Novel role for epalrestat: protecting against NLRP3 inflammasome-driven NASH by targeting aldose reductase

C57BL/6 mice were obtained from SPF Biotechnology Co., Ltd (Beijing, China). All animals were reared at SPF conditions, with 40–70% humidity and 12 h of light/12 h of dark per day.

Bone marrow-derived macrophages (BMDMs) were obtained from C57BL/6 mice (10 weeks old) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 1% penicillin/streptomycin (P/S) and 50 ng/mL murine macrophage colony-stimulating factor (M-CSF). THP1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% P/S. HEK-293 T cells were cultured in DMEM supplemented with 10% FBS and 1% P/S. All cells were incubated at 37 °C in a humidified atmosphere with 5% (v/v) CO₂.

Phorbol-12-myristate-13-acetate (PMA), dimethyl sulfoxide (DMSO), adenosine triphosphate (ATP), Nigericin, LPS, TRIzol, and ultrapure LPS were purchased from Sigma-Aldrich (Munich, Germany). Silicon dioxide (SiO₂), poly (I: C), poly (dA: dT), and Pam3CSK4 were obtained from InvivoGen (San Diego, USA). MCC950 was purchased from TopScience (Shanghai, China). Epalrestat, ponalrestat, ranirestat, and tolrestat were purchased from MedChemExpress (New Jersey, USA). MitoSOX was purchased from Invitrogen (Carlsbad, USA). Certified Fetal Bovine Serum (FBS) were obtained from VivaCell (Shanghai, China). Anti-mouse caspase-1 antibodies (AG-20B-0042) were purchased from Adipogen (San Diego, USA). Anti-human cleaved IL-1β (12,242), anti-human caspase-1 (4199S), anti-mouse IL-1β (12,507), and anti-NLRP3 (15101S) antibodies were purchased from CST (Boston, USA). Anti-ASC (sc-22514-R) antibodies were purchased from Santa Cruz Biotechnology (Dallas, USA). Anti-DDDK tag (20,543-1-AP) and anti-GAPDH (60,004-1-1G) antibodies were purchased from the Proteintech Group (Chicago, USA). Anti-β-actin (ab8226), anti-NEK7 (ab133514), and anti-aldose reductase (ab268058) antibodies were purchased from Abcam (Cambridge, UK). Color Prestained Protein marker (20AB01) was purchased from GenStar (Beijing, China). Salmonella was a gift from Dr. Tao Li of the National Center of Biomedical Analysis (Beijing, China).

The plasmids pCMV-Flag-Vector and pCMV-NLRP3-Flag were kindly provided by Dr. Tao Li from the National Center of Biomedical Analysis (Beijing, China).

THP1 and BMDMs were seeded in 24-well culture dishes at a density 1 × 10⁶ cells/mL or 1.5 × 10⁶ cells/mL and cultured overnight. Afterward, the cells were primed for 4 h with 50 ng/mL LPS. Then, the medium was replaced with Opti-MEM supplemented with epalrestat. After 1 h, the activation of NLRP3 was typically achieved through the following treatments: 5 mM ATP for 45 min, 7.5 μM nigericin for 30 min, 200 μg/mL SiO₂ for 6 h, or transfection with 1 μg/mL poly (I: C) using Lipofectamine 2000 for 6 h. NLRC4 or AIM2 inflammasome activation was accomplished using Salmonella or transfection with 1 μg/mL poly (dA: dT) using Lipofectamine 2000 for 6 h.

To detect caspase-1 p20 and IL-1β production, the cell supernatants were concentrated with TCA and then centrifuged at 12,000 ×g for 10 min at 4 °C. Next, the concentrated proteins were washed by turning up and down with acetone and centrifuged at 12,000 ×g for 5 min at 4 °C. The acetone-containing supernatant was discarded, the samples were placed in 1 × loading sample buffer, and the proteins were denatured at 105 ℃ for 15 min. The protein samples were then resolved on SDS–PAGE gels and transferred to a polyvinylidene difluoride (PVDF) membrane using a wet-transfer system. Next, the PVDF membranes were blocked in TBST (20 mM Tris–HCl [pH 7.6], 150 mM NaCl, and 0.1% [v/v] Tween-20) containing 5% (w/v) non-fat milk for 1 h at room temperature and then incubated with primary antibodies diluted in 5% (w/v) BSA in TBST overnight at 4 °C. Afterward, the membranes were washed thrice with TBST and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in 5% (w/v) non-fat milk in TBST for 1 h. Following three washes with TBST, the bands generated on the membrane were visualized on X-ray film using a chemiluminescent western blotting detection system.

To detect the expression of NLRP3, pro-IL-1β, ASC, and caspase-1 p45, the cell lysates were collected via direct lysis in a 1 × loading sample buffer. Western blot analysis was then performed as described above.

BMDMs were seeded overnight at a density of 1 × 10⁶ cells/mL in 96-well culture dishes. The cells were then incubated at 37 °C and treated with epalrestat for 24 h. After incubation, the spent medium was replaced with DMEM containing a with cell counting kit-8 (CCK-8) reagent and the cells were incubated for 30 min. The optical density (OD) values were then measured at 450 nm.

The release of LDH in the cell supernatants was measured using an LDH cytotoxicity assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions.

Cell culture supernatants, peritoneal lavage fluid, and mouse serum were collected. ELISA kits to detect mouse IL-1β (R&D Systems, SMLB00C), TNF-α (Dakewe, 1217202), and IL-6 (Dakewe, 1210602), and human IL-1β (Dakewe, 1110122) and TNF-α (Dakewe, 1117202) were used to detect the levels of the indicated cytokines according to the respective manufacturer’s instructions.

The activity of caspase-1 in the cell supernatants was measured using a Caspase-Glo^® 1 Inflammasome Assay (Promega, Beijing, China) according to the manufacturer’s instructions.

BMDMs were seeded in 12-well culture dishes at density of 1 × 10⁶ cells/mL overnight and then treated with 50 ng/mL LPS for 4 h. Next, the spent medium was replaced with Opti-MEM supplemented with epalrestat and incubated for 1 h. NLRP3, NLRC4, and AIM2 inflammasome activation was achieved using similar treatments as described above. Cells were lysed with a Triton buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, and EDTA-free protease inhibitor cocktail] and then centrifuged at 6000 × g for 10 min at 4 °C. Supernatants were then centrifuged at 6000 × g for 10 min. The pellet fractions were then washed with PBS and cross-linked with 2 mM disuccinimidyl suberate (DSS) in PBS for 30 min at 37 °C, followed by centrifugation at 6000 ×g for 15 min. Finally, the cross-linked pellets were dissolved in 1 × loading buffer and analyzed using a chemiluminescent western blotting detection system.

HEK-293 T cells were transfected with Flag-tagged plasmids (Flag-Vector and Flag-NLRP3) for 24 h and then treated with epalrestat for 6 h. Afterward, the cells were lysed with a lysis buffer (50 mM NaCl, 50 mM Tris, pH 7.8, 0.1% [v/v] Nonidet-P40, 5 mM EDTA and 10% [v/v] glycerol) containing an EDTA-free protease inhibitor cocktail and the cell lysates were collected and centrifuged at 12,000 rpm for 15 min. The supernatants were then immunoprecipitated with anti-Flag M2 affinity beads according to the manufacturer’s instructions. Cell lysates or immunoprecipitates were separated using a chemiluminescent western blotting detection system.

BMDMs were seeded in 12-well plates overnight and then treated with 50 ng/mL LPS for 4 h. The spent medium was then replaced with Opti-MEM supplemented with epalrestat. The cells were then incubated for 1 h and then treated with nigericin or ATP. To measure the intracellular K⁺ levels, the spent medium was aspirated before washing the cells three times with PBS. Ultrapure HNO₃ was then added to lyse the cells, and the lysates were boiled at 100 °C for 30 min. Intracellular K⁺ measurements were then performed via inductively coupled plasma mass spectrometry. To measure the intracellular Ca²⁺ levels, a trace showing ATP-induced Ca²⁺ flux was analyzed using a FLIPRT Tetra system (Molecular Devices, USA).

8 week-old male or female C57BL/6 mice were gavaged with epalrestat (120 mg/kg/day) or vehicle daily for 14 days. The body weight of the mice was measured daily. On the 15th day, after anesthetization, the serum of the mice was collected and assessed for AST, ALT, creatinine, TBIL, and glucose levels using commercial kits according to their respective manufacturer’s instructions.

8 week-old female C57BL/6 mice were gavaged with epalrestat (20 or 40 mg/kg), MCC950 (40 mg/kg), or vehicle for 1 h. After intraperitoneal injection with LPS (20 mg/kg) for 6 h, the mice were anesthetized, and the serum and peritoneal lavage fluids were collected. Cytokine levels in the lavage fluids and serum were detected using ELISA. The stained cells were analyzed using flow cytometry.

C57BL/6 mice (8 week-old, male) were fed with an MCD diet (518,810, Dyets, USA) or an identical diet supplemented with methionine and choline (MCS; 518,811, Dyets) for 6 weeks according to the manufacturer’s instructions. Afterward, the mice were randomly separated into groups and gavaged with epalrestat (20 mg/kg), MCC950 (20 mg/kg), or vehicle daily for a total of 5 days, and then 40 mg/kg every other day, for up to 6 weeks. Finally, the mice were anesthetized, and their liver and serum were collected for analysis.

Bioluminescence assays used for high-throughput screening showed that epalrestat might be a potential inhibitor of NLRP3 inflammasomes (Fig. 1A). As a further investigation of epalrestat's effects on the activation of the NLRP3 inflammasome, we determined its cytotoxicity in BMDMs. We found that epalrestat is not cytotoxic to BMDMs, even at concentrations up to 200 μmol/L (Fig. 1B). Studies have shown that activation of the NLRP3 inflammasome leads to the maturation and secretion of large amounts of pro-caspase-1 and pro-IL-1β [41, 42]. The results showed that epalrestat significantly inhibited caspase-1 activation, IL-1β maturation, and LDH release (Fig. 1C–G, Additional file 1: Fig. S1). Contrary to IL-1β, epalrestat has no effect on the secretion of TNF-α, an inflammasome-independent cytokine (Fig. 1C–G, Additional file 1) [18]. Similarly, epalrestat also significantly inhibited the production of caspase-1 and IL-1β and the nigericin-stimulated release of LDH in THP1 cells, but TNF-α expression was not affected (Fig. 1H–L). We also evaluated the expression of NLRP3 inflammasome complex proteins, including NLRP3, pro-IL-1β, caspase-1 p45, and ASC in the cell lysates. The results showed that epalrestat did not affect the expression of NLRP3 inflammasome complex proteins (Fig. 1C–L, Additional file 1: Fig. S1). Additionally, we compared the efficacy of epalrestat and other NLRP3 inhibitors, including sulforaphane, parthenolide and OLT1177. The results showed that epalrestat, sulforaphane, parthenolide and OLT1177 could inhibit the activation of NLRP3 inflammasome at the concentration of 40 μmol/L, and epalrestat is more effective than OLT1177 at this concentration (Additional file 2). These results confirm that epalrestat inhibits NLRP3 inflammasome activation in LPS-primed BMDMs and THP1 cells in vitro.

Pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) could activate the NLRP3 inflammasome, which involve nigericin, SiO2, MSU, ATP, and poly (I: C) [22, 43, 44]. Therefore, we investigated the role of epalrestat in NLRP3 inflammasome activation induced by other stimuli. Epalrestat inhibited the production of caspase-1 and IL-1β triggered by ATP, nigericin, SiO₂, and poly (I: C) in BMDMs (Fig. 2A, C–E). In accordance with previous studies, epalrestat did not affect the expression of NLRP3 inflammasome complex proteins in cell lysates or TNF-α in cell supernatants (Fig. 2A, C–E). These results indicate that epalrestat could inhibit NLRP3 inflammasome activation mediated by multiple agonists.

Caspase-11 can be activated by intracellular LPS or gram-negative bacteria to target noncanonical NLRP3 inflammasome activation. Therefore, the effect of epalrestat on the noncanonical NLRP3 inflammasome activation was test. As expected, epalrestat could suppress caspase-11-dependent caspase-1 cleavage and IL-1β secretion triggered by LPS after treatment in Pam3CSK4-primed BMDMs (Fig. 2A, C–E). Furthermore, epalrestat did not affect the levels of TNF-α in the cell supernatants or NLRP3 expression in the cell lysates (Fig. 2A, C–E). In conclusion, epalrestat, a broad NLRP3 inflammasome inhibitor, inhibited canonical and noncanonical NLRP3 inflammasome activation.

To further investigate that epalrestat is a specific NLRP3 inflammasome inhibitor, we evaluated its role in NLRC4 inflammasome and AIM2 inflammasome activation. The NLRC4 inflammasome can be activated by flagellin derived from bacteria, including Salmonella typhimurium, and the AIM2 inflammasome can be activated by double-stranded DNA [45, 46]. We verified that epalrestat does not affect NLRC4- or AIM2-dependent caspase-1 cleavage and the secretion of IL-1β from Salmonella typhimurium or poly (dA:dT) stimulation (Fig. 2B, F–H). Consistent with previous results, the expression of inflammasome complex proteins, including caspase-1 p45, pro-IL-1β, and ASC, and the secretion of TNF-α were not affected by epalrestat (Fig. 2B, F–H). Thus, these results showed that epalrestat was not affected the activation of NLRC4 and AIM2 inflammasomes.

Previous studies have shown that NLRP3 inflammasome activation in macrophages requires a two-step process, with the first phase (priming) being provided by microbes or endogenous molecules that can activate NF-kB to induce the expression of NLRP3 and pro-IL-1β, and the second phase (activation) being triggered by ATP, nigericin or MSU [43, 47‐49]. Meanwhile, other studies have shown that unlike ASC and caspase-1, the protein amounts of NLRP3 in resting macrophages are thought to be insufficient for NLRP3 activation [43, 47, 50, 51]. Next, we examined the effect of epalrestat on the expression of NF-κB-dependent NLRP3 and pro-IL-1β. The results suggested that epalrestat did not affect LPS-induced NLRP3 and pro-IL-1β expression when BMDMs were stimulated with epalrestat for 1 h after being pre-treated with LPS for 4 h or stimulated with epalrestat for 1 h before LPS treated for 4 h (Fig. 3A). Similarly, we also detect the production of TNF-α, and the results showed that epalrestat does not affect TNF-a production in these conditions (Fig. 3B). Thus, these results indicated that epalrestat does not suppress the NF-κB-dependent expression of NLRP3 or pro IL-1β and have no effect on the priming stage on the NLRP3 inflammasome activation.

As a key step, the adaptive protein ASC forms a single large perinuclear focus per cell during inflammasome activation [52‐54]. Therefore, we investigated whether ASC oligomerization is a key target in NLRP3 inflammasome activation during epalrestat treatment. Cytosolic fractions were cross-linked, which from cell lysates, and then visualized via immunoblotting to detect ASC monomers and higher-order complexes. Our results revealed that epalrestat could dose-dependently inhibit ASC oligomerization after stimulated with nigericin (Fig. 3C). The effect of other NLRP3 inflammasome stimulis in the presence or absence of epalrestat also was detected and found that ASC oligomerization could be suppressed upon multiple agonists-mediated NLRP3 inflammasome activation in LPS-primed BMDMs during epalrestat treatment (Fig. 3D). Consistent with previous results, ASC oligomerization induced by Salmonella typhimurium or poly (dA:dT) was not influenced by epalrestat treatment (Additional file 3). These results provide robust evidence that epalrestat does not affect ASC oligomerization during NLRC4 and AIM2 inflammasome activation. Moreover, the effect of epalrestat on the NLRP3 inflammasome activation is independent of ASC oligomerization, so we further investigate the upstream process of NLRP3 inflammasome activation to search for the mechanism by which epalrestat inhibits NLRP3 inflammasome activation.

We then investigated the mechanisms involved in NLRP3 inflammasome activation by epalrestat, such as K⁺ efflux, Ca²⁺ mobilization, and ROS generation [54‐56]. We first evaluated the effect of epalrestat on K⁺ efflux. The intracellular potassium levels could significantly decrease upon stimulation with nigericin. However, epalrestat does not influence this effect; this indicates that epalrestat inhibits NLRP3 inflammasome activation but not potassium efflux (Fig. 3E). In addition to NLRP3 inflammasome activation, flow cytometry was used to analyze ROS production. We did not observe a change in the ROS generated in BMDMs pre-treated with epalrestat and stimulated by nigericin, poly (dA:dT), or Salmonella typhimurium (Additional file 4). The results of the Ca²⁺ mobilization assay upon pre-treatment with epalrestat and stimulation with ATP also showed that epalrestat does not affect Ca²⁺ mobilization upstream of NLRP3 activation (Fig. 3F). Thus, NLRP3 activation is therefore postulated to be controlled by epalrestat acting downstream of K + efflux, Ca2 + flux, and ROS production.

To further elucidate whether epalrestat directly binds to the protein responsible for activating the NLRP3 inflammasome, such as NLRP3, caspase-1, NEK7, and ASC, epalrestat was conjugated with EAH-activated Sepharose (epalrestat-sepharose) and the control group was conjugated with control Sepharose (epalrestat-control). Epalrestat-interacting proteins were then subjected to a pull-down assay from the cell lysates for detection. And the results revealed that ASC, NLRP3, and caspase-1 were not pulled down by epalrestat-sepharose (Fig. 4A). Previous studies have demonstrated that epalrestat is a specific aldose reductase inhibitor. Thus, we detected whether aldose reductase could be pulled down by epalrestat-sepharose; the results showed that aldose reductase was not pulled down by epalrestat-sepharose. Next, we investigated whether aldose reductase interacts with NLRP3 during the activation of inflammasome. Flag-tagged NLRP3, and a semi-endogenous co-immunoprecipitation experiment were transfected into HEK-293 T cells using anti-Flag M2 beads. The co-immunoprecipitation experiment results showed that aldose reductase does not interact with NLRP3 upon epalrestat treatment (Fig. 4B). Conversely, NEK7 interacts with NLRP3 in HEK-293 T cells, promoting NLRP3 oligomerization and ASC recruitment to NLRP3; this interaction was not influenced by epalrestat treatment. Therefore, the mechanism underlying the effect of epalrestat on NLRP3 inflammasome activation does not involve the interaction between NLRP3 and aldose reductase.

To elucidate the mechanism of epalrestat on inhibiting NLRP3 inflammasome activation, we also tested whether aldose reductase is a critical target of NLRP3 inflammasome activation. Previous studies have reported that epalrestat, ponalrestat, ranirestat, and tolrestat are all aldose reductase inhibitors [57]. Thus, we evaluated their effects on NLRP3 inflammasome activation. Consistent with our expectation, the activation of caspase-1 and the maturation of IL-1β could be inhibited by epalrestat, ponalrestat, ranirestat, and tolrestat but not TNF-α, suggesting that aldose reductase inhibitors could suppress NLRP3 inflammasome activation (Fig. 4C–E). Taken together, aldose reductase is a key target of epalrestat to inhibit NLRP3 inflammasome activation.

To assess the effect of epalrestat in vivo, we employed NLRP3-dependent inflammatory models using LPS. Previous studies have reported that LPS injection in mice induced NLRP3-dependent IL-1β production and recruitment of inflammatory cells. Here, mice were intraperitoneally injected with LPS for 4 h after treatment with epalrestat or MCC950 for 1 h. Afterwards, serum and peritoneal lavage fluid were gathered to measure the concentration of inflammasome-independent cellular factors. The data showed that epalrestat and MCC950 dose-dependently suppressed IL-1β and TNF-α production (Fig. 5A–C). Similarly, epalrestat and MCC950 could inhibit LPS- triggered IL-1β expression in peritoneal lavage cells. Similar to the suppressive role of epalrestat on pro-inflammatory cytokines, peritoneal macrophages pre-treated with epalrestat could also be decrease, as measured via flow cytometry (Fig. 5D–E). These results suggest that epalrestat inhibits LPS-induced cytokine release through NLRP3 inflammasome activation in mice. Additionally, safety of epalrestat, which is currently available for clinical use in many countries, was also evaluated in vivo. Briefly, mice were gavaged daily for 14 days with epalrestat (120 mg/kg) at a three-fold higher dose than the one used for the LPS-triggered NLRP3 inflammasome activation experiments. We found that biochemical parameters, including plasma ALT, AST, creatinine, TBIL, and glucose, as well as body weight, were not influenced by epalrestat treatment, indicating that epalrestat is well-tolerated and safe in vivo (Fig. 5F–K).

Previous studies have reported that epalrestat is used to modify peripheral nerve function in diabetic patients, so we investigated whether new indications, such as for NASH, could be added. As reported, NASH could be caused by an MCD diet and the inflammatory response, and regulated by the sustained NLRP3 inflammasome activation. To estimate the influence of epalrestat on NASH, MCD or MCS diets were fed to mice to generate a NASH mouse model that can exhibit a few characteristics of NASH, including hepatocyte ballooning, hepatic steatosis, inflammatory cell infiltration of the liver lobules, and fibrosis. We observed that compared to MCS diet-treated mice, hepatic steatosis and fibrosis, and inflammatory factor infiltration were discovered in the MCD diet-treated mice. However, these pathological changes were significantly reduced after epalrestat treatment, as well as MCC950, an NLRP3 inflammasome inhibitor, when used as a positive control (Fig. 6A). Furthermore, the expression of ALT and AST in plasma, which were increased after MCD diet, were also alleviated by epalrestat or MCC950 treatment. Interestingly, treatment with a combination of epalrestat and MCC950 yielded similar effects to treatment with epalrestat or MCC950 alone in MCD diet-triggered NASH (Fig. 6B–C).

Next, we clarify whether epalrestat treatment is achieved by inhibiting NLRP3 inflammasome activation in NASH, so active caspase-1 expression and pro-inflammatory cytokine secretion were tested in liver tissue after epalrestat treatment. Consistent with the previous results, IL-1β and TNF-α mRNA expression, pro-inflammatory cytokine genes, were markedly decreased upon treatment with epalrestat or MCC950 (Fig. 6D, E). As expected, caspase-1 p20 expression in the liver tissue of MCD diet-treated mice was significantly reduced upon epalrestat or MCC950 treatment (Fig. 6G). In summary, we evaluated the mRNA transcription of genes involved in hepatic fibrogenesis. The mRNA levels of collagen 1 in the liver were reduced by epalrestat or MCC950 treatment (Fig. 6F). The results of the combination of epalrestat and MCC950 treatment on NASH also showed that caspase-1 p20 expression, and IL-1β and TNF-α mRNA levels in the liver tissue could be suppressed by the combination treatment and the therapeutic effect better than both administered separately (Fig. 6D–G). In conclusion, our results suggest that epalrestat alleviates liver inflammation and pathology in NASH by inhibiting NLRP3 inflammasome activation.