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

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

Gabsik Yang et al. Sci Rep. 2016.

Erratum in

Abstract

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.

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Figures

Figure 1

Figure 1. Oral administration of sulforaphane ameliorates high fat diet-induced hepatic steatosis in mice.

Mice were fed normal chow diet (NOR) or a high-fat diet (HFD) for 9 weeks. SFN (30 mg/kg/day) or orlistat (10 mg/kg/day) was administered once daily via oral gavage for 9 weeks. N = 8/group. (A) Liver weights (left panel), Liver weight/body weight (right panel). (B) Hepatic steatosis scores were determined with semi-quantitative pathological standards. (C) The liver tissues were stained with hematoxylin and eosin. (D) The hepatic levels of total cholesterol, triglycerides, and free fatty acids were determined. (E) ALT and AST serum levels were determined. (F) Serum homeostasis model assessment of insulin resistance (HOMA-IR) levels in mice. (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.

Figure 2

Figure 2. Oral administration of sulforaphane suppresses high fat diet-induced inflammasome activation in mouse livers.

Mice were fed normal chow diet (NOR) or a high-fat diet (HFD) for 9 weeks. Sulforaphane (SFN, 30 mg/kg/day) was administered once daily via oral gavage for 9 weeks. N = 8/group. (A) NLRP3, ASC and caspase-1 mRNA levels in the liver were determined by quantitative real time PCR. mRNA levels are expressed as the relative expression levels compared to the NOR + veh group for each gene. (B) The enzymatic activity of caspase-1 in liver homogenates was determined using a Caspase-1 assay kit and expressed as the relative activity to NOR alone. (C) IL-1β protein levels in liver homogenates were determined by ELISA. 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.

Figure 3

Figure 3. Sulforaphane suppresses activation of the NLRP3 inflammasome induced by saturated fatty acid in primary mouse hepatocytes.

After primary mouse hepatocytes were primed with LPS (500 ng/ml) for 4 hr, cells were treated with SFN for 1 hr at the indicated concentrations and further stimulated with palmitate (400 μM) for 16 hr. (A) Immunoblotting for caspase-1 (p10), IL-1β, pro-caspase-1, and pro-IL-1β was performed with cell culture supernatants and cell lysates. Cropped blots are presented and the full-length blots are included in the Supplementary Figure 1. The gels have been run under the same experimental conditions. (B) IL-1β secretion into the culture medium was determined by ELISA. N.D., not detected. (C) Cells were stained for ASC (green) and 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ml, blue) and assessed by confocal microscopy. Arrows indicate ASC speckles. Data are representative of three independent experiments. DIC, differential interference contrast. (D) The NAD+/NADH ratio in cell lysates was determined using a commercial kit. (E) Cells were stained with MitoSOX Red (4 mM) to detect mitochondrial superoxide and DAPI for nuclei staining and assessed by confocal microscopy. MitoSOX Red fluorescence was presented as relative fluorescence compared with the vehicle alone. (B,D,E) Values are presented as the mean ± SEM. #Significantly different from vehicle alone, p < 0.05. *Significantly different from palmitate alone, p < 0.05. Veh, vehicle.

Figure 4

Figure 4. Sulforaphane induces autophagy by the suppression of mTOR in hepatocytes.

(A,B) Primary mouse hepatocytes were treated with SFN as indicated (24 hr for (A); 20 μM for (B)). Cell lysates were analyzed with immunoblot analysis for LC3I, LC3II, and actin. (C) After primary mouse hepatocytes were treated with SFN (20 μM) for 24 hr, cells were assessed by transmission electron microscopy analysis (original magnification, 8000×). White arrows indicate the autophagosomes/autolysosomal structures. (D) After primary mouse hepatocytes were treated with SFN (20 μM) for 1 hr (except LC3 for 24 hr), cell lysates were analyzed by immunoblotting for LC3, p62, actin, phospho-mTOR(S2448), mTOR, phospho-p70S6K1(T389), and p70S6K1. (E) Mice were fed a normal chow (NOR) or a high-fat diet (HFD) for 9 weeks with or without daily oral administration of SFN (30 mg/kg/day) or orlistat (10 mg/kg/day) once per day for 9 weeks. Liver samples were analyzed by immunoblotting for LC3, IL-1β, actin, phospho-p70S6K1(T389) and p70S6K1. #1 and #2 represent independent liver samples. Veh, vehicle. (A,B,D,E) Cropped blots are presented and the full-length blots are included in the Supplementary Figures 2–5. The gels have been run under the same experimental conditions.

Figure 5

Figure 5. Sulforaphane induces activation of AMPK in hepatocytes.

(A,B) Primary mouse hepatocytes were treated with SFN (20 μM) for the indicated times. For (A), cell lysates were analyzed by immunoblotting for phospho-AMPK(Thr172) and AMPK. Cropped blots are presented and the full-length blots are included in the Supplementary Figure 6. The gels have been run under the same experimental conditions. For (B), the levels of phospho-AMPK(Thr172) were determined by ELISA kit and expressed as fold inductions compared with time 0. (C) After primary mouse hepatocytes were treated with SFN at the indicated concentrations for 1 hr, phospho-AMPK(Thr172) levels were determined by ELISA and expressed as fold inductions compared with the vehicle alone. (D) After primary mouse hepatocytes were treated with SFN at the indicated concentrations for 24 hr, AMP and ATP levels were determined. The AMP/ATP ratio was expressed relative to the vehicle alone. (E) After mice were orally administered SFN (20 mg/kg/day) for 5 days, AMP and ATP levels in the liver were determined. The AMP/ATP ratio was expressed relative to the vehicle alone. (B–E) Values are presented as the mean ± SEM. *Significantly different from the vehicle alone, p < 0.05.

Figure 6

Figure 6. Activation of AMPK by sulforaphane is associated with the induction of autophagy in primary mouse hepatocytes and mouse livers from high-fat diet-treated mice.

(A) After primary mouse hepatocytes were treated with SFN (20 μM) for 1 hr, cell lysates were analyzed by immunoblotting for phospho-AMPK(T172), AMPK, phospho-Ulk1(S317), phospho-Ulk1(S757), and Ulk1. (B) Mice were fed a normal chow (NOR) or a high-fat diet (HFD) for 9 weeks with or without daily oral administration of SFN (30 mg/kg/day) or orlistat (10 mg/kg/day) once per day for 9 weeks. Liver samples were analyzed by immunoblotting for phospho-AMPK(T172), AMPK, phospho-Ulk1(S317), and Ulk1. #1 and #2 represent independent liver samples. Veh, vehicle. (A, B) Cropped blots are presented and the full-length blots are included in the Supplementary Figures 7 and 8. The gels have been run under the same experimental conditions. (C) Differential regulation of signaling pathways involving AMPK, mTOR, p70S6K1, Ulk1, and autophagy by saturated fatty acid (palmitate) and SFN is depicted.

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