养阴方对HSP65攻击下ApoE^(-/-)小鼠Th17、Treg细胞失衡的作用研究

Effects of Yangyin Recipe on imbalance of Th17 and Treg cells in ApoE^(-/-) mice under HSP65 attack

目的考察在热休克蛋白(HSP)65攻击下养阴方对载脂蛋白E敲除(ApoE^(-/-))小鼠动脉粥样硬化(AS)的病变及辅助性T细胞17(Th17)、调节性T细胞(Treg)的影响及可能机制。方法将ApoE^(-/-)小鼠分别饲喂普通和高脂饲料,并分别sc无菌PBS或HSP65,以辛伐他汀为阳性药,研究养阴方对AS模型小鼠血脂、斑块、Th17细胞与Treg细胞比例(Th17/Treg)及相关炎症指标的影响。给药方式皆为ig给药,并以等体积生理盐水做对照。采用酯酶法检测血清总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL)及高密度脂蛋白(HDL)水平;油红O染色法测定主动脉斑块面积;流式细胞术测定抗凝血中IL-17A^+CD4^+Th17及Foxp3^+CD25^+CD4^+Treg细胞数量;ELISA法测定血清白细胞介素-6(IL-6)、IL-17、IL-2、转化生长因子-β(TGF-β)、IL-10水平;实时荧光定量PCR(q RT-PCR)法测定脾脏中Stat3、Stat5a、Foxp3和主动脉中Stat3、Stat5a mRNA表达;Western blotting法测定脾脏中磷酸化STAT3(pSTAT3)、磷酸化STAT5(pSTAT5)、FOXP3和主动脉中pSTAT3蛋白表达。结果养阴方使小鼠血清TC、TG、LDL水平显著降低(P<0.01),HDL显著升高(P<0.05);主动脉斑块面积明显减小(P<0.01);外周血IL-17A^+CD4^+Th17细胞显著减少(P<0.05),Foxp3^+CD25^+CD4^+Treg显著增加(P<0.05),Th17/Treg值大幅下降(P<0.01);血清IL-6水平显著下降(P<0.05),IL-2(P<0.01)、TGF-β(P<0.05)、IL-10(P<0.05)水平显著升高;脾脏pSTAT3蛋白水平下降(P<0.01),pSTAT5(P<0.01)和FOXP3(P<0.05)蛋白水平上升;主动脉Stat3 mRNA(P<0.01)和pSTAT3蛋白(P<0.05)水平下降。结论养阴方能够改善HSP65攻击下ApoE^(-/-)小鼠的高脂血症及AS,同时调节外周血中Th17/Treg的平衡和炎症因子的分泌从而改善炎症。养阴方对Th17、Treg的效应可能与减弱的IL-6/pSTAT3和增强的IL-2/pSTAT5信号有关。

Objective To investigate the effects of Yangyin Recipe on atherosclerosis(AS)lesions,helper T cells 17(Th17),and regulatory T cells(Treg)in apolipoprotein E knockout(ApoE?/?)mice under the heat shock protein(HSP)65 attack,and explore the potential mechanisms.Methods ApoE?/?mice were fed with normal and high-fat diets respectively,and injected with sterile PBS or HSP65.Simvastatin was used as the positive drug to study the effects of Yangyin Recipe on blood lipids,plaques,Th17/Treg cells and related inflammation indicators in AS model mice.Drugs were all intragastric administrated with equal volume of normal saline as a control.Serum total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL)and high-density lipoprotein(HDL)were detected by esterase method;The area of aortic plaque was determined by oil red O staining;The amounts of IL-17A+CD4+Th17 and Foxp3+CD25+CD4+Treg cells in anticoagulated blood were determined by flow cytometry;Serum IL-6,IL-17,IL-2,TGF-β,and IL-10 were determined by ELISA;mRNA expression of Stat3,Stat5a,Foxp3 in spleens and Stat3,Stat5a in aortas were detected by qPCR;Protein expression of phosphorylated STAT3(pSTAT3),phosphorylated STAT5(pSTAT5),FOXP3 in spleen,and pSTAT3 in aortas were determined by Western blotting.Results Under the influence of Yangyin Recipe,serum TC,TG,LDL levels decreased(P<0.01),and HDL increased(P<0.05);Aortic plaque area decreased remarkably(P<0.01);IL-17A+CD4+Th17 cells in peripheral blood decreased(P<0.05),Foxp3+CD25+CD4+Treg increased(P<0.05),and Th17/Treg ratio decreased drastically(P<0.01);Serum IL-6 decreased(P<0.05),IL-2(P<0.01),TGF-βand IL-10(P<0.05)increased;Protein levels of pSTAT3 in spleens decreased(P<0.01),but pSTAT5(P<0.01)and FOXP3(P<0.05)increased;Aortic Stat3 mRNA(P<0.01)and pSTAT3(P<0.05)protein decreased.Conclusion Yangyin Recipe can improve hyperlipidemia and AS in ApoE?/?mice during HSP65 attack,and regulate the balance of Th17/Treg in peripheral blood and the secretion of inflammatory factors to improve inflammation,which may be related to attenuate IL-6/pSTAT3 and enhance IL-2/pSTAT5 signaling.