一氧化氮抑制大鼠T淋巴细胞TNF-α和IL-6的释放并下调连接子蛋白40(Cx40)水平

Nitric oxide inhibits the release of TNF-α and IL-6 by down-regulating the expression of connexin 40 (Cx40) in rat T lymphocytes

目的探讨一氧化氮(NO)对淋巴细胞上连接蛋白40(Cx40)表达及肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)释放的影响。方法取WKY大鼠腹主动脉外周血,收集淋巴细胞进行体外培养,分为对照组、伴刀豆球蛋白A(Con A)处理组、Con A联合NO处理组。ELISA检测培养基上清中TNF-α和IL-6水平,免疫荧光技术检测淋巴细胞Cx40的表达和分布,流式细胞术检测淋巴细胞Cx40表达频率的变化,Western blot法检测淋巴细胞Cx40的蛋白水平。结果与对照组相比,Con A处理组TNF-α和IL-6水平升高,Con A联合NO处理组较Con A处理组降低;Cx40表达在淋巴细胞的细胞质和细胞核,与对照组相比,Cx40蛋白表达水平和表达频数均增加,与Con A处理组淋巴细胞相比,Con A联合NO处理组Cx40蛋白表达水平和表达频数均明显降低。结论 NO抑制促炎因子TNF-α和IL-6释放,同时伴随下调淋巴细胞上Cx40的表达,提示Cx40可能参与NO的抑炎作用。

Objective To investigate the effects of NO on the expression of connexin 40（ Cx40） on peripheral blood lymphocytes and the levels of TNF-α and IL-6 in the supernatant of culture medium. Methods Peripheral blood lymphocytes of Wistar-Kyoto（ WKY） rats were isolated from abdominal aorta and cultured in vitro,and then divided into control group,Con A group and Con A combined with NO group. ELISA was performed to test the levels of TNF-α and IL-6 in the supernatant of culture medium. Immunofluorescence technique was used to detect the expression and location of Cx40 on lymphocytes. Flow cytometry was used to determine the expression frequency of Cx40 on lymphocytes. Western blotting was conducted to examine the protein expression of Cx40 on lymphocytes. Results The levels of IL-6 and TNF-α were higher in the Con A group than in the control group,lower in the Con A combined with NO group than in the Con A group. Immunofluorescence showed that Cx40 was mainly expressed in the cytoplasm and the nucleus. The expression of Cx40 on lymphocytes was higher in the Con A group than in the control group,lower in the Con A combined with NO group than in the Con A group. The expression frequency of Cx40 on lymphocytes was higher in the Con A group than in the control group,lower in the Con A combined with NO group than in the Con A group. The protein expressions of Cx40 and Cx43 on lymphocytes were significantly higher in the Con A group than in the control group,lower in the Con A combined with NO group than in the Con A group.Conclusion NO inhibits the release of TNF-α and IL-6 from lymphocytes and down-regulates the expression of Cx40 on lymphocytes,suggesting that Cx40 on lymphocytes may be involved in the anti-inflammatory effect of NO.