| Objective:To investigate the possible molecular mechanism of resveratrol in alleviating the inflammatory response induced by MSU crystal,and to provide a theoretical basis for the treatment of gouty arthritis with resveratrol.Methods:(1)The peripheral blood samples of patients with gouty arthritis were collected,including 22 cases of acute gouty arthritis(AGA group)and 22 cases of non-acute gouty arthritis(NAGA group).Peripheral blood samples from 22 healthy controls(HCgroup)werecollected.Uric acid(UA),body mass index(BMI),and expression levels of TAK1 and p-TAK1 in peripheral blood mononuclear cells of all specimens were measured.(2)After peripheral blood mononuclear cells(PBMCs)of healthy were stimulated with MSU crystals at concentrations of 100,200 and 400 ug/ml for 24 h,we measured the expression levels of TAK1 and p-TAK1.(3)THP-1 monocyteswere treated with 100 ng/ml phorbol myristate acetate(PMA)to obtain THP-1-derived macrophages,and then stimulated with 200 ug/ml MSU crystal suspension.Meantime,different concentrations of resveratrol(Res)were treated for 24 h(with 2-hydroxypropyl-β-cyclodextrin solution),ELISA was used to detect the expression levels of inflammatory cytokines IL-1β and TNF-α and Western blot detection of TAK1 and p-TAK1 protein level.(4)C57BL/6 male mice were intraperitoneally injected with 25mg/ml MSU crystal suspension 120 ul to induce acute peritonitis mouse model.After 1h,15mg/kg concentration of Res was given.After 6h,peritoneal lavage fluid was counted by blood cell meter.The total number of exudate cells was measured by ELISA for Levels of inflammatory factors IL-1β and TNF-α.(5)Eight C57BL/6 male mices were randomly selected.After anesthesia,40 ul of MSU crystal suspension of 25 mg/ml was injected into the right foot pad as a model.The left footpad was injected with the same amount of sterile PBS as control.After 1 hour,the mice were treated with 15 mg/kg of Res.The control mice was treated with 2-hydroxypropyl-β-cyclodextrin solution.After 6 hours,the joint swelling was measured,and the mice were sacrificed 24 hours later.Total tissue was extracted from the joint tissue for Western blot or formalin-fixed HE staining to analyze the infiltration of inflammatory cells.Results:(1)There were significant differences in UA and BMI levels between AGA,NAGA and HC groups(P<0.05).The serum UA concentration in patients with AGA was significantly higher than that in patients with NAGA or HC(P<0.01).The level of UA in NAGA group was also significantly higher than that in HC group(P<0.05).The BMI of patients with AGA and NAGA was significantly higher than that of HC group(P<0.05).(2)There was no significant difference in TAK1 level in GA patients,but the levels of TAK1 and p-TAK1 were significantly higher than those in HC group.The level of p-TAK1 in AGA group was significantly higher than that in NAGA group(P<0.05).(3)After treatment of MHP crystals with THP-1 cells,the expression levels of TAK1 and p-TAK1 protein increased in a dose-dependent manner.(4)MSU stimulated THP-1-derived macrophages.The expression of p-TAK1,IL-1β and TNF-α in Res-treated group was lower than that in model group(P<0.05).(5)The cell count and inflammatory factors IL-1β and TNF-α in the peritoneal lavage fluid of the Res-treated group were significantly lower than those in the control group(P<0.05).(6)The swelling coefficient of the foot pad of the control group was larger than that of the Res treatment group.The histopathological analysis showed that the infiltration of inflammatory cells in the control group was significantly higher than that in the Res treatment group.Conclusion:(1)UA and BMI in patients with gouty arthritis are higher than those in healthy people,suggesting that obesity may increase the risk of gouty arthritis.(2)p-TAK1 may be involved in the inflammatory response of gout.(3)Combined with gout patient specimens,cell and animal experiments suggest that Res may inhibit MSU-induced inflammation by targeting TAK1. |
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