The NAMPT Inhibitor FK866 Attenuates DEN-Induced Liver Fibrosis in Mice

Abstract

Chronic liver disease (CLD) poses a significant global health challenge, and liver fibrosis is a crucial process in the pathogenesis of CLD. However, effective interventions to halt and reverse the progression of liver fibrosis remain elusive. This study investigated the potential of the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866 in treating diethylnitrosamine (DEN)-induced liver fibrosis in mice. We first demonstrated that DEN-induced hepatic fibrosis in mice was accompanied by upregulation of hepatic NAMPT and poly (ADP-ribose) polymerase 1 (PARP1) expression. Administration of FK866 inhibited the increase in alanine aminotransferase and aspartate aminotransferase levels and reversed the histopathological changes associated with DEN-induced liver fibrosis. It also suppressed the elevated expression of fibrotic markers, such as fibronectin, collagen IV, laminin, and α-smooth muscle actin. Further studies revealed that this therapeutic effect was achieved by inhibiting the NAD⁺ level, as well as the protein expression of NAMPT, PARP1, and inflammatory factors, including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α, and P65. In conclusion, FK866 exhibits therapeutic potential for the treatment of liver fibrosis.

INTRODUCTION

Chronic liver disease (CLD) is a major global health concern, causing approximately 2 million deaths annually.¹⁾ Liver fibrosis, a key event in the pathogenesis of CLD, is characterized by the excessive accumulation of extracellular matrix proteins during the wound-healing response to chronic liver injury. This accumulation distorts the hepatic structure, impairs parenchymal function, and can eventually lead to cirrhosis and even liver cancer.²⁾ Despite its importance, effective interventions to halt and reverse liver fibrosis, beyond etiological treatments, are currently lacking.³⁾ Thus, there is an urgent need to explore novel drugs for impeding or reversing this fibrotic process.

Inflammation is a major driver of liver fibrosis. The hepatic inflammatory response is a complex interplay of immune cells, cytokines, and chemokines, all of which contribute to the accumulation of extracellular matrix proteins and the development of liver fibrosis.⁴⁾ When immune cells are activated by inflammatory signals and recruited to the site of liver injury, pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 are released. As fibrosis progresses, a self-perpetuating cycle sustains the inflammatory response.⁵⁾

Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the salvage synthesis pathway of nicotinamide adenine dinucleotide (NAD⁺), has been implicated in various inflammatory diseases, including sepsis, rheumatoid arthritis, and diabetes.^6–8) Additionally, NAMPT has been identified as a marker of disease severity in pediatric inflammatory bowel disease.^9,10) Given its role in inflammation, we hypothesized that NAMPT could be a potential target for developing new drugs to treat liver fibrosis.

As is well known, NAD⁺ is an important coenzyme in cellular redox reactions and a substrate for enzymes such as sirtuins and poly (ADP-ribose) polymerases (PARPs).^11,12) Dehydrogenases catalyze the reduction of NAD⁺ to NADH upon accepting hydrogen atoms. The reverse reaction, the re-oxidation of NADH to NAD⁺, is facilitated by pathways including the respiratory chain. Reflecting the dynamic equilibrium between these 2 forms, the NAD⁺/NADH ratio provides a central readout of the cellular redox state and energy metabolism.¹³⁾ Notably, research indicates that restoring a balanced NAD⁺/NADH ratio can mitigate various pathological processes, including mitochondrial dysfunction, steatosis, inflammation, and oxidative stress.¹⁴⁾ Given this central role, the mechanisms governing NAD⁺ biosynthesis are crucial. In mammalian cells, the salvage pathway is the primary route for NAD⁺. NAMPT catalyzes the conversion of nicotinamide to nicotinamide mononucleotide, which is further converted to NAD⁺.^15,16) NAMPT influences the activity of NAD⁺-dependent enzymes and is involved in regulating the pathogenesis of metabolic disorders.¹⁷⁾ Recent evidence also suggests that NAMPT plays a crucial role in diet- and alcohol-induced hepatic steatosis.^18,19) However, the role NAMPT plays in the process of liver fibrosis is still largely unclear.

As a highly specific noncompetitive inhibitor of NAMPT, FK866 has relatively low toxicity; its inhibitor constants were calculated to be 0.4 nM for the enzyme/substrate complex (K(i)) and 0.3 nM for the free enzyme (K(i)′).²⁰⁾ Additionally, FK866-mediated inhibition of NAMPT has been extensively utilized as a pharmacological tool to elucidate the biological functions of NAMPT in inflammatory and metabolic disorders. Furthermore, some studies have demonstrated that FK866-mediated NAMPT inhibition attenuates inflammatory responses in various disease models, such as colitis,²¹⁾ acute lung injury,²²⁾ spinal cord injury,²³⁾ and rheumatoid arthritis.²⁴⁾ Therefore, this study employed FK866 to investigate the effects of NAMPT inhibition on liver fibrosis.

Diethylnitrosamine (DEN) is hepatotoxic and carcinogenic in many animals.²⁵⁾ Administration of DEN to mice induces hepatocellular injury, inflammation, and fibrosis.²⁶⁾ The use of DEN in mice to induce a mouse model of liver fibrosis has been widely used to study the pathogenesis of liver fibrosis and to evaluate therapeutic interventions.²⁷⁾

In this study, we used DEN to induce liver fibrosis in mice and then inhibited NAMPT activity with FK866 to investigate the role of NAMPT and NAD⁺-dependent enzymes in hepatic fibrosis.

MATERIALS AND METHODS

Animals and Experimental Design

Male C57BL/6 mice (aged 6–8 weeks with a body weight of 18–22 g) were purchased from the Experimental Animal Center of Chongqing Medical University. Under standard laboratory conditions, including a 12/12 h light/dark cycle, a relative humidity of 50–60%, and an environment of 23 ± 2°C, the mice had free access to standard rodent chow and water. The experimental procedures were approved by the Research Ethics Committee of Chongqing Medical University.

In the first part of the experiments, 18 mice were randomly allocated into 6 groups, with 3 mice in each group. DEN (Aladdin; Cat. No. N109570) was dissolved in sterilized water (concentration 0.1 mg/mL) to prepare the DEN solution. The DEN solution was administered orally to the mice (5 mg/kg/d).^28,29) At 0, 1, 3, 5, 7, and 9 weeks, 3 mice from one group were sacrificed.

In the second part of the experiments, 15 mice were randomly allocated into the control group, DEN group, and DEN + FK866 (MCE; Cat. No. HY-50876) group, with 5 mice in each group. FK866 was first dissolved in dimethyl sulfoxide to prepare a 200 mg/mL stock solution and stored at 20°C. For experiments, the FK866 stock solution was diluted in phosphate-buffered saline to 2 mg/mL and injected intraperitoneally into mice. The mice in the control group were only fed with distilled water, the mice in the DEN group were fed with DEN solution (5 mg/kg/d), and the mice in the DEN + FK866 group were fed with DEN (5 mg/kg/d) for 8 weeks and then injected with FK866 (10 mg/kg/d) for 7 d. According to previous research findings, FK866 exhibits no obvious toxicity and can exert its effects at this dose.^30–32) Mice in the control, DEN, and DEN + FK866 groups were sacrificed at week 9. Mice that died prematurely without completing the full experiment were excluded.

Liver Function Assessment

Liver function was assessed by quantifying alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. The blood samples were coagulated at room temperature and then centrifuged at 5000 × g and 4°C for 10 min to collect the serum. The serum levels of ALT and AST were determined using the corresponding kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China; Cat. No. C009-2-1) in accordance with the manufacturer’s guidelines.

Assessment of Hepatic NAD⁺ Content

NAD⁺ and NADH levels in liver tissue were measured using the NAD⁺/NADH colorimetric assay kit (Beyotime Biotechnology, Shanghai, China; Cat. No. S0175) according to the manufacturer’s instructions.

Histopathological Observation of the Liver

The liver samples were fixed in 4% paraformaldehyde. Then, the samples were dehydrated in 100% ethanol, infiltrated with soft paraffin, and embedded in molten paraffin according to standard procedures. Paraffin sections were cut into 5-µm-thick sections. Hematoxylin–eosin (H&E) and Masson’s trichrome staining were performed according to standard operating procedures, and the morphology was observed under a microscope.

Western Blot Analysis

Proteins were extracted from frozen liver tissue using RIPA lysis buffer supplemented with a 1 : 100 protease inhibitor cocktail, followed by the addition of a 1 : 100 phosphatase inhibitor cocktail. Protein concentrations were assessed using the BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL, U.S.A.; Cat. No. 23227). Protein samples were combined with 5 × loading buffer in a 1 : 4 ratio and boiled at 100°C for 10 min. Samples with identical protein content were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Nonspecific binding sites were blocked with skim milk powder dissolved in Tris-buffered saline with Tween-20 (TBST). Membranes were subsequently incubated overnight at 4°C with primary antibodies against NAMPT (Abcam, Cambridge, U.K.; Cat. No. Ab236874), PAPR1 (Proteintech, Rosemont, IL, U.S.A.; Cat. No. 13371-1-AP), fibronectin (FN; Proteintech; Cat. No. 15613-1-AP), laminin (LN; Sigma; Cat. No. MAB1905-I), collagen IV (Bioss; Cat. No. bs-0806R), α-smooth muscle actin (α-SMA) (Proteintech; Cat. No. 14395-1-AP), IL-1β (Proteintech; Cat. No. 16806-1-AP), IL-6 (Proteintech; Cat. No. 26404-1-AP), TNF-α (Proteintech; Cat. No. 17590-1-AP), P65 (Proteintech; Cat. No. 10745-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (Proteintech; Cat. No. 60004-1-Ig). Thereafter, the sections were incubated with horseradish peroxidase-conjugated anti-mouse/rabbit immunoglobulin G secondary antibody for 2 h. After incubation with primary and secondary antibodies, the membranes were washed with TBST. The proteins on the membrane were visualized using an enhanced chemiluminescence kit. Western blots were quantified using Image Lab (Bio-Rad, Hercules, CA, U.S.A.).

Statistical Analysis

The data were analyzed using GraphPad Prism 9.5.0 (GraphPad, San Diego, CA, U.S.A.). All quantitative values are presented as the mean ± standard deviation (S.D.). Student’s t-test was used to assess the differences between the 2 groups. One-way ANOVA was used to analyze differences among multiple groups, Dunnett’s test was used to examine the time-course effects, and Tukey’s test was used to assess pairwise effects. The correlation coefficient was calculated using Spearman’s correlation analysis. p < 0.05 was considered significantly different.

RESULTS

DEN-Induced Hepatic Fibrotic Injury Is Related to Elevated NAD⁺ Levels and Increased Expression of NAMPT and PARP1 in Mice

DEN has strong hepatic chemical toxicity. DEN-induced mouse models of liver fibrosis have been widely used to study the pathogenesis of liver fibrosis and evaluate potential therapeutic interventions. In this study, we also used DEN to induce a mouse model of liver fibrosis. The livers were collected at 0, 1, 3, 5, 7, and 9 weeks after DEN treatment, and H&E and Masson’s staining of liver sections were used to evaluate the pathologic morphology. The results showed that, before DEN treatment, the liver lobules of the mice had a clear structure, the hepatocyte cords were neatly arranged, the hepatic sinusoids were normal, and there were no abnormal hepatocytes or fibrous tissue proliferation. DEN treatment resulted in connective tissue hyperplasia, marked structural distortion, and fibrous collagen deposition between the portal vein and the lobules (Figs. 1A and 1B).

Fig. 1. DEN Treatment Resulted in Structural Distortion

(A) Livers stained with H&E. Representative histological sections were magnified (×100) and photographed. (B) Livers stained with Masson’s trichrome. Representative histological sections were magnified (×100) and photographed.

After liver injury, transaminases in hepatocytes are released into the blood, resulting in elevated levels of ALT and AST in the blood. Therefore, elevated ALT and AST indicate impaired liver function and are important liver function markers. The serum levels of AST and ALT in mice were measured at 0, 1, 3, 5, 7, and 9 weeks after DEN treatment, and the results showed that these levels increased gradually with prolonged DEN treatment (Figs. 2A and 2B). The results of staining and liver function marker assays indicated that hepatic fibrosis was induced in mice after 9 weeks of DEN treatment.

Fig. 2. DEN-Induced Hepatic Fibrosis in Mice Was Accompanied by Upregulation of Hepatic NAD⁺ Level, NAMPT and PARP1 Expression

(A) The serum ALT levels in each group (n = 3 mice per group). (B) The serum AST levels in each group (n = 3 mice per group). (C) The liver NAD⁺/NADH level in each group (n = 3 mice per group). (D) The liver NAD⁺ levels in each group (n = 3 mice per group). (E) The liver NADH levels in each group (n = 3 mice per group). (F) Protein expression of NAMPT. (G) Protein expression of PAPR1. (H) Relative protein expression of NAMPT in each group (n = 3 mice per group). (I) Relative protein expression of PARP1 in each group (n = 3 mice per group). (J) Correlation between ALT and NAMPT. (K) Correlation between AST and NAMPT. Data are presented as the mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001.

NAD⁺ is a coenzyme that is critical for many redox reactions within cells. It also serves as a substrate for sirtuins and PARPs, which are NAD⁺-dependent enzymes that are involved in inflammatory and fibrotic processes. Mice receiving DEN treatment gradually developed severe liver fibrosis, and hepatic NAD⁺ content was measured at 0, 1, 3, 5, 7, and 9 weeks; the results showed that DEN treatment led to a gradual increase in hepatic NAD⁺/NADH levels, among which the increase in NAD⁺ was the main contributor, while the change in NADH was not significant. In the time period designed for this experiment, NAD⁺/NADH levels increased with the duration of treatment, and the level of NAD⁺/NADH reached 7 times that of the first week in the ninth week (Figs. 2C–2E).

PARP1 is involved in the regulation of DNA repair, cell death, metabolism, and inflammatory responses. In these experiments, we found that the protein expression of PARP1 increased over time during DEN-induced hepatic fibrosis (Figs. 2G and 2I). It is noteworthy that there was a simultaneous elevation in the expression of NAMPT (Figs. 2F and 2H), which is the rate-limiting enzyme of the salvage NAD⁺ synthesis pathway, and PARP1, which is a NAD⁺-consuming enzyme, suggesting that elevated PARP1 may be associated with elevated NAMPT.

Correlation analysis of protein expression of NAMPT with serum AST and ALT levels at 0, 1, 3, 5, 7, and 9 weeks showed that NAMPT expression was significantly correlated with both ALT (r = 0.94, p < 0.05) and AST (r = 0.94, p < 0.05) levels (Figs. 2J and 2K). These findings suggest that the degree of liver function impairment and liver fibrosis in the DEN-induced liver fibrosis mouse model may be related to the increased NMAPT expression. Thus, we subsequently used FK866 to inhibit NAMPT and explore its role in liver fibrosis.

FK866 Alleviates DEN-Induced Liver Fibrosis in Mice

After 9 weeks of treatment with DEN, mice from the DEN group were sacrificed, and liver tissues were collected. H&E and Masson’s staining of liver tissue sections showed significant hepatic fibrosis features after the DEN challenge, whereas FK866 treatment attenuated these pathological changes (Fig. 3A). The serum levels of ALT and AST were also detected. The results showed that the serum levels of ALT and AST were significantly elevated, indicating significant liver function damage after DEN treatment. The serum levels of ALT and AST of the mice from the DEN + FK866 group were also evaluated. The results showed that the serum levels of ALT and AST decreased significantly after FK866 treatment compared with DEN alone (Figs. 3B and 3C). Furthermore, the Western blot results showed that the protein levels of fibrosis-associated molecules, such as LN, FN, collagen IV, and α-SMA, were elevated in DEN-treated mice with hepatic fibrosis, while these expressions were decreased after FK866 treatment (Fig. 3D), indicating the inhibitory effects of FK866 on DEN-induced hepatic fibrosis. The results indicated that FK866 treatment had a protective effect against DEN-induced liver function injury.

Fig. 3. FK866 Alleviated DEN-Induced Liver Fibrosis

(A) Livers stained with H&E and Masson’s trichrome. Representative histological sections were magnified (×100) and photographed. (B) The serum ALT levels in each group (n = 5 mice per group). (C) The serum AST levels in each group (n = 5 mice per group). (D) Protein expression of FN, LN, collagen IV, and α-SMA. Data are presented as the mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001.

FK866 Reduces Elevated Hepatic NAD⁺ Levels and Suppresses NAMPT and PARP1 Protein Expression

In accordance with the results in Figs. 4A–4C, the ratio of hepatic NAD⁺/NADH was higher in the DEN group than in the control group. In contrast, a 7-day FK866 treatment starting from week 8 significantly reduced the elevated ratio of NAD⁺/NADH, which was also mainly driven by the elevation of NAD⁺ (Figs. 4A–4C). Moreover, the expression levels of NAMPT and PARP1 were determined by Western blot, and the results showed that FK866 treatment suppressed their increased protein expression (Fig. 4D). The results indicated that FK866 inhibited NAD⁺ synthesis via downregulating the NAMPT expression. Subsequently, as a NAD⁺-consuming enzyme, the PARP1 expression was downregulated, which confirmed a NAD⁺ decline.

Fig. 4. FK866 Reduced the Elevation of Hepatic NAD⁺ Levels and Suppressed NAMPT and PARP1 Protein Expression

(A) The liver NAD⁺/NADH levels in each group (n = 5 mice per group). (B) The liver NAD⁺ levels in each group (n = 5 mice per group). (C) The liver NADH levels in each group (n = 5 mice per group). (D) Protein expression of PARP1 and NAMPT. Data are presented as the mean ± S.D. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001.

FK866 Inhibits the Protein Expression of Inflammatory Molecules in the Livers of Mice

To investigate the potential mechanisms underlying the anti-fibrotic properties of FK866, the protein expression levels of inflammatory molecules, including IL-1β, IL-6, TNF-α, and p65, were evaluated after a 7-day treatment course starting from week 8. The results showed that the protein levels of inflammatory molecules were increased after the DEN challenge and decreased due to FK866 administration (Fig. 5). Notably, prior studies indicate that FK866 monotherapy demonstrates no significant effect on inflammatory cytokines such as IL-1β, IL-6, and TNF-α,²¹⁾ indicating that FK866 had an inhibitory effect on DEN-induced liver inflammation. Therefore, the anti-fibrotic property of FK866 may be attributed to its inhibition of inflammatory molecule expression.

Fig. 5. FK866 Inhibited the Protein Expression of Inflammatory Molecules

Protein expression of P65, IL-1β, TNF-α, and IL-6.

DISCUSSION

CLD stands as a formidable global public health challenge, affecting more than 800 million people and causing about 2 million deaths each year.^1,33) The etiological spectrum of CLD is broad, encompassing viral infections like hepatitis B and C viruses, alcoholic liver disease stemming from excessive alcohol intake, and metabolic dysfunction-associated steatotic liver disease, previously known as non-alcoholic fatty liver disease.³⁴⁾ Decompensated cirrhosis, a major consequence of CLD, accounts for around 1 million deaths per year, and liver fibrosis is a crucial factor in the progression to cirrhosis,³⁵⁾ hepatitis serves as a critical driver of hepatic fibrosis.³⁶⁾ Fibrosis may be an attempt to limit the consequences of chronic liver injury, but it is also a key feature in the progression of CLD to cirrhosis.³⁷⁾ Currently, effective clinical treatments capable of halting and reversing liver fibrosis are scarce. Therefore, the search for novel and efficient drugs to decelerate this fibrotic process is of utmost urgency. In this study, we found that the elevated expression of NAMPT was in line with liver fibrosis progression. Notably, its inhibitor, FK866, exhibited anti-fibrotic effects, potentially paving the way for new liver fibrosis treatments.

Firstly, DEN was used to induce hepatic fibrosis in mice, which were sacrificed at different time points. It was found that the aggravated hepatic fibrosis was consistent with elevated NAD⁺ levels and NAMPT expression. As such, we postulated that FK866 could inhibit hepatic fibrosis via inhibiting the NAD⁺ synthesis pathway as well as the activity of NAD⁺-dependent enzymes.

In mammals, NAD⁺ is primarily synthesized via the salvage pathway, with NAMPT serving as the rate-limiting enzyme. FK866 is a specific inhibitor of NAMPT; 10 mg/kg is the effective dose at which FK866 exerts its effect with no obvious hepatotoxicity or nephrotoxicity,^30–32) and the NAD⁺ levels decline via inhibiting the activity of NAMPT. NAD⁺ is an essential cofactor in cellular redox reactions, an important co-substrate for sirtuins and PARPs, and plays an important role in regulating cellular homeostasis. PARP1 is the main isoform of the PARP family, which plays an important role in DNA repair, cell death, metabolism, and inflammatory responses.^38–41) It has been shown to mediate CCl₄-induced hepatocyte death, inflammation, and fibrosis.⁴²⁾ Overactivation of PARP promotes cell death and generates pro-inflammatory mediators. PARP inhibitors have been found to have tissue-protective and anti-inflammatory effects in animal models of various forms of inflammation.⁴²⁾ It has also been found that PARP-1 and PARP-2 regulate the metabolic functions of various organ systems in rodents, possibly by modulating NAD⁺ levels and thus Sirt1 activity.^38–41)

Nuclear factor-kappaB (NF-κB) is a family of transcription factors with crucial roles in numerous physiological and pathological processes. There are 2 distinct NF-κB pathways: the canonical and non-canonical pathways, each with different activation mechanisms. The canonical NF-κB pathway is activated in response to various external stimuli related to inflammation, immune response, cell proliferation, differentiation, and survival. Previous studies have shown that the activation of the canonical NF-κB pathway triggers the production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in the innate immune system, thereby initiating an inflammatory response. Conversely, these pro-inflammatory cytokines can also activate the canonical NF-κB pathway.⁴³⁾ It has been reported that inhibition of NAMPT by FK866 suppresses proinflammatory responses in macrophages,^44,45) while reducing inflammatory responses in many diseases, such as colitis,²¹⁾ acute lung injury,²²⁾ spinal cord injury,²³⁾ and rheumatoid arthritis.²⁴⁾ Moreover, some studies have indicated that the anti-inflammatory ability of FK866 is achieved by downregulating the NF-κB signaling pathway or inhibiting the NF-κB-dependent immune response.^21–23) In our study, FK866 treatment also inhibited the protein expression of p65, which was associated with the alleviation of liver inflammation and fibrosis. This effect may be due to the inhibition of the NAD⁺ synthesis pathway.

While we observed that FK866 possesses anti-inflammatory and anti-fibrotic effects, its specific impact on transforming growth factor β1 (TGF-β1), a key mediator of fibrosis,^46,47) remains unknown. Some studies have suggested that the excessive activation of PARP-1 can promote the secretion of TGF-β1 and activate the phosphorylation of Smad2/3. Inhibiting the excessive activation of PARP-1 can suppress TGF-β1, thereby inhibiting hepatic fibrosis. Combined with the results of this study, FK866 may also exert an anti-fibrotic effect by inhibiting NAD⁺ production to suppress PARP-1, which in turn inhibits TGF-β.⁴⁸⁾ These findings are highly consistent with our experimental results and provide important directions for our future investigations.

In conclusion, our findings suggest that FK866 has promising therapeutic potential for DEN-induced liver fibrosis, offering a novel approach for the treatment of liver fibrosis. However, further research, including preclinical and clinical studies, is needed to fully elucidate its efficacy and safety in humans and to explore its potential as a clinical treatment option.

DECLARATION

Conflict of Interest

The authors declare no conflict of interest.

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