A pharmacological inhibitor of NLRP3 inflammasome prevents non-alcoholic fatty liver disease in a mouse model induced by high fat diet

The activation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is closely associated with the development and progression of non-alcoholic fatty liver disease (NAFLD) induced by a high-fat diet. Therefore, we investigated whether oral administration of sulforaphane (SFN) prevented high-fat diet-induced NAFLD in mice by regulation of the NLRP3 inflammasome in the liver. Daily oral administrations of SFN reduced hepatic steatosis scores, serum ALT and AST levels, and hepatic levels of cholesterol, triglycerides, and free fatty acids in mice fed a high-fat diet. These were correlated with the suppression of NLRP3 inflammasome activation in the liver by SFN as evidenced by decrease in mRNA levels of ASC and caspase-1, caspase-1 enzyme activity, and IL-1β levels. SFN inhibited saturated fatty acid-induced activation of the NLRP3 inflammasome in primary mouse hepatocytes, accompanied by the restoration of mitochondrial dysfunction. The suppression of NLRP3 inflammasome by SFN was mediated by the regulation of AMP-activated protein kinase-autophagy axis. Our findings demonstrated that the suppression of NLRP3 inflammasome activation by an orally available small molecule inhibitor leads to the alleviation of the hepatic steatosis symptoms associated with NAFLD induced by a high-fat diet.

the pathogenesis of liver disease makes its inhibition an attractive target for the treatment of these disorders; in particular as only limited number of treatments is available for the liver disease 7 .
We attempted to find orally administrable small-molecule inhibitors that target the NLRP3 inflammasome for possible preventive application in the management of NAFLD. In this study, we found that sulforaphane (1-isothiocyanato-4-methylsulfinylbutane; SFN) suppressed the activation of the NLRP3 inflammasome activation induced by saturated fatty acid in hepatocytes. SFN is an organosulfur compound that exhibits potent anti-inflammatory activity and was recently reported to attenuate high fat diet-induced visceral adiposity, adipocyte hypertrophy, lipid accumulation 15 , and alcohol-induced steatosis 16 . However, whether SFN prevents NAFLD has not been investigated, and the underlying mechanism by which SFN improves hepatic steatosis is poorly understood. Therefore, we investigated whether SFN prevented high fat diet-induced NAFLD in mice and whether this prevention was mediated by regulation of the NLRP3 inflammasome in the liver.

Results
Oral administration of sulforaphane suppresses high fat diet-induced hepatic steatosis in mice mediated through the inhibition of inflammasome in liver. We investigated whether oral administration of SFN resulted in prevention of NAFLD associated with high fat intake. Mice were fed a normal chow (NOR) or high-fat diet (HFD) for 9 weeks with or without daily oral administration of SFN (30 mg/kg/day). Orlistat (10 mg/kg/day), an anti-obesity drug, was administered as a positive control. Consumption of high levels of fat resulted in the development of hepatic steatosis in mice, compared with mice fed NOR, as shown by an increase in liver weight, histological steatosis score, serum levels of ALT and AST, and hepatic levels of total cholesterol, triglycerides, and free fatty acids (Fig. 1). (G) For intraperitoneal glucose tolerance testing (IPGTT), glucose solutions (1 g/kg) were administered via intraperitoneal injection to mice after a 12-hr fasting. Blood glucose levels were measured at 0, 15, 30, 60, 90 and 120 min after the glucose challenge. Values are presented as the mean ± SEM (n = 8/group). # Significantly different from NOR alone, p < 0.05. *Significantly different from HFD + veh, p < 0.05. Veh, vehicle.
Daily oral administration of SFN for 9 weeks culminated in a reduction in the liver weight and hepatic steatosis scores increased by HFD (NOR + veh, 1.0; HFD + veh, 3.2; HFD + SFN, 1.3; HFD + orlistat, 2.4) (Fig. 1A,B). Consistently, histological examination indicated that lipid deposition in the livers of HFD-fed mice was inhibited by administration of SFN (Fig. 1C). SFN treatment decreased the levels of hepatic total cholesterol, triglycerides, and free fatty acids elevated by HFD (Fig. 1D). Furthermore, SFN treatment resulted in a reduction of the serum levels of ALT and AST increased by HFD (Fig. 1E). The suppressive effects of SFN on hepatic steatosis were comparable to orlistat, an anti-obesity drug. We examined whether suppression of hepatic steatosis by SFN was accompanied by the restoration of an HFD-induced metabolic disorder. The homeostasis model assessment of insulin resistance index (HOMA-IR) and glucose tolerance test indicated that oral administration of SFN prevented the insulin resistance induced by HFD (Fig. 1F,G).
To investigate whether the preventive effect of SFN on hepatic steatosis was associated with the suppression of the NLRP3 inflammasome in vivo, we determined the expression levels of NLRP3 inflammasome components (including NLRP3, ASC, and caspase-1) in the livers. ASC and caspase-1 expression was remarkably increased by HFD, whereas NLRP3 expression was slightly enhanced ( Fig. 2A). Oral administration of SFN inhibited HFD-induced upregulation of ASC and caspase-1 in the liver ( Fig. 2A). We further determined the enzymatic activity of caspase-1 and the protein level of IL-1β as hallmarks of inflammasome activation in the liver isolated from mice fed NOR or HFD for 9 weeks with or without SFN. HFD increased the enzymatic activity of caspase-1 and IL-1β protein levels in the liver, whereas oral administration of SFN reduced the increase of the caspase-1 activity and IL-1β protein level in the liver caused by HFD (Fig. 2B,C). These results demonstrate that oral administration of SFN resulted in the suppression of HFD-induced NLRP3 inflammasome activation in the mouse liver.
These results demonstrate that oral administration of SFN was effective in preventing HFD-induced hepatic steatosis and was beneficial in improving the insulin resistance induced by HFD. This prevention was potentially mediated by suppression of fat-induced NLRP3 inflammasome activation.
Sulforaphane suppresses the activation of the NLRP3 inflammasome induced by saturated fatty acid in primary hepatocytes. We investigated whether SFN inhibited the activation of the NLRP3 inflammasome induced by the saturated fatty acid, palmitate, in primary mouse hepatocytes. Palmitate-induced degradation of pro-caspase-1 to caspase-1(p10) as well as cleavage of pro-IL-1β to mature IL-1β in primary mouse hepatocytes, as determined by immunoblotting of caspase-1(p10) and IL-1β in cell supernatants (Fig. 3A). These results indicate that palmitate induced activation of the NLRP3 inflammasome in hepatocytes. SFN inhibited palmitate-induced production of caspase-1(p10) and mature IL-1β in hepatocyte cell supernatant (Fig. 3A). SFN suppressed palmitate-induced secretion of mature IL-1β into cell culture medium as determined by ELISA (Fig. 3B). Consistently, SFN prevented palmitate-induced oligomerization of ASC, another hallmark of inflammasome activation, as shown by speckle formation using confocal microscopy (Fig. 3C). These results demonstrate that SFN suppressed the activation of the NLRP3 inflammasome induced by saturated fatty acid in hepatocytes.
Mitochondrial dysfunction is the preceding event occurring with saturated fat-induced activation of the NLRP3 inflammasome 13,17,18 . Therefore, we investigated whether SFN prevented the mitochondrial dysfunction induced by palmitate in primary mouse hepatocytes. The ratio of oxidized to reduced nicotinamide adenine dinucleotide (NAD + /NADH) was decreased after treatment of palmitate due to mitochondrial dysfunction, while the NAD + /NADH ratio was recovered by SFN treatment (Fig. 3D). We next determined mitochondrial reactive oxygen species (ROS) content that can be increased by mitochondrial dysfunction and is crucial in inflammasome activation 19 . SFN reduced palmitate-induced production of mitochondrial ROS in hepatocytes (Fig. 3E). These results show that SFN prevented the mitochondrial dysfunction induced by saturated fatty acid, thereby suppressing NLRP3 inflammasome activation in hepatocytes. Sulforaphane induces activation of the AMPK-autophagy axis in mouse primary hepatocytes. Given that autophagy is linked to quality control of mitochondria by removing damaged or dysfunctional mitochondria, defective autophagy events lead to the accumulation of dysfunctional mitochondria, subsequently stimulating the activation of the NLRP3 inflammasome 17,20 . Recent studies have demonstrated essential protective function for autophagy during liver injury and death by inhibiting the generation of inflammasome-depenent IL-1β 21 . Immunoblotting results of LC3I conversion to LC3II indicated that SFN induced autophagosome formation in primary mouse hepatocytes (Fig. 4A,B). Transition electron microscopic analysis confirmed that autophagosome formation was induced by SFN in primary mouse hepatocytes (Fig. 4C). Activation of autophagy by SFN resulted in p62 degradation (Fig. 4D); p62 is an autophagy substrate that is degraded during autophagy activation. These suggest that SFN promotes autophagy. Mammalian target of rapamycin (mTOR) is a negatively regulator of autophagy. SFN reduced the phosphorylation of mTOR(Ser2448) in primary mouse hepatocytes (Fig. 4D), suggesting that SFN inhibited mTOR activation. SFN suppression of mTOR activation was confirmed by a decrease in p70S6K1 phosphorylation, which is a downstream substrate of mTOR (Fig. 4D).
To investigate whether SFN regulated fat-mediated decrease in autophagy, the effect of SFN on autophagy was examined in livers isolated from mice fed NOR, HFD, or HFD with SFN. Oral administration of SFN induced LC3II formation in the liver (Fig. 4E). Autophagy induction by SFN was correlated with the suppression of IL-1β generation (Fig. 4E). The suppression of mTOR in livers by SFN was confirmed by the reduction in the phosphorylation of p70S6K1, which was increased by HFD (Fig. 4E). The results show that oral administration of SFN prevented fat-induced impairment of autophagy in mouse liver. These results further demonstrate the regulation of autophagy-inflammasome axis by SFN both in vitro and in vivo.
Activation of AMP-activated protein kinase (AMPK) by saturated fatty acid is involved in the regulation of autophagy events. Therefore, we investigated whether SFN induced the activation of AMPK in hepatocytes. SFN induced phosphorylation of threonine 172 in the AMPK-α subunit in primary mouse hepatocytes in a time-and dose-dependent manner (Fig. 5A,B,C), suggesting that SFN activated AMPK in hepatocytes. SFN increased the AMP/ATP ratio in hepatocytes (Fig. 5D), suggesting that an increase in AMP and a decrease in ATP resulted in the SFN-induced activation of AMPK. To confirm the effect of SFN on hepatic energy changes in vivo, SFN (20 mg/kg) was orally administered to mice for 5 days, and the levels of adenine nucleotides were examined in mouse livers. Oral administration of SFN resulted in a decrease in hepatic ATP levels and an increase in hepatic AMP levels, with a 1.8-fold increase in the AMP/ATP ratio compared with vehicle treatment (Fig. 5E). These results indicate that SFN increased the ratio of AMP to ATP in hepatocytes, culminating in activation of AMPK both in vitro and in vivo.
To further confirm SFN activated the AMPK-autophagy axis in hepatocytes, we examined the regulation of the signaling pathways from AMPK to autophagy by SFN. AMPK acts as a positive regulator of autophagy by directly phosphorylating the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast Autophagy related-1 (ATG1), at Ser317. SFN induced the phosphorylation of Ulk1 at Ser317 in primary mouse hepatocytes (Fig. 6A). In contrast, the phosphorylation of Ulk1 at Ser757 was decreased by SFN (Fig. 6A). These suggest that SFN promotes autophagy by activating AMPK-Ulk1 pathway.
To investigate whether SFN regulated fat-mediated decrease in autophagy, the effect of SFN on signaling pathways from AMPK to autophagy was examined in the liver isolated from mice fed NOR, HFD, or HFD with SFN. HFD decreased the phosphorylation of AMPK(T172) and Ulk(S317) in the liver, whereas oral administration of SFN to mice reversed the HFD-induced downregulation of p-AMPK(T172) and p-Ulk1(S317) in liver tissues (Fig. 6B). These results demonstrate the activation of AMPK-autophagy pathway by SFN both in vitro and in vivo.
In addition, the results show that oral administration of SFN prevented fat-induced impairment of autophagy in mice via the activation of AMPK, followed by Ulk1 activation.
Collectively, our results demonstrate that SFN prevents HFD-induced hepatic steatosis mediated by the suppression of NLRP3 inflammasome activation in the liver. The activation of AMPK and autophagy by SFN leads to a recovery from the mitochondrial dysfunction induced by saturated fatty acids, thereby downregulating fat-induced activation of the NLRP3 inflammasome (Fig. 6C).

Discussion
These results suggest that regulating NLRP3 inflammasome activation with small molecule inhibitors is a potential therapeutic strategy to treat or prevent the pathological outcomes of NAFLD. Csak et al. 12 suggested that an increased influx of saturated fatty acids to the liver leads to activation of the NLRP3 inflammasome followed by IL-1β secretion and a subsequent inflammatory response. IL-1β is an important pro-inflammatory cytokine that amplifies inflammation and sensitizes hepatocytes to liver damage 10 . We identified SFN as a potent inhibitor of the NLRP3 inflammasome activation induced by a HFD or saturated fatty acid, as shown by a decrease in IL-1β and caspase-1 production in the liver. Our results demonstrate the inhibitory effects of SFN on NLRP3 inflammasome activation both in vitro in hepatocytes and an in vivo in an animal model.
Fatty acid-induced inflammasome activation in macrophages was NLRP3 dependent and involved increased mitochondrial production of reactive oxygen species (ROS) and decreased autophagy due to reduced AMPK activity 13 . A previous paper reported that metformin is a known AMPK activator that inhibits IL-1β production by macrophages of diabetic patients through AMPK activation in vitro 22 . Our results demonstrate that SFN activates AMPK and autophagy, whereas it suppresses the activation of the NLRP3 inflammasome and mitochondrial impairment in hepatocytes. These results are consistent with the hypothesis that AMPK activation attenuates inflammasome activation, which might involve rejuvenation of 'stressed' mitochondria, as dysfunctional mitochondria accelerate NLRP3 inflammasome activation 23 .
NAFLD is prevalent in women as well as men. Some studies showed that women had a higher prevalence as compared with men. Ayonrinde et al. reported in the Western Australian Pregnancy Cohort Study that women compared with men had a significantly higher prevalence of NAFLD and central obesity 24 . Interestingly, men with NAFLD showed more adverse metabolic phenotype and more severe liver injury than women with NAFLD 24 . In contrast, other population-based studies showed that NAFLD is significantly more prevalent in men than in women 25 . However, Younossi et al. reported that lean NAFLD was associated more with women 26 . These underscore the complexity of the influence of gender on the prevalence, severity, and phenotype of NAFLD. There are several factors suggested to contribute to the gender difference of NAFLD, including sex hormones, alcohol consumption, lifestyle, and environment. It is postulated that female hormones may protect against NAFLD since NAFLD is twice as common in postmenopausal women as in premenopausal women 27,28 . In our study, we chose male mice to exclude the influence of the female sex hormone cycle on the experiment because we intended to make the experimental system more consistent during 9-week diet study. It is important to compare any gender difference in SFN response because this would be the next step to advance towards the clinical application of our findings. It would be pursued in a future study to investigate whether there are gender-specific differences in the protective effect of SFN in NAFLD.
Collectively, our results demonstrate that SFN prevents HFD-induced hepatic steatosis mediated by the suppression of NLRP3 inflammasome activation in hepatocytes. The induction of autophagy by SFN leads to a recovery from the mitochondrial dysfunction induced by saturated fatty acid, thereby downregulating fat-induced activation of the NLRP3 inflammasome through AMPK-autophagy signaling. The subsequent decrease in IL-1β production in hepatocytes contributes to alleviating hepatic steatosis. These results suggest that modulation of NLRP3 inflammasome with small molecule inhibitors is a potentially effective strategy to prevent the pathological outcomes of NAFLD.

Materials and Methods
Ethics statement. All animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985). All the experimental procedures were carried out in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Catholic University of Korea (permission# 2014-015).
Animals. C57BL/6 mice (male) were purchased from Orient bio (Seoul, Korea) and were acclimated under specific pathogen-free conditions in an animal facility for at least a week before experimentation. The mice were housed in a 12:12-hr light/dark cycle, temperature (23 ± 3 °C) and humidity (40-60%) controlled room. All mice were allowed ad libitum access to their diet and water during the experimental period.  Preparation of primary mouse hepatocytes. Primary mouse hepatocytes were obtained by collagenase perfusion of livers as described previously 29 , with slight modifications. Mouse livers were perfused with phosphate buffered saline (PBS) containing type IV collagenase (100 U/ml; Worthington Biochemical, Lakewood, NJ) for digestion at a flow rate of 9 ml/min. Hepatocytes were isolated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 25 mM glucose and 10% fetal bovine serum (FBS). Viability was > 90% for all preparations, as determined by trypan blue staining. Prior to experimentation, cells were plated on collagen-coated (8 μg/cm 2 ) plates, maintained overnight in serum-free DMEM and used within 30 hr of plating.

Reagents. Purified lipopolysaccharides (LPS) from
High-fat diet study. C57BL/6 mice (males, 5 weeks-old) were randomly divided into four weight-matched groups (n = 8/group): normal chow diet (NOR), high-fat diet (HFD), HFD + SFN and HFD + orlistat. Mice were fed a HFD for 9 weeks, accompanied by daily oral administrations of either SFN (30 mg/kg) or orlistat (10 mg/kg). NOR (11.5 kcal%) and HFD (40 kcal%) were obtained from Research Diets, Inc. (New Brunswick, NJ, USA). Body weight and food intake were recorded every week. At the end of the 9-week period, all animals were fasted for 12 hr. On the following day, mice were anesthetized with Zoletil (Virbac, Carros Cedex, France), and blood samples were collected by cardiac puncture. Liver tissues were excised, rinsed, weighed, and stored at − 70 °C pending further analysis.
Immunoblot assay with hepatocytes and liver tissue. For immunoblotting of caspase-1 and IL-1β , hepatocytes were plated in 6-well plates (8 × 10 5 cells/ml) and primed with LPS (500 ng/ml) for 4 hr. Cells were washed with PBS to remove LPS before exposure to SFN to exclude any effect of SFN on LPS. Cells were treated with SFN for 1 hr and further stimulated with palmitate in serum-free medium. To detect caspase-1(p10) and IL-1β in cell culture supernatant, the supernatants were precipitated by adding one volume of methanol and 0.25 volumes of chloroform followed by centrifugation at 20,000 g for 10 min. The upper phase was discarded, and one volume of methanol was added. The mixture was centrifuged at 20,000 g for 10 min to obtain a protein pellet, which was dried at room temperature, resuspended in Laemmli buffer (0.25 M Tris-HCl, pH 6.8, 0.4% glycerol, 10% SDS, 0.2% 2-mercaptoethanol and 0.64% bromophenol blue) and subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). To detect pro-caspase-1 and pro-IL-1β , cell pellets were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 mM Na 3 VO 4 , 10 μg/ml aprotinin, and 10 μg/ml leupeptin) and processed for SDS-PAGE. Liver tissues isolated from experimental mice were homogenized at 4 °C in RIPA buffer and centrifuged at 10,000 g for 10 min at 4 °C; protein extracts processed for SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were probed with primary antibodies and their corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies. The bands were visualized using an enhanced chemiluminescence (ECL) system. Enzyme-linked immunosorbent assays. IL-1β levels in culture media or liver homogenate supernatants were determined using a DuoSet enzyme-linked immunosorbent assay (ELISA) kit (R&D systems) according to the manufacturer's instructions. Standard curve concentrations were 15.6 to 1000 pg/ml.

Confocal microscopy analysis.
Primary hepatocytes were plated overnight on cover slides in 24-well plates. After cells were fixed with methanol for 30 min, cells were blocked with 1% bovine serum albumin for 1 hr and incubated with an anti-ASC antibody (Santa Cruz Biotechnology Inc.) at 4 °C overnight. Cells were further incubated with Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen, Carlsbad, CA) for 2 hr at room temperature. To determine mitochondrial superoxide production, hepatocytes were stained with MitoSOX Red (4 mM; Invitrogen). Cells were co-stained with 4′ ,6-diamidino-2-phenylindole (DAPI, 1 μg/ml; Invitrogen) for nuclei staining. Slides were mounted in fluorescent mounting medium (Vector laboratories, Burlingame, CA). Samples were examined with an LSM710 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany). Images were obtained and analyzed with ZEN2011 software (Carl Zeiss).
Measurement of the NAD + /NADH ratio. Quantification of NAD and NADH was performed using a NAD + /NADH assay kit from Abcam (Cambridge, UK) according to the manufacturer's instructions. Livers were snap frozen in liquid nitrogen and extracted at 4 °C using the extraction buffer provided with the NAD + /NADH assay kit. NAD + and NADH values were normalized by their protein concentration.
Caspase-1 activity assay. The enzymatic activity of caspase-1 in mouse livers was measured using a Caspase-1 assay kit from Bio-vision (Milpitas, CA) according to the manufacturer's instructions. Fluorescence was recorded at 400 nm after excitation at 505 nm using a SpectraMaxM5 microplate reader (Molecular Devices, Sunnyvale, CA).