[Cellular and molecular mechanisms of anti-inflammatory effect of peroxisome proliferator-activated receptor α]

Objective: To investigate the cellular and molecular mechanisms of the anti-inflammatory effect of peroxisome proliferator-activated receptor α (PPARα). Methods: 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. Results: 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, P < 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, P < 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, P < 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, P < 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, P < 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, P < 0.05), IL-1β (4.277±0.407, 1.418±0.424, and 3.029±0.192, P < 0.01), IL-6 (4.175±0.549, 1.373±0.499, and 4.031±0.475, P < 0.05), CXCL-1 (8.199±1.149, 2.024±0.547, and 5.973±0.843, P < 0.05), and CXCL-10 (1.208±0.148, 0.206±0.069, and 0.798±0.170, P < 0.05). Conclusion: PPARα can promote cell autophagy and inhibit inflammatory response and may become a new therapeutic target for clinical prevention and treatment of inflammatory disease.