清热凉血汤含药血清对HaCaT细胞PKM2介导的糖酵解和细胞增殖、凋亡的调节作用

Regulatory Effect of Qingre Liangxue Decoction Containing Serum on PKM2 Mediated Glycolysis,Proliferation,and Apoptosis in HaCaT Cells

目的 探讨清热凉血汤(QRLXD)含药血清基于M2型丙酮酸激酶(PKM2)及其下游缺氧诱导因子-1α(HIF-1α)、葡萄糖转运蛋白1(GLUT1)介导糖酵解的信号通路,对活化的HaCaT细胞增殖、凋亡和炎症因子分泌的影响。方法 制备QRLXD含药血清与空白血清,构建肿瘤坏死因子-α(TNF-α)诱导HaCaT细胞银屑病模型,使用compound 3k(C3K)特异性抑制HaCaT细胞PKM2通路。原代培养HaCaT细胞,使用CCK-8法检测不同浓度(1、5、10、20、50、100μg/mL)QRLXD的含药血清对HaCaT细胞增殖的影响,并由此选择合适剂量用于后续实验。将HaCaT细胞分为空白对照(C)组、模型(M)组、C3K组、QRLXD低剂量(QRLXD-L)组、QRLXD中剂量(QRLXD-M)组、QRLXD高剂量(QRLXD-H)组,流式细胞术检测各组细胞的凋亡水平,蛋白质印迹法(Western Blot)检测PKM2、HIF-1α、GLUT1蛋白表达,实时荧光定量PCR (RT-PCR)检测细胞PKM2、HIF-1α、GLUT1 mRNA表达,发光法检测细胞的细胞内丙酮酸激酶(PK)活性、葡萄糖含量、腺嘌呤核苷三磷酸(ATP)水平及分泌乳酸水平,酶联免疫吸附测定法检测细胞白细胞介素-17(IL-17)和TNF-α分泌水平。结果 根据CCK-8结果,选择1、5、10μg/mL QRLXD含药血清设置为低、中、高剂量组,并干预24 h进行后续实验。与C组比较,M组细胞活力比值增加、凋亡率减少(P<0.01);PKM2、HIF-1α、GLUT1蛋白及m RNA表达升高(P<0.05,P<0.01);糖酵解相关指标葡萄糖含量、PK活性、乳酸及ATP含量均显著升高(P<0.01);炎症因子IL-17和TNF-α也明显增加(P<0.01)。与M组比较,C3K组及QRLXD-L、M、H组均能抑制细胞增殖且使细胞凋亡率增加(P<0.01),下调细胞中PKM2、HIF-1α、GLUT1蛋白及mRNA表达(P<0.05,P<0.01),细胞内PK活性、葡萄糖含量、ATP水平及培养液中乳酸水平均降低(P<0.05,P<0.01),细胞分泌的炎症因子IL-17、TNF-α显著减少(P<0.05,P<0.01),且含药血清的作用呈现剂量依赖性关系。与C3K组比较,各含药血清组抑制PKM2通道的作用不及C3K,但是其促进细胞凋亡的作用更强。结论 QRLXD含药血清改善活化的HaCaT细胞糖酵解及过度增殖水平,其机制与调控PKM2及其下游HIF-1α、GLUT1信号通路,进而抑制丙酮酸有关。

Objective To study the effects of Qingre Liangxue Decoction(QRLXD)containing serum on the glycolysis signaling pathway mediated by pyruvate kinase isozyme type M2(PKM2)and its downstream targets,hypoxia inducible factor-1α(HIF-1α)and glucose transporter 1(GLUT1),and its effects on theproliferation,apoptosis,and inflammatory cytokine secretion of activated HaCaT cells.Methods QRLXD containing serum and blank serum were prepared to construct tumor necrosis factor-α(TNF-α)-induced psoriasis model in HaCaT cells,and the PKM2 pathway of HaCaT cells was specifically inhibited by using compound 3k(C3K).HaCaT cells were cultured in primary culture,and the effects of QRLXD containing serum with different concentrations(1,5,10,20,50,100μg/mL)of QRLXD on HaCaT cell proliferation were detected using CcK-8 assay,and the appropriate dose was thus selected for subsequent experiments.HaCaT cells were divided into blank control(C),model(M),compound 3k(C3K),QRLXD low dose(QRLXD-L),QRLXD medium dose(QRLXD-M),and QRLXD high dose(QRLXD-H)groups,and apoptosis levels of cells in each group were detected by flow cytometry.The protein expressions of PKM2,HIF-1α,and GLUT1 were detected by Western Blot,mRNA expressions of PKM2,HIF-1α,and GLUT1 were detected by Real-time fluorescence PCR.And the activities of intracellular pyruvate kinase(PK),glucose,adenine nucleoside triphosphate(ATP),and secretion of lactic acid were detected by luminescence assay.The secretion levels of IL-17 and TNF-αwere determined by ELISA.Results Based on the CCK-8 results,1,5,and 10μg/mL QRLXD containing serum were selected to be set as low,medium,and high dose groups and intervened for 24 h for subsequent experiments.Compared with group C,the cell viability value increased and apoptosis rate decreased in group M(P<0.01);the protein and mRNA expressions of PKM2,HIF-1α,and GLUT1 significantly increased(P<0.05,P<0.01);and the glycolysisrelated indices of glucose content,PK activity,lactate acid,and ATP content significantly increased(P<0.01);inflammatory factors IL-17 and TNF-αalso significantly increased(P<0.01).Compared with group M,cell proliferation were inhibited and apoptosis rate increased in C3K,QRLXD-L,M,and H groups(P<0.01),mRNA and protein expressions of PKM2,HIF-1α,and GLUT1 down-regulated(P<0.05,P<0.01),and intracellular PK activity,glucose content,ATP level,and lactate acid level in the culture medium all decreased(P<0.05,P<0.01),and the inflammatory factors IL-17 and TNF-αsecreted by the cells were significantly reduced(P<0.05,P<0.01),and the effects of QRLXD-containing serum showed a dose-dependent relationship.Compared with C3K group,the inhibitory effect of QRLXD containing serum groups on PKM2 channel was not as strong as C3K,but its promotion of cell apoptosis was stronger than the latter.Conclusion QRLXD containing serum improved the glycolysis and over-proliferation level of activated HaCaT cells,and its mechanism was related to regulating PKM2 and its downstream HIF-1αand GLUT1 signaling pathways,thus further inhibiting pyruvate.