小檗碱对结核抗原持续刺激致T细胞耗竭的干预作用

Berberine intervenes T-cell exhaustion caused by persistent Mycobacterium tuberculosis antigen stimulation

目的探究小檗碱对结核分枝杆菌抗原持续刺激致T细胞耗竭的影响。方法应用结核抗原持续刺激小鼠模拟临床结核分枝杆菌慢性感染病理过程:卡介苗感染C57BL/6小鼠1周后,使用结核抗原ESAT6-CFP10(EC)和Mtb10.4-HspX(MH)持续刺激4次,每次间隔1周,建立T细胞耗竭模型。利用该模型进行干预治疗:结核抗原刺激期间每天给予不同剂量小檗碱灌胃治疗,末次抗原刺激后1周,流式细胞术检测抗原特异性T细胞分泌γ干扰素的水平和脾脏CD4^(+)和CD8^(+)T淋巴细胞表面抑制性受体PD-1的表达;ELISA检测血清中γ干扰素的水平;生化试剂盒检测脾脏淋巴细胞糖酵解代谢产物丙酮酸和乳酸的浓度;小檗碱体外作用于Jurkat T细胞后检测丙酮酸和乳酸的浓度。结果结核抗原持续刺激致抗原特异性T细胞分泌γ干扰素的水平降低(P<0.05),CD4^(+)和CD8^(+)T细胞表达PD-1增多(P<0.05),提示发生T细胞耗竭。不同剂量的小檗碱治疗后,减弱了T细胞的持续活化,血清γ干扰素水平降低(P<0.01),CD4^(+)和CD8^(+)T细胞PD-1表达减少(P<0.05)。体外实验表明小檗碱抑制Jurkat T细胞的糖酵解(P<0.01)。结论结核抗原持续刺激诱导T细胞耗竭,而小檗碱治疗可抑制T细胞的糖酵解,调节T细胞活化,预防T细胞耗竭。

Objective To explore the effect of berberine on T-cell exhaustion caused by continuous Mycobacterium tuberculosis(M.tuberculosis)antigen stimulation.Methods C57BL/6 mice were continuously stimulated with M.tuberculosis antigens to mimic the immunopathological process of T-cell exhaustion in clinical tuberculosis patients.Mice were infected with high-doses of Bacille Calmette-Guérin for one week,and then continuously stimulated with M.tuberculosis antigens ESAT6-CFP10(EC)and Mtb10.4-HspX(MH)for four weeks,one time every week.The mice were treated with different doses of berberine by gavage on the above model.One week after the last antigen stimulation,flow cytometry was used to detect interferon-γsecreted by antigen-specific T cells and the expression of inhibitory receptor PD-1 on the surface of CD4^(+)and CD8^(+)T lymphocytes in spleen,serum interferon-γwas detected by ELISA.The contents of glycolytic metabolite pyruvate and lactic acid in spleen lymphocytes were determined by the biochemical kit.Jurkat T cells were treated with berberine in vitro,and the contents of pyruvate and lactic acid in the supernatant of Jurkat T cells culture was detected by the biochemical kit.Results Continuous stimulation of M.tuberculosis antigens induced the interferon-γsecretion by antigen-specific T cells and PD-1 expression on CD4^(+)and CD8^(+)T cells decreased(P<0.05),indicating T-cell exhaustion.Different doses of berberine reduced the sustained activation of T cells,the serum interferon-γand expression of PD-1 on CD4^(+)and CD8^(+)T cells were decreased(P<0.05).In vitro experiments showed that berberine inhibited glycolysis of Jurkat T cells(P<0.01).Conclusion Continuous M.tuberculosis antigens stimulation could induce T-cell exhaustion,while berberine treatment inhibited T cells glycolysis and regulated the activation of T cells,preventing T-cell exhaustion.