Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry of Agriculture, College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot, 010018, China.
Department of Veterinary Medicine, College of Animal Science and Technology, Hebei North University, Zhangjiakou, 075131, Hebei, China.
Cell Stress Chaperones. 2021 Mar;26(2):387-401. doi: 10.1007/s12192-020-01184-z. Epub 2021 Jan 6.
Liver damage is the most severe complication of heat stress (HS). Hydrolyzed camel whey protein (CWP) possesses bioactive peptides with obviously antioxidant and anti-inflammatory activities. The current study aims to investigate whether CWP that is hydrolyzed by a simulated gastrointestinal digestion process, named S-CWP, protects BRL-3A hepatocytes from HS-induced damage via antioxidant and anti-inflammatory mechanisms. BRL-3A cells were pretreated with S-CWP before being treated at 43 °C for 1 h, and the levels of the cellular oxidative stress, inflammation, apoptosis, biomarkers for liver function, the activities of several antioxidant enzymes, and the cell viability were analyzed. The expression level of pivotal proteins in correlative signaling pathways was evaluated by western blotting. We confirmed that S-CWP alleviated HS-induced hepatocytes oxidative stress by decreased reactive oxygen species (ROS), nitric oxide (NO), 8-Hydroxy-2'-deoxyguanosine (8-OHdG), lipid peroxidation (LPO), protein carbonylation (PCO), and the activities of NADPH oxidase while enhanced superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), heme oxygenase-1 (HO-1) activities, and GSH content. S-CWP suppressed HS-induced inflammatory response by reducing the phosphorylation of NF-κB p65, the expression of NLRP3, and caspase-1 and finally alleviated caspase-3-mediated apoptosis. S-CWP also alleviated HS-induced hepatocyte injury by reducing alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels and restoring Heat Shock Protein 70 (HSP70) expression. Furthermore, S-CWP treatment significantly enhanced the expression of NF-E2-related nuclear factor erythroid-2 (Nrf2) and HO-1. The antioxidant and anti-inflammatory effects of S-CWP were weakened by ML385, a specific Nrf2 inhibitor. Additionally, zinc protoporphyrin (ZnPP), a specific HO-1 inhibitor, significantly reversed S-CWP-induced reduction in the phosphorylation of NF-κB p65. Thus, our results revealed that S-CWP protected against HS-induced hepatocytes damage via activating the Nrf2/HO-1 signaling pathway and inhibiting NF-κB/NLRP3 axis.
肝损伤是热应激（HS）最严重的并发症。水解骆驼乳清蛋白（CWP）具有生物活性肽，具有明显的抗氧化和抗炎活性。本研究旨在探讨经模拟胃肠消化过程水解的 CWP（S-CWP）是否通过抗氧化和抗炎机制保护 BRL-3A 肝细胞免受 HS 诱导的损伤。在将 BRL-3A 细胞用 43°C 处理 1 小时之前，用 S-CWP 预处理细胞，并分析细胞氧化应激、炎症、细胞凋亡、肝功能生物标志物、几种抗氧化酶的活性以及细胞活力。通过蛋白质印迹法评估相关信号通路中关键蛋白的表达水平。我们证实 S-CWP 通过降低活性氧（ROS）、一氧化氮（NO）、8-羟基-2'-脱氧鸟苷（8-OHdG）、脂质过氧化（LPO）、蛋白羰基化（PCO）和 NADPH 氧化酶的活性来减轻 HS 诱导的肝细胞氧化应激，同时增强超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）、血红素加氧酶-1（HO-1）的活性和 GSH 含量。S-CWP 通过降低 NF-κB p65 的磷酸化、NLRP3 和半胱天冬酶-1 的表达，最终减轻半胱天冬酶-3 介导的细胞凋亡，从而抑制 HS 诱导的炎症反应。S-CWP 还通过降低丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）和碱性磷酸酶（ALP）水平和恢复热休克蛋白 70（HSP70）表达来减轻 HS 诱导的肝细胞损伤。此外，S-CWP 处理显著增强了 NF-E2 相关核因子红细胞-2（Nrf2）和 HO-1 的表达。特异性 Nrf2 抑制剂 ML385 削弱了 S-CWP 的抗氧化和抗炎作用。此外，特异性 HO-1 抑制剂锌原卟啉（ZnPP）显著逆转了 S-CWP 诱导的 NF-κB p65 磷酸化减少。因此，我们的结果表明，S-CWP 通过激活 Nrf2/HO-1 信号通路和抑制 NF-κB/NLRP3 轴来保护细胞免受 HS 诱导的损伤。
