Cyanidin-3-O-glucoside improves non-alcoholic fatty liver disease by promoting PINK1-mediated mitophagy in mice

Abstract

Background and purpose: Identifying safe and effective compounds that target to mitophagy to eliminate impaired mitochondria may be an attractive therapeutic strategy for non-alcoholic fatty liver disease. Here, we investigated the effects of cyanidin-3-O-glucoside (C3G) on non-alcoholic fatty liver disease (NAFLD) and the underlying mechanism.

Experimental approach: Non-alcoholic fatty liver disease was induced by a high-fat diet for 16 weeks. C3G was administered during the last 4 weeks. In vivo, recombinant adenoviruses and AAV8 were used for overexpression and knockdown of PTEN-induced kinase 1 (PINK1), respectively. AML-12 and HepG2 cells were used for the mechanism study.

Key results: C3G administration suppressed hepatic oxidative stress, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation and steatosis and improved systemic glucose metabolism in mice with NAFLD. These effects of C3G were also observed in palmitic acid-treated AML-12 cells and hepatocytes from NAFLD patients. Mechanistic investigations revealed that C3G increased PINK1/Parkin expression and mitochondrial localization and promoted PINK1-mediated mitophagy to clear damaged mitochondria. Knockdown of hepatic PINK1 abolished the mitophagy-inducing effect of C3G, which blunted the beneficial effects of C3G on oxidative stress, NLRP3 inflammasome activation, hepatic steatosis and glucose metabolism.

Conclusion and implications: These results demonstrate that PINK1-mediated mitophagy plays an essential role in the ability of C3G to alleviate NAFLD and suggest that C3G may be a potential drug candidate for NAFLD treatment.

FIGURE 1

Cyanidin‐3‐O‐glucoside (C3G) ameliorates hepatic steatosis and improves systemic glucose metabolism. (a–g, i–k) Mice were fed either a chow diet or an high fat diet fat (HFD) or 16 weeks. C3G‐treated mice were fed an high fat diet for 12 weeks and then fed an high fat diet containing 0.2% C3G for another 4 weeks (n = 9 mice per group). (a–c) Body weight, liver weight and LW/BW ratio. (d) Gross anatomical views of representative mouse liver. (e) Representative images of H&E and Oil‐red O staining of liver sections (original magnification 20×). (f) Hepatic triglyceride content. (g) mRNA abundance of genes related to cholesterol synthesis and efflux and fatty acid uptake, synthesis and β‐oxidation in the liver. (h) triglyceride content in hepatocytes from healthy controls and patients with non‐alcoholic fatty liver disease (NAFLD). Hepatocytes were treated with or without 100‐μM C3G for 12 h (n = 6). (i, j) Fasting blood glucose and insulin levels. (k) HOMA‐IR indexes. Data were expressed as the mean ± SEM. ^* P < 0.05; ^# P < 0.05

FIGURE 2

Cyanidin‐3‐O‐glucoside (C3G) inhibits NLRP3 inflammasome activation. (a–e) Mice were treated as described in Figure 1 (n = 9 mice per group). (a, b) Content of IL‐1β and IL‐18 in the livers and blood from different groups. (c–e) mRNA abundance of NLRP3, Caspase‐1, IL‐1β and IL‐18, the protein abundance of NLRP3, Pro‐caspase‐1 and IL‐1β and the activity of Caspase‐1 in the livers from different groups. (f–i) mRNA abundance of NLRP3, Caspase‐1, IL‐1β and IL‐18 in hepatocytes from healthy controls and patients with NAFLD. Hepatocytes were treated with or without 100‐μM C3G for 12 h (n = 6). Data were expressed as the mean ± SEM. ^* P < 0.05

FIGURE 3

Cyanidin‐3‐O‐glucoside (C3G) attenuates hepatic oxidative stress and clears damaged mitochondria. (a–f, i–o) Mice were treated as described in Figure 1 (n = 9 mice per group). (a–e) H₂O₂ and MDA content, activities of SOD, CAT and GSH‐Px in the livers from different groups. (f) mRNA abundance of PGC‐1α, NRF‐1, NRF‐2 and TFAM in the livers from different groups. (g, h) ROS content in hepatocytes from healthy controls and patients with non‐alcoholic fatty liver disease (NAFLD). Hepatocytes were treated with or without 100‐μM C3G for 12 h (n = 6). (i) Representative TEM images of the liver. Autophagosomes (arrow). (j) Number of autophagosomes in the TEM images of the liver. (k) Hepatic protein abundance of TOM20 and COX IV in the livers from different groups. (l) Representative images of hepatic IHC staining for TOM20 and COX IV (original magnification 40×). (m–o) Energy expenditure in different groups. Data were expressed as the mean ± SEM. ^* P < 0.05

FIGURE 4

Cyanidin‐3‐O‐glucoside (C3G) restores ) high fat diet (HFD)‐impaired hepatic mitophagy. (a–d) Mice were treated as described in Figure 1 (n = 9 mice per group). (a) Protein abundance of p62, LC3, PINK1 and Parkin in the livers from different groups. (b) Representative images of hepatic IHC staining for LC3, p62, PINK1 and Parkin in different groups (original magnification 40×). (c, d) Protein levels of PINK1, Parkin and LC3 in mitochondrial or cytoplasmic fractions of the livers from different groups. (e) Protein abundance of p62, LC3, PINK1, Parkin, TOM20 and COX IV in hepatocytes from healthy controls and patients with non‐alcoholic fatty liver disease (NAFLD). Hepatocytes were treated with or without 100‐μM C3G for 12 h (n = 6). Data were expressed as the mean ± SEM. ^* P < 0.05

FIGURE 5

Knockdown of PINK1 blocks cyanidin‐3‐O‐glucoside (C3G)‐induced hepatic mitophagy. (a) Schematic of the experimental design. (b–e) Mice were treated as shown in Figure 6a (n = 9 mice per group). (b, c) Protein levels of PINK1, Parkin and LC3 in mitochondrial or cytoplasmic fractions of the livers from different groups. (d) Hepatic protein abundance of TOM20 and COX IV in the livers from different groups. (e) Representative images of hepatic IHC staining for TOM20 and COX IV in different groups (original magnification 40×). Data were expressed as the mean ± SEM. ^* P < 0.05

FIGURE 6

Cyanidin‐3‐O‐glucoside (C3G) induces mitophagy via PINK1 in hepatocytes. (a, b) Representative images of PINK1 and TOM20, LC3 and TOM20 immunofluorescence staining in AML‐12 cells (n = 6). (c) Representative images of GFP‐LC3 and COX IV immunofluorescence staining in AML‐12 cells (n = 6). (d, e) Protein levels of PINK1, Parkin and LC3 in mitochondrial or cytoplasmic fractions in HepG2 cells (n = 6). Data were expressed as the mean ± SEM. ^* P < 0.05

FIGURE 7

PINK1‐mediated mitophagy is required for the beneficial effects of C3G on non‐alcoholic fatty liver disease (NAFLD). Mice were treated as shown in Figure 6a (n = 9 mice per group). (a) Representative images of H&E and Oil‐red O staining of liver sections (original magnification 20×). (b) Hepatic triglyceride (TG) content in the indicated groups. (c, d) Fasting blood glucose and insulin levels. (e) HOMA‐IR indexes. (f, g) Protein abundance of NLRP3, Pro‐Caspase‐1 and IL‐1β and the activity of Caspase‐1 in the livers from different groups. (h, i) H₂O₂ and MDA content in the livers from different groups. Data were expressed as the mean ± SEM. ^* P < 0.05

FIGURE 8

Proposed model for the beneficial effects of cyanidin‐3‐O‐glucoside (C3G) on non‐alcoholic fatty liver disease (NAFLD). C3G up‐regulated PINK1 and Parkin expression, which localized on damaged mitochondrial and enhanced autophagic flux simultaneously. Subsequently, damaged mitochondrial was engulfed by mitophagosome and degraded by mitophagolysosome, which further ameliorates hepatic steatosis, oxidative stress and NLRP3 inflammasome activation