淫羊藿苷对Dahl盐敏感大鼠心肌重塑模型外周血树突状细胞及Th17/Treg平衡的影响

Effect of Icariin on Peripheral Blood Dendritic Cells and Th17/Treg Balance in Myocardial Remodeling Model of Dahl Salt-sensitive Rats

目的:探讨淫羊藿苷介导维生素D系统对Dahl盐敏感大鼠心肌重塑模型外周血树突状细胞(DCs)表型及辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡的影响。方法:将50只SPF级Dahl盐敏感大鼠分为模型组、维生素D组(3×10-5mg·kg^(-1)·d^(-1))、淫羊藿苷高、中、低剂量组(给药剂量分别为120、60、30 mg·kg^(-1)·d^(-1)),10只Dahl盐抵抗大鼠作为正常组。通过给予含8%Na Cl高盐饲料喂养构建心肌重塑模型。造模6周后,除正常组和模型组给予等体积超纯水灌胃外,连续给药6周。采用心脏超声心动图、苏木素-伊红(HE)及马松(Masson)染色法观察大鼠心脏病理变化和纤维化改变;酶联免疫吸附测定法(ELISA)检测大鼠血清25-羟基维生素D_(3)[25(OH)D_(3)]、N-末端脑钠肽前体(NT-Pro BNP)、白细胞介素(IL)-17、转化生长因子(TGF)-β_(1)、IL-12、IL-10水平;流式细胞术检测大鼠外周血DCs表型及Th17/Treg细胞比例;实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)分别检测大鼠外周血树突状细胞维生素D受体(VDR)、1α-羟化酶(CYP27B1)、24-羟化酶(CYP24A1) m RNA及蛋白表达。结果:与正常组比较,模型组大鼠发生心肌细胞排列紊乱,胞浆肥大肿胀等病理改变;心肌胶原纤维增生显著、心肌纤维排列较紊乱;血清25(OH)D_(3)、IL-10水平降低,血清IL-17、TGF-β_(1)、IL-6、IL-12、NT-Pro BNP含量明显升高(P<0.05);外周血DCs细胞高表达共刺激分子CD40、CD80、CD86及主要组织相容性复合体Ⅱ(MHC-Ⅱ),低表达CD11、CD11b(P<0.05);外周血Th17细胞比例明显升高,Treg细胞比例明显降低,Th17/Treg值明显升高(P<0.05);外周血树突状细胞CYP24A1 m RNA表达量和蛋白明显升高,CYP27B1、VDR m RNA和蛋白表达明显降低(P<0.05)。与模型组比较,各给药组心肌纤维排列相对规整,心肌细胞肿胀明显减轻,心肌组织病理形态均有不同程度的改善;心肌组织病理改变均有不同程度的改善和减轻;明显降低血清NT-Pro BNP、IL-17、TGF-β_(1)、IL-6、IL-12含量,明显升高血清25(OH)D_(3)、IL-10含量(P<0.05);明显降低大鼠外周血树突状细胞共刺激分子CD40、CD80、CD86及MHC-Ⅱ表达量,升高CD11、CD11b分子表达(P<0.05);明显降低外周血Th17细胞比率、Th17/Treg值,升高Treg细胞比率(P<0.05);明显降低大鼠外周血树突状细胞CYP24A1 m RNA和蛋白表达量、明显提高VDR、CYP27B1 m RNA和蛋白表达(P<0.05)。结论:淫羊藿苷可通过调控维生素D系统调节外周血树突状细胞表型及Th17/Treg细胞比例,从免疫平衡角度发挥改善心肌重塑的作用。

Objective:To investigate the effect of icariin(ICA)-mediated vitamin D system on peripheral blood dendritic cells(DCs)and helper T cells 17(Th17)/regulatory T cells(Treg)balance in myocardial remodeling model of Dahl salt-sensitive rats.Method:Fifty SPF Dahl salt-sensitive rats were divided into model group,vitamin D group(3×10-5 mg·kg^(-1)·d^(-1)),and high-,medium-,and low-dose ICA groups(120,60,30 mg·kg^(-1)·d^(-1)),and 10 Dahl salt-resistant rats were used as normal group.The myocardial remodeling model was established by feeding rats with a high-salt diet containing 8%NaCl.After six weeks of modeling,the normal group and the model group were given an equal volume of ultrapure water by gavage,and other groups were continuously administrated for six weeks.Cardiac echocardiography,hematoxylin-eosin(HE)staining,and Masson staining were used to observe the pathological changes in cardiac structure and fibrosis.The levels of serum 25(OH)D_(3),B-type N-terminal pro-brain natriuretic peptide(NT-ProBNP),interleukin(IL)-17,transforming growth factor(TGF)-β_(1),IL-12,and IL-10 were detected by enzyme-linked immunosorbent assay(ELISA).The phenotype of peripheral blood DCs and the ratio of Th17/Treg cells of rats were detected by flow cytometry.Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot were used to detect the mRNA and protein expressions of vitamin D receptor(VDR),1α-hydroxylase(CYP27B1),and 24-hydroxylase(CYP24A1)in peripheral blood DCs of rats.Result:Compared with the control group,the rats in the model group had pathological changes such as disordered arrangement of myocardial cells and cytoplasmic hypertrophy and swelling.Myocardial collagen fibers proliferated significantly,and the arrangement of myocardial fibers was disordered.The levels of serum 25(OH)D_(3) and IL-10 were significantly decreased,and the levels of serum IL-17,TGF-β_(1),IL-6,IL-12,and NT-ProBNP were significantly increased(P<0.05).The costimulatory molecules CD40,CD80,CD86,and MHC-Ⅱwere highly expressed in the peripheral blood DCs,and the expression of CD11 and CD11b was lower(P<0.05).The proportion of Th17 cells in the peripheral blood was significantly increased,and the proportion of Treg cells was decreased.The ratio of Th17/Treg was increased(P<0.05).The mRNA and protein expressions of CYP24A1 in peripheral blood DCs increased,and the mRNA and protein expressions of CYP27B1 and VDR decreased(P<0.05).Compared with the model group,the arrangement of myocardial fibers in each drug administration group was relatively regular,and the swelling of myocardial cells was significantly reduced.The pathological morphology of myocardial tissue was improved to varying degrees.The pathological changes in myocardial tissue were improved and alleviated to varying degrees.The drug could reduce the serum levels of NT-ProBNP,IL-17,TGF-β_(1),IL-6,and IL-12 and increase the level of serum 25(OH)D_(3) and IL-10(P<0.05).The expression of costimulatory molecules CD40,CD80,CD86,and MHC-Ⅱin the peripheral blood DCs of rats was decreased,and the expression of CD11 and CD11b molecules was increased(P<0.05).The drug could reduce the proportion of Th17 cells in peripheral blood and the ratio of Th17/Treg cells and increase the proportion of Treg cells(P<0.05).It could decrease the mRNA and protein expressions of CYP24A1 in peripheral blood DCs of rats and elevate the mRNA and protein expression of VDR and CYP27B1(P<0.05).Conclusion:ICA can regulate the phenotype of peripheral blood DCs and the ratio of Th17/Treg cells by regulating the vitamin D system and play a role in improving myocardial remodeling from the perspective of immune balance.