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OA北大核心CSTPCD

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-1 α(HIF-1 α)and glucose transporter 1(GLUT1),and its effects on the proliferation,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(1,5,10,20,50,100 µg/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-1 α,and GLUT1 were detected by Western Blot,mRNA expressions of PKM2,HIF-1 α,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-α were determined by ELISA.Results Based on the CCK-8 results,1,5,and 10 µg/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-1 α,and GLUT1 significantly increased(P＜0.05,P＜0.01);and the glycolysis-related indices of glucose content,PK activity,lactate acid,and ATP content significantly increased(P＜0.01);inflammatory factors IL-17 and TNF-α also 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-1 α,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-α secreted 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-1 α and GLUT1 signaling pathways,thus further inhibiting pyruvate.