Resmetirom Ameliorates NASH-Model Mice by Suppressing STAT3 and NF-κB Signaling Pathways in an RGS5-Dependent Manner

Abstract

Resmetirom, a liver-directed, orally active agonist of THR-β, could play a favorable role in treating NASH, but little is known about the underlying mechanism. A NASH cell model was established to test the preventive effect of resmetirom on this disease in vitro. RNA-seq was used for screening, and rescue experiments were performed to validate the target gene of the drug. A NASH mouse model was used to further elucidate the role and the underlying mechanism of resmetirom. Resmetirom effectively eliminated lipid accumulation and decreased triglyceride (TG) levels. In addition, repressed RGS5 in the NASH model could be recovered by resmetirom treatment. The silencing of RGS5 effectively impaired the role of resmetirom. In the NASH mouse model, obvious gray hepatization, liver fibrosis and inflammation, and increased macrophage infiltration were observed in liver tissues, while resmetirom almost returned them to normal conditions as observed in the control group. Pathological experimental data also confirmed that resmetirom has great potential in NASH treatment. Finally, RGS5 expression was suppressed in the NASH mouse model, but it was upregulated by resmetirom treatment, while the STAT3 and NF-κB signaling pathways were activated in NASH but inhibited by the agent. Resmetirom could improve NASH by recovering RGS5 expression and subsequently inactivating the STAT3 and NF-κB signaling pathways.

Keywords: NASH; NF-κB; RGS5; STAT3; resmetirom.

Figure 1

Resmetirom effectively improved NASH in cell models generated using oleic acid. (A), HepG2 and NCTC 1469 cells were treated with the indicated doses of oleic acid for 48 h, and Oil red O staining experiment was performed to determine the intracellular lipid accumulation. (B), ELISA assay was performed to detect the intracellular TG levels in cells treated as in (A). (C), HepG2 and NCTC 1469 cells were treated with the indicated concentrations of resmetirom for 48 h, MTT assay was used to assess the cytotoxicity of the drug. (D), HepG2 and NCTC 1469 cells were pre-incubated with the indicated concentrations of resmetirom for 48 h, followed by another 48 h treatment with the indicated doses of oleic acid to establish the NASH cell model, and Oil red O staining experiment was performed to determine the intracellular lipid accumulation. (E), ELISA assay was performed to detect the intracellular TG levels in cells treated as in (D). Data are presented by mean ± SD for three independent experiments. * p < 0.05; ** p < 0.01 vs. control.

Figure 2

mRNA sequencing was performed to elucidate the mRNA expression profile in the resmetirom-treated NASH cell model. (A), HepG2 cells were pre-incubated with 0 or 100 μM resmetirom for 48 h, followed by treatment with 0- or 1.2-mM oleic acid for another 48 h, and Oil red O staining experiment was performed to determine the intervention effect of the drug. (B), Total RNA was extracted for mRNA-sequencing assay, differentially expressed genes (DEGs, fold change > 2, p < 0.05) were obtained, and volcano plots were generated using DEGs between NASH (NASH cell model) vs. control (left panel), and NASH_R (NASH cell model treated with 100 μM resmetirom) vs. NASH (right panel). (C), Heat maps were produced for DEGs between NASH vs. control (upper panel), and NASH_R vs. NASH (lower panel). (D), GO enrichment analysis was performed in DEGs between NASH vs. control (upper panel), and NASH_R vs. NASH (lower panel). (E), KEGG enrichment analysis was performed in DEGs between NASH vs. control (upper panel), and NASH_R vs. NASH (lower panel).

Figure 3

Resmetirom might ameliorate NASH in an RGS5-dependent manner. (A) The expressions of the top five DEGs were further validated by qPCR. (B), HepG2 cells were treated with indicated concentrations of oleic acid for 48 h, Western blot assay was used to determine the expression of RGS5 protein. The protein level was analyzed and is presented in the histogram in the right panel. (C), HepG2 cells were pre-incubated with 0 or 100 μM resmetirom for 48 h, followed by treatment with 0- or 1.2-mM oleic acid for another 48 h, and the indicated protein expressions were determined by Western blot assays. The protein level was analyzed and is presented in the histogram in the right panel. (D), HepG2 cells were transfected with RGS5 siRNA or NC siRNA for 24 h, and qPCR experiment was performed to examine the silence efficiency. (E), Total proteins were extracted in cells treated as in (D), and Western blot assay was performed to detect the RGS5 expression. The protein level was analyzed and is presented in the histogram in the right panel. (F), HepG2 cells were pre-incubated with 0 or 100 μM resmetirom for 48 h and transfected with RGS5 siRNA-2 or NC for 24 h, followed by treatment with 0- or 1.2-mM oleic acid for another 48 h, and Oil red O staining experiment was performed to determine the cellular lipid accumulation. (G), Total proteins were extracted in cells treated as in (F), and Western blot assay was performed to detect the expressions of the indicated proteins. The protein level was analyzed and is presented in the histogram in the right panel. Data are presented by mean ± SD for three independent experiments. * p < 0.05; ** p < 0.01 vs. control.

Figure 4

Establishment of NASH mouse models. (A), C57BL/6J mice (male, 6 weeks old) were fed with normal or AMLN diets, and body weights were measured every eight days. (B), After about 25 weeks feeding, one mouse from each group was sacrificed (left panel), livers were removed, and presented in right panel. (C), Serum was obtained from blood from caudal vein for detection of HDL-C, LDL-C, TG, and T-chol by ELISA assay. (D), Sections of livers from mice in B were used to perform the Oil red O staining assay to determine the lipid accumulation in liver tissue. (E), Sections of livers from mice in B were used in Masson staining experiment to confirm fibrosis status in liver tissues. (F), H&E staining was used to determine the pathological change in livers. (G), IHC assay was performed to confirm the pan-macrophage marker F4/80 in livers. (H), qPCR analysis of genes associated with liver fibrosis. (I), qPCR analysis of genes associated with inflammation. Data are presented by mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 vs. control.

Figure 5

Resmetirom significantly suppressed NASH in mouse model generated by AMLN diet. After finishing the establishment of the NASH mouse models, all mice were continually feeding as previously, while sixteen mice of NASH group were treated with either a low dose (3 mg/kg, eight mice) or high dose (5 mg/kg, eight mice) of resmetirom every day. (A), Body weights were measured every four days. (B), After 48 days of treatment, mice were sacrificed and the presented mouse for each group is shown. The arrows indicate the positions of the liver. (C), Livers were removed and presented. (D), The ratios of livers to body weights were calculated and presented. (E), IHC assay was performed to confirm the pan-macrophage marker F4/80 in livers, and the infiltration of macrophages is quantified and statistically analyzed in right panel. (F), qPCR analysis of genes associated with liver fibrosis. (G), qPCR analysis of genes associated with inflammation. Data are presented by mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 vs. control.

Figure 6

Resmetirom effectively inhibited lipid accumulation in NASH mouse model. (A), Sections of livers from mice in Figure 5 were used to perform the Oil red O staining assay to determine the lipid accumulation in liver tissue. (B), HE staining assay was used to confirm the pathological change in livers. (C), TUNEL assays were used to evaluate the cell apoptosis in liver tissues. (D), Masson staining assays were used to evaluate the fibrosis status in liver tissues. For all experiments, representative pictures are shown.

Figure 7

Resmetirom restored RGS5 expression and repressed the activation of STAT3 signaling pathway in NASH mouse model. (A), Sections of livers from mice in Figure 5 were used to perform the IHC assay to determine the RGS5 expression in liver tissue. Representative data are shown. (B), Total protein was extracted from livers in Figure 5, Western blot assay was performed to detect the expression of the indicated proteins. The protein level was analyzed and is presented in the histogram in the lower panel. (C), The potential underlying mechanism of how resmetirom improves NASH in a mouse model. Data are presented by mean ± SEM for three independent experiments. * p < 0.05; ** p < 0.01 vs. control.