N-乙酰半胱氨酸对双酚A诱导的猪肾细胞凋亡和炎症反应的抑制作用  

Inhibitory Effect of N-acetylcysteine on Bisphenol A-Induced Apoptosis and Inflammatory Response in Porcine Kidney Cells

作　　者：陶文静 张子婷 刘媛 宋丹 李向臣 TAO WenJing;ZHANG ZiTing;LIU Yuan;SONG Dan;LI XiangChen(College of Animal Science and Technology/College of Veterinary Medicine,Zhejiang A&F University/Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,Hangzhou 311300)

机构地区：[1]浙江农林大学动物科技学院/动物医学院/浙江省畜禽绿色生态健康养殖应用技术研究重点实验室,杭州311300

出　　处：《中国农业科学》2023年第3期549-558,共10页Scientia Agricultura Sinica

基　　金：浙江省自然科学基金(LQ22C170001);浙江农林大学科研发展基金(22020FR080);国家科技基础条件平台子课题(TZDWZYK2019-08)。

摘　　要：【背景】双酚A(BPA)广泛应用于塑料材料工业制造,其从塑料制品中渗出,暴露在食物、水、土壤和空气等多种环境介质中,导致动物长期接触,并经过胎盘和母乳传递给后代,干扰动物生长和发育,对动物生长性能和生产效率产生不利影响。N-乙酰半胱氨酸(NAC)作为公认的强效抗氧化剂,能够调节氧化应激、细胞凋亡、炎症等多种病理生理过程。然而,NAC对BPA诱导的猪肾细胞损伤的调节作用尚不清楚。【目的】探究抗氧化剂NAC在BPA诱导的PK15细胞凋亡和炎症反应中的潜在作用,为NAC在对抗BPA诱导的猪肾细胞损伤中的应用研究提供科学依据。【方法】选取PK15细胞为试验材料,采用相应的抗氧化检测试剂盒分别测定细胞内过氧化氢酶(CAT)、总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性变化;设置不同浓度NAC(0、2、5和10 mmol·L^(-1))预处理,并与BPA共处理PK15细胞,CCK-8检测细胞活力以筛选最佳的NAC作用浓度;实时荧光定量PCR(qRT-PCR)检测凋亡相关基因(BAX、BCL-2和Caspase3)表达和炎症相关基因(IL-8、IL-6、IL-1β和TNF-α)mRNA相对表达量,蛋白免疫印迹检测凋亡相关蛋白(BAX、BCL-2、cleaved-Caspase3)水平;免疫荧光染色检测凋亡细胞数量和核因子κB(NF-κB)核易位。【结果】与对照组相比,BPA处理显著降低了细胞内CAT、T-SOD和GSH-Px活性(P<0.05)。CCK-8结果显示,与对照组相比,BPA显著降低PK15细胞活力,而不同浓度的NAC均能显著促进PK15细胞活力(P<0.05),且与BPA单独处理相比,5和10 mmol·L^(-1)NAC预处理均能显著促进PK15细胞活力(P<0.05);qRT-PCR和蛋白免疫印迹结果显示,BPA处理显著增加了BAX、Caspase3 mRNA相对表达量以及蛋白表达水平,减少了BCL-2 mRNA相对表达量和蛋白表达(P<0.05),而NAC预处理能够减少BPA诱导增加的BAX、Caspase3基因和蛋白表达,增加BCL-2基因和蛋白表达;Hoechst 33258荧光染色显示,BPA处理的细�【Background】Bisphenol A(BPA)is widely used in the industrial manufacturing of plastic materials,which seeps out from plastic products and exposes to various environmental media such as food,water,soil,and air,resulting in long-term exposure of animals.It is passed to offspring through the placenta and breast milk,interfering with animal growth and development and adversely affecting animal growth performance and production efficiency.N-acetylcysteine(NAC),as a recognized potent antioxidant,can regulate various pathophysiological processes,such as oxidative stress,apoptosis,and inflammation.However,the regulatory role of NAC on BPA-induced porcine kidney cell injury remains unclear.【Objective】This study aimed to explore the potential role of the antioxidant NAC on BPA-induced apoptosis and inflammatory responses in PK15 cells.【Method】PK15 cells were selected as experimental materials,and the activities of catalase(CAT),total superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px)were measured by the corresponding antioxidant detection kits.PK15 cells were treated with different concentrations of NAC(0,2,5,10 mmol·L^(-1))and then co-treated with BPA,and then the cell viability was detected by CCK-8 to select the optimal concentration of NAC.The expression of apoptosis-related genes(BAX,BCL-2 and Caspase3)and inflammatory genes(IL-8,IL-6,IL-1βand TNF-α)as well as protein expression were detected by real-time quantitative PCR(qRT-PCR)and western blotting.The number of apoptotic cells and nuclear factor kappa B(NF-κB)nuclear translocation were detected by immunofluorescence staining.【Result】The results showed that BPA significantly reduced the activities of CAT,T-SOD and GSH-Px in PK15 cells,compared with the control group(P<0.05).CCK-8 results showed that BPA significantly decreased PK15 cell viability in contrast to control group,whereas the different concentrations of NAC significantly promoted cell viability,and 5,10 mmol·L^(-1)NAC pretreatment significantly promoted cell viability when

关 键 词：双酚A N-乙酰半胱氨酸 细胞凋亡 炎症反应 PK15细胞 氧化应激 

分 类 号：S859.8[农业科学—临床兽医学]