Study On The Mechanism Of "Clearing Heat,Removing Dampness And Dredging Collaterals Method" Based On The Function Of Cyr61 In Acute Gouty Arthritis

ObjectiveTo determine the efficacy and safety of the method of "clearing heat,removing dampness and dredging collaterals" in the clinical treatment of acute gouty arthritis.It is confirmed that Cyr61,as an important pro-inflammatory factor,is involved in the inflammatory process of acute gout.It is concluded that the method of "clearing heat,removing dampness and dredging collaterals" may reduce the expression levels of IL-1β,IL-6 and TNF-α by down-regulating Cyr61,thus treating acute gouty arthritis.MethodsPart Ⅰ: A randomized,double-blind,controlled study was conducted in patients with acute gouty arthritis of rheumatic stagnant heat syndrome.After 5 days of treatment with "clearing heat,removing dampness and dredging collaterals",the pain score of affected joints,tenderness and swelling of joints,the overall evaluation of patients and researchers on treatment response,Cyr61,IL-1β,IL-6,TNF-α and safety indexes were observed.Part Ⅱ: The rat gout model was induced by monosodium urate(MSU)suspension,and the Cyr61 sh RNA lentivirus was constructed and randomly divided into groups.The local joints were observed by gross observation and histopathology,the levels of Cyr61,IL-1β,IL-6 and TNF-α mRNA were detected by Real-time PCR,and the protein expression of the above indexes was detected by immunohistochemistry and ELISA.Part Ⅲ: Rat fibroblast-like synoviocytes(FLS)were cultured in vitro.The drugcontaining serum of traditional Chinese medicine and Cyr61 sh RNA lentivirus acted on FLS,then exogenous Cyr61 protein was used to stimulate FLS,and NF-κ B signal pathway inhibitor was added to observe the apoptosis morphology and cell cycle of each group.Real-time PCR,Western blot and ELISA techniques were used to compare the expression of Cyr61,IL-1β,IL-6,TNF-α mRNA and protein.Cyr61 was overexpressed with lentivirus before the treatment with Huzhang Tongfeng granule,and the expression of IL-1β,IL-6 and TNF-α were detected by ELISA.Results1.After the traditional Chinese medicine treatment of "clearing heat,removing dampness and dredging collaterals method",the VAS score,tenderness and swelling score of the affected joint in the treatment group were significantly lower than those in the baseline period(P<0.05),there was no statistical difference in the overall evaluation of the two groups of patients and researchers on the treatment response(P>0.05).Cyr61,IL-1β,IL-6 and TNF-α in the treatment group were significantly lower than those in the baseline period(P<0.05),there was no significant difference in the difference of safety indexes between the two groups before and after treatment(P>0.05).2.In vivo,the mRNA and protein levels of Cyr61,IL-1β,IL-6 and TNF-α in synovial tissue and serum of gout model rats were significantly increased(P<0.05 verse normal group).After the expression of Cyr61 was inhibited by lentivirus,the mRNA and protein levels of Cyr61,IL-1β,IL-6 and TNF-α were significantly decreased(P<0.05 verse normal group).Huzhang Tongfeng granule of "clearing heat,removing dampness and dredging collaterals method" group could down regulate Cyr61 mRNA(P<0.05 verse model group)and protein level(P<0.001 verse model group).Both Qingre Lishi formula(P<0.001 verse model group)and Tongluo formula(P<0.001 verse model group)could down regulate Cyr61 protein level,but had no effect on Cyr61 mRNA expression(P>0.05).Huzhang Tongfeng granule could down regulate IL-1β protein level(P<0.001 verse model group),IL-6 mRNA(P<0.05 verse model group)and protein level(P<0.01 verse model group),TNF-α mRNA and protein level(P<0.05 verse model group);Qingre Lishi formula could down regulate IL-1β protein level(P<0.01 verse model group),IL-6mRNA(P<0.05 verse model group)and protein level(P<0.01 verse model group);Tongluo formula could down regulate IL-6 mRNA level(P<0.001 verse model group),TNF-α mRNA level(P<0.05 verse model group).3.In vitro,the apoptosis rate of FLS were significantly increased in Huzhang group,Qingre Lishi group,Tongluo group and lentivirus group(P<0.001 verse model group).In the three groups treated with traditional Chinese medicine,the degree of increment of apoptotic cells was in the order of Huzhang group > Qingre Lishi group > Tongluo group,there was significant difference(P<0.001).Compared with the model group,the number of cells in the G1 phase of Huzhang group,Qingre Lishi group,Tongluo group and the lentivirus group increased(P<0.001),while the number of cells in the S phase decreased significantly(P<0.001),and this effect was more obvious in the Huzhang group(P<0.001).MSU could induce FLS to secrete IL-1β,IL-6 and TNF-α at the levels of mRNA and protein translation(P<0.001 verse normal group),and up-regulate the expression of Cyr61 mRNA and protein(P<0.001 verse normal group).The expression of IL-1β,IL-6 and TNF-α mRNA and protein in FLS were significantly stimulated by Cyr61(P<0.05).In the presence of NF-κB inhibitor,the mRNA and protein expressions of IL-1 β,IL-6 and TNF-α in FLS stimulated by Cyr61 combined with MSU were significantly decreased(P<0.05).Immunofluorescence analysis showed that the nuclear transcription level of NF-κB protein in MSU combined with Cyr61 group was higher than that in Cyr61 group(P<0.05).The mRNA and protein expressions of Cyr61(P<0.001 verse model group),IL-1β(P<0.001 verse model group),IL-6(P<0.01 verse model group)and TNF-α(P<0.001 verse model group)in Huzhang group were significantly decreased;the mRNA and protein expressions of Cyr61(P<0.001 verse model group),IL-1β(P<0.01 verse model group)and TNF-α(P<0.001 verse model group)in Qingre Lishi group were significantly decreased,and the mRNA expression of IL-6(P<0.01 verse model group)was significantly decreased;the mRNA expression levels of Cyr61(P<0.01 verse model group),IL-1β(P<0.01 verse model group)and IL-6(P<0.05 verse model group)in Tongluo group were significantly decreased.The expression of IL-1β(P<0.01 verse Huzhang group),IL-6(P<0.001 verse Huzhang group)and TNF-α(P<0.01 verse Huzhang group)were significantly increased after the treatment with Huzhang Tongfeng granule and Cyr61 overexpression with lentivirus.Conclusions1.The method of "clearing heat,removing dampness and dredging collaterals" can effectively relieve the clinical symptoms and signs of acute gouty arthritis,and reduce the expression levels of Cyr61,IL-1β,IL-6 and TNF-α without obvious adverse reactions.2.As an important pro-inflammatory factor,Cyr61 protein participates in the inflammatory process of acute gouty arthritis and promotes the secretion of IL-1β,IL-6and TNF-α in a partially dependent way of NF-κB.3.The method of "clearing heat,removing dampness and dredging collaterals" may reduce the expression levels of IL-1β,IL-6 and TNF-α by down-regulating Cyr61 to treat acute gouty arthritis.