三七总皂苷调控HIF-1α/PDHK1通路介导的CD4+T细胞有氧氧化促进Treg分化的机制

Mechanism of Panax Notoginseng Saponins Regulates HIF-1α/PDHK1 Pathway-mediated Aerobic Oxidation of CD4+ T Cells to Promote Treg Differentiation

关键词:  三七总皂苷  HIF-1α  PDHK1  Treg  CD4⁺T  FOXP3

Key words:  panax notoginseng saponins  HIF-1α  PDHK1  Treg  CD4⁺T  FOXP3

摘要:

目的 观察三七总皂苷(panax notoginseng saponins，PNS)调控HIF-1α/PDHK1通路介导的有氧氧化，探讨其对促进Naive CD4⁺T细胞向Treg细胞分化的机制。方法 磁珠分选C57BL/6小鼠脾脏Naive CD4⁺T细胞，诱导其向Treg细胞分化并进行体外培养，将Naive CD4⁺T细胞分为PNS处理组(5、10、20 μg·mL^-1)、PNS联合HIF-1α抑制剂(PX-478)组，并设置对照组，采用流式细胞术检测Treg细胞分化比例，Western blotting检测HIF-1α、PDHK1蛋白表达，实时荧光定量PCR检测HIF-1α、PDHK1、FOXP3 mRNA的表达，酶联免疫吸附实验检测细胞培养上清液中IL-10的水平。结果 PNS可显著增加Treg细胞比例，并增加其IL-10的分泌水平，使细胞中FOXP3 mRNA的表达增加；同时抑制HIF-1α、PDHK1蛋白及mRNA的表达。10 μmol·L^-1的PX-478处理细胞后再以10 μg·mL^-1的PNS干预细胞，其对PDHK1、FOXP3的表达及Treg细胞的分化比例的调控作用与单独使用10 μmol·L^-1的PX-478处理细胞无明显差异。结论 PNS通过HIF-1α抑制PDHK1的表达而增强Naive CD4⁺T细胞的有氧氧化，促进其向Treg细胞分化。

Abstract:

OBJECTIVE To observe the regulation of aerobic oxidation mediated by HIF-1α/PDHK1 pathway by PNS, and to explore its mechanism of promoting the differentiation of Naive CD4⁺T cells into Treg cells. METHODS Naive CD4⁺T cells were isolated from the spleen of C57BL/6 mice by magnetic beads and induced to differentiate into Treg cells for in vitro culture. Naive CD4⁺T cells were divided into PNS treatment group(5, 10, 20 μg·mL^-1), PNS combined with HIF-1α inhibitor(PX-478) group, and the control group was set up. The proportion of Treg cells differentiation was detected by flow cytometry. The expression of HIF-1α and PDHK1 protein was detected by Western blotting. The expression of HIF-1α, PDHK1 and FOXP3 mRNA was detected by real-time fluorescence quantitative PCR. The level of IL-10 in cell culture supernatant was detected by enzyme-linked immunosorbent assay. RESULTS PNS could significantly increase the proportion of Treg cells and the secretion level of IL-10, and increase the expression of FOXP3 mRNA in cells. At the same time, the expression of HIF-1α and PDHK1 protein and mRNA was inhibited. When the cells were treated with 10 μmol·L^-1 PX-478 and then treated with 10 μg·mL^-1 PNS, the expression of PDHK1 and FOXP3 and the differentiation ratio of Treg cells were not significantly different from those treated with 10 μmol·L^-1 PX-478 alone. CONCLUSION PNS can reduce the expression level of PDHK1 by HIF-1α to enhance the aerobic oxidation of Naive CD4⁺T cells and promote their differentiation into Treg cells.