栀子苷调节PI3K/AKT/mTOR信号通路在动脉粥样硬化形成过程中对Th17/Treg功能的影响

Effect of Geniposide Regulating PI3K/AKT/mTOR Signaling Pathway on Th17/Treg Function during Atherogenesis

目的:观察栀子苷对载脂蛋白E缺乏(ApoE^(-/-))小鼠Th17/调节性T(Treg)细胞失衡的影响及其作用机制。方法:将50只纯合子ApoE^(-/-)雌性小鼠随机分为对照组、模型组和栀子苷低剂量组、栀子苷中剂量组、栀子苷高剂量组。对照组小鼠喂养普通饲料,模型组和栀子苷组小鼠喂养高脂饲料。从第8周开始,栀子苷各剂量组每日灌胃栀子苷(25、50、100 mg/kg),连续8周。试验结束时,采用油红O染色评估主动脉及其根部动脉粥样硬化(AS)病变面积比。采用定量逆转录聚合酶链式反应(RT-PCR)分析主动脉组织肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-17A和IL-10 mRNA表达;采用流式细胞仪分析脾脏中Th17和Treg细胞百分比;蛋白免疫印迹法(Western Blot)检测主动脉组织磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关蛋白表达。结果:油红O染色病变显示,栀子苷中剂量组、栀子苷高剂量组病变百分比低于模型组(P<0.05)。与对照组比较,模型组主动脉TNF-α、IL-6和IL-17A mRNA表达水平升高(P<0.05);栀子苷各剂量组主动脉TNF-α、IL-6和IL-17A mRNA表达水平降低(P<0.05)。与对照组比较,模型组主动脉抗炎细胞因子IL-10 mRNA表达水平降低(P<0.05);栀子苷各剂量组主动脉抗炎细胞因子IL-10 mRNA表达水平升高(P<0.05)。与对照组比较,模型组小鼠脾脏中Th17细胞百分比升高,Treg细胞百分比降低(P<0.05)。栀子苷处理恢复了AS小鼠Th17和Treg细胞的平衡。栀子苷抑制PI3K的表达及AKT和mTOR的磷酸化,MHY1485(mTOR活化剂)减弱了栀子苷对T细胞分化的影响。结论:栀子苷抗AS作用机制可能与抑制PI3K/AKT/mTOR信号引起的Treg细胞增多和Th17细胞减少有关。

Objective:To observe the effect of Geniposide on Th17/regulatory T(Treg)cell imbalance in apolipoprotein E deficient(ApoE^(-/-))mice and its mechanism.Methods:Fifty female homozygous ApoE^(-/-)mice were randomly divided into control group,model group,geniposide low-dose group,geniposide medium-dose group and geniposide high-dose group.From the 8th week,each dose group was given daily gavage of geniposide(25,50,100 mg/kg)for 8 weeks respectively.At the end of the experiment,the lesion area ratio of atherosclerosis(AS)in the aorta and its roots was assessed by oil red O staining.Quantitative reverse transcription polymerase chain reaction(RT-PCR)was used to detect the mRNA expression of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-17A and IL-10 in aortic tissue.The percentage of Th17 and Treg cells in spleen was analyzed by flow cytometry.Western Blot was used to detect phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signals in aortic tissue.Results:Oil red O staining showed that pathological percentage in the geniposide medium-dose group and geniposide high-dose group was lower than that in the model group(P<0.05).Compared with the control group,the mRNA expressions of TNF-α,IL-6,and IL-17A in aorta of the model group increased(P<0.05),and the mRNA expressions of TNF-α,IL-6,and IL-17A in aorta of geniposide dose groups decreased(P<0.05).Compared with the control group,the percentage of Th17 cells increased and the percentage of Treg cells decreased in the spleens of the model group(P<0.05).Geniposide restored the balance of Th17 and Treg cells in AS mice.Geniposide inhibited the expression of PI3K and the phosphorylation of AKT and mTOR,and MHY1485(mTOR activator)attenuated the effect of geniposide on T cell differentiation.Conclusion:The mechanism of anti-AS action of geniposide might be related to the increase of Treg cells and the decrease of Th17 cells induced by inhibition of PI3K/AKT/mTOR signaling pathway.