Antihepatofibrotic effect of Guizhifuling pill on carbon tetrachloride-induced liver fibrosis in mice

Abstract

Objective: To evaluate the protective effects and the underlying mechanism of Guizhifuling pill (, GZFL) on carbon tetrachloride (CCl)-induced hepatic fibrosis in mice.

Methods: Male ICR mice by intraperitoneally administered with 20% CCl (mixed 1∶4 in soybean oil) to induce liver fibrosis. Mice that underwent CCl were orally with GZFL. Using hematoxylin and eosin and Masson staining to examine the pathological changes in liver tissue. Serum biochemical parameters, antioxidant enzyme activity and proinflammatory cytokines was assessed. Nuclear factor-kappaB (NF-κB) pathway and nuclear factor-erythroid 2-related factor 2 (Nrf2) family members were evaluated by Western blotting.

Results: Our findings indicated that GZFL could effectively suppress the progression of liver fibrosis in mice, which was determined based on the improvement in liver function and reduction of collagen deposition. GZFL treatment also decreased the level of cytokines and increased the activity of antioxidant enzymes in liver tissue. Moreover, GZFL exerted anti-inflammatory and antioxidant effects through regulating the Nrf2-mediated antioxidant system and inhibiting the NF-κB pathway.

Conclusions: GZFL may prevent the progression of liver fibrosis by regulating the Nrf2/ heme oxygenase-1 and NF-κB signaling pathways, thereby highlighting its role in the management of liver fibrosis.

Keywords: Guizhifuling pill; NF-E2-related factor 2; NF-kappa B; heme oxygenase-1; liver fibrosis.

Figure 1. GZFL attenuates CCl₄-induced liver injury and fibrosis in mice

A: representative livers at the time of sacrifice; B: HE staining of representative liver tissues (B1-B5:original magnification ×100; B6-B10: partially enlarged pictures × 400); C: Masson's trichrome staining of liver sections (C1-C5:original magnification ×100; C6-C10: partially enlarged pictures × 400). Vacuoles (), hepatic sinusoids (), inflammatory cells (), necrotic hepatocytes (), and connective tissue hyperplasia () in the HE staining figure. Control: intraperitoneal injection with normal saline, twice a week for 4 weeks; gavaged with normal saline daily, 4 weeks. Model: intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with normal saline daily, 4 weeks. Colchicine: positive group, intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with colchicine (0.1 mg/kg) daily, 4 weeks. L-GZFL: intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with GZFL (150 mg/kg) daily, 4 weeks. H-GZFL: intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with GZFL (300 mg/kg) daily, 4 weeks. GZFL: Guizhifuling pill; CCl₄: carbon tetrachloride; HE: hematoxylin and eosin.

Figure 2. Western blotting to determine the protein expression of Nrf2-mediated antioxidant signaling

A: Western blotting to determine the expression of Nrf2, NQO1, HO-1, and GCLM in mouse liver; B: Nrf2 protein expression; C: HO-1 protein expression; D: NQO1 protein expression; E: GCLM protein expression. Control: intraperitoneal injection with normal saline, twice a week for 4 weeks; gavaged with normal saline daily, 4 weeks. Model: intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with normal saline daily, 4 weeks. Colchicine: positive group, intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with colchicine (0.1 mg/kg) daily, 4 weeks. L-GZFL: intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with GZFL (150 mg/kg) daily, 4 weeks. H-GZFL: intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with GZFL (300 mg/kg) daily, 4 weeks. Data are presented as mean ± standard deviation (n = 10). GZFL: Guizhifuling pill; CCl₄: carbon tetrachloride; Nrf2: nuclear factor-erythroid 2-related factor 2; NQO1: nicotinamide quinone oxidoreductase 1; HO-1: heme oxygenase-1; GCLM: glutamate-cysteine ligase modifier subunit; GAPDH: glyceraldehyde-3-phosphate. ^a P < 0.01 vs Control; ^b P < 0.01 vs Model.

Figure 3. Determination of protein expression in the NF-κB signaling pathway using Western blotting

A: Western blotting to determine the expression of IKKα, IκBα, NF-κB p50, NF-κB p65, and p-NF-κB p65 in mouse livers; B: IKKα protein expression; C: IκBα protein expression; D: NF-κB p50 protein expression; E: NF-κB p65 and p-NF-κB p65 protein expression; F: TLR4 protein expression. Control: intraperitoneal injection with normal saline, twice a week for 4 weeks; gavaged with normal saline daily, 4 weeks. Model: intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with normal saline daily, 4 weeks. Colchicine: positive group, intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with colchicine (0.1 mg/kg) daily, 4 weeks. L-GZFL: intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with GZFL (150 mg/kg) daily, 4 weeks. H-GZFL: intraperitoneal injection with CCl₄, twice a week for 4 weeks; gavaged with GZFL (300 mg/kg) daily, 4 weeks. Data are presented as mean ± standard deviation (n = 10). GZFL: Guizhifuling pill; CCl₄: carbon tetrachloride; TLR4: toll-like receptor 4; NF-κB: nuclear factor-kappa B; IKKα: IKK alpha; p-NF-κB p65: phospho-NF-κB p65; GAPDH: Glyceraldehyde-3-phosphate. ^a P < 0.01 vs Control; ^b P < 0.01 vs Model.