Combination of sorafenib and gadolinium chloride (GdCl3) attenuates dimethylnitrosamine(DMN)-induced liver fibrosis in rats

Abstract

Background/aims: Liver sinusoidal endothelial cells (SECs), hepatic stellate cells (HSCs) and Kupffer cells (KCs) are involved in the development of liver fibrosis and represent a potential therapeutic target. The therapeutic effects on liver fibrosis of sorafenib, a multiple tyrosine kinase inhibitor, and gadolinium chloride (GdCl3), which depletes KCs, were evaluated in rats.

Methods: Liver fibrosis was induced in rats with dimethylnitrosamine, and the effects of sorafenib and/or GdCl3 in these rats were monitored. Interactions among ECs, HSCs and KCs were assessed by laser confocal microscopy.

Results: The combination of sorafenib and GdCl3, but not each agent alone, attenuated liver fibrosis and significantly reduced liver function and hydroxyproline (Hyp). Sorafenib significantly inhibited the expression of angiogenesis-associated cell markers and cytokines, including CD31, von Willebrand factor (vWF), and vascular endothelial growth factor, whereas GdCl3 suppressed macrophage-related cell markers and cytokines, including CD68, tumor necrosis factor-α, interleukin-1β, and CCL2. Laser confocal microscopy showed that sorafenib inhibited vWF expression and GdCl3 reduced CD68 staining. Sorafenib plus GdCl3 suppressed the interactions of HSCs, ECs and KCs.

Conclusion: Sorafenib plus GdCl3 can suppress collagen accumulation, suggesting that this combination may be a potential therapeutic strategy in the treatment of liver fibrosis.

Fig. 1

Effects of sorafenib and/or GdCl3 on histological changes of rat livers. a Histological images of rat livers stained with HE (magnification X200). b Histological images of rat livers stained with Sirius red (magnification X100). c qualification of Fig. 1b. d Liver Hyp content. The number in HE, Sirius red staining, and Hyp detection was as the same as the animal number in each group. All results are expressed as mean ± S.D. *, p < 0.05; ** p < 0.01 vs 4 W. Control, vehicle alone; 4 W, DMN for 4 weeks; S, DMN for 4 weeks plus sorafenib for 2 weeks; G, DMN for 4 weeks plus GdCl3 for 2 weeks; S + G, DMN for 4 weeks plus sorafenib and GdCl3 for 2 weeks

Fig. 2

Effect of sorafenib and/or GdCl3 blockage on angiogenesis in DMN-induced liver fibrosis in vivo. a vWF immunohistochemistry (magnification X400, n = 4). b Levels of mRNAs encoded by genes related to angiogenesis (n = 6). c the Expression of CD31, VEGF, Ang1, VCAM1 was analyzed using western blot (n = 6). Control, vehicle alone; 4 W, DMN for 4 weeks; S, DMN for 4 weeks plus sorafenib for 2 weeks; G, DMN for 4 weeks plus GdCl3 for 2 weeks; S + G, DMN for 4 weeks plus sorafenib and GdCl3 for 2 weeks. The negative control consisted of samples from DMN-treated animals in the absence of primary antibody. All results reported as mean ± S.D. *p < 0.05; **p < 0.01 vs 4 W

Fig. 3

Effects of sorafenib and/or GdCl3 blockage on in vivo expression of proinflammatory cytokines in liver fibrosis. a CD68 immunohistochemistry (magnification X 400, n = 4). b Levels of mRNA encoded by genes encoding proinflammatory cytokines in rats with DMN-induced fibrosis (n = 6). c The Expression of CD68, TNF-α, IL1β, CCL2 was analyzed using western blot (n = 6). Control, vehicle alone; 4 W, DMN for 4 weeks; S, DMN for 4 weeks plus sorafenib for 2 weeks; G, DMN for 4 weeks plus GdCl3 for 2 weeks; S + G, DMN for 4 weeks plus sorafenib and GdCl3 for 2 weeks. Results reported as mean ± S.D. *p < 0.05; **p < 0.01 vs 4 W

Fig. 4

Effects of sorafenib and/or GdCl3 on in vivo expression of pro-fibrotic factors in rat liver fibrosis. a α-SMA immunohistochemistry (magnification X 200, n = 4). b Levels of mRNA encoded by genes encoding factors associated with fibrosis sis (n = 6). c the Expression of α-SMA, TIMP1, TGF-β1, COL1 was analyzed using western blot (n = 6). Control, vehicle alone; 4 W, DMN for 4 weeks; S, DMN for 4 weeks plus sorafenib for 2 weeks; G, DMN for 4 weeks plus GdCl3 for 2 weeks; S + G, DMN for 4 weeks plus sorafenib and GdCl3 for 2 weeks. Results reported as mean ± S.D. * p < 0.05; ** p < 0.01 vs 4 W

Fig. 5

Effects of sorafenib and/or GdCl3 on interactions of HSCs, KCs and SECs in DMN-induced liver fibrosis in rats. Hepatic cryosections were stained with antibodies against α-SMA (blue), CD68 (red), and vWF (green) (magnification X400, n = 3). 4 W, DMN for 4 weeks; S, DMN for 4 weeks plus sorafenib for 2 weeks; G, DMN for 4 weeks plus GdCl3 for 2 weeks; S + G, DMN for 4 weeks plus sorafenib and GdCl3 for 2 weeks