摘要
目的:探究四神丸含药血清对人结肠癌细胞HCT116有氧糖酵解的影响和作用机制。方法:采用细胞增殖与活性检测(CCK-8)法检测四神丸含药血清(2.5%、5%、10%)处理结肠癌HCT116细胞24、48、72 h的细胞活力;微量法检测细胞培养液中乳酸浓度和细胞内葡萄糖含量,以及己糖激酶(HK)和果糖-6-磷酸激酶(PFK)活性;实时荧光定量聚合酶链式反应(Realtime PCR)检测葡萄糖转运酶1(GluT1)mRNA含量;蛋白免疫印迹法(Western blot)检测GluT1、甲基转移酶样3(MettL3)蛋白表达;免疫荧光染色法检测细胞GluT1表达;比色法检测N6-甲基腺苷(m^(6)A)RNA甲基化水平。结果:与空白组比较,2.5%、5%、10%四神丸含药血清24 h对HCT116细胞活力均无明显影响,10%四神丸含药血清在48 h可明显抑制HCT116细胞活力(P<0.05),故后续实验选用10%四神丸含药血清,给药时间为48 h。与空白组比较,10%四神丸含药血清能够降低乳酸生成(P<0.05),并下调细胞对葡萄糖摄取(P<0.05),并降低糖酵解关键限速酶HK和PFK的活性(P<0.05),同时四神丸含药血清还可降低GluT1蛋白(P<0.01)及其mRNA表达(P<0.05),并减少表达GluT1的细胞比例(P<0.01)。与空白组比较,四神丸含药血清还能降低MettL3蛋白含量(P<0.05)及m^(6)A RNA甲基化水平(P<0.01)。结论:四神丸能够抑制结肠癌细胞糖酵解,其作用机制可能与降低MettL3过表达,抑制m^(6)A RNA甲基化,下调GluT1及细胞内HK、PFK等有氧糖酵解相关酶的活性有关。
Objective:To explore the effect and mechanism of Sishenwan-containing serum on aerobic glycolysis in human colon cancer HCT116 cells.Method:Cell counting kit-8(CCK-8)was used to detect the cell viability of colon cancer HCT116 cells after treatment with Sishenwan-containing serum(2.5%,5%,and 10%)for 24,48,72 h.The concentration of lactic acid,the content of intracellular glucose,and the activity of hexokinase(HK)and fructose-6-phosphate kinase(PFK)in the cell culture medium were detected by the micro-method.The content of glucose transporter 1(GluT1)mRNA was detected by Real-time quantitative polymerase chain reaction(Real-time PCR).The protein expression of GluT1 and methyltransferase-like 3(MettL3)was detected by Western blot.The expression of GluT1 in cells was detected by immunofluorescence and the level of N6-methyladenosine(m^(6)A)RNA methylation was detected by colorimetry.Result:Compared with the normal serum,2.5%,5%,and 10%Sishenwan-containing serum had no significant effect on the viability of HCT116 cells at 24 h,while 10%Sishenwan-containing serum showed a significant inhibitory effect on the viability of HCT116 cells at 48 h(P<0.05).Hence,10%Sishenwan-containing serum was used in subsequent experiments,and the intervention time was 48 h.Compared with the normal serum,10%Sishenwan-containing serum could reduce lactate production(P<0.05),down-regulate glucose uptake(P<0.05),and blunt the activities of HK and PFK,the key rate-limiting enzymes of glycolysis(P<0.05).Meanwhile,10%Sishenwan-containing serum could decrease the expression of GluT1 protein(P<0.01)and mRNA(P<0.05)and reduce the proportion of cells expressing GluT1(P<0.01).Compared with the normal serum,Sishenwan-containing serum also decreased the protein content of MettL3(P<0.05)and the methylation level of m^(6)A RNA(P<0.01).Conclusion:Sishenwan can inhibit glycolysis in colon cancer cells,and its inhibitory mechanism may be related to reducing MettL3 overexpression,inhibiting m^(6)A RNA methylation,and down-regulating GluT1 and the activities of intracellular aerobic glycolysis-related enzymes such as HK and PFK.
作者
蒋义芳
黄娅
肖冲
周舒雯
郑黎鹂
由凤鸣
JIANG Yifang;HUANG Ya;XIAO Chong;ZHOU Shuwen;ZHENG Lili;YOU Fengming(Traditional Chinese Medicine(TCM)Regulating Metabolic Diseases Key Laboratory of Sichuan Province,Hospital of Chengdu University of TCM,Chengdu 610075,China;Cancer Institute,Chengdu University of TCM,Chengdu 610072,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2023年第19期26-33,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金青年科学基金项目(82204951)
四川省科技厅项目(2020JDRC0102)
四川省中医药重点学科建设项目(2100601)。
