Antioxidant, anti-inflammatory and anti-fibrotic effects of Boswellia serrate gum resin in CCl4-induced hepatotoxicity

Abstract

The present study aims to investigate the potential antioxidant, anti-inflammatory and anti-fibrotic effects of Boswellia serrate (BS) gum resin against carbon tetrachloride (CCl₄)-induced liver damage. Four groups consisting of eight rats each were designated: Group I, normal healthy control; group II, CCl₄-induced liver fibrosis; group III, CCl₄-induced liver fibrosis followed by BS treatment daily for two weeks; and group IV, CCl₄-induced liver fibrosis followed by silymarin treatment daily for two weeks. Expression of tumor necrosis factor-α (TNF-α) and nuclear factor κB (NF-κB), interleukin-6 (IL-6), transforming growth factor-β (TGF-β) and cyclooxygenase-2 (COX-2) were assessed, in addition to histopathological and fibrotic changes in liver tissues isolated from the rats. BS significantly ameliorated CCl₄-induced increases in serum aspartate (AST) and alanine transaminase (ALT) levels, reduced lactate dehydrogenase (LDH) activities in addition to restoring total bilirubin, triglyceride and albumin levels. BS treatment also alleviated oxidative stress and improved total antioxidant capacity in the liver, and reduced the expression of TNF-α, NF-κB, TGF-β, IL-6 and COX-2. On a histopathological level, BS treatment also exhibited antifibrotic activity. In conclusion, these findings suggest that BS contains potentially hepatoprotective effects against CCl4-induced liver injury via its antioxidant, anti-inflammatory and antifibrotic characteristics.

Keywords: boswellia serrata; cyclooxygenase-2; hepatoprotective; interleukin-6; liver fibrosis; transforming growth factor-β.

Figure 1.

Total antioxidant activity (mM/mg protein) as measured in liver homogenates from the four test groups. A significant reduction in total antioxidant activity in hepatic tissues was observed following CCl₄ administration compared with healthy control. BS administration significantly improved CCl₄-induced inhibition of total antioxidant activity in an effect that was similar to silymarin. Each value represents the mean ± SEM with 8 rats/group. ***P<0.001 vs. Control and ###P<0.001 vs. CCl₄. CCl₄, carbon tetrachloride; BS, Boswellia serrata gum extract.

Figure 2.

Catalase activity (units/mg protein) in liver homogenates from the four test groups. Catalase activity in hepatic tissues showed a significant reduction upon CCl₄ administration compared with healthy control. BS administration significantly ameliorated CCl₄-induced inhibition of catalase activity in an effect that was similar to silymarin. Each value represents the mean ± SEM with 8 rats/group. ***P<0.001 vs. Control and ###P<0.001 vs. CCl₄. CCl₄, carbon tetrachloride; BS, Boswellia serrata gum extract.

Figure 3.

Malondialdehyde (nM/mg protein) levels in liver homogenates from the four test groups. Using malondialdehyde levels as marker, lipid peroxidation in hepatic tissues increased significantly following CCl₄ treatment compared with healthy control. BS administration significantly alleviated CCl₄-induced lipid peroxidation to levels similar to that produced by silymarin. Each value represents the mean ± SEM with 8 rats/group. ***P<0.001 vs. control group and ###P<0.001 vs. CCl₄. MDA, malondialdehyde; CCl₄, carbon tetrachloride; BS, Boswellia serrata gum extract.

Figure 4.

Effect of BS treatment on the expression of nuclear NF-κB and TNF-α levels in liver tissues from the four test groups. (A) Western blot analysis of nuclear NF-κB protein expression using β-actin as an internal loading control. (B) Reverse-transcription-semi quantitative PCR analysis of TNF-α mRNA expression using GAPDH as the internal loading control. A significant increase in NF-κB as well as TNF-α levels was observed after CCl₄ treatment which was significantly reversed after BS treatment. (C) Quantified densitometry analysis of (A). (D) Quantified densitometry analysis of (B). Each value represents mean ± SEM for 8 rats/group. ***P<0.001 vs. Control and ###P<0.001 vs. CCl₄. CCl₄, carbon tetrachloride; BS, Boswellia serrata gum extract; TNF-α, tumor necrosis factor-α.

Figure 5.

Expression of TGF-β and IL-6 in the serum of the four treatment groups. (A) CCl₄ treatment resulted in a significant increase in the serum levels of TGF-β and (B) IL-6 compared with healthy controls. Administration of BS or silymarin after CCl₄ treatment partially but significantly restored the serum levels of both markers compared with CCl₄. TGF-β, transforming growth factor-β; IL-6, interleukin-6; CCl₄, carbon tetrachloride; BS, Boswellia serrata gum extract.

Figure 6.

Immunohistochemical staining of COX-2. (A) Liver tissue sections from control animals showing almost no staining for COX-2. (B) Prominent COX-2 expression can be observed in liver tissue sections from the CCl₄-treated group compared with healthy controls. (C) Liver tissue sections from the BS-treated group or (D) the silymarin group exhibited weaker staining for COX-2 compared with the CCl₄-treated group. Arrows indicated COX-2 stained cells. Magnification, ×400. COX-2, cyclooxygenase-2; CCl₄, carbon tetrachloride; BS, Boswellia serrata gum extract.

Figure 7.

Histological examination of liver sections from the four treatment groups following H&E staining. (A) Liver tissue sections from healthy control show normal architecture of hepatocytes. (B) CCl₄ treatment resulted in damaged cells, shrunken nuclei, portal infiltration with inflammatory cells (arrows), cystic dilatation of bile duct and fibroplasia in the portal triad and centrilobular congestion. (C) BS treatment resulted in restoration of the normal architecture showing Kupffer cells activation and mild vacuolization (arrows) with the absence of congestion. (D) Similar observations as that produced by B.S with limited focal apoptosis (arrows) can be seen in tissues from the silymarin treatment group. Magnification, ×400. (E) Quantified fibrosis and (F) inflammation scores. Values represent the mean ± SEM of histopathological scoring. **P<0.001 vs. Control and #P<0.001 vs. CCl₄. CCl₄, carbon tetrachloride; BS, Boswellia serrata gum extract.

Figure 8.

Histological examination of liver sections from the four treatment groups stained with Masson's trichrome. (A) Liver sections from healthy control showed little staining for collagen fibers. (B) Carbon tetrachloride treatment exhibited moderate fibrous septa formation with positive histochemical staining for collagen fibers. (C) Boswellia serrata gum extract treatment resulted in marked reductions in collagen fiber bundles, in a similar way to that observed in (D) tissues following silymarin treatment. Magnification, ×400. Arrows indicate collagen fibers in the different test groups.