基于自噬途径探究白花蛇舌草乙醇提取物对结肠癌SW480细胞凋亡情况的影响

Effect of Hedyotis Diffusa Ethanol Extract on Apoptosis of Colon Cancer SW480 Cells Based on Autophagy Pathway

目的探讨白花蛇舌草乙醇提取物对结肠癌SW480细胞自噬和凋亡的调节作用,为临床治疗结肠癌提供新的途径。方法将结肠癌SW480细胞完全培养后接种于6孔板,设置对照组与低、中、高剂量组,分别加入含有0μg/ml、25μg/ml、50μg/ml、100μg/ml的白花蛇舌草乙醇提取物进行干预。采用噻唑蓝(MTT)法检测细胞增殖情况,计算细胞增殖抑制率(IR);DNA断裂的原位末端标记(TUNEL)法检测细胞凋亡情况,计算细胞凋亡率;AO染色观察细胞自噬的情况;Western blot法检测相关蛋白表达。结果低、中、高剂量组24 h、48 h、72 h后IR水平均高于对照组,差异有统计学意义(P<0.05);随着作用时间的延长和浓度的提高,白花蛇舌草乙醇提取物对结肠癌SW480细胞增殖的抑制作用越明显,在24 h、48 h、72 h的半数抑制浓度(IC50)分别为(35.78±2.52)μg/ml、(24.59±2.74)μg/ml和(18.54±2.12)μg/ml。低、中、高剂量组细胞凋亡率均明显高于对照组,差异有统计学意义(P<0.05),随着浓度的提高,白花蛇舌草乙醇提取物促进结肠癌SW480细胞凋亡的作用越明显。低、中、高剂量组细胞内发出红色荧光的酸性自噬囊泡的比重明显高于对照组。与对照组比较,低、中、高剂量组细胞干预72 h后自噬相关蛋白Beclin-1、BNIP3、Atg 5表达均明显增加,p62表达明显降低,差异有统计学意义(P<0.05)。结论白花蛇舌草乙醇提取物对结肠癌SW480细胞增殖抑制作用显著,其机制可能与诱导细胞自噬和凋亡,共同促进细胞死亡有关。

Objective To investigate the regulatory effect of Hedyotis diffusa ethanol extract on autophagy and apoptosis of colon cancer SW480 cells,so as to provide a new way for clinical treatment of colon cancer.Methods Colon cancer SW480 cells were completely cultured and seeded on 6-well plate.The control group and the low,medium and high dose group were set up,and added respectively 0μg/ml,25μg/ml,50μg/ml,100μg/ml of Hedyotis diffusa ethanol extract.MTT method was used to detect cell proliferation and calculate cell proliferation inhibition rate(IR).Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL).Autophagy was observed by AO staining.The protein expression was detected by Western blot.Results The IR levels of the low,medium,and high-dose groups were higher than those of the control group after 24 h,48 h,and 72 h,and the difference was statistically significant(P<0.05).With the extension of the action time and the increase of concentration.The inhibitory effect of the ethanol extract of Diffusa on the proliferation of colon cancer SW480 cells is more obvious.The IC50 at 24 h,48 h and 72 h is(35.78±2.52)μg/ml and(24.59±2.74)μg/ml and(18.54±2.12)μg/ml,respectively.The apoptosis rate of the low,medium and high dose groups was significantly higher than that of the control group,and the difference was statistically significant(P<0.05),indicating that Hedyotis diffusa ethanol can significantly promote the apoptosis of colon cancer SW480 cells.With the increase of concentration,the the promotion effect was more obvious.The proportion of acidic autophagic vesicles that emit red fluorescence in the cells of the low,medium,and high dose groups was significantly higher than that of the control group,indicating that the level of autophagy increased.The expressions of autophagy-related proteins Beclin-1,BNIP3 and Atg 5 were significantly increased after 72 hours of cell intervention in the low,medium,and high dosegroups,and the expression of p62 was significantly decreased.Compared with the control group,the difference was statistically significant(P<0.05).Conclusion The ethanol extract of Hedyotis diffusa has significant inhibitory effect on the proliferation of colon cancer SW480 cells,and its mechanism may be related to the induction of autophagy and the promotion of apoptosis.