Punicalagin relieves hepatic injury by antioxidation and enhancement of autophagy in diet-induced nonalcoholic steatohepatitis

Abstract

Hepatic injury induced by many factors play a central role in the pathogenesis of liver diseases. Punicalagin (PUN) is a major antioxidant polyphenolic compound extracted from pomegranates. The aim of this study was to investigate the potential role of PUN on liver injury induced by nonalcoholic steatohepatitis (NASH). Therefore, the effects and mechanistic action of PUN on NASH mouse model induced by choline-deficient, L-amino acid- defined, high-fat (CDAAH) diet were investigated in vivo. Wild-type or nuclear erythroid 2-related factor 2 (Nrf2) KO mice were fed with CDAAH diet to induce NASH and then treated with PUN (100, 300 or 500 mg kg^{- 1} day^{- 1}) by gavage for 12 weeks. Blood and liver samples were collected to assess liver function, oxidative stress, inflammation, and autophagy pathological status. The results showed that 300 mg/kg PUN was the optimal concentration for relieving hepatic injury in NASH, characterized by decreased activities of serum alanine transaminase, aspartate aminotransferase, and liver lactate dehydrogenase activity and histopathological structural damage, and showed a hepatoprotective effect against NASH. PUN significantly reduced the level of liver inflammation and Txnip-NLRP3 signaling pathway in NASH mice. PUN reduced oxidative stress by reducing liver malondialdehyde levels and the accumulation of reactive oxygen species (ROS) and increasing liver superoxide dismutase and glutathione peroxidase activity. PUN may also attenuate oxidative stress and induce autophagy through the p62/Nrf2 and AMPK/mTOR/ULK1 pathway. More importantly, we found that these protective effects of PUN were attributed to enhanced antioxidant, anti-inflammatory and autophagy activity, which was mediated by the activation of the Nrf2 pathway using Nrf2 KO mice. In summary, the present results indicate that PUN successfully relieved NASH-induced liver damage by upregulating Nrf2 signaling and autophagy.

Keywords: Autophagy; NASH; Nrf2; Oxidative stress; Punicalagin; Txnip-NLRP3 signaling.

Fig. 1

PUN alleviated liver injury in diet-induced NASH. (A) Serum ALT and AST levels and liver hydroxyproline concentrations. Data are presented as the mean ± SD (n = 10, from three independent experiments). (B, C) The H&E staining of liver tissues in the Control, vehicle and PUN (100, 300, 500 mg/kg) groups (original magnification, 200×). (D-E) Detection of ALT and AST concentrations in serum of PUN-treated mice after NASH. (F, G) H&E and Masson staining of liver tissues (original magnification, 200×). Similar results were obtained from three independent experiments. All data are presented as the mean ± SEM (n = 6 in each group). *P < 0.05 and **P < 0.01 vs. Control group; ##P < 0.01 vs. NASH group.

Fig. 2

PUN attenuated hepatic inflammatory response in mice with NASH. (A-C) Effects of PUN on NASH-induced serum TNF-α, IL-6, and IL-1β generation. Similar results were obtained from three independent experiments. (D-G) Effects of PUN on NLRP3, ASC, cleaved-caspase-1, and mature-IL-1β protein expression was measured by western blotting and quantification of protein expression was performed by densitometric analysis. Similar results were obtained from three independent experiments. All data are presented as the mean ± SEM (n = 6 in each group). *p < 0.05 and **P < 0.01 vs. Control group; ##P < 0.01 vs. NASH group.

Fig. 3

PUN enhanced the antioxidant activities in the liver injury model induced by NASH. (A-C) The hepatic concentration of GSH, MDA and hepatic SOD activity after CDAAH-induced NASH with or without intragastric infusion of PUN. (D-E) Cellular ROS estimated using the probe DCFH-DA by fluorescence microscopy. All data are presented as means ± SEM (n = 6 in each group). *P < 0.05 and **P < 0.01 vs. the NASH group.

Fig. 4

PUN upregulated the p62/Nrf-2 signaling in mice with NASH-induced liver injury. (A-C) Immunofluorescence stained analysis of Nrf2 and p-p62 in liver tissues (original magnification, 200×). (D-G) Liver tissue was analyzed by western blot for assessment of nuclear abundance of Nrf2, HO-1 and phosphorylation of p62 at ser349. β-actin was used as an internal control. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 6 in each group). ^*P < 0.05 and ^**P < 0.01 vs. Control group; ^#P < 0.05 and ^##P < 0.01 vs. NASH group.

Fig. 5

PUN regulates up-regulated autophagy via the AMPK/mTOR/ULK1 signaling pathway. (A-E) Effects of PUN on protein abundance of Beclin-1, LC3, Atg5 and Atg7 measured by western blotting. (F-I) Effects of PUN on protein abundance of p-AMPK, p-mTOR and p-ULK1 measured by western blotting. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 6 in each group). ^*P < 0.05 and ^**P < 0.01 vs. Control group; ^#P < 0.05 and ^##P < 0.01 vs. NASH group.

Fig. 6

PUN improved liver injury caused by NASH dependent upon Nrf2 deficiency in mice. WT and Nrf2^−/− mice were injected intraperitoneally with PUN (300 mg/kg) for 24 h followed by challenge with NASH. (A-B) Blood serum collected assessment of ALT and AST activities. (C-F) Liver tissues serum TNF-α, IL-6, and IL-1β and ROS generation. Similar results were obtained from three independent experiments. (G, H) Representative histological sections of liver were stained with H&E and Masson (magnification×400). Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 6 in each group). ^*P < 0.05 and ^**P < 0.01 vs. Control group; ^#P < 0.05 and ^##P < 0.01 vs. NASH group.

Fig. 7

PUN improved Txnip-NLRP3 signaling pathway caused by NASH dependent upon Nrf2 deficiency in mice. WT and Nrf2^−/− mice were injected intraperitoneally with PUN (300 mg/kg) for 24 h followed by challenge with NASH. (A-F) Effects of PUN on protein abundance of Txinp, NLRP3, ASC, Cleaved-Caspase1 and Mature-IL-1β measured by western blot. β-actin was used as an internal control. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 6 in each group). ^*P < 0.05 and ^**P < 0.01 vs. Control group; ^#P < 0.05 and ^##P < 0.01 vs. NASH group.

Fig. 8

PUN improved Autophagy caused by NASH dependent upon Nrf2 deficiency in mice. WT and Nrf2^−/− mice were injected intraperitoneally with PUN (300 mg/kg) for 24 h followed by challenge with NASH. (A-E) Effects of PUN on protein abundance of Beclin-1, Atg5, Atg7, LC3, p-AMPK, p-ULK1 and p-mTOR measured by western blot. β-actin was used as an internal control. Similar results were obtained from three independent experiments. All data are presented as means ± SEM (n = 6 in each group). ^*P < 0.05 and ^**P < 0.01 vs. Control group; ^#P < 0.05 and ^##P < 0.01 vs. NASH group.

Fig. 9

Punicalagin Relieves Hepatic Injury by Antioxidation and Enhancement of Autophagy in diet-induced Nonalcoholic Steatohepatitis.