补阳还五汤提取物调控肺癌细胞对乏氧环境适应性的作用及机制

Buyang Huanwu decoction extracts regulate the adaptability to hypoxic environment in lung cancer cells and its mechanism

目的探讨补阳还五汤提取物(Buyang Huanwu decoction,BHD)调控肺癌细胞对乏氧环境适应性的作用及潜在机制。方法将肺癌细胞Calu-1和A549暴露在缺氧环境中16 h后使用或不使用1 mg/mL BHD处理,分为Control组(使用同体积的PBS处理)和BHD组,检测肺癌细胞的增殖水平并通过RNA-Seq检测基因表达的差异。选取下调倍数在4倍以上的基因进行筛选,鉴定调控肺癌细胞增殖的关键基因。转染过表达空载体或HIF-α的过表达质粒后,分为Vector组和HIF-α组,检测肺癌细胞的增殖水平。同时,检测Control组和BHD组的耗氧量、葡萄糖水平、乳酸水平和谷氨酰胺水平。结果BHD显著降低了Calu-1和A549的增殖水平(P<0.05)。BHD显著调控多种基因的mRNA水平下调。通过siRNA敲低筛选上述表达水平下调4倍的基因对细胞增殖水平的影响,发现敲低HIF-α时肺癌细胞Calu-1和A549的增殖水平显著下降(P<0.05)。过表达HIF-α后,肺癌细胞Calu-1和A549的增殖水平显著上升(P<0.05)。BHD处理16 h后,相比于Control组,肺癌细胞Calu-1和A549的葡萄糖水平显著上升(P<0.05)、乳酸水平显著下降(P<0.05)、谷氨酰胺水平无显著变化(P>0.05)。BHD处理的同时过表达HIF-α后,相比于Control组,肺癌细胞Calu-1和A549的增殖水平无显著变化(P>0.05),耗氧量无显著变化(P>0.05),葡萄糖水平和乳酸水平无显著变化(P>0.05)。结论BHD通过下调HIF-α的表达量,减少肺癌细胞的糖酵解水平,降低肺癌细胞对乏氧环境的适应性,抑制肺癌细胞的增殖。

Objective To explore the effect of Buyang Huanwu decoction(BHD)in regulating the adaptability to hypoxic environment in lung cancer cells and its mechanism.Methods Lung cancer cells Calu-1 and A549 were exposed to hypoxia for 16 h with or without 1 mg/mL BHD treatment,and divided into control group(treated with the same volume of PBS)and BHD group.Proliferation levels of lung cancer cells was detected,and differences in gene expression was detected by RNA-Seq.The genes with down-regulated folds of more than 4 times were selected for screening,and the key genes regulating the proliferation of lung cancer cells were identified.After transfection with overexpressing empty vector or HIF-αoverexpression plasmid,they were divided into vector group and HIF-αgroup,and the proliferation level of lung cancer cells was detected.At the same time,the oxygen consumption,glucose level,lactate level and glutamine level were detected in the control group and the BHD group.Results BHD significantly decreased the proliferation levels of Calu-1 and A549(P<0.05).BHD significantly regulated the down-regulation of mRNA levels of multiple genes.The effects of the above-mentioned genes whose expression levels were down-regulated by 4 times on cell proliferation were screened by siRNA knockdown,and it was found that the proliferation levels of lung cancer cells Calu-1 and A549 were significantly decreased when HIF-αwas knocked down(P<0.05).After overexpression of HIF-α,the proliferation levels of lung cancer cells Calu-1 and A549 were significantly increased(P<0.05).After 16 h of BHD treatment,compared with the control group,the glucose levels of Calu-1 and A549 lung cancer cells were significantly increased(P<0.05),the lactate levels were significantly decreased(P<0.05),and the glutamine levels had no significant changes(P>0.05).After BHD treatment and overexpression of HIF-α,compared with the control group,the proliferation levels of Calu-1 and A549 in lung cancer cells did not change significantly(P>0.05),the oxygen consumption did not change significantly(P>0.05),the glucose levels did not change significantly(P>0.05),and lactate levels were not significantly changed(P>0.05).Conclusion BHD can reduce the glycolysis level of lung cancer cells by down-regulating the expression of HIF-α,reduce the adaptability of lung cancer cells to hypoxic environment,and finally inhibit the proliferation of lung cancer cells.