氯沙坦通过调节Treg/Th17细胞免疫平衡减轻慢性肾病小鼠动脉粥样硬化

Losartan inhibited atherosclerosis in experimental renal failure by regulating Treg/Th17 cells immune balance

目的探讨氯沙坦对慢性肾病(CKD)小鼠动脉粥样硬化(AS)改变及对T细胞(Th17/Treg)免疫平衡的影响。方法将ApoE^(-/-)小鼠分为对照组、SNx组和氯沙坦组,用5/6肾切除法(SNx)构建ApoE^(-/-)小鼠CKD模型,对照组2次手术仅暴露双肾,不切除肾脏。造模后第5周开始灌胃,给予氯沙坦组小鼠30 mg/(kg·d)剂量的氯沙坦连续12周,其余小鼠给予等体积0.5%羧甲基纤维素钠。造模16周后,测小鼠外周血清尿素氮和肌酐含量。取小鼠主动脉根部行HE染色测量AS斑块面积;流式细胞仪检测脾脏辅助性T细胞17 (Th17)及调节性T淋巴细胞(Treg)的比例;用ELISA法检测小鼠外周血清中IL-17、IL-6、TGF-β1及IL-10细胞因子的水平。结果 SNx组小鼠血清尿素氮和肌酐水平明显高于对照组(P <0. 01),表明CKD造模成功。与SNx组比较,氯沙坦组肾功能明显改善,血清尿素氮和肌酐水平明显更低(P <0. 05)。对照组、SNx组及氯沙坦组主动脉根部AS斑块面积分别为(16.35±3. 72)×10~4μm^2,(28. 64±5. 86)×10~4μm^2和(22. 76±3. 97)×10~4μm^2,表明氯沙坦能显著降低CKD小鼠AS斑块面积(P<0. 05)。对照组、SNx组及氯沙坦组小鼠脾脏中Treg细胞比例分别为(4. 34±0. 93)%,(1.78±0.56)%,(2. 68±0. 58)%,氯沙坦能明显增高CKD小鼠Treg细胞的比例(P <0. 01)。对照组、SNx组及氯沙坦组小鼠脾脏中Th17细胞比例分别为(0. 11±0. 06)%,(0. 67±0. 12)%,(0. 37±0. 08)%,表明氯沙坦能明显降低CKD小鼠Th17细胞比例(P <0. 01)。与对照组相比,SNx组小鼠外周血清中细胞因子IL-17、IL-6的水平明显更高(P <0.01),IL-10及TGF-β1的水平明显更低(P<0. 01),氯沙坦干预后TGF-β1和IL-10水平升高,IL-17和IL-6水平降低(P <0. 05)。结论氯沙坦抑制Th17细胞亚群分化,促进Treg细胞亚群分化,可能通过调节Treg/Th17细胞免疫平衡从而减轻CKD小鼠动脉粥样硬化。

Objective To explore the effects and mechanisms of losartan on atheroscleorsis and T cell （Treg/Thl7） immune balance of CKD apolipoprotein E knockout （ ApoE -/- ） mice. Methods The model of CKD was induced by a 5/6 nephrectomy （SNx） in male ApoE -/- mice. ApoE -/- mice were ran- domly allocated into 3 subgroups： the control group, SNx group and losartan group. The fifth week after building model the mice in losartan group were taken losartan at a dose of 30 mg/（ kg · d） by intragastric administration for 12 weeks. While the other mice were treated with the same volume of 0. 5% sodium car- boxymethylcellulose. Sixteen weeks after nephrectomy, the serum levels of urea and creatinine were determined. Haematoxylin Eosin （HE） staining were used to observe the general morphology changes of atherosclerotic plaque. Flow cytometry was performed to detect the proportion of T cells （ Treg/Th17 ） in spleen. Enzyme linked immunosorbent assay （ELISA） was performed to detect the level of cytokines in serum such as interleukin （IL）-17, IL-6, transforming growth factor-β1 （TGF-β1） and IL-10. Results Sixteen weeks after nephrectomy, the result of serum creatinine and urea nitrogen showed the CKD animal model es- tablished successfully. Losartan could improved the renal function of CKD mice. The size of aortic root plaques in control group, SNx group, and losartan group are （16. 35 ±3.72） × 10 4 μm2, （28. 64 ±5.86） × 10 4 μm2 and （ 22. 76 ± 3.97 ） ×10 4 μm2 respectively, indicating that losartan treatment significantly decreased the size of aortic root plaques of the CKD mice （ P 〈 0. 05 ）. The proportion of Treg cells in the spleen of control group, SNx group, and losartan group are （4. 34 ± 0. 93 ） % , （ 1.78 ±0. 56 ） % , and （2. 68 ± 0. 58 ）% respectively, indicating that losartan treatment significantly increased the proportion of Treg cells of CKD mice （P 〈 0. 01 ）. The proportion of Th17 cells in the spleen of control group, SNx group, and losartan group are （ 0. 11 ± 0. 06 ） % , （ 0. 67 ± 0. 12 ） %, （ 0. 37 ± 0. 08 ） % respectively, indicating that losartan treatment significantly decreased the proportion of Thl7 cells of CKD mice （ P 〈 0. 01 ）. Compared with control mice, the level of eytokines TGF-β1 and IL-10 in the serum of the SNx group mice significantly decreased （P 〈 0. 01 ） , and the level of cytokines IL-17 and IL-6 in the serum of the SNx group mice signifi- cantly increased （ P 〈 0. 01 ）, indicating that such effects could be significantly regulated by losartan （ P 〈 0. 05 ）. Conclusions Losartan inhibited the differentiation of Th17 cell subsets, promoted the differentiation of Treg, and alleviated atherosclerosis in CKD ApoE -/- mice by modulating the immune imbalance of the Treg/Th17 cell.