Hawthorn Herbal Preparation from Crataegus oxyacantha Attenuates In Vivo Carbon Tetrachloride -Induced Hepatic Fibrosis via Modulating Oxidative Stress and Inflammation

Abstract

Hawthorn (HAW) is a herbal preparation extracted from Crataegus oxyacantha. HAW has cardioprotective, antioxidants, anti-inflammatory, and anti-hypotensive effects. HAW's effect on hepatic fibrosis remains, however, unknown. This study evaluated the impact of HAW on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats and elucidated its mechanisms. HAW reduced liver index and the serum liver enzyme markers and reduced liver damage, and fibrosis as confirmed by histopathological scoring of hematoxylin-eosin staining. Collagen deposition was reduced in HAW group compared to CCl4 group as confirmed by Masson staining, hydroxyproline content, and both mRNA and protein levels of alpha-smooth muscle actin, collagen 1 and 3. HAW also down regulated the gene expressions of inflammatory markers including interleukin-IL-1β, tumor necrosis factor-α, transforming growth factor-β 1, nuclear factor kappa-B, and cyclooxygenase-2 and decreased the myeloperoxidase activity. The effects of HAW was also associated with decreased levels of hepatic oxidative stress markers (malondialdehyde and P.Carbonyl) and with increased activity of superoxide dismutase. Those effects are possibly mediated by blocking the pro-oxidant machinery and down regulating the inflammatory and profibrotic responses. Finally, chlorogenic acid, epicatechin, rutin, vitexin quercetin, and iso quercetin were identified as the major species of polyphenols of the HAW herbal preparation used here. Therefore, HAW's potent protecting effects against liver fibrosis predicts a significant beneficial application.

Keywords: antioxidants; carbon tetrachloride; hawthorn; liver fibrosis.

Figure 1

Effects of Hawthorn (HAW) on CCl4-induced liver damage in rats. (A) representative images of hematoxylin and eosin (H & E)-stained hepatic sections of the different groups (100×), Scale bar equals 100 µm. Histopathological evidence of CCl4-induced liver damage in rats showing area of necrosis with inflammatory cell infiltration (arrow), and diffuse fatty change (star), (B) severity score of necrosis, (C) inflammation, and (D) fatty metamorphosis. (E) liver weight, (F) body weight, and (G) liver/body weight ratio. Serum markers of liver damage aspartate aminotransferase (AST) (H), alanine aminotransferase (ALT) (I), gamma-glutamyl transferase (GGT) (J) activities and total bilirubin (K), and albumin (L) levels. Data are presented as means ± SEM. (n = 6 per group). a is p < 0.05 vs. control group, b is p < 0.05 vs. CCl4 group, and c is p < 0.05 vs. HAW group.

Figure 2

Effects of HAW on the CCl4-induced liver fibrosis rat model. (A) representative images of Masson staining (blue area) staining, 100× magnification, scale bar equals 200 µm. (B) Representative images of immunohistochemically expression of collagen-1 (brown area). Scale bar equals 100 µm, (C) severity score of Masson, and (D) Masson-positive area expression as a percentage of the total area (n = 5 per group) using the free software NIH ImageJ. (E) collagen-1 expression area as a percentage of the total area (n = 6 per group). (F) hydroxyproline (HP) level in the liver. (G) representative images of immunohistochemically expression of collagen-3 in the liver (brown area) scale bar equals 100 µm. (H) collagen-3 expression area as a percentage of the total area (n = 3 per group). (I,J) Analysis of type 1 and 3 collagen mRNA expression in the liver by quantitative RT-PCR (n = 3 per group). Results were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. Data are presented as means ± SEM (n = 6 in each group). a is p < 0.05 vs. control group, b is p < 0.05 vs. CCl4 group, and c is p < 0.05 vs. HAW group.

Figure 3

Effects of HAW on the CCl4-induced hepatic stellate cells (HSC) activation in liver fibrosis model. HAW inhibited HSC activation in liver fibrosis. (A) representative images of immunohistochemically expression of α-SMA in the liver was determined by immunohistochemistry (brown area), 100× magnification, scale bar equals 100µm. In CCL4 section marked staining for alpha-smooth muscle actin (α-SMA) is found along with the fibrous septa. (B) the expression of α-SMA cells in each section was calculated by counting the number of brown staining, α-SMA-positive cells in five fields per section at 400× magnification, n = 6 per group). (C) the mRNA expression levels of α-SMA were detected by q-RTPCR. Data are presented as means ± SEM, n = 3 per group. a is p < 0.05 vs. control group, b is p < 0.05 vs. CCl4 group, and c is p < 0.05 vs. HAW group.

Figure 4

HAW inhibited the overexpression of inflammatory factors in rats. (A) myeloperoxidase (MPO) activity, (n = 6 per group) and mRNA expression of (B) IL-1β, (C) tumor necrosis factor (TNF-α), (D) TGF β, (E) nuclear factor kappa-B (NF-kB), and (F) cyclooxygenase-2 (COX-2) in liver tissues (n = 3 per group). Results of mRNA expression were normalized to GAPDH mRNA. Data are presented as means ± SEM). a is p < 0.05 vs. control group, b is p < 0.05 vs. CCl4 group, and c is p < 0.05 vs. HAW group.

Figure 5

HAW inhibited oxidative stress markers in rats. (A) malondialdehyde (MDA) content (B) P.Carbonyl content and (C) superoxide dismutase (SOD) activity in liver tissues. Data are presented as means ± SEM (n = 6 in each group). a is p < 0.05 vs. control group, b is p < 0.05 vs. CCl4 group, and c is p < 0.05 vs. HAW group.

Figure 6

The typical HPLC-UV chromatogram of compositional polyphenols presented in HAW extract.

Figure 7

A graphical abstract highlighting the protective effect of HAW in CCl4-induced liver fibrosis by blocking the pro-oxidant machinery and the downregulation of the inflammatory and profibrotic responses. The protective effects include reducing deposition. Liver toxicity, improving the liver histology and decreasing collagen.