芦丁对人结肠癌SW480细胞凋亡的促进作用及其作用机制

Promotion effect of rutin on apoptosis of human colon cancer SW480 cells and its mechanism

目的:探讨芦丁对人结肠癌(CRC)SW480细胞凋亡的影响,阐明芦丁在CRC治疗中的作用及其可能的分子机制。方法:毒性与基因比较数据库(CTD)和GeneCards数据库预测与芦丁相关的交集mRNA,基因本体(GO)和京都基因与基因组百科全书(KEGG)数据库预测交集mRNA的生物学功能及作用通路,采用String数据库寻找与Notch-1相关作用蛋白;以人CRC SW480细胞作为研究对象,分别给予0.1、1.0和10.0μmol·L^(-1)γ-分泌酶抑制剂DAPT初步筛选,进而给予1、2、4、8和10μmol·L^(-1)DAPT干预,筛选出芦丁与DAPT的最适给药浓度。实验分为空白对照组、芦丁组(22μmol·L^(-1))、DAPT组(1μmol·L^(-1))和芦丁+DAPT组(22μmol·L^(-1)芦丁+1μmol·L^(-1)DAPT);MTT法检测SW480细胞增殖活性,Hoechst33258核染色观察各组细胞凋亡形态,流式细胞术检测各组SW480细胞凋亡率,实时荧光定量PCR(RT-qPCR)法检测各组细胞中Notch-1 mRNA表达水平,Western blotting法检测各组细胞中,Notch-1、含半胱氨酸的天冬氨酸蛋白水解酶3(caspase-3)、含半胱氨酸的天冬氨酸蛋白水解酶9(caspase-9)、B细胞淋巴瘤(Bcl-2)和Bcl-2相关X蛋白(Bax)的蛋白表达水平。结果:CTD和GeneCards数据库共有芦丁的34个交集mRNA;GO和KEGG数据库预测出交集mRNA与CRC细胞凋亡过程相关;String数据库中与Notch-1相关蛋白16个[相互作用分数(score)>0.42];芦丁与DAPT的最适给药浓度分别为22μmol·L^(-1)和1μmol·L^(-1)。与空白对照组比较,芦丁组、DAPT组和芦丁+DAPT组细胞凋亡率明显升高(P<0.05),细胞中Notch-1 mRNA和蛋白表达水平均明显降低(P<0.05),细胞中caspase-3、caspase-9和Bax蛋白表达水平均明显升高(P<0.05),Bcl-2蛋白表达水平明显降低(P<0.05)。芦丁组与DAPT组细胞凋亡率比较差异无统计学意义(P>0.05)。与芦丁组和DAPT组比较,芦丁+DAPT组细胞凋亡率明显升高(P<0.05),caspase-3、caspase-9和Bax蛋白表达水平明显升高(P<0.05),而Bcl-2蛋白表达水平明显降低(P<0.05)。结论:生物信息学分析及实验研究结果均表明芦丁能促进CRC细胞凋亡,这一作用可能是通过靶向调控Notch-1调节丝裂原活化蛋白激酶(MAPK)信号通路来实现的。

Objective:To explore the effect of rutin on the colorectal cancer(CRC)SW480 cells,and to clarify the effect of rutin in the treatment of CRC and its possible molecular mechanism.Methods:The Comparative Toxicogenomics(CTD)and GeneCards databases were used to predict the intersection mRNA related to rutin;Gene Ontdogy(GO)and Kyoto Encyclopedia of Genes and Genones(KEGG)databases were used to predict the biological function and action pathways of the intersection mRNA;String database was used to find the Notch-1 related proteins.The human CRC SW480 cells were used as the subjects,0.1,1.0,and 10.0μmol·L^(-1)DAPT were given for preliminary screening,then 1,2,4,8 and10μmol·L^(-1)DAPT were given to intervene,and the optimal concentrations of rutin and DAPT were screened out.The human CRC SW480 cells were divided into blank control group,rutin group(22μmol·L^(-1)),DAPT group(1μmol·L^(-1))and rutin+DAPT group(22μmol·L^(-1)rutin+1μmol·L^(-1)DAPT).MTT method was used to detect the cell proliferation activities;Hoechst33258 nuclear staining was used to observe the apopotic morphology of cells,and flow cytometry was used to detect the apoptotic rates;real-time fluorescence quantitative PCR(RT-qPCR)method was performed to detect the expression levels of Notch-1 mRNA in the SW480 cells in various groups;Western blotting method was used to determine the protein expression levels of Notch-1,caspase-3,caspase-9,B-cell lymphoma 2(Bcl-2),and Bcl-2associated X protein in the SW480 cells in various groups.Results:In CTD and GeneCards databases,there were a total of 34 intersection mRNA;the GO and KEGG databases predicted that the intersection mRNA was related to the process of colon cancer apoptosis;String database verified 16 Notch-1 related proteins(score>0.42);the optimal administration concentrations of rutin and DAPT were 22μmol·L^(-1)and 1μmol·L^(-1);compared with blank control group,the apoptotic rates in rutin group,DAPT group and rutin+DAPT group were increased significantly(P<0.05),the expression levels of Notch1 mRNA and protein were significantly reduced(P<0.05),the expression levels of caspase-3,caspase-9,and Bax proteins in the cells were increased significantly(P<0.05),while the expression levels of Bcl-2 protein were decreased significantly(P<0.05).There was no significant difference in the apoptotic rate between rutin group and DAPT group(P>0.05).Compared with rutin group and DAPT group,the apoptotic rate in rutin+DAPT group was significantly increased(P<0.05),the expression levels of caspase-3,caspase-9,and Bax proteins were increased significantly(P<0.05),while the expression level of Bcl-2 protein was decreased significantly(P<0.05).Conclusion:The results of bioinformatics analysis and experiment show that rutin can promote the apoptosis of CRC cells,which may be achieved by targeting Notch-1 and regulating MAPK signal pathway.