TIGAR对酮病奶牛脂肪组织氧化应激的影响

Effects of TIGAR on oxidative stress in adipose tissue of ketotic cows

本试验分为体内试验和体外试验两部分。体内试验选取健康和酮病奶牛各10头,采集脂肪组织。ELISA结果显示,与健康奶牛相比,临床酮病奶牛脂肪组织中丙二醛(malonaldehyde,MDA)含量和活性氧(reactive oxygen species,ROS)活性较高,谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)和超氧化物歧化酶(superoxide dismutase,SOD)活性较低。Western blot结果显示,与健康组相比,酮病组脂肪组织中TIGAR蛋白表达水平显著上升,Nrf2-HMOX1信号通路蛋白表达水平显著上调,氧化应激相关指标SOD1、过氧化氢酶(catalase,CAT)和谷胱甘肽-S-转移酶(glutathione S-transferase,GST)的蛋白质水平显著上升。体内试验结果表明,酮病奶牛体内脂肪组织发生氧化应激。体外试验在分离培养牛原代脂肪细胞的基础上,分别通过腺病毒沉默或过表达TIGAR以及体外采用添加H_(2)O_(2)作用2 h,构建牛脂肪细胞氧化应激模型,来检测TP53诱导细胞凋亡调节因子(TP53 induces glycolysis and apoptosis factors,TIGAR)对牛脂肪细胞氧化应激的影响。Western blot结果显示,与对照组相比,H_(2)O_(2)组表现为氧化应激增强,核相关因子2(nuclear correlation factor 2,Nrf2)通路中Nrf2及血红素加氧酶1(hemeoxygenase-1,HMOX1)表达降低,相关氧化应激蛋白表达量降低;与H_(2)O_(2)组相比,过表达TIGAR加H_(2)O_(2)组Nrf2-HMOX1的蛋白表达水平上调,相关氧化应激蛋白SOD1、CAT和GST表达量上升。体外试验结果表明,过表达TIGAR缓解H_(2)O_(2)诱导的脂肪细胞氧化应激。与H_(2)O_(2)组相比,沉默TIGAR加H_(2)O_(2)组,Nrf2-HMOX1信号通路及氧化应激相关蛋白SOD1、CAT和GST的蛋白表达下调。结果表明,沉默TIGAR加剧H_(2)O_(2)给脂肪细胞带来的氧化应激。

This experiment was divided into two parts:in vivo and in vitro.In the in vivo experiment,10 healthy and 10 ketotic cows were selected,and adipose tissues were collected.ELISA results showed that malonaldehyde(MDA)content and reactive oxygen species(ROS)activity were higher in adipose tissues of cows with clinical ketotic compared with healthy cows,and glutathione peroxidase(GPP)activity was higher than that of cows with clinical ketotic.Western blot results showed that compared with the healthy group,adipose tissue of the ketotic group showed a significant increase in the expression level of TP53 induces glycolysis and apoptosis factors(TIGAR)protein,a significant up-regulation in the expression level of Nrf2-HMOX1 signaling pathway protein,and a significant increase in the expression level of oxidative stress protein in adipose tissue of the ketotic group.The expression level of TIGAR protein was significantly up-regulated,the protein level of Nrf2-HMOX1 signaling pathway was significantly up-regulated,and the protein levels of oxidative stress-related indexes SOD1,catalase(CAT)and glutathione S-transferase(GST)were significantly increased.It indicated that oxidative stress occurred in the adipose tissue of ketotic cows in vivo.In vitro experiments were carried out to detect the effect of TIGAR on oxidative stress in bovine adipocytes by adenoviral silencing or overexpression of TIGAR in isolated and cultured bovine primary adipocytes as well as by the addition of hydrogen peroxide in vitrofor 2h.The Western blot results showed that the hydrogen peroxide group showed enhanced oxidative stress compared with the control group,and the nuclear correlation factor 2was significantly increased.correlation factor 2(Nrf2)pathway,decreased expression of Nrf2and hemeoxygenase-1(HMOX1),and decreased expression of related oxidative stress proteins in the hydrogen peroxide group;compared with the hydrogen peroxide group,the protein expression levels of Nrf2-HMOX1and related oxidative stress proteins were up-regulated in the group with overexpression of TIGAR and hydrogen peroxide,and the expression levels of related oxidative stress proteins were up-regulated in the group with overexpression of TIGAR and hydrogen peroxide.Expression level was upregulated,and the expression of related oxidative stress proteins SOD1,CAT and GST increased.This indicates that overexpression of TIGAR alleviated hydrogen peroxide-induced oxidative stress in adipocytes.Protein expression of Nrf2-HMOX1signaling pathway and oxidative stress-related proteins SOD1,CAT and GST were down-regulated in the silencing TIGAR plus hydrogen peroxide group compared to the hydrogen peroxide group.It indicates that silencing TIGAR exacerbated the oxidative stress caused by hydrogen peroxide to adipocytes.