阿奇霉素对胃癌细胞增殖、凋亡及炎症因子表达水平的影响

Influence of azithromycin on proliferation and apoptosis of gastric cancer cells and inflammatory factor expression level

目的探讨阿奇霉素对人胃癌细胞(AGS细胞)上清液中炎症因子表达、增殖和凋亡的影响及核转录因子-κB(NF-κB)信号通路的调控作用。方法体外培养AGS细胞,将其分为对照组(不进行干预)和不同水平(12.5、25.0、50.0、100.0μg/mL)阿奇霉素组,干预24 h,筛选阿奇霉素最适水平用于后续实验。细胞分组:对照组、阿奇霉素组(50.0μg/mL阿奇霉素)、阳性药物组(50.0μg/mL 5-氟尿嘧啶)、抑制剂组(50.0μg/mL阿奇霉素+1.0μmol/L NF-κB通路抑制剂BAY11-7082)和激活剂组(50.0μg/mL阿奇霉素+1.0μmol/L NF-κB通路激动剂Prostratin),干预24 h。采用细胞计数试剂盒-8(CCK-8)检测细胞活力;采用酶联免疫吸附试验(ELISA)测定细胞上清液中的炎症因子[白细胞介素(IL)-10及IL-1β]水平;采用5-乙炔基-2′脱氧尿嘧啶核苷(EdU)测定细胞增殖率;采用Hoechst33258染色试剂盒测定细胞凋亡率;采用蛋白免疫印迹(WB)法测定增殖细胞核抗原(PCNA)、半胱氨酸蛋白酶-3(Caspase-3)及NF-κB通路相关蛋白表达水平。结果用CCK-8检测AGS细胞活力,根据实验结果选择50.0μg/mL阿奇霉素用于后续实验。与对照组比较,阿奇霉素组和阳性药物组AGS细胞上清液中IL-1β水平、细胞增殖率、PCNA表达水平、磷酸化(p)NF-κB p65/NF-κB p65和p-IκBα/IκBα明显降低(P<0.05),IL-10水平、细胞凋亡率和Caspase-3表达水平明显升高(P<0.05);与阿奇霉素组比较,抑制剂组中BAY11-7082的出现增强了阿奇霉素对AGS细胞的作用(P<0.05),激活剂组中Prostratin的出现则削弱了阿奇霉素对AGS细胞的作用(P<0.05)。结论阿奇霉素能抑制AGS细胞的炎症和增殖,并诱导其凋亡,其作用机制可能与阻滞NF-κB通路信号转导有关。

Objective To investigate the influence of azithromycin on the expression of inflammatory factors in supernatant,proliferation and apoptosis of human gastric cancer cell(AGS)and its regulation effect on nuclear transcription factor-κB(NF-κB)signaling pathway.Methods The AGS cells were cultured in vitro and divided into the control group(non-intervention)and different levels(12.5,25.0,50.0,100.0μg/mL)of azithromycin groups.After 24 h of intervention,the optimal azithromycin level was screened out for the follow-up experiment.Then AGS cells were divided into the control group,azithromycin group(50.0μg/mL azithromycin),positive drug group(50.0μg/mL 5-fluorouracil),inhibitor group(50.0μg/mL azithromycin+1.0μmol/L NF-κB pathway inhibitor BAY11-7082)and activator group(50.0μg/mL azithromycin+1.0μmol/L NF-κB passway activator Prostratin).The intervention lasted for 24 h.The cell counting kit 8(CCK-8)was used to detect the cell viability.The levels of supernate inflammatory factors interleukin(IL-10)and IL-1βwere determined by enzyme-linked immunosorbent assay(ELISA).The cell proliferation rate was determined by 5-acetyne-2'deoxyuracil nucleoside(EdU).The Hoechst33258 staining kit was used to determine the apoptosis rate.The expression levels of proliferating cell nuclear antigen(PCNA),Caspase-3 and NF-κB pathway-related proteins were determined by Western blotting(WB).Results The activity of AGS cells was detected by CCK-8,and 50.0μg/mL azithromycin was selected for the subsequent experiment.Compared with the control group,the level of IL-1β,cell proliferation rate,expression level of PCNA,phosphorylation(p)-NF-κB p65/NF-κB p65 and p-IκBα/IKκBαprotein in AGS cells of the azithromycin group and positive drug group were significantly decreased(P<0.05),and the level of IL-10,cell apoptosis rate and expression level of Caspase-3 were significantly increased(P<0.05).Compared with the azithromycin group,the BAY11-7082 appearance in the inhibitor group enhanced the effect of azithromycin on AGS cells(P<0.05),while the Prostratin appearance in the activator group weakened the effect of azithromycin on AGS cells(P<0.05).Conclusion Azithromycin could inhibit the inflammation and proliferation of AGS cells and induce their apoptosis,its mechanism may be related to block NF-κB pathway signal transduction.