Deficiency of DJ-1 Ameliorates Liver Fibrosis through Inhibition of Hepatic ROS Production and Inflammation

Abstract

Liver fibrosis is a global health problem and previous studies have demonstrated that reactive oxygen species (ROS) play important roles in fibrogenesis. Parkinson disease (autosomal recessive, early onset) 7 (Park7) also called DJ-1 has an essential role in modulating cellular ROS levels. DJ-1 therefore may play functions in liver fibrogenesis and modulation of DJ-1 may be a promising therapeutic approach. Here, wild-type (WT) and DJ-1 knockout (DJ-1 KO) mice were administrated with carbon tetrachloride (CCl4) to induce liver fibrosis or acute liver injury. Results showed that DJ-1 depletion significantly blunted liver fibrosis, accompanied by marked reductions in liver injury and ROS production. In the acute CCl4 model, deficiency of DJ-1 showed hepatic protective functions as evidenced by decreased hepatic damage, reduced ROS levels, diminished hepatic inflammation and hepatocyte proliferation compared to WT mice. In vitro hepatic stellate cells (HSCs) activation assays indicated that DJ-1 has no direct effect on the activation of HSCs in the context of with or without TGFβ treatment. Thus our present study demonstrates that in CCl4-induced liver fibrosis, DJ-1 deficiency attenuates mice fibrosis by inhibiting ROS production and liver injury, and further indirectly affecting the activation of HSCs. These results are in line with previous studies that ROS promote HSC activation and fibrosis development, and suggest the therapeutic value of DJ-1 in treatment of liver fibrosis.

Keywords: DJ-1; ROS; inflammation; lipid peroxidation.; liver fibrosis; liver injury.

Figure 1

DJ-1 deficiency attenuates CCl4-induced liver fibrosis in Vivo. (A) Representative Sirius red staining and α-SMA immunohistochemistry of livers from WT mice and DJ-1 KO mice after CCl4 injection for 8 weeks (200× magnification). Bar graphs show quantification of them (n = 6-9). The data are shown as the mean ± SEM. *P<0.05. (B) α-SMA, TIMP-1, Col1α1, Col3α1, TIMP-2, MMP-2 mRNA expression in livers was analyzed by qPCR after CCl4 injection for 8 weeks (n = 6-9). All gene expression levels were normalized to housekeeping control GAPDH. *P<0.05. (C) Protein expression of α-SMA in whole livers from mice treated with CCl4 for 8 weeks was analyzed by immunoblotting. Bar graph shows quantification of it (n = 4). ***P<0.001. (D) TGFβ mRNA expression in livers of WT and DJ-1 KO mice after CCl4 injection for 4 weeks or 8 weeks was analyzed by qPCR (n = 6-9). The data are shown as the mean ± SEM. *P<0.05. RFC: relative fold change. (E) Phosphorylation levels of Smad2 in livers from WT mice and DJ-1 KO mice treated with CCl4 for 8 weeks were analyzed by immunohistochemistry. Bar graph shows quantification of it (n = 7). *P<0.05.

Figure 2

DJ-1 has no direct effect on primary HSCs activation in vitro. (A) mRNA levels of activated HSCs markers were analyzed by qPCR after isolated HSCs cultured for 1d, 4d and 7d (n = 4-5). (B) mRNA levels of TIMP-1, α-SMA, Col1α1 in isolated HSCs of the two groups were analyzed after TGFβ treatment for 24h. The data are shown as the mean ± SEM. RFC: relative fold change.

Figure 3

Deficiency of DJ-1 attenuates liver damage and ROS generation in CCl4-induced acute liver injury. (A) HE staining and serum ALT levels were displayed at different time points after CCl4 injection (n = 4-7). *P<0.05, **P<0.01, ***P<0.001. (B) F4/80 and MPO Immunochemistry were performed in liver sections of WT mice and DJ-1 KO mice after CCl4 injection for 36h and 48h (400× magnification). (C) Cleaved-caspase 3 in liver sections from WT mice and DJ-1 KO mice after CCl4 injection for 36h was shown (n = 3) (200× magnification). Quantification of the positive areas was also performed (n = 3). **P<0.01. (D) mRNA levels of IL-6, TNF-α cytokines were determined by qPCR 36h post CCl4 injection. **P<0.01, ***P<0.001. (E) Immunochemistry for PCNA and Ki67 were performed after CCl4 injection for 36h and 48h respectively (200× magnification). Bar graphs show quantification of them (n = 5). **P<0.01, ***P<0.001. (F) ROS production was visualized by DCFH-DA probe in frozen liver sections from WT mice and DJ-1 KO mice 24h after CCl4 treatment (200× magnification). (G) MDA content in liver tissue homogenates was evaluated between WT mice and DJ-1 KO mice 24h after CCl4 injection. *P<0.05. (H) Gene expression of CYP2E1 in two groups was displayed 24h and 36h after CCl4 injection.

Figure 4

DJ-1 deletion suppresses ROS production and lipid peroxidation in CCl4-induced chronic liver injury. (A) Serum ALT and AST levels were determined in WT mice and DJ-1 KO mice after CCl4 injection for 8 weeks (n = 6-9). *P<0.05, **P<0.01. (B) Cleaved-caspase 3 in liver sections from WT mice and DJ-1 KO mice after CCl4 injection for 8 weeks was shown (n = 3) (200× magnification). Quantification of the positive areas was also performed (n = 3). *P<0.05. (C) Immunochemistry for MPO and CD11b were performed in liver sections of WT mice and DJ-1 KO mice after CCl4 injection for 8 weeks (400× magnification). Bar graphs show quantification of them, *P<0.05. (D) Gene expression of cytokines IL-6, TNF-α in the whole liver tissues was analyzed by qPCR after CCl4 injection for 8 weeks. *P<0.05. RFC: relative fold change. (E) ROS production was visualized in frozen liver sections and (F) MDA content was evaluated in liver tissue homogenates from WT mice and DJ-1 KO mice treated with CCl4 for 4 weeks (200× magnification). (G) Gene expression of CYP2E1 in two groups was displayed after 8 weeks CCl4 injection. (H) ROS production in isolated Kupffer cells, hepatocytes, HSCs was shown with mice treated with CCl4 for 4 weeks (100× magnification). (I) mRNA levels of HMOX-1 in cultured Kupffer cells, hepatocytes, HSCs were analyzed by qPCR with mice treated with CCl4 for 4 weeks. *P<0.05. RFC: relative fold change.