摘要
目的:观察健脾养正方通过下调丙酮酸激酶同工酶M2(PKM2)蛋白表达而抑制有氧糖酵解过程,从而对大肠癌HCT116细胞的促凋亡和抑制上皮间质转化(EMT)的作用机制。方法:采用噻唑蓝(MTT)比色法检测健脾养正方对大肠癌HCT116细胞增殖的影响;通过流式细胞术检测健脾养正方(2. 0,4. 0,8. 0 g·L^-1)对HCT116细胞诱导凋亡的影响;通过细胞划痕和细胞侵袭实验(Transwell)观察健脾养正方(2. 0,4. 0,8. 0 g·L^-1)对HCT116细胞的迁移和侵袭能力的影响;通过乳酸(LD)测试盒和葡萄糖测定试剂盒分别检测健脾养正方(2. 0,4. 0,8. 0 g·L^-1)对HCT116细胞糖酵解代谢的影响;通过蛋白免疫印迹法(Western blot)检测各组细胞凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax)和EMT相关蛋白上皮型钙黏蛋白(E-cadherin),神经型钙黏蛋白(N-cadherin),波形蛋白(Vimentin)及糖酵解关键蛋白PKM2的表达;结果:MTT比色法显示,与空白组比较,健脾养正方作用HCT116细胞48 h,随着药物浓度增加,健脾养正方对HCT116细胞增殖抑制效应也增加,且在质量浓度为4. 0 g·L^-1时,HCT116细胞抑制率在53. 87%左右,从而选取健脾养正方2. 0,4. 0,8. 0 g·L^-1作为低、中、高剂量组进行研究;细胞流式结果显示,与空白组比较,健脾养正方低、中、高剂量组均明显诱导HCT116细胞凋亡(P<0. 05),且随给药浓度增加,诱导细胞凋亡作用更明显(P<0. 05);细胞划痕和Transwell显示,与空白组比较,健脾养正方低、中、高剂量组均具有抑制HCT116细胞迁移和侵袭作用(P<0. 05),随给药浓度增加,作用更明显(P<0. 05);乳酸和葡萄糖的测定显示,与空白组比较,随给药浓度增加,健脾养正方低、中、高剂量组细胞产生的乳酸量逐渐减少(P<0. 05),葡萄糖利用量亦逐渐降低(P<0. 05);Western blot显示,与空白组比较,健脾养正方低、中、高剂量组E-cadherin,Bax蛋白表达量上调(P<0. 05),N-cadherin,Vimentin,Bcl-2及PKM2蛋白表达量下调(P<0. 05)。结论:健脾养正方可有效诱导大肠癌HCT116细胞凋亡及抑制EMT,其机制可能与其通过下调PKM2蛋白表达而抑制HCT116细胞的有氧糖酵解途径有关。
Objective: To investigate the mechanism of Jianpi Yangzheng recipe in inhibiting aerobic glycolysis by down-regulating the expression of pyruvate kinase isoenzyme M2(PKM2)protein,in order to promote apoptosis and inhibite epithelial-mesenchymal transition(EMT)in HCT116 cells of colorectal cancer.Method: The effect of different concentrations of Jianpi Yangzheng recipe on HCT116 cell proliferation was detected by methylthiazolyldiphenyl-tetrazolium bromide(MTT)colorimetry. Flow cytometry was used to detect the effect of different concentrations of Jianpi Yangzheng recipe(2. 0, 4. 0, 8. 0 g·L^-1)on HCT116 cell apoptosis. The effect of Jianpi Yangzheng recipe(2. 0,4. 0,8. 0 g·L^-1)on the migration and invasion ability of HCT116 cells was observed by cell scratch and cell invasion assay(Transwell). The effect of different concentrations of Jianpi Yangzheng recipe(2. 0,4. 0,8. 0 g·L^-1)on glycolysis metabolism of HCT116 cells were detected by lactic acid(LD)test kit and glucose assay kit,respectively. Western blot was used to detect the expressions of apoptosis-related proteins,like B lymphocyte tumor-2 gene(Bcl-2),Bcl-2 related X protein(Bax)and EMT-related proteins,like epithelial cadherin(E-cadherin),neurogenic cadherin(N-cadherin),Vimentin,and PKM2,the key protein of glycolysis,in each group. Result:MTT assay showed that,compared with the blank group,HCT116 cells were treated with Jianpi Yangzheng recipe for 48 h. With the increase of drug concentration,the inhibitory effect of Jianpi Yangzheng recipe on the proliferation of HCT116 cells was also increased;and when the concentration was 4. 0 g·L^-1,the inhibition rate of HCT116 cells was about53. 87%. Therefore,2. 0,4. 0,8. 0 g·L^-1 were selected as low,medium and high-dose groups for the study. The cell flow cytometry results indicated that,compared with the blank group,the low,medium and high-dose groups all significantly induced the apoptosis of HCT116 cells(P<0. 05),and the effect in inducing apoptosis was more obvious with the increase of drug concentration(P<0. 05). Cell scratch and Transwell showed that,compared with the blank group,all the groups had an inhibitory effect on migration and invasion of HCT116 cells(P<0. 05),and the effect was more significant with the increase of drug concentration(P<0. 05). The determination of lactic acid and glucose indicated that compared with the blank group,with the increase of drug concentration,the amount of lactic acid produced by cells in each group gradually decreased(P<0. 05),while the glucose dosage also gradually decreased(P<0. 05). Western blot showed that,compared with the blank group,the protein expressions of E-cadherin and Bax were up-regulated in groups with different concentrations,whereas the protein expressions of N-cadherin,Vimentin,Bcl-2 and PKM2 were down-regulated(P<0. 05).Conclusion: Jianpi Yangzheng recipe can effectively induce the apoptosis of HCT116 cells and inhibit EMT in colorectal cancer. The possible mechanism may be related to the inhibition of aerobic glycolysis pathway of HCT116 cells by down-regulating PKM2 protein expression.
作者
王红星
吴坚
袁梦云
张星星
陈敏
刘沈林
WANG Hong-xing;WU Jian;YUAN Meng-yun;ZHANG Xing-xing;CHEN Min;LIU Shen-lin(Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2020年第16期93-100,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81973782)
国家自然科学基金青年科学基金项目(81704031)
第三届国医大师传承工作室及全国名中医传承工作室建设项目(国中医药办人教函〔2018〕119号)
江苏省研究生科研创新计划项目(KYCX19_1205,KYCX19_1214)。
