富硒乳酸菌对黄曲霉毒素B_(1)诱导鸡小肠上皮细胞凋亡的影响

Effect of Selenium-Enriched Lactic Acid Bacteria on Apoptosis of Chicken Small Intestinal Epithelial Cells Induced by Aflatoxin B_(1)

试验旨在探究富硒乳酸菌对黄曲霉毒素B_(1)(AFB_(1))对鸡小肠上皮细胞(CSIEC)凋亡的影响。将体外培养的CSIEC细胞分为对照组(C)、AFB_(1)(300μmol/L AFB_(1))、AFB_(1)+0.01 Se(300μmol/L AFB_(1)+0.01μmol/L富硒乳酸菌,以Se计)、AFB_(1)+0.05 Se和AFB_(1)+0.1 Se组,待细胞长至60%~70%汇合时,AFB_(1)+0.01 Se、AFB_(1)+0.05 Se和AFB_(1)+0.1 Se组培养液更换为含对应Se浓度的培养液,对照组和AFB_(1)组更换为正常培养液,8 h后向含AFB_(1)组加入300μmol/L AFB_(1),继续培养24 h。用CCK-8法检测细胞存活率,ELISA法检测培养液中乳酸脱氢酶(LDH)的活性,TUNEL法检测细胞凋亡率,Western blotting检测线粒体通路和内质网通路相关蛋白Bcl-2、Bax、PERK、ATF4、CHOP、GRP78、p53和p21的表达。细胞存活率检测结果表明,与对照组相比,其余各组CSIEC存活率均极显著降低(P<0.01);与AFB_(1)组相比,加硒各组存活率均极显著升高(P<0.01)。LDH活性检测结果表明,与对照组相比,其余各组LDH活性均极显著升高(P<0.01);与AFB_(1)组相比,加硒各组LDH活性均极显著降低(P<0.01)。细胞凋亡率检测结果表明,与对照组相比,AFB_(1)组TUNEL阳性细胞数极显著升高(P<0.01),且与加硒各组差异均不显著(P>0.05)。Bcl-2/Bax表达量检测结果表明,与对照组相比,AFB_(1)组Bcl-2/Bax极显著降低(P<0.01),AFB_(1)+0.05 Se组显著升高(P<0.05)。内质网应激通路相关蛋白表达量检测结果表明,与对照组相比,AFB_(1)组PERK、CHOP、GRP78和p53的表达量均极显著升高(P<0.01);与AFB_(1)组相比,加硒各组PERK、GRP78表达量均极显著降低(P<0.01),AFB_(1)+0.05 Se和AFB_(1)+0.1 Se组CHOP和p53的表达量均极显著或显著降低(P<0.01;P<0.05)。因此,富硒乳酸菌能够缓解AFB_(1)诱导的CSIEC凋亡。

In order to investigate the effect of selenium-enriched lactic acid bacteria on the apoptosis of chicken small intestinal epithelial cells(CSIEC)induced by aflatoxin B_(1)(AFB_(1)),CSIEC cultured in vitro were divided into 5 groups:control group(C),AFB_(1) group(300μmol/L AFB_(1)),AFB_(1)+0.01 Se group(300μmol/L AFB_(1)+0.01μmol/L selenium-enriched lactic acid bacteria,in terms of Se),AFB_(1)+0.05 Se group and AFB_(1)+0.1 Se group.When the cells grew to 60%-70%confluence,the culture medium of AFB_(1)+0.01 Se,AFB_(1)+0.05 Se and AFB_(1)+0.1 Se groups was replaced with the culture medium containing the corresponding Se concentration,and C and AFB_(1) groups were replaced with normal culture medium.After 8 h,300μmol/L AFB_(1) was added to the AFB_(1)-containing groups,and the culture was continued for 24 h.CCK-8 was used to detect the antagonistic effect of selenium-enriched lactic acid bacteria on the cytoactive of CSIEC induced by AFB_(1).ELISA was used to detect the change of lactate dehydrogenase(LDH)activity in the culture medium.TUNEL was used to observe the effect of selenium-enriched lactic acid bacteria against AFB_(1) on cell apoptosis rate.The protein levels of Bcl-2/Bax,PERK,ATF4,CHOP,GRP78,p53 and p21 associated with mitochondrial and endoplasmic reticulum stress pathways were detected by Western blotting.The results showed that the cytoactive of CSIEC in the other groups was extremely significantly lower than that in control group(P<0.01),and those in selenium-added groups were extremely significantly higher than those in AFB_(1) group(P<0.01).The LDH activities in the other groups were extremely significantly higher than those in control group(P<0.01),and LDH activities in selenium-added groups were extremely significantly lower than those in AFB_(1) group(P<0.01).Compared with control group,the number of TUNEL positive cells in AFB_(1) group was extremely significantly increased(P<0.01);and no significant difference were found between selenium-added groups and AFB_(1) group(P>0.05).Compared with control group,the protein expression levels of Bcl-2/Bax in AFB_(1) group were extremely significantly decreased(P<0.01),and that in AFB_(1)+0.05 Se group was increased(P<0.05).Endoplasmic reticulum stress pathway protein levels showed that the protein levels of PERK,CHOP,GRP78 and p53 in AFB_(1) group were extremely significantly higher than those in control group(P<0.01),protein levels of PERK and GRP78 in selenium-added groups were significantly lower than those in AFB_(1) group(P<0.01).Protein levels of CHOP and p53 in AFB_(1)+0.05 Se and AFB_(1)+0.1 Se groups were extremely significantly lower or significantly lower than those in AFB_(1) group(P<0.01;P<0.05).Therefore,selenium-enriched lactic acid bacteria could alleviate the apoptosis of CSIEC induced by AFB_(1).