Clusterin inhibits lipopolysaccharide induced liver injury

Abstract

Lipopolysaccharide (LPS) exacerbates liver injury by activating various inflammatory pathways. Clusterin, a glycoprotein involved in lipid transport, and cytoprotection, is known to have inhibitory effects on liver steatosis and fibrosis. In this study, we investigated the role of clusterin in regulating LPS-induced liver injury and its effects on liver injury in C57BL/6 mice and clusterin knockout mice injected with LPS for 3 h. Primary Kupffer cells (KCs) and hepatocytes (HCs) were isolated from these mice and examined using immunohistochemistry, real-time RT-PCR, ELISA, and western blot analysis to assess the effects of clusterin. Clusterin deficiency significantly exacerbated LPS-induced liver injury, as evidenced by increased inflammatory cell infiltration, elevated serum alanine aminotransferase and aspartate aminotransferase levels, and upregulated expression of pro-inflammatory cytokines and components of the NLRP3 inflammasome. By contrast, overexpression of clusterin in primary Kupffer cells and hepatocytes significantly reduced these inflammatory markers. Furthermore, the protective mechanism of clusterin involved inhibition of the STAT3 signaling pathway. These findings suggest that clusterin is a useful therapeutic target to modulate cytokine production and key inflammatory signaling pathways in inflammatory liver diseases.

Keywords: Clusterin; LPS; Liver inflammation; NLRP3 inflammasome; STAT3.

Fig. 1

Effect of clusterin deficiency on LPS-induced liver injury in mice. (A) Representative images of H&E staining of liver tissue sections from wild-type and Clu-KO mice injected with LPS for 3 h. (B) Serum levels of ALT and AST in WT and Clu-KO mice treated with and without LPS. (C) Representative real-time PCR analysis of pro-inflammatory cytokines in WT and Clu-KO mice treated with and without LPS. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2

Effect of clusterin deficiency on NLRP3 inflammasome activation in LPS-injected mice. (A) ELISA analysis of IL1-β and IL-18 levels in serum from wild-type and Clu-KO mice injected with LPS for 3 h. (B) Representative real-time PCR analysis of IL-1β and NLRP3 in WT and Clu-KO mice treated with and without LPS. (C) Immunohistochemical staining for IL-1β in liver sections of LPS-treated Clu-KO mice. (D) Representative western blot analysis of NLRP3 and ASC proteins in the liver of Clu-KO mice after LPS injection. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

Effects of clusterin deficiency on the inflammatory response in isolated primary Kupffer cells (KC) and hepatocytes (HC). (A, B) Representative real time PCR analysis of NLRP3, IL1-β, IL-6 and TNF-α in KC and HC cells isolated from WT and Clu-KO mice. Cells were treated with LPS (1 µg/ml) for 2 h and incubated for 24 h after media change. (C) ELISA of IL1-β secretion by KC and HC cells isolated from WT and Clu-KO mice and treated with and without LPS. (D) Representative western blot analysis of NLRP3 protein levels in HCs isolated from WT and Clu-KO mice and treated with and without LPS. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4

Effects of clusterin on inflammatory markers in LPS-treated Kupffer cells (KC) and hepatocytes (HC). (A, B) Real-time PCR analysis of the effects of clusterin on LPS-induced expression of IL-1β, IL-1α, iNOS, TNF-α, and IL-6 in KCs and HCs. Cells were treated with LPS (1 µg/ml) or infected with Ad-clusterin virus for 2 h, and incubated for 24 h after media change. (C, D) Western blot analysis of the effects of clusterin on LPS-induced expression of NLRP3, IL-1β, and cleaved caspase 1 in KCs and HCs. *p < 0.05, **p < 0.01, *** p < 0.001.

Fig. 5

Effects of clusterin on STAT3 signaling upon LPS-induced liver injury. (A) Immunohistochemical analysis of STAT3 nuclear translocation in liver tissues of WT and Clu-KO mice injected with LPS. **p < 0.01. (B) Representative western blot analysis of p-STAT3 in the liver of WT and Clu-KO mice injected with LPS. *p < 0.05. (C) Representative western blot analysis of p-STAT3 in HCs isolated from WT and Clu-KO mice and treated with LPS. (D, E) Western blot analysis of the effects of clusterin on LPS-induced p-STAT3 in KCs and HCs.

Fig. 6

Effects of clusterin on NLRP3 inflammasome activity in THP-1 macrophages. (A) Representative real time PCR analysis of NLRP3 and IL1-β in THP-1 cells. Cells were infected with Ad-Clusterin virus for 2 h, incubated for 18 h, and treated with LPS (1 µg/ml) or LPS/ATP (1 mM) for 6 h. *p < 0.05, **p < 0.01. (B) ELISA of IL1-β secretion by THP-1 cells treated with LPS or LPS/ATP. *p < 0.05, **p < 0.01. (C, D) Representative western blot analysis of NLRP3, IL1-β and caspase1 in THP-1 cells treated with LPS or LPS/ATP. (E) Representative western blot analysis of p-STAT3 in THP-1 cells.