A Herbal Formula, CGXII, Exerts Antihepatofibrotic Effect in Dimethylnitrosamine-Induced SD Rat Model

Abstract

We aimed to evaluate the antihepatofibrotic effects of CGXII, an aqueous extract which is composed of A. iwayomogi, A. xanthioides, and S. miltiorrhiza, against dimethylnitrosamine- (DMN-) induced hepatofibrosis. Male Sprague Dawley rats were intraperitoneally injected with 10 mg/kg of DMN for 4 weeks (three consecutive days weekly). Rats were orally given distilled water, CGXII (50 or 100 mg/kg), or dimethyl dimethoxy biphenyl dicarboxylate (50 mg/kg) daily. DMN injection caused substantial alteration of total body weight and liver and spleen mass, whereas they were notably normalized by CGXII. CGXII treatment also markedly attenuated the elevation of serum aspartate aminotransferase and alanine aminotransferase levels, hepatic lipid peroxidation, and protein carbonyl contents. Collagen accumulation in hepatic tissue evidenced by histopathological analysis and quantitative assessment of hepatic hydroxyproline was ameliorated by CGXII. Immunohistochemistry analysis revealed decreased α-smooth muscle actin supporting the antihepatofibrotic effect of CGXII. The profibrogenic cytokines transforming growth factor-β, platelet-derived growth factor-β, and connective tissue growth factor were increased by DMN injection. Administration of CGXII normalized the protein and gene expression levels of these cytokines. Our findings suggest that CGXII lowers the levels of profibrogenic cytokines and thereby exerts antifibrotic effects.

Figure 1

Chemical composition analysis based on high-performance liquid chromatography (HPLC). The water extract of CGXII and their standard compounds were subjected to HPLC. Chromatogram of reference compound mixtures (a) and CGXII (b). See Table 1.

Figure 2

Histopathological analysis and immunohistochemical staining. Following DMN injection, rats were orally given distilled water, CGXII (50 mg and 100 mg/kg), or DDB (50 mg/kg) daily for 4 weeks. The liver tissues were examined using hematoxylin and eosin (a), Masson's trichrome (b), and immunohistochemistry for α-SMA (c); pathophysiologic examinations were performed under light microscopy (100x magnification). The inflammation scores (d), METAVIR scores (e), and the α-SMA positive cells (f) were analyzed. Data are expressed as the mean ± SD (n = 6). ^### p < 0.001, compared with the normal group; ^∗ p < 0.05 and ^∗∗∗ p < 0.001, compared with the DMN group.

Figure 3

Contents of hydroxyproline, malondialdehyde, and protein carbonyl. Following DMN injection, rats were orally given distilled water, CGXII (50 mg and 100 mg/kg), or DDB (50 mg/kg) daily for 4 weeks. Hydroxyproline (a), malondialdehyde (MDA) (b), and protein carbonyl (c) content in the liver tissues. Data are expressed as the mean ± SD (n = 6). ^### p < 0.001, compared with the normal group; ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001, compared with the DMN group.

Figure 4

Determination of profibrogenic cytokines and TIMP-1. Following DMN injection, rats were orally given distilled water, CGXII (50 mg and 100 mg/kg), or DDB (50 mg/kg) daily for 4 weeks. Quantitative analysis of TGF-β1 (a), PDGF-BB (b), CTGF (c), and TIMP-1 (d) was performed in the liver tissues using ELISA kits. Data are expressed as the mean ± SD (n = 6). ^### p < 0.001, compared with the normal group; ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001, compared with the DMN group.

Figure 5

mRNA expression of hepatic fibrosis molecules. Real-time PCR was performed to determine the mRNA levels of TGB-β1, PDGF-BB, CTGF, Col 1a1 and TIMP-1, MMP-2 and BAMBI, and Smad7 in hepatic tissues. Expression was normalized as a ratio to β-actin. Data are expressed as the mean ± SD (n = 6). ^## p < 0.01 and ^### p < 0.001, compared with the normal group. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^∗∗∗ p < 0.001, compared with the DMN group. ^¶Both BAMBI and Smad7 were normalized in the DMN group, and the others were expressed as fold changes, which were normalized in the normal group.