消癌解毒方含药血清增强NK细胞杀伤结肠癌的作用及机制

Xiaoai Jiedu Prescription-containing Serum Enhances Lethal Effect of NK Cells on Colon Cancer Cells

目的:探讨消癌解毒方含药血清对自然杀伤(NK)细胞杀伤结肠癌细胞的增强作用及分子机制。方法:消癌解毒方含药血清(0.1%、0.5%、1%、5%、10%)处理HCT-116细胞、NK-92MI细胞24 h,噻唑蓝(MTT)比色法检测其对细胞增殖的影响。选取低体积分数(0.1%、0.5%、1%)含药血清处理共培养的HCT-116细胞、NK-92MI细胞24 h,钙黄绿素-乙酰甲酯/碘化丙啶(Calcein-AM/PI)双染检测NK细胞对结肠癌细胞杀伤作用;流式细胞仪检测结肠癌细胞凋亡;蛋白免疫印迹法(Western blot)检测凋亡、信号传导及转录激活蛋白4(STAT4)通路相关蛋白表达;酶联免疫吸附测定法(ELISA)检测干扰素(IFN)-γ分泌。结果:MTT比色法显示,与空白组比较,消癌解毒方含药血清(5%、10%)能有效抑制HCT-116、NK-92MI细胞增殖(P<0.01),但消癌解毒方含药血清(0.1%、0.5%、1%)对细胞增殖无明显影响。与空白组比较,消癌解毒方含药血清(0.1%、0.5%、1%)呈浓度依赖性的增强NK细胞对结肠癌细胞的杀伤活性,并诱导结肠癌细胞凋亡(P<0.01)。Western blot显示,与空白组比较,消癌解毒方含药血清能下调B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关xl蛋白(Bcl-xl)表达,上调Bcl-2相关X蛋白(Bax)表达(P<0.05,P<0.01);与共培养组比较,消癌解毒方含药血清(0.1%、0.5%、1%)能激活p-STAT4磷酸化,促进IFN-γ表达(P<0.05)。ELISA显示,与空白组比较,消癌解毒方含药血清(0.1%、0.5%、1%)能提高IFN-γ分泌量(P<0.01)。结论:消癌解毒方含药血清增强NK细胞杀伤结肠癌细胞活性的机制可能与激活STAT4通路,增加NK细胞IFN-γ分泌量,下调Bcl-xl、Bcl-2表达,上调Bax表达,进而促进结肠癌细胞凋亡相关。

Objective:To explore the influence of Xiaoai Jiedu prescription(XJP)-containing serum on natural killer(NK)cells′lethal effect on colon cancer cells and the molecular mechanism.Method:XJP-containing serum(0.1%,0.5%,1%,5%,10%)was used to treat HCT-116 cells and NK-92MI cells respectively for 24 h,and methyl thiazolyl tetrazolium(MTT)assay was employed to detect cell proliferation.Then,low-concentration(0.1%,0.5%,1%)XJP-containing serum was selected to treat co-cultured HCT-116 cells and NK-92MI cells for 24 h and calcein acetoxymethyl ester/propidium iodide(Calcein-AM/PI)was applied to detect the killing effect of NK cells on colon cancer cells.Flow cytometry was used to detect apoptosis of colon cancer cells,Western blot the expression of apoptosis-related proteins and signal transducer and activator of transcription 4(STAT4)pathway-related proteins,and enzyme-linked immunosorbent assay(ELISA)the secretion of interferon(IFN)-γ.Result:High-concentration(5%,10%)XJP-containing serum inhibited the proliferation of HCT-116 and NK-92MI cells(P<0.01),while low-concentration(0.1%,0.5%,1%)XJP-containing serum had no obvious influence on cell proliferation compared with the blank group.As compared with the blank group,low-concentration XJP-containing serum enhanced the killing activity of NK cells against colon cancer cells in a concentration-dependent manner(P<0.01),and induced apoptosis of colon cancer cells(P<0.01).Moreover,XJP-containing serum(0.1%,0.5%,1%)down-regulated the expression of B-cell lymphoma 2(Bcl-2)and B-cell lymphoma-extra large(Bcl-xl),and up-regulated the expression of Bcl-2-associated X(Bax)compared with the blank group(P<0.05,P<0.01).Compared with the co-culture group,XJP-containing serum(0.1%,0.5%,1%)increased the expression of p-STAT4 and IFN-γ(P<0.05).ELISA result showed that XJP-containing serum(0.1%,0.5%,1%)raised IFN-γsecretion(P<0.01).Conclusion:XJP-containing serum can enhance the activity of NK cells to kill colon cancer cells.The mechanism is the likelihood that it activates STAT4 pathway,increases IFN-γsecretion by NK cells,down-regulates the expression of Bcl-xl and Bcl-2,and up-regulates the expression of Bax,thereby promoting the apoptosis of colon cancer cells.