Remdesivir attenuates high fat diet (HFD)-induced NAFLD by regulating hepatocyte dyslipidemia and inflammation via the suppression of STING

Abstract

High-fat diet (HFD) is a predisposing factor for metabolic syndrome-related systemic inflammation and non-alcoholic fatty liver disease (NAFLD). However, there is still no effective therapeutic treatment for NAFLD. Here, we showed that remdesivir (RDV, GS-5734), as a broad-spectrum antiviral nucleotide prodrug with anti-inflammatory effects, was effective for attenuating HFD-induced metabolic disorder and insulin resistance. Results revealed that the liver weight, hepatic dysfunction and lipid accumulation were markedly increased compared with that of the Control group, while that of the RDV group exhibited significant reduction, accompanied by the improved signaling pathway regulating fatty acid metabolism. In agreement with reduced lipid deposition, RDV supplementation suppressed the systematic and hepatic inflammation, as evidenced by reduction of inflammatory cytokines and the blockage of nuclear factor κB (NF-κB) signaling. In addition, stimulator of interferon genes (STING) and its down-streaming factor interferon regulatory factor 3 (IRF3) were greatly increased in livers of HFD-fed mice, which were considerably restrained by RDV treatment. The in vitro analysis suggested that RDV functioned as an inhibitor of STING, contributing to the suppression of dyslipidemia and inflammation induced by palmitate (PA). However, PA-triggered lipid deposition and inflammatory response was further accelerated in hepatocytes with STING over-expression. Notably, RDV-attenuated lipid disorder and inflammation were significantly abrogated by the over-expression of STING in PA-stimulated hepatocytes. Taken together, these findings indicated that RDV exhibited protective effects against NAFLD development mainly through repressing STING signaling, and thus could be considered as a potential therapeutic strategy.

Keywords: Dyslipidemia; Inflammation; NAFLD; Remdesivir; STING.

Fig. 1

Remdesivir attenuates metabolic syndrome in HFD-fed mice. (A) Body weight of mice. (B) Fat mass calculation. (C) Total energy intake in each group. (D) Assessments of serum leptin. (E) H&E staining of white adipose tissue sections. Scale bar, 100 μm. (F) Average adipocyte area in white adipose tissue. Measurements of (G) fasting blood glucose and (H) insulin levels. (I) Results for HOMA-IR in each group. (J) OGTT (left panel) and ITT (right panel) results for the determination of insulin resistance. (K) Serum endotoxin was tested. Data are means ± SEM (n = 9/group). ∗p < 0.05 and ∗∗p < 0.01 versus the Con group; ⁺p < 0.05 and ⁺⁺p < 0.01 versus the HFD group.

Fig. 2

Remdesivir alleviates hepatic function and lipid accumulation in HFD-induced mice. (A) Liver weight of mice. (B) The ratio of liver weight to body weight. (C) AST, ALT, (D) TG, TC, (E) LDL-C and HDL-C contents in serum were measured. (F) Liver TG, TC and NEFA levels were assessed. (G) H&E (up panel) and Oil Red O (down panel) staining for hepatic sections. Scale bar, 50 μm. (H) Results for inflammation score, ballooning score and steatosis score. (I) RT-qPCR results for genes in liver associated with fatty acid synthesis, uptake and β-oxidation. (J) Western blot analysis for proteins associated with fatty acid metabolism in liver samples. Data are means ± SEM (n = 6/group). ∗p < 0.05 and ∗∗p < 0.01 versus the Con group; ⁺p < 0.05 and ⁺⁺p < 0.01 versus the HFD group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Remdesivir inhibits inflammatory response in liver of HFD-fed mice. (A) Serum inflammatory factors were measured by ELISA. (B) RT-qPCR analysis of genes associated with inflammation in liver tissues. (C) IHC staining of F4/80 in hepatic sections. Scale bar, 50 μm. (D) RT-qPCR results for STING in liver tissues. (E) Western blot results for STING, p-TBK1, p-IRF3, IFN-β and p-NF-κB in hepatic samples. (F–I) Kupffer cells and hepatocytes were treated with PA (250 μM) or LPS (100 ng/ml) for 24 h in the absence or presence of RDV (10 μM). Then, all cells were collected for the following analysis. (F,G) RT-qPCR analysis of genes related to inflammatory factors in cells. (H,I) Western blotting results for STING protein expression levels in the treated cells. Data are means ± SEM (n = 6 in each group for in vivo study; n = 3 in each group for in vitro study). ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001 versus the Con group; ⁺p < 0.05 and ⁺⁺p < 0.01 versus the HFD, PA or LPS group.

Fig. 4

Remdesivir-regulated lipid accumulation and inflammation is dependent on STING in PA-incubated hepatocytes. (A) The hepatocytes were transfected with siSTING for 24 h, followed by PA (250 μM) treatment for another 24 h. Western blot analysis was then used for transfection efficacy determination. (B–E) The hepatocytes were transfected with siSTING for 24 h, followed by PA (250 μM) treatment for another 24 h with or without RDV (10 μM). (B) Oil Red O staining of cells. Scale bar, 25 μm. RT-qPCR measurements of genes associated with (C) lipid metabolism and (D) inflammatory response. (E) Western blot results for p-TBK1, p-IRF3, IFN-β and p-NF-κB in the treated hepatocytes. (F) The hepatocytes were transfected with oeSTING for 24 h to over-express STING, and then were exposed to PA (250 μM) for another 24 h. Western blotting was applied for transfection efficiency calculation. (G) Oil Red O staining of hepatocytes. Scale bar, 25 μm. The mRNA expression levels of genes associated with (H) lipid metabolism and (I) inflammatory response were assessed by RT-qPCR. (J) Western blot results for p-TBK1, p-IRF3, IFN-β and p-NF-κB in the treated hepatocytes. Data are means ± SEM (n = 3/group). ∗∗p < 0.01 and ∗∗∗p < 0.001 versus the Con group; ⁺p < 0.05, ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001 versus the PA group; ^#p < 0.05 and ^##p < 0.01 versus the PA + RDV group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)