梓醇干预PKM2/LDHA表达调控Th17细胞分化的机制研究

Mechanism of catalpol regulating Th17 cell differentiation by interfering PKM2/LDHA expression

目的研究梓醇通过干预丙酮酸激酶M2(PKM2)、乳酸脱氢酶A(LDHA)表达进而影响辅助性T细胞17(Th17)分化的机制。方法从C57BL/6小鼠脾脏中分选出naive CD4^(+)T细胞,通过加入定向分化刺激剂诱导72 h使naive CD4^(+)T细胞向Th17细胞分化。在诱导分化的同时,分别用0(定向对照)、20、40、80μg/mL梓醇对细胞进行处理。采用流式细胞术检测细胞中Th17细胞分化比例,采用比色法检测细胞培养上清液中丙酮酸、乳酸水平,采用实时荧光定量-逆转录聚合酶链式反应(qRT-PCR)法检测细胞中视黄酸相关孤儿受体γt(RORγt)、PKM2、LDHA mRNA表达情况,采用Western blot法检测细胞中PKM2、LDHA、信号转导和转录激活因子3(STAT3)、磷酸化STAT3(p-STAT3)蛋白表达情况。结果与定向对照组比较,经20、40、80μg/mL梓醇作用72 h后,细胞中Th17细胞分化比例分别降低了6.74%、8.41%、9.24%;细胞培养上清液中丙酮酸、乳酸水平,细胞中PKM2、LDHA、RORγt mRNA表达水平以及细胞中PKM2、LDHA蛋白表达水平和STAT3磷酸化水平均显著降低(P<0.05)。结论梓醇可通过下调PKM2、LDHA表达来降低糖酵解水平,进而抑制Th17细胞分化。

OBJECTIVE To investigate the mechanism of catalpol affecting the differentiation of helper T cell 17(Th17)by interfering the expressions of pyruvate kinase M2(PKM2)and lactate dehydrogenase A(LDHA).METHODS The naive CD4^(+)T cells were selected from the spleen of C57BL/6 mice,and were differentiated into Th17 cells by adding directional differentiation stimulants for 72 hours.At the same time,the cells were treated with 0(directed control),20,40 and 80μg/mL catalpol.The flow cytometry was used to detect the proportion of Th17 cell differentiation in cells;the colorimetric method was adopted to detect the levels of pyruvate and lactate in cell culture supernatant;mRNA expressions of retinoid-related orphan nuclear receptor gamma t(RORγt),PKM2 and LDHA were detected by qRT-PCR method;Western blot was used to detect the expression levels of PKM2,LDHA,signal transducer and activator of transcription 3(STAT3),and phosphorylated STAT3(p-STAT3)proteins in cells.RESULTS Compared with the directed control group,after 72 hours of treatment with 20,40,80μg/mL catalpol,the differentiation ratio of Th17 cells were decreased by 6.74%,8.41%,9.24%,and the levels of pyruvate and lactate in the cell culture supernatant,the mRNA expressions of PKM2,LDHA and RORγt as well as the protein expressions of PKM2 and LDHA and the phosphorylation of STAT3 were significantly reduced(P<0.05).CONCLUSIONS Catalpol can reduce the glycolysis level by down-regulating the expressions of PKM2 and LDHA,thereby inhibiting the differentiation of Th17 cells.