Antihepatofibrotic effect of Guizhifuling pill (桂枝茯苓丸) on carbon tetrachloride-induced liver fibrosis in mice

doi:10.19852/j.cnki.jtcm.20220707.003

Abstract

Abstract:

OBJECTIVE: To evaluate the protective effects and the underlying mechanism of Guizhifuling pill (桂枝茯苓丸, GZFL) on carbon tetrachloride (CCl₄)-induced hepatic fibrosis in mice.

METHODS: Male ICR mice by intraperitoneally administered with 20% CCl₄ (mixed 1∶4 in soybean oil) to induce liver fibrosis. Mice that underwent CCl₄ 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.

Key words: liver fibrosis, NF-E2-related factor 2, heme oxygenase-1, NF-kappa B, Guizhifuling pill

XU Baogui, ZHENG Jiawen, TIAN Xiaoxiao, YUAN Falei, LIU Zhongliang, YANG Zuisu, DING Xianjun. Antihepatofibrotic effect of Guizhifuling pill (桂枝茯苓丸) on carbon tetrachloride-induced liver fibrosis in mice[J]. Journal of Traditional Chinese Medicine, 2022, 42(5): 715-722.

Figures/Tables 8

Table 1 Changes of body weight in each group (g)

Time (weeks)	Control	Model	Colchicine	L-GZFL	H-GZFL
Week 1	24.12±0.15	23.92±0.13	23.77±0.07	23.65±0.06	23.92±0.24
Week 2	29.84±0.34	28.65±0.41	27.89±0.64	28.42±0.55	28.32±0.30
Week 3	32.07±0.20	29.90±0.76	30.04±0.83	28.96±0.17	29.75±0.45
Week 4	34.01±0.52	29.59±0.56	30.05±0.62	29.78±1.10	29.76±0.64
Week 5	34.73±0.70	30.98±1.08	31.64±1.14	31.38±0.71	31.07±0.78
Week 6	35.32±0.41	31.66±0.56	32.52±0.98	33.27±0.72	33.28±1.45
Week 7	35.81±0.89	32.18±0.40	34.28±0.70	35.09±0.12	34.26±0.97
Week 8	37.56±1.07	33.35±0.89	36.83±0.89	36.08±0.23	35.25±0.81
Week 9	37.84±0.94	33.21±0.43	35.68±1.09	35.80±1.02	35.89±0.52

Table 2 Change in liver index and the expression of hydroxyproline of mice

Item	Control	Model	Colchicine	L-GZFL	H-GZFL
Liver index	3.793±0.181	5.416±0.093^a	4.163±0.216^b	4.220±0.082^b	4.357±0.195^ab
Hyp (μg/mg)	0.064±0.003	0.329±0.004^a	0.174±0.005^ab	0.179±0.007^ab	0.089±0.004^ab

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.

Table 3 Effect of GZFL on liver indices in mice

Liver index	Control	Model	Colchicine	L-GZFL	H-GZFL
ALT (U/L)	27.05±4.64	48.75±3.04^a	34.60±3.88^b	39.23±1.59^ab	43.02±4.02^a
AST (U/L)	35.02±5.78	59.23±3.26^a	42.23±2.64^b	49.85±4.24^a	55.30±3.59^a
γ-GT (U/L)	5.01±0.17	6.49±0.33^a	5.13±0.34^b	5.60±0.55	5.95±0.31
ALB (g/L)	70.89±5.04	54.02±3.12	66.64±5.77	62.02±2.14	61.24±6.88

Table 4 Effect of GZFL on antioxidant capacities and MDA levels in liver tissues

Antioxidants marker	Control	Model	Colchicine	L-GZFL	H-GZFL
MDA	2.52±0.08	3.32±0.16^a	2.66±0.24^b	2.82±0.08^ab	2.95±0.16^ab
SOD	208.53±15.13	172.36±18.60^a	197.53±11.62	194.12±9.82	185.82±22.23
GSH-Px	106.87±2.38	79.63±3.43^a	100.95±6.63^b	97.26±5.01^b	99.64±8.16^b
T-AOC	6.87±0.42	3.45±0.20^a	5.96±0.20^b	4.94±0.53^ab	4.18±0.13^a
CAT	6.12±0.63	4.40±0.31^a	5.27±0.52	5.23±0.30	5.20±0.21

Table 5 Effect of GZFL on the levels of proinflammatory cytokines in liver tissue (pg/mL)

Proinflammatory cytokine	Control	Model	Colchicine	L-GZFL	H-GZFL
IL-1β	36.8±1.1	61.7±1.5^a	41.8±1.6^b	49.7±2.5	55.8±2.4^a
IL-6	73.7±3.1	111.0±1.8^a	84.7±3.4	88.8±2.5	97.6±2.4
TNF-α	61.8±3.5	98.0±4.0^a	66.0±4.2^a	72.4±2.7^a	81.3±3.9^a

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.

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