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. 2020 Nov 5:7:546445.
doi: 10.3389/fmed.2020.546445. eCollection 2020.

Adiponectin Inhibits NLRP3 Inflammasome Activation in Nonalcoholic Steatohepatitis via AMPK-JNK/ErK1/2-NFκB/ROS Signaling Pathways

Zhixia Dong  1 , Qian Zhuang  1 , Xin Ye  1 , Min Ning  1 , Shan Wu  1 , Lungen Lu  2 , Xinjian Wan  1
Affiliations

Adiponectin Inhibits NLRP3 Inflammasome Activation in Nonalcoholic Steatohepatitis via AMPK-JNK/ErK1/2-NFκB/ROS Signaling Pathways

Zhixia Dong et al. Front Med (Lausanne). .

Abstract

Adiponectin, an adipose-derived adipokine, possesses a hepatoprotective role in various liver disorders. It has been reported that hypoadiponectinemia can affect with the progression of non-alcoholic fatty liver diseases (NAFLD). Inflammasome activation has been recognized to play a major role during the progression of NAFLD. This research aimed to explore the effect of adiponectin on palmitate (PA)-mediated NLRP3 inflammasome activation and its potential molecular mechanisms. Male adiponectin-knockout (adiponectin-KO) mice and C57BL/6 (wild-type) mice were fed a high-fat-diet (HFD) for 12 weeks as an in vivo model of non-alcoholic steatohepatitis (NASH). Serum biochemical markers, liver histology and inflammasome-related gene and protein expression were determined. In addition, the hepatocytes isolated from wide type mice were exposed to PA in the absence or presence of adiponectin and/or AMPK inhibitor. The activation of NLRP3 inflammasome was assessed by mRNA and protein expression. Furthermore, ROS production and related signaling pathways were also evaluated. In the in vivo experiments, excessive hepatic steatosis with increased NLRP3 inflammasome and its complex expression were found in adiponectin-KO mice compared to wild-type mice. Moreover, the expression levels of NLRP3 inflammasome pathway molecules (NFκB and ROS) were upregulated, while the phosphorylation levels of AMPK, JNK, and Erk1/2 were downregulated in adiponectin-KO mice compared with wild-type mice. In the in vitro study, PA increased lipid droplet deposition, NF-kB signaling and ROS production. Additionally, PA significantly promoted NLRP3 inflammasome activation and complex gene and protein expression in hepatocytes. Adiponectin could abolish PA-mediated inflammasome activation and decrease ROS production, which was reversed by AMPK inhibitor (compound C). Furthermore, the results showed that the inhibitory effect of adiponectin on PA-mediated inflammasome activation was regulated by AMPK-JNK/ErK1/2-NFκB/ROS signaling pathway. Adiponectin inhibited PA-mediated NLRP3 inflammasome activation in hepatocytes. Adiponectin analogs or AMPK agonists could serve as a potential novel agent for preventing or delaying the progression of NASH and NAFLD.

Keywords: AMPK; NAFLD; NLRP3 inflamamasome; adiponectin; hepatocytes.

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Figures

Figure 1
Figure 1
Adiponectin deficiency aggravates liver injury and steatosis as well as sensitizes to HFD-induced NLRP3 inflammasome activation. Four-week-old male wild type (WT) and adiponectin-knockout (APN KO) mice were fed either ND or HFD for 12 weeks. Liver tissue and serum samples were harvested at baseline, 4w, 8w, and 12w, respectively. (A) The body weight and the levels of adiponectin after HFD (B) the serum levels of ALT, AST, and the concentrations of TG and CHOL were determined by colorimetric assay at baseline, 4w, 8w, and 12w. (C) H&E staining and oil red staining in liver tissue sections for evaluation of inflammation and steatosis at different stages, the semi-quantitative analysis of ORO staining was performed by image J. (D) NLRP3, ASC, Capase1, IL1β, IL18, TNFα, and IL6 mRNA expression in liver tissue were detected by qPCR. (E) NLRP3, caspase1, and IL1β immunohistochemical staining in liver tissue sections from mice-fed with HFD at 12w. Results was presented as mean ± SEM, n = 6 mice per group. Differences between two groups were compared using a Student's t-tests. Differences between multiple groups were compared using one-way analysis of variance. ***p < 0.001, **p < 0.01.
Figure 2
Figure 2
The effects of adiponectin deficiency on ROS production and NFκB/AMPK/MAPK signaling pathways. (A) The ROS levels in liver tissue (B) NFκB and (C–E) AMPK/ p-AMPK, JNK/ p-JNK and ErK1/2/p-ErK1/2 protein expression was detected by quantitative analysis of WB, quantitative data were normalized by actin. The same image for actin has been reused in this figure because the loading control and involved protein was from the same batch of samples, in the case of loading equivalent amounts of total protein, actin were only detected once on a separated membrane to observe whether the internal parameter bands are consistent among the samples. Results was presented as mean ± SEM, n = 6 mice per group, Differences between two groups were compared using a Student's t-tests. Differences between multiple groups were compared using one-way analysis of variance **p < 0.01, *p < 0.05.
Figure 3
Figure 3
Adiponectin alleviates PA-mediated NLRP3 inflammasome expression in hepatocytes. Hepatocytes were isolated from mice and cultured for 48 h, then cells were serum-starved for 6 h and exposed to palmitic acid (PA, 300 μmol/ml) for 24 h. For inhibition experiment, adiponectin (APN, 10 μg/ml) was used 2 h prior to palmitic acid treatment. (A) Lipid droplet deposition assay was determined using BODIY fluorescence dye; the expression of NLRP3 was detected by immunofluorescence. (B–H) The expression levels of NLRP3 inflammasome complex genes such as NLRP3/ASC/Caspase1/IL1β/IL18 and other inflammatory makers (TNFα and IL6) were determined by qPCR. Results are presented as means ± SEM, all experiments were performed at least three times and at least in triplicate. Differences between two groups were compared using a Student's t-tests. Differences between multiple groups were compared using one-way analysis of variance. ***p < 0.001, **p < 0.01.
Figure 4
Figure 4
AMPK inhibitor reverses the inhibitory role of adiponectin on PA-induced NLRP3 inflammasome expression. Hepatocytes were pre-treated with APN and/or Compound C for 2 h prior to incubation with PA for 24 h. (A) NLRP3, (B) caspase1, (C) ASC, (D) IL-1β, (E) IL-18, (F) TNFα and (G) IL6 mRNA expression were detected by qPCR. Results are presented as means ± SEM, all experiments were performed at least three times and at least in triplicate. Differences between two groups were compared using a Student's t-tests. Differences between multiple groups were compared using one-way analysis of variance. ***p < 0.001, **p < 0.01, *p < 0.05.
Figure 5
Figure 5
AMPK inhibitor attenuates adiponectin-mediated hepatoprotective effects against PA-mediated NLRP3 inflammasome activation. Hepatocytes were pre-treated with APN 2 h prior to incubation with PA for 24 h. For AMPK inhibition experiment, Compound C (CC, 10 μM) were using 2 h prior to adiponectin. Cell and culture supernatant were harvested for further experiment (A) Western blot images and quantified expression of NLRP3 (B), Procaspase1, Caspase1 (C), pro-IL1β and IL1β (D). Quantitative data were normalized by actin. (E–H) The concentration of inflammatory cytokines IL1β, IL18, TNFα, and IL6 in cell culture supernatant were measured by ELISA. Results was presented as mean ± SEM, all experiments were performed at least three times and at least in triplicate. Differences between two groups were compared using a Student's t-tests. Differences between multiple groups were compared using one-way analysis of variance. **p < 0.01, *p < 0.05.
Figure 6
Figure 6
Adiponectin plays a protective role against PA-mediated NLRP3 inflammasome activation via AMPK-JNK/Erk1/2-NFκB/ROS signaling pathways. Hepatocytes were pretreated with APN 2 h prior to incubation with PA for 24 h. For AMPK inhibition experiment, Compound C (10 μM) were using 2 h prior to adiponectin. (A) Western blot images and quantified levels of (B–E) total AMPK and phosphorylated AMPK, total JNK, and phosphorylated-JNK, total ErK1/2 and phosphorylated-ErK1/2, total NFκB p65, and phosphorylated-p65. Quantitative data were normalized by actin. (F) The ROS production in hepatocytes was detected by BODIPY fluorescence dye. Results was presented as mean ± SEM, all experiments were performed at least three times and at least in triplicate. Differences between two groups were compared using a Student's t-tests. Differences between multiple groups were compared using one-way analysis of variance. **p < 0.01, *p < 0.05.
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