Hepatotoxicity is a commonly encountered clinical diagnosis with various notable etiologies. To name a few, there are drug-induced, environmental exposure to toxins or chemicals, infective, metabolic, and auto-immune. Almost two-thirds of the drug withdrawals from the pharmaceutical market had the label of hepatic injury risk associated. The drug-induced liver injury is mostly gradual to happen, predictable, and dose-dependent. However, environmental toxin or chemical exposure led to hepatotoxicity is sudden in onset and of unpredictable nature. The water, food, and air contamination in today’s fast-paced life put one in an environment containing harmful chemicals and toxins. It is nearly impossible to scrutinize the surrounding environment for possible hepatotoxin and act accordingly.
Carbon tetrachloride (CCl4) is a commonly used hepatotoxicity-inducing agent. It induces the same picture of chemical/toxin exposure hepatotoxicity in the experimental animals as observed in the clinical practice. CCl4 undergoes bio-transformation via the CYP2E1 enzyme. It gets converted into trichloromethyl peroxy free radicals, chloroform, and hexachloroethane. The trichloromethyl peroxy moiety gets covalently bonded to the cellular macromolecules of nucleic acid, protein, and lipid metabolism. Protein synthesis in the hepatic cell is halted because of hypo-methylation of ribosomal RNA. Many therapeutic options are continuously being explored for the therapeutic potential for protection of liver injury. A recent meta-analysis by our group concluded the efficacy of N-acetyl cysteine (NAC) and refuted claims of benefit in silymarin and silibinin in anti-tubercular drug-related hepatotoxicity. NAC is one of the limited options available in liver injury management. The opposite effect of NAC before and after alcohol use raises safety and efficacy concerns. Therefore, hunting for a safer and more effective therapeutic agent is justifiable. Ayurveda, an ancient medical science, has always practiced plant-derived remedial solutions for almost all ailments. Convolvulus pluricaulis, a commonly found plant in northern India, is being explored for its many health benefits. It has shown hypolipidemic, anti-diabetic, anti-epileptic, anti-ulcer, and anti-infective potential. A study performed by Garg et al. found scopoletin to be an active phyto-chemical in the C. pluricaulis plant extract.
With this background information, we performed a study to explore therapeutic potential of the scopoletin pure form in the carbon tetrachloride-induced hepatotoxicity model in Wistar rats. The study also tried to elucidate further information on the duration of the carbon tetrachloride-induced hepatotoxicity model in Wistar rats. The information is significantly limited on the longevity and therefore the hurdle of testing potential therapeutic agents.
MATERIALS AND METHODS
The scopoletin phyto-chemical (pure form) and olive oil (vehicle for CCl4) were procured from Sigma Aldrich, USA. CCl4 (AR grade) was purchased from LOBA Chem. Pvt. Ltd.
In compliance with the ‘Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA) guidelines,’ all animal experiments were performed. Wistar rats of either sex with a weight between 250 and 300 g were utilized. Rats were placed in standard polypropylene cages (two rats in each cage). Regular chow diet and water were given ad libitum, and a 12 hours light-dark cycle was followed. The relative humidity and temperature were sustained at 55 ± 5% and 22 ± 2°C, respectively. The animals were conditioned to laboratory conditions 10 days before the experimental procedures. The study protocol got approval from the Institutional Animal Ethics Committee (IAEC) (761/IAEC/110 held on 27/10/2020) of the Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh. The study abided by ARRIVE 2.0 guidelines of animal experimentation.
Overall, 36 rats were utilized in this present investigation and were allocated into six groups (n = 6). Before the experiment, animals fasted overnight with free access to water. Three days prior to model induction (CCl4 single dose), the baseline LFT levels were measured. Thereafter, the study groups were assigned as
Group 1 – Normal control (NC) and received no treatment throughout the study period.
Group II – Experimental control (EC) and received carbon tetrachloride (single i.p. injection 0.5 ml/Kg mixed in olive oil in 1:1 ratio) as an hepatotoxicity-inducing agent via the intra-peritoneal route. It was administered when companion groups had received three doses of study medications.
Groups III, IV, V, and VI received scopoletin 1 mg/kg, 5 mg/kg, 10 mg/kg, and NAC (150 mg/kg) once daily at 9 am in the morning, respectively, 3 days before CCl4 administration till day 7.
Groups III, IV, and V were labeled as low dose (LD), medium dose (MD), and high dose (HD) scopoletin groups, respectively, whereas group I and group VI were labeled as normal control (NC) and positive control (PC) groups, respectively.
Three hours after the last dose of study medications (scopoletin or NAC), CCl4 was administered in groups II–VI. Twenty-four hours later, the blood samples were collected by retro-orbital puncture using ketamine anesthesia. Serum samples were assessed for liver function test analysis. Overall, rat blood sampling was conducted on day -3, day 1, and day 7 for the liver function test analysis.
Bio-chemical assessment in the blood samples
Serum was separated after centrifugation of blood samples at 8000 rpm for 5 mins and stored at -800C until analysis. Liver function test (LFT) parameters including aspartate amintotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, total protein, and albumin were determined using Roche bio-chemical test kits with a semi-auto analyzer by colorimetric methods.
Oxidative stress assessment
All rats were sacrificed on the last day of the experiment. The liver tissue was dissected, weighed, and minced under cold conditions. After that, it was homogenized in 0.1 M phosphate buffer saline (PBS) with pH of 7.4 for composing 10% (w/v) liver homogenate. This homogenate was used for evaluation of reduced glutathione (GSH) and malondialdehyde (MDA) levels.
The liver tissue was carefully removed and fixed in 10% formalin solution. For histological studies, tissues were inserted in paraffin and stained with hematoxylin and eosin. The sections were examined under a microscope. Cell necrosis, central vein congestion, portal triad inflammation, and sinusoid dilatation were examined for liver injury.
The data achieved were stated as a mean ± standard error of mean (SEM). Statistical analysis was performed using Graph pad prism version 8 in which one-way ANOVA followed by post-hoc Tukey’s test was applied.
Liver function test assessment
(a) On baseline: At baseline, no statistically significant difference was found in LFT parameters such as AST, ALT, ALP, total bilirubin, total protein, and albumin (P > 0.05) among all groups in inter-group comparison.
(b) Effect of various treatments on LFT after 24 hrs of CCl4 dosing: The CCl4-induced hepatotoxicity model was developed in rats on day 1, and it was clear by marked elevation in serum AST (1526.66 ± 60.72 in EC vs 143.86 ± 10.81 in NC; P < 0.0001), ALT (894.83 ± 52.47 in EC vs 52.95 ± 2.16 in NC; P < 0.001), ALP levels (285.16 ± 22.21 in EC vs 124.16 ± 12.22 in NC, P < 0.05), and total bilirubin (0.216 ± 0.035 in EC vs 0.055 ± 0.005 in NC, P < 0.05). However, the serum protein (6.01 ± 0.26 in EC vs 6.84 ± 0.12 in NC) and albumin levels (3.13 ± 0.122 in EC vs 4.18 ± 0.18 in NC) were observed to be reduced non-significantly in the EC in comparison to NC group. The PC group indicated a significant reduction in AST (838 ± 48.64, P < 0.001) and ALT levels (515 ± 33.22, P < 0.05) in comparison to the EC. However, PC could not produce a significant change in ALP (179.5 ± 14.05 vs 285.16 ± 22.21 in EC), total protein (6.29 ± 0.08 vs 6.01 ± 0.26 in EC), albumin (2.975 ± 0.08 vs 3.13 ± 0.122 in EC), and total bilirubin levels (0.16 ± 0.01 vs 0.216 ± 0.03 in EC) (P > 0.05).
In the LD (1 mg/kg scopoletin) group, a significant reduction was observed in AST levels (901.83 ± 72.75, P < 0.05), whereas a non-significant difference in ALT (620.66 ± 55.60), ALP (255.66 ± 17.40), total bilirubin (0.1865 ± 0.023), total protein (6.6 ± 0.085), and albumin levels (2.85 ± 0.05) was noted when compared to the EC group. The study observed the MD (5 mg/Kg scopoletin) group showing a significant reduction in AST levels (727.33 ± 29.15, P < 0.001) and ALT levels (532.66 ± 24.23, P < 0.01). However, a non-significant difference was found in total bilirubin (0.184 ± 0.008), total protein (6.82 ± 0.11), albumin (2.94 ± 0.085), and ALP levels (235 ± 7.9) in comparison with the EC group. The HD (10 mg/Kg scopoletin) group caused a significant reduction in AST (923.16 ± 50.19, P < 0.01) in comparison to the EC group. However, a non-significant difference in ALT (628.66 ± 45.35), ALP (183 ± 14.17), total bilirubin (0.2131 ± 0.022), total protein (6.55 ± 0.121), and albumin levels (2.91 ± 0.07) was observed with HD scopoletin in comparison to the EC group.
(a) Effect of various treatments on LFT on Day 7: On the 7 day follow-up, the EC group saw a fall in AST (197.33 ± 11.20), ALT (72.83 ± 3.47), and total bilirubin levels (0.19 ± 0.02), whereas an increase was observed in ALP (364.16 ± 29.15), and total protein levels (7.46 ± 0.19) compared to the NC group. The PC group showed no significant difference in AST (220.83 ± 8.34 vs 197.33 ± 11.2 in EC), ALT (59 ± 2.67 vs 72.83 ± 3.47 in EC), total bilirubin (0.14 ± 0.01 vs 0.19 ± 0.023 in EC), albumin (3.03 ± 0.07 vs 3.51 ± 0.11 in EC), total protein (5.95 ± 0.07 vs 7.46 ± 0.19 in EC), and ALP levels (292.5 ± 14.41 vs 364.16 ± 29.15 in EC).
In MD group rats, a significant reduction was found in total bilirubin (0.12 ± 0.01, P < 0.05), whereas the HD treatment depicted a significant decrease in ALP levels (292 ± 16.6, P < 0.05). In the rest of the scopoletin-treated rats, a non-significant difference was found in AST (139.66 ± 10.85 in LD, 186 ± 18.5 in MD, and 192.83 ± 6.40 in HD), ALT (62.5 ± 1.76 in LD, 66.83 ± 3.19 in MD, and 63.66 ± 3.02 in HD), total bilirubin, (0.145 ± 0.007 in LD, and 0.15 ± 0.005 in HD), total protein (7.31 ± 0.06 in LD, 7.26 ± 0.17 in MD, and 7.13 ± 0.18 in HD), albumin (3.15 ± 0.06 in LD, 3.21 ± 0.08 in MD, and 3.27 ± 0.05 in HD), and ALP levels (335.33 ± 35.35 in LD, and 326.83 ± 17.56 in MD) [Table 1].
Effects on Oxidative stress markers (GSH and MDA)
(a) Effects on MDA levels: MDA production up-regulated significantly in EC in comparison to the NC group (EC 0.3727 ± 0.06 vs NC 0.1947 ± 0.016) (P < 0.05). The PC group showed an insignificant decline in MDA levels in comparison to the EC group. Scopoletin treatments caused a significant reduction in MDA levels as compared to the EC group (0.1821 ± 0.019 in LD, 0.1714 ± 0.018 in MD, and 0.1701 ± 0.012 in HD) (P < 0.01 in all three groups).
(b) Effects on GSH levels: Administration of carbon tetrachloride significantly depleted GSH levels (35.50 ± 3.68 in EC vs 54.75 ± 5.35 in NC; P < 0.05). NAC treatment significantly increased GSH levels as compared to carbon tetrachloride alone (53.68 ± 6.82, P < 0.05). Scopoletin treatment significantly up-regulated the GSH levels in carbon-tetrachloride-treated rats (57.44 ± 5.11 in LD, 53.70 ± 6.27 in MD, and 65.25 ± 5.096 in HD) (P < 0.05 in all three groups) [Figure 1a].
In the EC group, liver tissues showed occasional hepatocyte necrosis, diffused fatty changes, micro-vesicular steatosis, portal triad inflammation, and central vein congestion. Minimal central vein congestion and mild fatty changes were observed in the positive control. However, the scopoletin-treated rats showed the changes, which appeared similar to those of the normal control [Figure 1b].
This study was executed for exploration of therapeutic potential of scopoletin in hepatotoxicity management. Scopoletin, an active phyto-chemical, has been identified in several plant species. To name a few, there are Datura metel, Mallotus resinosus, Solanum nigram, and Viburnum prunifolium. The roots and leaves of these plants contain scopoletin, which has shown its worth in hypolipidemic, anti-diabetic, and neuro-logical indications.[8,10,11] The world is slowly moving towards the ancient medical science of India, that is, Ayurveda. It emphasizes upon the healing nature of various plant extracts used for a multitude of illnesses. Many allopathic medicines in the current era are also of plant origin. The limited treatment options available in liver injury management and the opposite effects of NAC observed on liver functions, especially before and after alcohol use, directed the investigators to explore a relatively new, potentially safer, and effective phyto-chemical in the present study.
CCl4 induces hepatocyte injury in the cell membrane, leading to leakage of AST, ALT, and ALP in the vascular compartment. It mimics the chemical/toxin/xenobiotic-induced liver injury in clinical practice. This type of liver injury is less understood in terms of the clinical management of patients. The present study observed a rise in AST, ALT, and bilirubin levels coupled with a fall in total protein and albumin levels. It was noted 24 hours after the CCl4 injection. The positive control used in the study, NAC, was chosen for the present study based upon its concrete role as a hepatoprotective agent. NAC prevented the significant rise in LFT parameters and simultaneously produced a rise in GSH in the current study also. Similar changes were noted with all three doses of scopoletin. Further, it was obvious from the histopathological findings that the hepatocytes, central vein, and portal triad nearly normalized in scopoletin groups, contrary to the group which received CCl4. The scopoletin medium-dose (i.e., 5 mg/kg) effect was more prominent as compared to a low dose and high dose as it halted the rise in both AST and ALT. The dose-dependent action could not be observed in the current study.
The doses of 1, 5, and 10 mg/kg scopoletin oral administration were adopted from a study by Chang et al., demonstrating the anti-inflammatory effects of scopoletin with the same doses in the carrageenan (Carr)-induced paw edema model. Several studies have happened using scopoletin in either the crude form or polyherbal preparation form. In contrast, the present study used scopoletin in its pure form. The present study used olive oil as a vehicle for CCl4 administration because of the liver injury minimizing effect that may be responsible for the absence of any mortality in the experimental control group.
The CCl4-induced liver injury model induction time, that is, 24 h, is very well known. It makes the experiment suitable for the evaluation of any study medication in acute liver injury management. Because the liver injury effect produced by CCl4 is reversible, the effects wane off in some time. To the best of our knowledge, there exists no literature on the duration of CCl4-induced liver injury in rats. It would be prudent to know if only a few doses can be administered or if a week-long therapy can be assessed in CCl4-induced liver injury in rats. The novelty of the current study lies in ascertaining the duration of CCl4-induced liver injury in rats. Based upon bio-chemical assessment on day 7, the changes in LFT were near normal, guiding the researcher groups on not using the model beyond 5 to 7 days, post-induction. The present study may help direct the early-phase exploration of scopoletin in addition to the human pharmacokinetics and evaluation of various formulations.
The results of the current study illustrate that scopoletin has effective hepatoprotective action upon carbon tetrachloride-induced hepatic injury in rats.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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