载牛乳铁蛋白肽-壳聚糖纳米粒对过氧化氢诱导的奶牛乳腺上皮细胞氧化损伤的保护作用

Protective Effects of Bovine Lactoferrin Chitosan Nanoparticles on Oxidative Damage of Dairy Cow Mammary Epithelial Cells Induced by Hydrogen Peroxide

本试验以载牛乳铁蛋白肽-壳聚糖纳米粒(BLfcin-NPs)为添加剂,研究其对过氧化氢(H_(2)O_(2))诱导的奶牛乳腺上皮细胞(BMECs)氧化损伤的保护作用。采用单因素完全随机试验设计,以BMECs为模型,采用不同浓度[0(对照组)、100、200、400、600、800、1000μmol/L]H_(2)O_(2)对BMECs分别处理0、2、4、6、8、12 h,确定构建BMECs氧化损伤模型的最佳H_(2)O_(2)浓度及作用时间。以500μg/mL的BLfcin-NPs预处理BMECs 12 h后用400μmol/L的H_(2)O_(2)处理6 h,检测BMECs内线粒体膜电位(MMP)、抗氧化指标以及抗氧化与凋亡相关基因mRNA相对表达量。结果显示:1)与0μmol/L H_(2)O_(2)处理的对照组相比,不同浓度H_(2)O_(2)作用6 h后显著或极显著降低BMECs活力(P<0.05),并对细胞产生毒性,并且在400μmol/L的H_(2)O_(2)作用下BMECs晚期凋亡率极显著升高(P<0.01)。400、600、800μmol/L的H_(2)O_(2)作用6 h后,BMECs内ROS含量增加,且呈剂量依赖性。2)与未进行BLfcin-NPs预处理的H_(2)O_(2)诱导的氧化损伤BMECs细胞相比,BLfcin-NPs预处理显著提高了BMECs内线粒体膜电位下降比例(P<0.05),显著升高了谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性(P<0.05),并显著降低了丙二醛(MDA)含量(P<0.05);此外,BLfcin-NPs预处理还显著上调了BMECs内抗氧化相关基因核因子E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)和醌氧化还原酶1(NQO1)的mRNA相对表达量(P<0.05),同时显著上调了凋亡相关基因B淋巴细胞瘤-2(Bcl-2)的mRNA相对表达量(P<0.05),并显著下调了Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶-3(Caspase-3)的mRNA相对表达量(P<0.05)。综上所述,BLfcin-NPs通过调控Nrf2信号通路中关键基因的表达缓解H_(2)O_(2)诱导的BMECs氧化损伤。

This experiment used bovine lactoferrin chitosan nanoparticles(BLfcin-NPs)as an additive to explore its protective effect on hydrogen peroxide(H_(2)O_(2))-induced oxidative damage in dairy cow mammary epithelial cells(BMECs).Adopting a single-factor completely randomized experimental design,using BMECs as a model,treated with different concentrations of H_(2)O_(2)(0,100,200,400,600,800 and 1000μmol/L)for different treatment time(0,2,4,6,8 and 12 h),in order to determine the optimum H_(2)O_(2) concentration and action time for BMECs oxidative damage model.BMECs were pretreated for 12 h by 500μg/mL BLfcin-NPs,and then treated with 400μmol/L H_(2)O_(2) for 6 h.The mitochondrial membrane potential(MMP),antioxidant indexes,and the mRNA relative expression levels of antioxidant-related and apoptosis-related genes of BMECs were measured.The results showed as follows:1)compared the control group which treated with 0μmol/L H_(2)O_(2),different concentrations of H_(2)O_(2) treated 6 h significantly or extremely significantly decreased the viability of BMECs(P<0.05)and had cytotoxicity on cells,causing oxidative damage,and the apoptosis rate at the late stage was extremely significantly increased when BMECs treated with 400μmol/L H_(2)O_(2)(P<0.01).ROS content in BMECs was increased when BMECs treated 6 h with 400,600 and 800μmol/L H_(2)O_(2) in a dose dependent manner.2)BLfcin-NPs pretreatment significantly increased the decline ratio of MMP of oxidative damaged BMECs induced by H_(2)O_(2)(P<0.05),and at the same time significantly increased the activities of glutathione peroxidase(GSH-Px),catalase(CAT)and superoxide dismutase(SOD)(P<0.05)and significantly reduced malondialdehyde(MDA)content(P<0.05);moreover,BLfcin-NPs pretreatment significantly up-regulated the mRNA relative expression levels of antioxidant-related genes in BMECs such as nuclear factor red blood cell related factor 2(Nrf2),heme oxygenase-1(HO-1)and quinone oxidoretase-1(NQO-1)(P<0.05),significantly up-regulated the mRNA relative expression level of apoptosis-related gene B-cell lymphoma 2(Bcl2)(P<0.05),and significantly down-regulated the mRNA relative expression level of apoptosis-related genes Bcl2-associated X protein(Bax)and cysteinyl aspartate specific protease-3(Caspase-3)(P<0.05).In summary,BLfcin-NPs alleviate the oxidative damage caused by H_(2)O_(2) to BMECs by regulating the expression of key genes in the Nrf2 signaling pathway.