of TRPA1 was measured by immunoassay4.The KOA apoptotic model was established in chondrocytes in vitro,and then the expression of TRPA1 in chondrocytes was assessed by polymerase chain reaction and western blot,and the functionality of TRPA1 channel by Ca2+ influx measurements.Meanwhile,chondrocyte apoptosis rate,mitochondrial transmembrane potential,and the expression of apoptosis-related proteins were measured after pharmacological inhibition of TRPA15.The KOA animal model was established in rats in vivo.The left limb bearing capacity,cold pain threshold and histological changes were observed,as well as the production of IL-6,MMP-1,MMP-3 in synovium and apoptosis rate in cartilage in rats were measured after inhibition of TRPA1.6.The KOA animal model was established in rats in vivo.The healthy rats were used as the blank group,the KOA rats as the model group and the-KOA rats treated by,Wenjing Huoxue External Application "as the observation group.The left limb bearing capacity and cold pain threshold were assessed,the histological changes were observed,as well as the expressions of TRPA1 in DRG,synovium and cartilage in rats were measured.Results1.The point estimate was large for differences in PPT and CPT between knee OA participants and controls.However,no significant differences in HPT were observed between knee O A participants and controls.2.In the lipopolysaccharide(LPS)-treated synoviocytes,the expression of IL-1?,TNF-a and IL-6 was significantly up-regulated.The supernatant from inflammatory synoviocytes upregulated the fluorescence intensity in neurons,and the upregulations were significantly attenuated after pharmacological inhibition of TRPA1.3.After being induced by LPS,the gene and protein expression of TRPA1 was increased,and Ca2+influx mediated by TRPA1 in synoviocytes was also enhanced.In addition,pharmacological inhibition and gene silencing of TRPA l downregulated the production of IL-6,MMP-1,and MMP-3 in LPS-treated synoviocytes.4.After being induced by IL-1?,the gene and protein expression of TRPA1 was increased,and Ca2+ influx mediated by TRPA1 in chondrocytes was also enhanced.Pharmacological inhibition of TRPA1 downregulated the apoptotic rate in IL-1?-treated rat chondrocytes.In addition,the membrane potential depolarization was improved and significantly increased expression of apoptosis-related proteins also reduced by the TRPA1 antagonist.5.In the KOA rats,the left limb bearing capacity and cold pain threshold were significant down-regulated,as well as the synovial lesion and cartilage damage were more serious,accompanied with the increased expression of TRPA1 in DRG,synovium and cartilage.The decreased left limb bearing capacity and cold pain threshold were effectively restored after pharmacological inhibition of TRPA1.Meanwhile,synovial inflammation and cartilage damage were also reduced by pharmacological inhibition of TRPA1.The markedly increased expression of IL-6,MMP-1,MMP-3 in synovium,as well as the chondrocyte apoptosis rate was also reduced by the TRPA1 antagonist.6."Wenjing Huoxue External Application" up-regulated the left limb bearing capacity and cold pain threshold and alleviated the synovial lesion and cartilage damage in rats with KOA.Meanwhile,"Wenjing Huoxue External Application" also siginificantly down-regulated the protein expression of TRPA1 in DRG,synovium and cartilage in model rats.Conclusion1.Evidence from this meta-analysis suggests that the cold hyperalgesia is present in knee joint with KOA,happened to coincide with the clinical features described as"the pain was deteriorated by cold,but relieved by warm" in KOA patients.2.Our result suggested that the activation of TRPA1 channel-cold hyperalgesia-pain pathway mediated the pathogenesis of pain in OA joint,which was regarded the molecular mechanism of "cold evil causes pain" in bone rheumatism.3.Our result suggested that the activation of TRPA 1 channel in OA joint participated in the pathogenesis of synovial lesion and cartilage damage,which was regarded the molecular mechanism of "cold evil aggravates disease" in bone rheumatism.4.Therapeutic mechanism of "Wenjing Huoxue external application" in treating KOA was related to the regulation of TRPA1 protein expression in DRG,synovium and cartilage,which provided a modern theoretical basis for treating "cold evil" induced bone rheumatism with therapeutic principle of "cold disease was treated by hot medicine"5.The clinical effect of "Wenjing Huoxue external application" in treating KOA is well proved.In addition to its advantages including fewer side effects and high patient's compliance,therefore "warming channel and activating blood circulation external application" is extremely valuable. |
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