基于ROS-MAPK-NFκb通路探究黄芩苷正丁酯缓解断奶仔猪肠炎的分子机制

In this project, the enteritis model of weaned piglets is replicated by LPS, and the n-butyl ester of baicalin (low, medium and high dose) is used for the treatment of enteritis. The jejunum of the weaned piglets is selected to measure the secretion of inflammatory factors, the activities of antioxidant enzyme and MAPK protein expression. IPEC-J2 cells are treated to establish the complex model of intestinal inflammation and oxidative stress with LPS and H2O2. The oxidative stress responses (including the ROS and MDA content, Nrf2 protein in the nucleus, HO-1 and NQO 1 protein expression), the MAPK protein expression (ERK, JNK and P38 protein expression and its phosphorylation) and the inflammation responses (including TLR4 protein expression, P65 protein expression in the nucleus and its phosphorylation, inflammation cytokines expression such as TNF-α，IL-1β and IL-6) will be detected by several molecular biology technologies in IPEC-J2 cells. In the complex model, H2O2 or NAC will be added to prove that ROS regulates the expression of MAPK by detecting the protein expression of MAPK, MAPK inhibitors will be added to prove that MAPK regulates the NF-κb signaling pathway by detecting P65 protein expression and the secretion of inflammatory factors. In the complex model, the n-butyl ester of baicalin (low, medium and high dose) will be added to prove its pharmacology function by detecting the oxidative stress, MAPK expression and inflammatory responses in IPEC-J2 cells. The MAPK protein expression in the jejunum of piglets will be also measured for verification. This project attempts to to explain the mediation of MAPK protein in the process of inflammation and oxidative stress, and the molecular mechanism of the n-butyl ester of baicalin regulating intestinal inflammation and oxidative stress responses by ROS-MAPK-NFκb signal pathway in weaned piglets.

本项目用LPS复制断奶仔猪肠炎模型，并低、中、高浓度的黄芩苷正丁酯给予治疗，检测断奶仔猪空肠的炎性因子、抗氧化物酶活性以及MAPK蛋白的表达；用LPS和H2O2处理猪肠道上皮细胞（IPEC-J2）建立炎症氧化应激复合模型，用分子生物学等手段检测IPEC-J2细胞氧化应激，MAPK的表达以及炎症反应。在模型中添加H2O2或NAC检测细胞MAPK的表达证明ROS对MAPK的调控作用；添加MAPK抑制剂检测下游P65蛋白磷酸化、入核及炎性因子的表达证明MAPK对NF-κb信号通路的调控。在复合模型中添加低、中、高剂量黄芩苷正丁酯，检测黄芩苷正丁酯对IPEC-J2细胞的氧化应激、MAPK表达和炎症反应的调控。本项目试图解释MAPK在炎症和氧化应激中的作用以及黄芩苷正丁酯基于ROS-MAPK-NFκb信号通路对断奶仔猪肠道炎症和氧化应激的调控机理。

断奶仔猪应激是制约规模化养猪业发展的关键问题，仔猪断奶应激会伴随氧化应激、炎症反应、肠道屏障功能损伤。为探明黄芩苷正丁酯对断奶仔猪肠道氧化应激和炎症反应的调控及其分子作用机制，本项目从以下四个方面展开研究：MAPK 信号通路在IPEC-J2细胞氧化应激和炎症反应的调控作用；黄芩苷对肠炎模型猪IPEC-J2细胞和小鼠空肠抗炎抗氧化作用及机制；TNF-a诱导MODE-K细胞发生氧化应激及黄芩苷的调控作用；黄芩苷正丁酯对肠炎模型小鼠空肠和猪IPEC-J2细胞的抗炎抗氧化作用及分子机制。.LPS刺激IPEC-J2细胞后，细胞内产生了大量的活性氧（ROS），激活细胞的炎症反应和氧化应激，ROS含量下降后细胞炎症反应降低，ROS可通过下调MAPK家族蛋白中JNK、ERK、P38蛋白，阻断P38蛋白磷酸化的方式，减缓IPEC-J2细胞的炎症反应，表明MAPK 信号通路介导氧化应激和炎症反应。在LPS构建的小鼠空肠炎症模型中，黄芩苷（推荐剂量200 mg/kg BW）可通过抑制NF-κB信号通路发挥抗炎症作用，通过激活Nrf2信号通路发挥抗氧化作用。LPS刺激的IPEC-J2细胞后，黄芩苷可通过下调ROS/ERK/P-P38、抑制NF-κB的信号通路在IPEC-J2细胞发挥抗炎作用，通过激活Nrf2/HO1信号通路发挥抗炎、抗氧化作用。在LPS诱导的MODE-K细胞条件下中，黄芩苷经TLR4/TNF-α/ROS/HO1途径调控细胞的炎症反应和氧化应激进程。本项目制备了黄芩苷正丁酯，经薄层色谱、高效液相色谱、液质联用等方式鉴定纯度为纯度为97%。在LPS诱导的肠炎模型小鼠十二指肠和空肠，黄芩苷正丁酯可以通过TLR4/NF-κB信号发挥抗炎作用, 在猪IPEC-J2细胞内，黄芩苷正丁酯可通过ROS/Nrf2/HO1信号通路发挥抗氧化作用，表明黄芩苷正丁酯通过TLR4/NF-κB和ROS/Nrf2/HO1信号通路对肠炎模型小鼠空肠和猪IPEC-J2细胞发挥抗炎抗氧化作用。.本文阐释了ROS/MAPK信号通路，TNF-α/ROS/HO1途径在断奶仔猪肠道中氧化应激和炎症反应间的作用，探明了黄芩苷和黄芩苷正丁酯对肠道抗炎、抗氧化的作用机制，这为临床治疗仔猪早期断奶应激综合征奠定了理论依据。