Ivermectin Attenuates CCl4-Induced Liver Fibrosis in Mice by Suppressing Hepatic Stellate Cell Activation

Abstract

Liver fibrosis, a common liver dysfunction with high morbidity and mortality rates, is the leading cause of cirrhosis and hepatocellular carcinoma, for which there are no effective therapies. Ivermectin is an antiparasitic drug that also has been showing therapeutic actions in many other diseases, including antiviral and anticancer actions, as well as treating metabolic diseases. Herein, we evaluated the function of ivermectin in regulating liver fibrosis. Firstly, carbon tetrachloride (CCl₄)-injected Balb/c mice were used to assess the antifibrosis effects of ivermectin in vivo. Further, CFSC, a rat hepatic stellate cell (HSC) line, was used to explore the function of ivermectin in HSC activation in vitro. The in vivo data showed that ivermectin administration alleviated histopathological changes, improved liver function, reduced collagen deposition, and downregulated the expression of profibrotic genes. Mechanistically, the ivermectin treatment inhibited intrahepatic macrophage accumulation and suppressed the production of proinflammatory factors. Importantly, the ivermectin administration significantly decreased the protein levels of α-smooth muscle actin (α-SMA) both in vivo and in vitro, suggesting that the antifibrotic effects of ivermectin are mainly due to the promotion of HSC deactivation. The present study demonstrates that ivermectin may be a potential therapeutic agent for the prevention of hepatic fibrosis.

Keywords: TGF-β1; hepatic stellate cells; inflammation; ivermectin; liver fibrosis.

Figure 1

Effects of ivermectin on CCl₄-induced hepatic pathological and biochemical parameters in mice: (A) liver tissues were stained with hematoxylin and eosin (HE) for the histopathological analysis (original magnifications, ×200); (B) liver index values; (C,D) plasma AST and ALT levels. Data are expressed as means ± SEM (n = 6–8/group). Note: *** p < 0.001 vs. control group, # p < 0.05, ## p < 0.01 vs. CCl₄-treated model group.

Figure 2

Ivermectin inhibited fibrosis in CCl₄-treated mice: (A) Sirius red staining (original magnification, ×200) of liver tissues; (B) quantitation of fibrotic areas in the liver; (C) hydroxyproline levels in the liver. Data are expressed as means ± SEM (n = 6–8/group). Note: *** p < 0.001 vs. control group, ## p < 0.01, ### p < 0.001 vs. CCl₄-treated model group.

Figure 3

Anti-inflammatory effects of ivermectin in liver tissues from mice who received a CCl₄ injection: (A) hepatic F4/80 detection via immunohistochemistry (IHC) staining (original magnification, ×200) in liver tissues; (B) F4/80-positive area; (C–G) mRNA expression levels of IL-6, TNF-α, IL-1β, MCP-1, and RANTES in liver tissues. Data are expressed as means ± SEM (n = 6–8/group). Note: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. CCl₄-treated model group.

Figure 4

Impacts of ivermectin on the CCl₄-induced elevation of profibrogenic genes in the liver: (A–E) mRNA expression levels of α-SMA, CTGF, Col1α1, Col3α1, and TGF-β1 in liver tissues; (F) protein expression levels of Col1α1 and Col3α1 in the liver. Data are expressed as means ± SEM (n = 6–8/group). Note: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group, # p < 0.05, ## p < 0.01 vs. CCl₄-treated model group.

Figure 5

Effects of ivermectin on HSC activation in the liver in CCl₄-treated mice: (A) IHC staining of α-SMA in the liver; (B) α-SMA-positive areas; (C) the Western blot analysis of α-SMA protein expression in the liver. Data are expressed as means ± SEM (n = 6–8/group). Note: **** p < 0.0001 vs. control group, #### p < 0.0001 vs. CCl₄-treated model group.

Figure 6

Impacts of ivermectin on cell viability in CFSC cells: (A) bright-field images of CFSC cells; (B) cell viability of CFSC cells assessed using a cell counting kit-8 (CCK-8) assay. Data are expressed as means ± SEM (n = 3). Note: *** p < 0.001.

Figure 7

Effects of ivermectin on TGF-β1-induced HSC activation: (A) IHC staining of α-SMA in CFSC cells that received TGF-β1 stimulation and ivermectin treatment; (B) α-SMA intensity of fluorescence; (C) the Western blot analysis of α-SMA protein expression in CFSC cells. Data are expressed as means ± SEM (n = 3). Note: *** p < 0.001 vs. control group, # p < 0.05, ### p < 0.001 vs. TGF-β1-treated model group.