摘要
为探究姜黄素(Cur)对玉米赤霉烯酮(ZEA)诱导的猪肾上皮细胞(PK-15)凋亡的影响及其可能的保护机制,体外培养PK-15细胞,给予玉米赤霉烯酮和不同浓度姜黄素(6.25、12.5、25μmol/L)处理24 h,通过MTT法测定姜黄素和ZEA对PK-15细胞活力的影响;荧光显微镜观察Hoechst 33342活细胞染色判断细胞凋亡情况;JC-1法检测细胞线粒体膜电位变化;试剂盒检测细胞上清液中LDH活性;实时荧光定量PCR测定细胞中的Bax、Bcl-2和Caspase-3的mRNA表达水平;Western blot测定Bax、Bcl-2和Caspase-3蛋白表达水平.结果表明,浓度在25μmol/L以下的姜黄素对PK-15细胞几乎没有毒性,ZEA的半数抑制浓度(IC50)为36.55μg/mL;姜黄素极显著提高ZEA降低的PK-15细胞活力(P<0.01),缓解ZEA引起的细胞凋亡,极显著降低ZEA诱导的LDH活性(P<0.01),显著降低ZEA引起的线粒体膜电位丢失(P<0.01);ZEA极显著(P<0.01)升高细胞中Bax和Caspase-3的mRNA和蛋白表达,极显著(P<0.01)降低Bcl-2的mRNA和蛋白表达;与ZEA组比较,+Cur6.25组的Bax mRNA表达量和Bax、Caspase-3蛋白表达均极显著(P<0.01)降低,Bcl-2的蛋白表达极显著(P<0.01)降低,+Cur12.5组显著降低了Bax和Caspase-3的mRNA表达(P<0.05),极显著(P<0.01)降低Bax和Caspase-3的蛋白表达,极显著升高了Bcl-2的mRNA表达和蛋白表达(P<0.01),+Cur25组的Bcl-2的mRNA表达量和蛋白表达量极显著(P<0.01)的升高,Caspase-3的mRNA表达和蛋白表达极显著(P<0.01)降低.本研究表明ZEA诱导PK-15细胞发生凋亡,姜黄素可通过抑制线粒体凋亡途径减轻其诱导的细胞凋亡,从而起到保护作用提高细胞活力.
The purpose of this study was to investigate the effect of curcumin(Cur)on porcine renal epithelial cell(PK-15)apoptosis induced by ZEA and its possible protective mechanism.PK-15 cells were cultured in vitro and treated with zearalenone and various concentrations of curcumin(6.25,12.5,and 25μmol/L)for 24 h.The effects of curcumin and ZEA on PK-15 cell viability were determined by MTT assay.Hoechst 33342 live-cell staining was performed using fluorescence microscopy to determine cell apoptosis.Changes in mitochondrial membrane potential were detected using the JC-1 method.The LDH activity in cell supernatants was detected using a dedicated assay kit.The mRNA expression levels of Bax,Bcl-2,and Caspase-3 were determined by real-time quantitative PCR.The levels of Bax,Bcl-2,and Caspase-3 protein expression were determined by western blotting.The results showed that curcumin had almost no toxicity toward PK-15 cells at concentrations below 25μmol/L,and the IC50 of ZEA was 36.55μg/mL.Curcumin significantly increased(P<0.01)PK-15 cell viability,alleviated apoptosis induced by ZEA,significantly reduced(P<0.01)ZEA-induced LDH activity,and significantly reduced(P<0.01)ZEA-induced mitochondrial membrane potential loss.ZEA significantly increased(P<0.01)the mRNA expression and protein levels of Bax and Caspase-3 and significantly(P<0.01)decreased the mRNA expression and protein levels of Bcl-2.The expression of Bax mRNA,Bax,Caspase-3,and Bcl-2 in the+Cur6.25 group decreased significantly(P<0.01),the mRNA expression of Bax and Caspase-3 was significantly decreased in the+Cur12.5 group(P<0.05),the protein expression of Bax and Caspase-3 was significantly decreased(P<0.01),and the mRNA and protein expression of Bcl-2 were significantly increased(P<0.01),the mRNA and protein expression of Bcl-2 in the+Cur25 group were significantly increased(P<0.01),while the mRNA and protein expression of Caspase-3 were significantly decreased(P<0.01).These results suggest that ZEA could induce PK-15 cell apoptosis and that curcumin could reduce the apoptosis of PK-15 cells induced by ZEA by inhibiting the mitochondrial membrane changes.
作者
卢春亭
肖仕鑫
钟佩云
周珈羽
邓辉
张凯照
黄小红
崔红杰
LU Chunting;XIAO Shixin;ZHONG Peiyun;ZHOU Jiayu;DENG Hui;ZHANG Kaizhao;HUANG Xiaohong;CUI Hongjie(University Key Laboratory for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province,Fuzhou 350002,China;Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health,Fuzhou 350002,China)
出处
《应用与环境生物学报》
CAS
CSCD
北大核心
2023年第2期357-363,共7页
Chinese Journal of Applied and Environmental Biology
基金
福建省科技厅重大专项专题项目(2021NZ029008)
福建省自然科学基金项目(2021J01080)
福建省中青年教师教育科研项目(JAT210066)
横向课题项目(KH190363A)资助
