Hepatocyte Growth Factor Mediates the Antifibrogenic Action of Ocimum bacilicum Essential Oil against CCl4-Induced Liver Fibrosis in Rats

Abstract

The current investigation aimed to evaluate the antifibrogenic potential of Ocimum basilicum essential oil (OBE) and further to explore some of its underlying mechanisms. Three groups of rats were used: group I (control), group II (CCl4 model) and group III (OBE-treated) received CCl4 and OBE 2 weeks after the start of CCl4 administration. Oxidative damage was assessed by the measurement of MDA, NO, SOD, CAT, GSH and total antioxidant capacity (TAC). Liver fibrosis was assessed histopathologically by Masson's trichrome staining and α-smooth muscle actin (α-SMA) immunostaining. Expression of hepatocyte growth factor (HGF) and cytochrome P450 (CYP2EI isoform) was estimated using real-time PCR and immunohistochemistry. OBE successfully attenuated liver injury, as shown by histopathology, decreased serum transaminases and improved oxidative status of the liver. Reduced collagen deposition and α-SMA immuopositive cells indicated an abrogation of hepatic stellate cell activation by OBE. Furthermore, OBE was highly effective in stimulating HGF mRNA and protein expression and inhibiting CCl4-induced CYP2E1 down-regulation. The mechanism of antifibrogenic action of OBE is hypothesized to proceed via scavenging free radicals and activating liver regeneration by induction of HGF. These data suggest the use of OBE as a complementary treatment in liver fibrosis.

Keywords: CYP2E1; HGF; Ocimum basilicum; antioxidant; liver fibrosis; α-SMA.

Figure 1

Histopathological examination of livers from different groups. (A–C) H & E staining; (D–E) Masson’s trichrome. (A) The control group (I) showing normal cellular architecture (H & E ×100); (B) CCl₄ group (II) showing extensive hepatocellular necrosis, marked fatty degeneration, dilation of portal blood vessels and marked fibrous bridging with collagen septa formation (H & E ×100); (C) CCl₄ + OBE group (III) showing moderate centrilobular hepatic necrosis with mild vacuolar degeneration of hepatocytes and thin collagenous septa formation (H & E ×100); (D) The control group (I) showing normal collagen fibers in the area of portal and central vein (Masson’s trichrome ×100); (E) CCl₄ group (II) showing extensive collagen deposition (arrow) and pseudolobular formation (Masson’s trichrome ×100); (F) CCl₄ + OBE group (III) showing slight accumulation and spread of collagen fibers (arrow) (Masson’s trichrome ×100).

Figure 2

Pattern of immunohistochemistry staining for α-SMA in the liver of different groups. (A) The control group (I) showing normal expression of α-SMA positive staining in the smooth musculature of the blood vessels (arrow) (×400); (B) CCl₄ group (II) showing intense immunopostive reaction (arrow) in myofibroblasts cells (×400); (C) CCl₄ + OBE group (III) showing reduction in the areas of immunopostive cells (arrow) (×400); (D) Bar chart represents the α-SMA immunopositivity expressed as % area. Mean values with different superscripts are significantly different (p < 0.05).

Figure 3

Pattern of immunohistochemistry staining for HGF in the liver of different groups. (A) The control group (I) showing weak immunopostive reaction (×400); (B) CCl₄ group (II) showing immunopostive staining in number of cells (×400); (C) CCl₄ + OBE group (III) showing intense immunopostive reaction (×400); (D) Bar chart represents the HGF immunopositivity expressed as area %. Mean values with different superscripts are significantly different (p < 0.05).

Figure 4

Expression level of HGF (A) and CYP2E1 (B) genes in the different groups. Group I: vehicle control; group II: CCl₄; group III: OBE-treated. The steady-state levels of mRNA in the liver were analyzed by real-time PCR assay. Values are presented as means ± SD (n = 6). GAPDH was used as an invariant internal control for calculating mRNA-fold changes. Mean values with different letters are significantly different (p < 0.05).