Jiao M J, Zhou L, Ren F, Wang Y D, Shen C, Duan Z P, Zhao C Y
Department of Infectious Diseases, Third Affiliated Hospital, Hebei Medical University, Shijiazhuang 050051,China.
Beijing Artificial Liver Treatment and Training Center, Beijing YouAn Hospital, Capital Medical University, Beijing 100069, China.
Zhonghua Gan Zang Bing Za Zhi. 2016 Dec 20;24(12):916-920. doi: 10.3760/cma.j.issn.1007-3418.2016.12.008.
To investigate the cellular and molecular mechanisms of the anti-inflammatory effect of peroxisome proliferator-activated receptor α (PPARα). Firstly, bone marrow-derived macrophages (BMDMs) were randomly divided into control group, LPS group, WY14643 10 μmol/L group, WY14643 25 μmol/L group, and WY14643 50 μmol/L group using a random number table. Secondly, BMDMs were randomly divided into LPS group, WY14643+LPS group, and 3-MA+WY14643+LPS group. Primary BMDMs were stimulated by LPS (20 ng/ml) to establish the cellular model of inflammation. The selective agonist of PPARα WY14643 was administered at doses of 10, 25, and 50 μmol/L (50 μmol/L for the second part of the experiment) at 2 hours before model establishment. The autophagy inhibitor 3-MA was administered at a dose of 10 mmol/L at 2 hours before model establishment. The cells in the control group were treated with dimethylsulfoxide (DMSO) at the same dose. The cells were transfected with GFP-LC3 plasmids at 24 hours before model establishment. The cells were harvested at 6 hours after LPS stimulation and related tests were performed. Green fluorescent protein was measured under a fluorescence microscope to evaluate autophagy activity. Quantitative real-time PCR was used to measure tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and mRNA expression of chemokine-1 (CXCL-1) and chemokine-10 (CXCL-10). Western blot was used to measure PPARα and autophagy-related proteins LC3, ATG-5, ATG-7, and LAMP-1. A one-way analysis of variance was used for comparison between groups, and the LSD-t test was used for comparison between any two groups. In vitro, PPARα activation inhibited LPS-induced inflammatory response in primary macrophages in a dose-dependent manner. The results of gene expression showed that the relative expression of TNF-α, IL-1β, IL-6, CXCL-1, and CXCL-10 was as follows in the control group, LPS group, WY14643 10 μmol group, WY14643 25 μmol group, and WY14643 50 μmol group: TNF-α (0.085±0.009, 4.065±0.544, 3.281±0.368, 1.780±0.293, and 0.781±0.303, < 0.01), IL-1β (0.081±0.017, 0.776±0.303, 0.225±0.154, 0.161±0.068, and 0.101±0.025, < 0.05), IL-6 (0.041±0.011, 0.189±0.014, 0.144±0.033, 0.126±0.013, and 0.048±0.015, < 0.01), CXCL-1 (0.051±0.011, 0.515±0.145, 0.356±0.078, 0.257±0.068, and 0.069±0.030, < 0.01), and CXCL-10 (0.126±0.068, 0.831±0.093, 0.508±0245, 0.474±0.047, and 0.204±0.021, < 0.05). In vitro, PPARα activation promoted autophagy in vitro in a dose-dependent manner. The results of Western blot and fluorescence microscopy in the control group, LPS group, WY14643 10 μmol group, WY14643 25 μmol group, and WY14643 50 μmol group showed that the expression of autophagy-related proteins and autophagosome formation gradually increased with the increasing concentration of WY14643. In vitro, WY14643 inhibited autophagy, promoted inflammatory response in primary macrophages, and reversed the anti-inflammatory effect of PPARα. The results of gene expression showed that the relative expression of TNF-α, IL-1β, IL-6, CXCL-1, and CXCL-10 was as follows in the LPS group, WY14643+LPS group, and 3-MA+WY14643+LPS group: TNFα (4.327±0.478, 1.218±0.424, and 3.901±0.447, < 0.05), IL-1β (4.277±0.407, 1.418±0.424, and 3.029±0.192, < 0.01), IL-6 (4.175±0.549, 1.373±0.499, and 4.031±0.475, < 0.05), CXCL-1 (8.199±1.149, 2.024±0.547, and 5.973±0.843, < 0.05), and CXCL-10 (1.208±0.148, 0.206±0.069, and 0.798±0.170, < 0.05). PPARα can promote cell autophagy and inhibit inflammatory response and may become a new therapeutic target for clinical prevention and treatment of inflammatory disease.
为研究过氧化物酶体增殖物激活受体α（PPARα）抗炎作用的细胞和分子机制。首先，采用随机数字表法将骨髓来源的巨噬细胞（BMDMs）随机分为对照组、LPS组、10 μmol/L WY14643组、25 μmol/L WY14643组和50 μmol/L WY14643组。其次，将BMDMs随机分为LPS组、WY14643 + LPS组和3 - MA + WY14643 + LPS组。用LPS（20 ng/ml）刺激原代BMDMs以建立炎症细胞模型。在模型建立前2小时，分别以10、25和50 μmol/L的剂量（实验第二部分为50 μmol/L）给予PPARα的选择性激动剂WY14643。在模型建立前2小时，以10 mmol/L的剂量给予自噬抑制剂3 - MA。对照组细胞用相同剂量的二甲基亚砜（DMSO）处理。在模型建立前24小时，将细胞用GFP - LC3质粒转染。在LPS刺激后6小时收获细胞并进行相关检测。在荧光显微镜下测量绿色荧光蛋白以评估自噬活性。采用定量实时PCR检测肿瘤坏死因子 - α（TNF - α）、白细胞介素 - 1β（IL - 1β）、白细胞介素 - 6（IL - 6）以及趋化因子 - 1（CXCL - 1）和趋化因子 - 10（CXCL - 10）的mRNA表达。采用蛋白质免疫印迹法检测PPARα以及自噬相关蛋白LC3、ATG - 5、ATG - 7和LAMP - 1。组间比较采用单因素方差分析，任意两组间比较采用LSD - t检验。体外实验中，PPARα激活以剂量依赖方式抑制LPS诱导的原代巨噬细胞炎症反应。基因表达结果显示，对照组、LPS组、10 μmol WY14643组、25 μmol WY14643组和50 μmol WY14643组中TNF - α、IL - 1β、IL - 6、CXCL - 1和CXCL - 10的相对表达分别为：TNF - α（0.085±0.009、4.065±0.544、3.281±0.368、1.780±0.293和0.781±0.303，P<0.01），IL - 1β（0.081±0.017、0.776±0.303、0.225±0.154、0.161±0.068和0.101±0.025，P<0.05），IL - 6（0.041±0.011、0.189±0.014、0.144±0.033、0.126±0.013和0.048±0.015，P<0.01），CXCL - 1（0.051±0.011、0.515±0.145、0.356±0.078、0.257±0.068和0.069±0.030，P<0.01），CXCL - 10（0.126±0.068、0.831±0.093、0.508±0.245、0.474±0.047和0.204±0.021，P<0.05）。体外实验中，PPARα激活以剂量依赖方式促进体外自噬。对照组、LPS组、10 μmol WY14643组、25 μmol WY14643组和50 μmol WY14643组的蛋白质免疫印迹和荧光显微镜结果显示，自噬相关蛋白的表达和自噬体形成随WY14643浓度增加而逐渐增加。体外实验中，WY14643抑制自噬，促进原代巨噬细胞炎症反应，并逆转PPARα的抗炎作用。基因表达结果显示，LPS组、WY14643 + LPS组和3 - MA + WY14643 + LPS组中TNF - α、IL - 1β、IL - 6、CXCL - 1和CXCL - 10的相对表达分别为：TNFα（4.327±0.478、1.218±0.424和3.901±0.447，P<0.05），IL - 1β（4.277±0.407、1.418±0.424和3.029±0.192，P<0.01），IL - 6（4.175±0.549、1.373±0.499和4.031±0.475，P<0.05），CXCL - 1（8.199±1.149、2.024±0.547和5.973±0.843，P<0.05），CXCL - 10（1.208±0.148、0.206±0.069和0.798±0.170，P<0.05）。PPARα可促进细胞自噬并抑制炎症反应，可能成为临床预防和治疗炎症性疾病的新治疗靶点。
