Fluorofenidone Attenuates BLM-induced Pulmonary Inflammation And Fibrosis In Mice Via Inhibiting The NALP3/IL-1R1/MyD88Signaling Pathway

Objective:To determine whether FD can attenuate bleomycin-induced pulmonary inflammation and fibrosis through suppressing activation of NALP3inflammasome and IL-1R1/MyD88pathway, and exert its mechanisms of aitifibrotic effects.Methods:1. Eighty male adult mice (C57BL/6) were randomly divided into five groups as the following (each group includes sixteen mice):the control group (Control), the bleomycin group (BLM), the BLM+fluorofenidone group (FD), the BLM+Z-YVAD-FMK group (Cpl inhibitor) and the BLM+Anakinet group (IL-1Ra).2. The model of pulmonary fibrosis was established through intra-tracheally instillation with bleomycin (5mg/kg) except Control group by instillation the same dose of normal saline.24hs before the establishment of the model, the FD group was forced fed by fluorofenidone (500mg/kg, once per day), the Cp1inhibitor group was intraperitoneal injection with YVAD-FMK (10mg/kg) at8hs and4hs before the establishment of the model, the IL-1Ra group was intraperitoneal injection with Anakinet (100ug, twice per day). Stochastically eight mice in each group were killed separately on the second and14th day after instillation. Their pathological section of left lung tissues were harvested for hemotoxylin and eosin stain and Masson’s trichrome stain, the right lung tissues were preserved by liquid nitrogen. The IL-1β, MCP-1, MPO, IL-6, α-SMA, Fibronectin, Collagen I mRNA expressions were detected by Real-time PCR. The IL-1β, MCP-1, MPO, IL-6protein expressions were detected by ELISA. The caspase1, IL-1R1, MyD88, α-SMA, Fibronectin protein expressions were detected by Western Blot.Results:1. Compared with the control group, BLM group presents a large amount of inflammatory cells infiltrated in the pulmonary tissue, and the mean inflammation score is significantly higher than the control group on the second day and14th day after treated with BLM (P<0.001). The number of inflammatory cells infiltrated and the mean inflammation score are both decreased after FD, Cpl inhibitor, IL-1Ra treatment (P<0.001).2. On the time point of14th day, compared with the control group, BLM group exerts excessive deposition of mature collagen in the interstitium, distorted pulmonary architecture, and higher the mean fibrosis score (P<0.001). After treatment of FD, Cpl inhibitor, IL-1Ra, the deposition of collagen is significantly reduced, and the mean fibrosis score declines (P<0.001), and there is no significant difference among the three treatment group..3. ELISA and/or Real-time PCR show that compared with control group, the protein and mRNA levels of IL-1β\MCP-1, MPO, IL-6are elevated in BLM group on second day and14th day after treatment with BLM (P<0.001). Through treatment of FD, Cp1inhibitor, IL-1Ra, the protein and mRNA levels of IL-1β, MCP-1, MPO, IL-6descend significantly (P<0.05), and on the protein level of IL-6, the inhibiting effect of FD is superior than that of Cp1inhibitor (P<0.05).4. Western and/or Real time PCR show that compared with the control group, the protein and mRNA levels of a-SMA, Fibronectin, Collagen I are significantly enhanced in BLM group on the14day (P<0.01). The protein and mRNA levels of a-SMA, Fibronectin, Collagen I are significantly decreased when aiministration with FD, Cpl inhibitor, or Il-1Ra (P<0.05), and on the protein levels of a-SMA and Fibronectin, the inhibiting effect of FD is superior than that of Cp1inhibitor (P<0.05).5. Western blot shows enhanced levels of active casepasel, IL-1R1and MyD88proteins in BLM group when compared with the control group onsecond day and14th day after treated with BLM (P<0.001) The level of active caspasel both decrease significantly after FD and Cpl inhibitor treatment (P<0.01), and there is no significant difference between two treatment group. The levels of IL-1R1, MyD88proteins decline after FD and IL-1Ra treatment (P<0.01), and there is no significant difference between two treatment group. However, the expression of active caspasel protein cannot be inhibited through I1-1Ra treatment (P>0.05), and the levels IL-1R1, MyD88protein are not reduced after Cp1inhibitor treatment (P>0.05)Figures:40, Tables:19, References:37.Conclusions:1. Fluorofenidone attenuates BLM-induced pulmonary inflammation and fibrosis of mice.2. Fluorofenidone attenuates BLM-induced pulmonary inflammation and fibrosis of mice through inhibiting the NALP3/IL-1R1/MyD88signaling pathway.