摘要
目的探讨柚皮素对Jurkat T细胞免疫功能的影响及其基于JAK3-STAT5信号通路的作用机制。方法以Jurkat T细胞为研究对象。采用CCK8法检测细胞增殖率[对照组、柚皮素低浓度组(50μmol/L)、柚皮素中浓度组(100μmol/L)和柚皮素高浓度组(200μmol/L)];流式细胞术分析白细胞介素-2(IL-2)、γ干扰素(IFN-γ)分泌及外泌体膜表面细胞毒性T淋巴细胞相关抗原4(CTLA-4)、程序性死亡受体1(PD-1)表达[激动剂组(PHA 10μg)、抑制剂组(BD7501μmol/L)、对照组、柚皮素低浓度组(50μmol/L)、柚皮素中浓度组(100μmol/L)];RT-qPCR检测Cyclin-D1、Bcl-2 mRNA水平[对照组、柚皮素低浓度组(50μmol/L)、柚皮素中浓度组(100μmol/L)];Western blot法检测JAK3、STAT5磷酸化及下游蛋白表达水平[激动剂组(PHA 10μg)、抑制剂组(BD7501μmol/L)、对照组、柚皮素低浓度组(50μmol/L)、柚皮素中浓度组(100μmol/L)]。结果与对照组比较,柚皮素低、中浓度组中细胞增殖率升高(P<0.05),而柚皮素高浓度组细胞增殖率降低(P<0.05)。与对照组比较,柚皮素低浓度组、柚皮素中浓度组、激动剂组IL-2和IFN-γ分泌水平升高,抑制剂组IL-2和IFN-γ分泌水平降低(P<0.05)。与对照组比较,柚皮素低浓度组外泌体CTLA-4表达量降低,柚皮素中浓度组、激动剂组外泌体CTLA-4和PD-1表达量降低,抑制剂组外泌体CTLA-4和PD-1表达量升高(P<0.05)。与对照组比较,柚皮素低、中浓度组Cyclin-D1 mRNA表达量升高,柚皮素中浓度组Bcl-2 mRNA表达量升高(P<0.05)。与对照组比较,柚皮素低浓度组、柚皮素中浓度组、激动剂组JAK3和STAT5磷酸化水平、CyclinD1和Bcl-2蛋白表达升高,抑制剂组JAK3和STAT5磷酸化水平、CyclinD1和Bcl-2蛋白表达降低(P<0.05)。结论柚皮素对Jurkat T细胞功能具有双向调节作用:低浓度促进增殖及细胞因子分泌,高浓度抑制增殖;其机制可能与抑制免疫检查点分子表达、激活JAK3-STAT5信号通路及调控增殖相关基因表达有关,柚皮素可能作为T细胞免疫调节的潜在候选分子。
Objective To investigate the effects of naringin on the immune function of Jurkat T cells and its mechanism of action based on the JAK3-STAT5 signalling pathway.Methods Jurkat T cells were used as the study subjects.Cell proliferation rates were measured using the CCK8 assay(control group,low-concentration naringin group[50μmol/L],medium-concentration naringin group[100μmol/L],and high-concentration naringin group[200μmol/L]);flow cytometry was used to analyse the secretion of interleukin-2(IL-2)and interferon-γ(IFN-γ)and the expression of cytotoxic T lymphocyte-associated antigen 4(CTLA-4)and programmed death receptor 1(PD-1)on the surface of exosomes(agonist group[PHA 10μg],inhibitor group[BD7501μmol/L],control group,low-concentration naringin group[50μmol/L],and medium-concentration naringin group[100μmol/L]);RT-qPCR was used to detect the mRNA levels of Cyclin-D1 and Bcl-2(control group,low-concentration naringin group[50μmol/L],medium-concentration naringin group[100μmol/L]);Western blot analysis of JAK3 and STAT5 phosphorylation and downstream protein expression levels(agonist group[PHA 10μg],inhibitor group[BD7501μmol/L],control group,low-concentration naringin group[50μmol/L],and medium-concentration naringin group[100μmol/L]).Results Compared with the control group,the cell proliferation rate was increased in the low-concentration naringin group and medium-concentration naringin group(P<0.05),while it was decreased in the high-concentration naringin group(P<0.05).Compared with the control group,the secretion levels of IL-2 and IFN-γin the low-concentration naringin group,the medium-concentration naringin group and the agonist group were increased,while the secretion levels of IL-2 and IFN-γin the inhibitor group were decreased(P<0.05).Compared with the control group,the expression level of CTLA-4 in exosomes in the low-concentration naringin group was decreased,the expression levels of CTLA-4 and PD-1 in exosomes in the medium-concentration naringin group and the agonist group were decreased,and the expression levels of CTLA-4 and PD-1 in exosomes were increased in the inhibitor group(P<0.05).Compared with the control group,the expression level of Cyclin-D1 mRNA in the low-concentration naringin group and the medium-concentration naringin group was increased,and the expression level of Bcl-2 mRNA in the medium-concentration naringin group was increased(P<0.05).Compared with the control group,the phosphorylation levels of JAK3 and STAT5,and the expressions of CyclinD1 and Bcl-2 proteins in the low-concentration naringin group,the medium-concentration naringin group,and the agonist group were increased,while the phosphorylation levels of JAK3 and STAT5,and the expressions of CyclinD1 and Bcl-2 proteins were decreased in the inhibitor group(P<0.05).Conclusion Naringin exerts a bidirectional regulatory effect on Jurkat T cell function:low concentrations promote proliferation and cytokine secretion,while high concentrations inhibit proliferation.The mechanism may be related to the inhibition of immune checkpoint molecule expression,activation of the JAK3-STAT5 signalling pathway,and regulation of proliferation-related gene expression.Naringin may serve as a potential candidate molecule for T cell immune regulation.
作者
路鹏鹏
罗淑敏
徐芳
王一越
李伟华
LU Pengpeng;LUO Shumin;XU Fang;WANG Yiyue;LI Weihua(Beijing Institute of Hepatology,Beijing You’an Hospital,Capital Medical University,Beijing 100069,China)
出处
《中国医药导报》
2025年第21期1-7,共7页
China Medical Herald
基金
国家自然科学基金资助项目(82274447)
首都卫生发展科研专项项目(首发2024-2-1152)。
