摘要
旨在研究HO-1对猪小肠上皮细胞(IPEC-J2)氧化损伤的缓解作用。利用不同浓度H_(2)O_(2)(100、200、300、400、500、600、1000μmol/L)分别处理IPEC-J2细胞6、12、24 h,利用CCK-8法检测细胞活力,确定H_(2)O_(2)处理细胞的浓度。将IPEC-J2细胞分为对照组(CT)、H_(2)O_(2)组、HO-1+H_(2)O_(2)组,每组3个重复。RT-qPCR检测IPEC-J2细胞中IL-1α、IL-6、IL-8、Keap1、Nrf2 mRNA的相对表达丰度;ELISA检测IPEC-J2细胞中丙二醛(MDA)含量、过氧化氢酶(CAT)活性;化学荧光法检测细胞中活性氧(ROS)水平。结果显示,随着H_(2)O_(2)浓度的增加,IPEC-J2细胞活力降低,400μmol/L H_(2)O_(2)处理12 h,600μmol/L H_(2)O_(2)处理6 h或24 h,IPEC-J2细胞活力均降至50%以下。根据细胞活力下降50%的原则,后续选用400μmol/L H_(2)O_(2)处理IPEC-J2细胞12 h。与CT组相比,H_(2)O_(2)组IPEC-J2细胞中的IL-1α(P<0.05)、IL-6(P<0.01)和IL-8(P<0.01)mRNA相对表达丰度增加,细胞中ROS荧光强度增强(P<0.01)和MDA含量(P<0.001)升高。与H_(2)O_(2)组相比,HO-1+H_(2)O_(2)组IPEC-J2细胞中IL-6(P<0.05)、IL-1α(P<0.01)和IL-8(P<0.05)mRNA相对表达丰度降低,细胞中ROS荧光强度减弱(P<0.05)和MDA含量显著下降(P<0.01)。Keap1/Nrf2信号结果显示,H_(2)O_(2)上调Keap1 mRNA表达(P<0.01),下调Nrf2 mRNA表达(P<0.01),降低CAT活性(P<0.01);HO-1与H_(2)O_(2)共处理,IPEC-J2细胞中Keap1 mRNA表达下调(P<0.01),Nrf2 mRNA表达上调(P<0.01),CAT活性升高(P<0.01)。提示400μmol/L H_(2)O_(2)能诱导IPEC-J2细胞发生氧化损伤,HO-1能在一定程度上缓解H_(2)O_(2)诱导IPEC-J2细胞的氧化损伤,HO-1能激活H_(2)O_(2)抑制的Keap1/Nrf2信号,促进抗氧化酶CAT的产生,发挥抑炎、抗氧化作用。
The aim of this experiment is to investigate the alleviative effect of HO-1 on oxidative damage in pig intestinal epithelial cells(IPEC-J2).Different concentrations of H_(2)O_(2)(100,200,300,400,500,600,1000μmol/L)were used to treat IPEC-J2 cells for 6,12,and 24 h,respectively.The treated concentration of H_(2)O_(2)was determined using CCK-8 method to detect cell viability.IPEC-J2 cells were divided into control group(CT),H_(2)O_(2)group,and HO-1+H_(2)O_(2)group,with 3 replicates in each group.The relative expression abundances of IL-1α,IL-6,IL-8,Keap1,Nrf2 mRNA were detected by RT-qPCR.The contents of malondialdehyde(MDA)and the activity of catalase(CAT)in IPEC-J2 cells were detected by ELISA.The level of reactive oxygen species(ROS)in cells was detected by chemical fluorescence method.The results showed that the activity of IPEC-J2 cells decreased with the increase of H_(2)O_(2)concentration.When treated with 400μmol/L H_(2)O_(2)for 12 h and 600μmol/L H_(2)O_(2)for 6 or 24 h,the activity of IPEC-J2 cells decreased below 50%.According to the principle of a 50%decrease in cell viability,IPEC-J2 cells were subsequently treated with 400μmol·L-1 H_(2)O_(2)for 12 h.Compared with the CT group,the IL-1α(P<0.05),IL-6(P<0.01),and IL-8(P<0.01)mRNA relative expression abundances were increased,the fluorescence intensity of ROS(P<0.01)and MDA content(P<0.01)were increased in IPEC-J2 cells of the H_(2)O_(2)group.Compared with the H_(2)O_(2)group,the IL-1α(P<0.01),IL-6(P<0.05),and IL-8(P<0.05)mRNA relative expression abundances of IPEC-J2 cells in the HO-1+H_(2)O_(2)group decreased,at the same time,ROS fluorescence intensity(P<0.05)and MDA content(P<0.01)in cells significantly decreased.The Keap1/Nrf2 signal results showed that H_(2)O_(2)upregulated Keap1 mRNA expression(P<0.01),downregulated Nrf2 mRNA expression(P<0.01),and decreased CAT activity(P<0.01).Co-treatment of HO-1 and H_(2)O_(2),downregulating of Keap1 mRNA expression(P<0.01),upregulating of Nrf2 mRNA expression(P<0.01),and elevating of CAT activity(P<0.01)in IPEC-J2 cells.These results suggested that 400μmol/L H_(2)O_(2)induced oxidative damage in IPEC-J2 cells.HO-1 alleviated the oxidative damage induced by H_(2)O_(2)in IPEC-J2 cells.HO-1 activated the Keap1/Nrf2 signal,which was inhibited by H_(2)O_(2),and promoted the production of antioxidant enzyme CAT,exerting anti-inflammatory and antioxidant effects.
作者
吴魏
杨涵琳
刘一
张赛宇
何俊辉
杨彦宾
郭爽
王月影
李和平
WU Wei;YANG Han-lin;LIU Yi;ZHANG Sai-yu;HE Jun-hui;YANG Yan-bin;GUO Shuang;WANG Yue-ying;LI He-ping(Key Laboratory of Animal Biochemistry and Nutrition/Ministry of Agriculture and Rural Affairs,Key Laboratory of Animal Growth and Development of Henan Province/College of Verterinary Medicine,Henan Agricultural University,Zhengzhou,Henan,450046,China)
出处
《动物医学进展》
北大核心
2024年第7期64-70,共7页
Progress In Veterinary Medicine
基金
国家自然科学基金面上项目(32272958)
河南省科技攻关计划项目(222102110402)。
