Fig 2: DAT displays anti-inflammatory and antiviral effects in vitro. A The fecal DAT levels of mice treated with vehicle (PBS) or TFA for 1 week were measured by mass spectroscopy (n = 3 mice per group). MH-S cells were untreated (PBS) or treated with DAT and then infected with PR8 (MOI = 0.01). The mRNA levels of (B) viral, (C) IL-1β, (D) IL-6, (E) TNF-α, (F) IFN-β, (G) Mx2, (H) Oas1, and (I) Oas2 were determined by real-time PCR. β-actin was used as a control. J Phosphorylated and total levels of p38, ERK1/2, and JNK were measured in MH-S cells by western blot. K Immunoblotting of RIG-I, MAVS, total and phosphorylated TBK1, and IRF3 measured in MH-S cells. The intensities of bands relative to the control were measured with ImageJ software, and the results are shown below. The value are shown as means ± SD of three individual trials. *P < 0.05, **P < 0.01

Fig 3: Mitochondrial uncoupler CCCP inhibits type I IFN responses. (A) 293T cells were transfected with RFP-Parkin alone or together with GFP-Mito, 24 h post-transfection, the cells were untreated or treated with CCCP (10 μM) for 6 h and subjected to IF analysis. Scale bar is 10 μm. (B) HEK293 cells were transfected with a luciferase reporter of the IFN-β promoter, ISRE promoter, and NF-κB promoter. 24 h post-transfection, the cells were untreated or treated with CCCP (10 μM) for 2 h, then left unstimulated or stimulated with SeV for 10 h, and luciferase assays were performed. (C) HEK293 cells were untreated or treated with CCCP (10 μM) for different times, unstimulated or stimulated with SeV for 10 h. Then quantitative RT-PCR analysis of IFNB1, ISG15, and IFIT1 mRNA. (D) HEK293 cells were untreated or treated with different concentrations of CCCP for 2 h, then unstimulated or stimulated with SeV for 10 h, and subjected to quantitative RT-PCR analysis. (E) HEK293 cells were co-transfected with IFN-β luciferase reporter and RIG-I-N, MDA5-N, MAVS, TBK1, IKKε, IRF3, or IRF3-5D for 24 h, then untreated or treated with CCCP (10 μM) for 12 h, and subjected to luciferase assays. (F) HEK293 cells were untreated or treated with CCCP (10 μM) for 2 h, then unstimulated or stimulated with SeV for different times, and the whole cell extracts were subjected to IB analysis. The data represent the average of three independent experiments and were analyzed by unpaired t-test. All data represent the mean ± S.D. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 4: HSP27 enhances encephalomyocarditis virus (EMCV)-triggered RIG-I-like receptor (RLR)/melanoma differentiation-associated gene 5 (MDA5) signaling pathway. (A) A549 cells were transfected with either 150 nmol of siHSP27-Mix or siNC for 36 h before infecting with EMCV at an multiplicity of infection (MOI) of 0.01 for 36 h. The protein expression of MDA5, MAVS, TBK1, p-TBK1, IRF3, p-IRF3, HSP27, and VP1 were analyzed by immunoblotting. GAPDH was used as a loading control. (B) A549 cells were transfected with pCMV-Myc or Myc-HSP27 (0.5 µg) for 24 h before infecting with EMCV at an MOI of 0.01 for 36 h. The protein expression of MDA5, MAVS, TBK1, p-TBK1, IRF3, p-IRF3, Myc, and VP1 were analyzed by immunoblotting. GAPDH was used as a loading control.

Fig 5: HET0016 as a lipid peroxidation inhibition protects against sepsis.A Levels of plasma MDA from healthy control or patients. B Spearman correlation analysis for plasma MDA and D-lactate or APACHE II score in all patients (n = 28). C Kaplan–Meier analysis according to a cutoff value calculated from ROC analysis. D Representative histopathological section images, immunohistochemical or fluorescent images in the intestinal tissues of patients with sepsis. The scale bar represents 50 µm. The quantified fluorescence intensity of 4-HNE was shown in the lower left corner. E Survival analysis of the indicated mice in CLP-induced sepsis with or without treatment of 10 mg/kg HET0016, 20 mg/kg JSH-23, or 10 mg/kg Carnosol at 2 h before CLP and 12, 24, 48, and 72 h after CLP (n = 15 per group). F Immunoblot analysis of p-TBK1, TBK1, p-P65, and P65 in PBMCs from patients with sepsis or healthy control, supplemented with or without HET0016 (5 µM) for 24 h (n = 4 per group). G qPCR analysis of Il-6, Il-1b, and Tnf mRNA in PBMCs from patients with sepsis or healthy control, supplemented with or without HET0016 (5 µM) for 24 h (n = 6 per group). H Production of MDA in PBMCs from patients with sepsis supplemented with or without HET0016 (5 µM) for 24 h (n = 6 per group). Data are shown as mean ± SD, and analysis by one-way ANOVA test. Data in (G, H) are analyzed by paired Student’s t-test. PBMCs peripheral blood mononuclear cells, MODS multiple organ dysfunction syndromes, MDA malondialdehyde, FTH ferritin heavy chain, GPX4 glutathione peroxidase 4, 4-HNE 4-hydroxynonenal. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.