Hepatoprotective effects of chamazulene against alcohol-induced liver damage by alleviation of oxidative stress in rat models

2.1 Chemicals and reagents

Chamazulene (analytical standard), ethanol (purity ≥99.8%), biochemical analysis kits, ELISA assay kit, haematoxylin and eosin (H&E), and all other chemicals and reagents were either purchased from Sigma-Aldrich (St Louis, MO, USA) or Pure One Biotechnology Ltd (Shanghai, China).

2.2 Experimental animals

Thirty male Wistar rats weighing 150–180 g (6–8 weeks old) were randomly divided into five groups with six rats (n = 6) per group. The rats were acclimatized for 1 week before the initiation of experiment, placed in plastic cages at room temperature with fresh air ventilation, and provided free access to water and chow ad libitum. The experiment was started at the beginning of June 2018 in the medical laboratory of Sichuan Academy of Medical Sciences.

1. Ethical approval: The research related to animal use complied with all the relevant national regulations and institutional policies for the care and use of animals. The animal ethical committee of Sichuan Provincial People’s Hospital gave approval for the animal experiment (ethical number: scrmyy2019072301).

2.3 Treatment protocol

Rats were divided into control group, ethanol-administered alcoholic model group (EtOH), low dose and high dose of chamazulene plus ethanol-treated groups, and silymarin plus ethanol-treated positive control group. Dosages for chamazulene and silymarin were chosen from preliminary tests on antioxidant activities, and duration of treatment and protocols were followed according to the previous study on alcoholic liver disease models by Ge et al. [5]. The control group received oral administration of saline in distilled water throughout the experiment. All other groups received oral administration of ethanol (50% v/v) for 6 weeks at the dose of 8 mL/kg body weight (b.w.) per day for the first 2 weeks, followed by 12 mL/kg b.w. per day for the remaining 4 weeks. Ethanol dosage for liver injury was chosen based on a similar study performed by Ge et al. [5] after confirmation through preliminary tests. Chamazulene was orally administered 1 h before the administration of ethanol at 25 mg/kg b.w. per day for the low-dose group and 50 mg/kg b.w. per day for the high-dose group for the whole 6 weeks. The positive control group rats were orally treated with silymarin at 50 mg/kg b.w. per day followed by ethanol following a similar procedure for 6 weeks. On the 43rd day after 24 h of last ethanol administration, the rats were anaesthetized to collect blood samples for serum biochemical analysis and the liver tissues were dissected for biochemical and histopathological analyses. The excised wet liver was weighed for liver index measurement. Liver index (%) was calculated by the formula: (liver weight [g]/body weight of rat [g]) × 100%. Blood samples were left to clot at room temperature and centrifuged at 3,000 rpm for 10 min to obtain serum for enzymatic (aspartate aminotransferase [AST], alanine aminotransferase [ALT], and alkaline phosphatase [ALP]) and cytokine (tumour necrosis factor-α [TNF-α] and interleukin-6 [IL-6]) analyses. Excised liver tissues were cleaned and homogenized (10%) in freshly prepared sodium phosphate-buffered saline for liver enzymatic and non-enzymatic antioxidant analyses. Portions of liver tissues were fixed in 10% formaldehyde buffer for histopathological studies. The remaining samples were frozen and stored at −80°C.

2.4 Determination of lipid peroxidation, reduced glutathione (GSH), and antioxidant enzymes in tissue

Biochemical analysis of tissue enzymes, GSH, and lipid peroxidation levels was performed according to the method of Li et al. [16]. Tissue homogenates (10%) were centrifuged at 3,000 rpm for 10 min at 4°C to obtain the supernatant for the determination of lipid peroxidation, GSH, glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD). Lipid peroxidation levels were analyzed using a commercial kit based on the formation of thiobarbituric acid reactive substances in conjugation with malondialdehyde (MDA) following the method of Ayidin et al. [17]. The GSH levels were assayed using a commercial kit based on the formation of 5-thiol-2-nitrobenzoic acid. The activities of antioxidant enzymes (SOD, CAT, and GPx) were determined using commercially available assay kits following the instructions provided by the manufacturer (Pure One Biotechnology Ltd) according to the method of Bacanli et al. [18]. The biochemical results were spectrophotometrically analyzed using an UV-Vis digital spectrophotometer (Shimadzu UV-1900, Japan). Total protein concentration in the liver tissues was measured by a standard Bradford protein assay.

2.5 Determination of serum AST, ALT, and ALP

The activities of serum biomarker enzymes for liver damage such as ALP, AST, and ALT were analyzed using commercial kits according to the instructions given by the manufacturer, following the guidelines of Gnanaraj et al. [3] and Li et al. [16]. The results for serum biomarker enzymes were given as units per litre of serum.

2.6 Determination of pro-inflammatory cytokines using the ELISA assay

The activities of serum pro-inflammatory cytokines (TNF-α and IL-6) were determined using ELISA assay kits (Pure One Biotechnology Ltd) according to the instructions given in the reagent kits with reference to the protocols of Xu et al. [6]. The ELISA assay results for the activities of pro-inflammatory cytokines were expressed as picograms per millilitre of serum.

2.7 Evaluation of liver histopathological changes

Liver tissues fixed in 10% formaldehyde buffer were dehydrated and fixed in paraffin wax, sliced to 5 µm thick films, and stained with H&E. The sections of stained liver tissues were observed under a light microscope for the evaluation of fatty changes, lymphocytic infiltration, ballooning degeneration, and hepatocellular necrosis following the method of Ge et al. [5].

2.8 Statistical analysis

All the values are expressed as mean ± standard error of mean. Statistical package for social sciences software (SPSS 17.0, USA) was used for statistical significance testing. Analysis of variance was performed together with Dunnett’s multiple comparison test for analysing the difference between groups. Statistical significance was considered valid for p values less than 0.05.

3.1 Effects of chamazulene on the liver index of ethanol-induced liver injury in rats

The final body weight and liver index of alcoholic liver injury model rats are given in Table 1. The liver index of the ethanol-induced liver injury model group shows a significantly increased value compared to that of the normal control group. Chamazulene significantly reduced the liver index in a dose-dependent manner as compared to the ethanol control group (p < 0.05). Silymarin also showed a significantly lower liver index as compared to the alcoholic liver model group (p < 0.05).

3.2 Chamazulene restores GSH and antioxidant enzyme activities in alcoholic liver injury

The levels of GSH and activities of antioxidant enzymes (SOD, GPx, and CAT) in ethanol-induced liver injury are shown in Figure 1. It is clearly shown that GSH was largely consumed in the ethanol-induced model group as compared to the normal control group (p < 0.05). Chamazulene significantly restored the GSH levels comparably better than the ethanol-induced model group (p < 0.05). Silymarin also showed significantly increased levels of GSH as compared to those of the ethanol-induced model group (p < 0.05). The activities of antioxidant enzymes, SOD, CAT, and GPx, were drastically depleted in the ethanol-induced model group (p < 0.05), but chamazulene increased the activities of antioxidant enzymes at both 25 mg/kg b.w. and 50 mg/kg b.w. doses (p < 0.05). The results were comparable to positive control silymarin.

Figure 1

Effect of chamazulene on the activities of non-antioxidant GSH and antioxidant enzymes (GPx, CAT, and SOD) against ethanol-induced liver injury. Results are displayed as the mean analysis of six rats (n = 6) and standard error of mean. The symbol ‘a’ represents significance (p < 0.05) as compared to the normal control group, whereas the symbol ‘b’ represents significance (p < 0.05) as compared to the ethanol control group. GSH = reduced glutathione; GPx = glutathione peroxidase; CAT = catalase; SOD = superoxide dismutase.

3.3 Chamazulene prevents MDA formation and decreases serum ALP, AST, and ALT levels

The effects of chamazulene on MDA formation and serum biomarker enzymes for liver injury such as ALP, AST, and ALT are shown in Figure 2. The MDA level was clearly high in the ethanol-induced model group compared to that of the normal control group (p < 0.05). Chamazulene at both 25 mg/kg b.w. and 50 mg/kg b.w. doses significantly prevented MDA formation in comparison to the ethanol-induced model group (p < 0.05). The activities of serum biomarker enzymes (AST, ALT, and ALP) were significantly high in the ethanol-induced model group compared to those of the normal control group (p < 0.05). Chamazulene dose dependently was able to suppress the levels of serum AST, ALT, and ALP compared to the ethanol-induced model group (p < 0.05). Silymarin significantly reduced the levels of MDA, ALP, AST, and ALP in contrast to the ethanol-induced model group (p < 0.05).

Figure 2

Effect of chamazulene on the levels of MDA formation and serum liver injury marker enzymes (ALP, AST, and ALT) against ethanol-induced liver damage. Results are displayed as the mean analysis of six rats (n = 6) and standard error of mean. The symbol ‘a’ represents significance (p < 0.05) as compared to the normal control group, whereas the symbol ‘b’ represents significance (p < 0.05) as compared to the ethanol control group. MDA = malondialdehyde; ALP = alkaline phosphatase; AST = aspartate aminotransferase; ALT = alanine aminotransferase.

3.4 Preventive effects of chamazulene on the levels of pro-inflammatory cytokines

ELISA assay results for the levels of pro-inflammatory cytokines (TNF-α and IL-6) in ethanol-induced liver injury are shown in Figure 3. The levels of TNF-α and IL-6 were significantly elevated in the serum of the ethanol-induced liver damage group as compared to those of the normal control group (p < 0.05). Chamazulene was able to suppress the levels of TNF-α and IL-6 in the serum of the 25 mg/kg b.w. treated group and 50 mg/kg b.w. treated group in contrast to the ethanol-induced model group (p < 0.05). Silymarin also suppressed the serum levels of TNF-α and IL-6 in comparison to the ethanol-induced model group (p < 0.05).

Figure 3

Effect of chamazulene on pro-inflammatory cytokines (TNF-α and IL-6) against ethanol-induced liver damage. Results are displayed as the mean analysis of six rats (n = 6) and standard error of mean. The symbol ‘a’ represents significance (p < 0.05) as compared to the normal control group, whereas the symbol ‘b’ represents significance (p < 0.05) as compared to the ethanol control group. TNF-α = tumour necrosis factor-α; IL-6 = interleukin-6.

3.5 Histopathological changes in alcoholic liver injury due to chamazulene administration

Histopathological alterations (H&E) in the alcoholic liver injury model and protective effect of chamazulene are shown in Figure 4. The morphological arrangement of liver cells in the normal control group showed healthy signs with regular hepatocyte arrangements, distinct nucleus, and sinusoids. In contrast, the hepatocytes of the ethanol-induced model group showed extensive derangement with loss of sinusoidal spaces, inflammatory signs with cell infiltrations were observed, necrotic cells were found with loss of nuclear integrity, ballooning degeneration, and fatty changes were clearly visible. Chamazulene administration reversed the morphological changes caused by ethanol-induced liver damage by restoring the hepatocyte arrangements, reduced inflammatory cell infiltration, and preserved sinusoidal spaces. The silymarin-treated group showed positive morphology of liver with arrangement of hepatocytes almost similar to normal liver.

Figure 4

H&E staining of ethanol-induced liver damage and the protective effects of chamazulene at 100× magnification. (A) Control group: normal arrangement of hepatocytes and sinusoidal spaces; (B) alcohol control group: heavy loss of sinusoidal spaces, presence of necrotic cells, lymphocytic infiltration, ballooning degeneration, and fatty changes are observed, indicated by black arrows; (C) chamazulene (25 mg/kg b.w. + EtOH) group: there are several necrotic hepatocytes and lymphocytic infiltrations present, indicated by black arrows but mostly sinusoidal spaces show recovery; (D) chamazulene (50 mg/kg b.w. + EtOH) group: hepatocytes show signs of protection against ethanol-induced liver injury with distinct sinusoidal spaces; and (E) positive control group: silymarin as a positive control significantly protected the liver against ethanol-induced injury which shows arrangement of hepatocytes almost similar to normal liver.