Repurposing of the analgesic Neurotropin for MASLD/MASH treatment

Abstract

Background: The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has increased in recent decades. Approximately 25% of patients with MASLD progress to metabolic dysfunction-associated steatohepatitis, which is characterized by hepatic steatosis plus hepatocyte damage, inflammation, and fibrosis. We previously reported that Neurotropin (NTP), a drug used for relieving pain in Japan and China, inhibits lipid accumulation in hepatocytes by preventing mitochondrial dysfunction. We hypothesized that inhibiting hepatic steatosis and inflammation by NTP can be an effective strategy for treating MASLD and tested this hypothesis in a MASLD mouse model.

Methods: Six-week-old C57BL/6NJ male mice were fed a normal diet and normal drinking water or a high-fat diet with high fructose/glucose water for 12 weeks. During the last 6 weeks, the mice were also given high-dose NTP, low-dose NTP, or control treatment. Histologic, biochemical, and functional tests were conducted. MitoPlex, a new proteomic platform, was used to measure mitochondrial proteins, as mitochondrial dysfunction was previously reported to be associated with MASLD progression.

Results: NTP inhibited the development of hepatic steatosis, injury, inflammation, and fibrosis induced by feeding a high-fat diet plus high fructose/glucose in drinking water. NTP also inhibited HSC activation. MitoPlex analysis revealed that NTP upregulated the expression of mitochondrial proteins related to oxidative phosphorylation, the tricarboxylic acid cycle, mitochondrial dynamics, and fatty acid transport.

Conclusions: Our results indicate that NTP prevents the development of hepatic steatosis, injury, and inflammation by preserving mitochondrial function in the liver and inhibits liver fibrosis by suppressing HSC activation. Thus, repurposing NTP may be a beneficial option for treating MASLD/metabolic dysfunction-associated steatohepatitis.

FIGURE 1

Hepatic steatosis and injury are reduced by NTP treatment in a mouse MASLD/MASH model. (A) Protocol for the mouse MASLD/MASH model. Mice were fed a normal chow diet with normal drinking water (ND/NW) or a high-fat diet with high fructose/glucose drinking water (HFD/HFGW) for a total of 12 weeks. After 6 weeks of diet feeding, the mice also received vehicle control, OCA (30 mg/kg/d), low-dose NTP (NTP-L; 50 NU/kg/d), or high-dose NTP (NTP-H;100 NU/kg/d) for an additional 6 weeks. N = 9–10 in each group. (B) Body weight changes during the 12-week experimental period. (C) Liver weight at week 12. (D, E) Serum ALT and AST levels at week 12. (F) Hematoxylin & eosin staining of liver tissues. Representative pictures are shown. Data are shown as mean ± SD. Significance was determined using 2-way ANOVA, with the Bonferroni post hoc analysis. **p < 0.005. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; MASH, metabolic dysfunction–associated steatohepatitis; MASLD, metabolic dysfunction–associated steatotic liver disease; NTP, Neurotropin; NU, Neurotropin units; OCA, obeticholic acid.

FIGURE 2

Hepatic lipogenesis is suppressed by NTP treatment in a mouse MASLD/MASH model. Mice were fed a normal chow diet with normal drinking water (ND/NW) or a high-fat diet with high fructose/glucose drinking water (HFD/HFGW) for 12 weeks. Vehicle, OCA, or NTP was administered for the last 6 weeks. N = 9–10 per group. (A) Oil red O staining of liver tissues. Representative pictures are shown. (B) Quantification of Oil Red O–positive areas at week 12. (C) Liver triglyceride levels at week 12. (D–G) Hepatic Scd, Srebf1, Dgat1, and Dgat2 mRNA expression at week 12 was determined by quantitative real-time PCR. Data are shown as mean ± SD. Significance was determined using 2-way ANOVA, with Bonferroni post hoc analysis. *p < 0.05, **p < 0.005. Abbreviations: MASH, metabolic dysfunction–associated steatohepatitis; MASLD, metabolic dysfunction–associated steatotic liver disease; NTP, Neurotropin; NTP-H, high-dose Neurotropin; NTP-L, low-dose Neurotropin; NU, Neurotropin units; OCA, obeticholic acid; pos., positive.

FIGURE 3

NTP treatment suppresses MASLD/MASH-induced hepatic inflammation in mice. Mice were fed a normal chow diet with normal drinking water (ND/NW) or a high-fat diet with high fructose/glucose drinking water (HFD/HFGW) for 12 weeks. Vehicle, OCA, or NTP was administered for the last 6 weeks. N = 9–10 per group. (A) Immunofluorescence results of liver tissues at week 12, with staining for F4/80 shown in red and for DNA (Hoechst stain) shown in blue. Representative pictures are shown. (B) Quantification of F4/80 staining at week 12. (C–G) Hepatic Cxcl1, Ccl2, Cxcl5, Tnf, and Il6 mRNA expression at week 12 was determined by quantitative real-time PCR. Data are shown as mean ± SD. Significance was determined using 2-way ANOVA, with the Bonferroni post hoc analysis. *p < 0.05, **p < 0.005. Abbreviations: MASH, metabolic dysfunction–associated steatohepatitis; MASLD, metabolic dysfunction–associated steatotic liver disease; NTP, Neurotropin; NTP-H, high-dose Neurotropin; NTP-L, low-dose Neurotropin; NU, Neurotropin units; OCA, obeticholic acid; pos., positive.

FIGURE 4

NTP treatment inhibits MASLD/MASH-induced intracellular inflammatory pathways in mice. Protein expression was examined using the lysates from livers of mice fed a normal chow diet with normal drinking water (ND/NW) or a high-fat diet with high fructose/glucose drinking water (HFD/HFGW) for 12 weeks, with treatment of Vehicle control or NTP for the last 6 weeks. Immunoblots for phospho-JNK, total JNK, phospho-ERK, total ERK, phospho-p38, total p38, and β-actin. Three representative protein lysates of each condition were used for immunoblotting. Representative images of 3 independent experiments are shown. Abbreviations: ERK, extracellular signal-regulated kinase; JNK, Jun kinase; MASH, metabolic dysfunction–associated steatohepatitis; MASLD, metabolic dysfunction–associated steatotic liver disease; NTP, Neurotropin.

FIGURE 5

NTP treatment reduces MASLD/MASH-induced hepatic fibrogenesis in mice. Mice were fed a normal chow diet with normal drinking water (ND/NW) or a high-fat diet with high fructose/glucose drinking water (HFD/HFGW) for 12 weeks. Vehicle, OCA, or NTP was administered for the last 6 weeks. N = 9–10 per group. (A) Reticulin staining of liver tissues at 12 weeks. Stained collagen is shown in black. Representative pictures are shown. (B) Quantification of reticulin staining at 12 weeks. (C–G) Hepatic Col1a1, Col3a1, Col4a1, Timp1, and Tgfb1 mRNA expression at 12 weeks was determined by quantitative real-time PCR. Data are shown as mean ± SD. Significance was determined using 2-way ANOVA, with the Bonferroni analysis. *p < 0.05, **p < 0.005. Abbreviations: MASH, metabolic dysfunction–associated steatohepatitis; MASLD, metabolic dysfunction–associated steatotic liver disease; NTP, Neurotropin; NTP-H, high-dose Neurotropin; NTP-L, low-dose Neurotropin; NU, Neurotropin units; OCA, obeticholic acid; pos., positive.

FIGURE 6

TGF-β–induced fibrogenic response is inhibited by NTP treatment in primary HSCs. Mouse primary HSCs were isolated from Col1a1-green fluorescent protein (GFP) reporter transgenic mice. One hour after pretreatment with vehicle or NTP (0.2 or 0.4 NU/mL), HSCs were treated with 5 ng/mL TGF-β for 24 hours. (A) GFP signals and immunofluorescent staining for α-SMA were visualized by fluorescent microscopy. Representative pictures are shown. (B) GFP fluorescence intensity was measured by FACS. Representative results from 3 independent experiments are shown. (C–E) Acta2, Col1a1, and Serpine1 mRNA expression was determined by quantitative real-time PCR. N = 5 in each group. Data are shown as mean ± SD. Significance was determined using 2-way ANOVA with the Bonferroni post hoc analysis. *p < 0.05, **p < 0.005. Representative results from 3 independent experiments are shown. Abbreviations: NTP, Neurotropin; NU, Neurotropin units; OCA, obeticholic acid; pos., positive.

FIGURE 7

Altered expression of proteins and genes related to mitochondrial function in MASLD/MASH is preserved by NTP treatment. (A) Mitochondrial protein expression was measured using the MitoPlex platform. Protein extracts from liver tissues of our mouse MASLD/MASH model were used. The mice were treated with vehicle, OCA, or NTP. N = 5 per group. The heatmap visualized the 25 top-ranked proteins based on 1-way ANOVA. (B) Comparison of Log2-fold changes in 31 mitochondrial proteins in the liver tissues of mice treated with NTP, compared to vehicle control, as reported by MitoPlex. Significance was determined using an unpaired Student t test. *p < 0.05, ***p < 0.001. N = 5 per group. (C–F) Hepatic mRNA expression of Ppargc1b, Ogg1, Cox7c, and Cox8c was determined by quantitative real-time PCR. N = 4–5 per group. Data are shown as mean ± SD. Significance was determined using 2-way ANOVA with the Bonferroni post hoc analysis. *p < 0.05, **p < 0.005. (G) Immunoblots for PGC-1β, phospho-AMPKα, total AMPKα, and β-actin using the lysates from liver tissues of our mouse MASLD/MASH model. Three representative protein lysates of each condition were used for immunoblotting. Representative images of 3 independent experiments are shown. Abbreviations: ETC, electron transport chain; HFD/HFGW, high-fat diet with high fructose/glucose drinking water; MASH, metabolic dysfunction–associated steatohepatitis; MASLD, metabolic dysfunction–associated steatotic liver disease; ND/NW, normal chow diet with normal drinking; NTP, Neurotropin; NTP-H, high-dose Neurotropin; OCA, obeticholic acid; TCA, tricarboxylic acid.

FIGURE 8

NTP prevents lipid accumulation and improves mitochondrial function in human hepatocytes. Primary human hepatocytes were pretreated with vehicle or NTP (0.2 or 0.4 NU/mL) for 1 hour, followed by treatment with 300 μM palmitic acid (PA) and 300 μM oleic acid (OA) or control (BSA) for an additional 24 hours. Cellular lipid accumulation was examined using Oil Red O staining. (A) Representative microscopic images of Oil Red O staining. (B) Quantification of Oil Red O staining. Data are shown as mean ± SD of 8 high-power fields (×200). Significance was determined using 2-way ANOVA with the Bonferroni post hoc analysis. *p < 0.05, **p < 0.005. Similar results were obtained in 3 independent experiments. Representative results are shown. (C, D) Mitochondrial respiration measured by Seahorse assay in primary human hepatocytes treated with vehicle or 300 μM PA plus 300 μM OA for 25 hours. (C) Kinetics of oxygen consumption rate (OCR) after sequential compound injections. (D) Bar chart highlighting the differences in basal respiration, ATP production, maximal respiration, and spare respiratory capacity between groups. N = 5 per group. Data are shown as mean ± SD. Significance was determined using 2-way ANOVA with the Bonferroni post hoc analysis. *p < 0.05, **p < 0.005. (E, F) Mitochondrial respiration measured by the Seahorse assay in primary human hepatocytes treated with vehicle or NTP (0.2 or 0.4 NU/mL) for 25 hours before measuring OCR. (E) Kinetics of OCR after sequential compound injections. (F) Bar chart highlighting differences in basal respiration, ATP production, maximal respiration, and spare respiratory capacity between groups. N = 5 per group. Data are shown as mean ± SD. Significance was determined using 2-way ANOVA with the Bonferroni post hoc analysis. *p < 0.05, **p < 0.005. (G, H) Mitochondrial respiration measured by Seahorse assay in primary human hepatocytes treated with vehicle or NTP (0.2 or 0.4 NU/mL) for 1 hour, followed by treatment with 300 μM PA and 300 μM OA for an additional 24 hours before determining OCR. (G) Kinetics of OCR after sequential compound injections. (H) Bar chart highlighting differences in basal respiration, ATP production, maximal respiration, and spare respiratory capacity between groups. N = 5 per group. Data are shown as mean ± SD. Significance was determined using 2-way ANOVA with the Bonferroni post hoc analysis. *p < 0.05, **p < 0.005. Representative results from 3 independent experiments are shown. Abbreviations: BSA, bovine serum albumin; FCCP, fluoro-carbonyl cyanide phenylhydrazone; NTP, Neurotropin; pos., positive.