Hepatoprotective Mechanism of Apigenin via Suppression of Oxidative Inflammatory Signaling and Apoptosis against Hepatotoxicity Induced by CCl₄ in Rats

Environmental toxicants are ubiquitous and have been implicated in the pathophysiology of chronic diseases and organ damage (Famurewa et al., 2022). Carbon tetrachloride is among these environmental pollutants that inadvertently enter the human body and trigger pathologies. It is an exogenous industrial solvent with a strong affinity for the liver and it has been recognized as a hepatotoxicant (Zhou et al., 2020). In spite of its hepatotoxic and nephrotoxic stress in humans and experimental animals, it is still being used in dry cleaning, fumigation of grains, insecticide and filling fire extinguishers (Que et al., 2022). The hepatic metabolism of CCl₄ via the action of cytochrome P450-dependent monooxygenases results in the production of its hepatic metabolites, trichloromethyl (CCl₃) and trichloromethyl peroxyl (•OOCCl₃) reactive oxygen species (ROS) (El-Hadary and Hassanien, 2016). The metabolites are potent free radicals that trigger subsequent reactive oxygen species (ROS) generation. Hepatic diseases, including hepatic cirrhosis and  fibrosis have been associated with ROS effect (El-Hadary and Hassanien, 2016). The prevailing mechanism of CCl₄-induced hepatotoxic damage is consistent with the potential of its oxidative metabolites to cause hepatic antioxidant impairment leading to oxidative stress,  and pro-inflammatory activation Yang et al., (2018). Oxidative stress is capable of initiating membrane lipid peroxidation and, finally cell death (Zhou et al., 2020). The trichloromethyl radical can react with sulfhydryl groups of glutathione enzymes and other protein thiols to cause deficit in glutathione metabolism and consequently reduces cell activities of superoxide dismutase and catalase (Almatroodi et al., 2020). Existing literature has implicated the involvement of oxidative pro-inflammation and apoptosis in CCl₄-induced hepatic pathologies Yue et al., (2020).

A robust body of literature reports that natural antioxidants are efficacious in preventing oxidative stress-related liver pathologies (Almatroodi et al., 2020). Apigenin (4,5,7-trihydroxyflavone) is a natural polyphenolic flavone widely found in fruits, herbs and vegetables (Shankar et al., 2017). Systematic investigations on the health benefits of APG have shown its several pharmacological effects, including antioxidant, anti-inflammatory, antidiabetic, anti-Alzheimer’s disease, anticancer, antiviral and antihypertensive (DeRango-Adem and Blay, 2021 Ahmad et al., (2019) and Wu et al., 2021). Therefore, the study herein was designed to explore APG’s possible hepatoprotective effect and mechanism against CCl₄-induced hepatotoxicity in rats.

Chemicals
 
Carbon tetrachloride (Cat. No. 56-23-5) was purchased from Loba Chemie (India), olive oil (Cat. No. EL 40-105) was purchased from AGROVIM (Greece) and apigenin (Cat. No. 520-36-5) was purchased from Matrix Scientific (Columbia, SC, USA). Reagent kits for liver function markers (ALT: E-BC-K235-S and AST: E-BC-K236-M). The kits for SOD (SOD: Cat. No. SD 2521), CAT (CAT: Cat.No. CA 2517) and GPx (GPx: Cat. No. GP 2524) activities and MDA (MDA: Cat. No. MD 2529) level were obtained from BioDiagnostics, Giza, Egypt. The MAO kit (EMAO-100) was purchased from BioAssay Systems, CA, USA. The kits for cytokines were procured from OriGene Technologies Inc., Rockville, MD and MyBioSource, Inc., San Diego, USA, while ELISA kits for apoptosis markers were obtained from PEVIVA, USA.
 
Experimental animals
 
Thirty male rats (weighing 200-220 g)  were used which were obtained from the Faculty of Science at King Faisal University, Kingdom of Saudi Arabia. The experimental design used in this study was approved by the Department of Chemistry Research and Ethics Committee, College of Science, King Faisal University, Kingdom of Saudi Arabia, with reference number KFU-REC/2020-09-02. The rats were housed in a laboratory animal room under standard management conditions of a temperature of 20-25°C and the exposure time to light per day was 12 hours.
 
Experimental design
 
Following 14 days of acclimatization, rats were randomly divided into six groups (n=5/group).
Group I (Control): Rats received an intraperitoneal (i.p.) injection of olive oil (3 ml/kg b.w.).
Group II (CCl4): Rats received CCl4 (30% in olive oil) (3 ml/kg b.w., i.p) [20].
Group III (APG + CCl4): Rats received APG (10 mg/kg, orally) + CCl4 (3 ml/kg b.w., i.p).
Group IV (APG + CCl4): Rats received APG (20 mg/kg, orally) + CCl4 (3 ml/kg bw, ip).
Group V (APG): Rats received APG (10 mg/kg b.w, orally).
Group VI (APG): Rats received APG (20 mg/kg b.w, orally).

The treatment was performed twice per week for seven consecutive weeks. The doses of CCl₄ and APG doses were chosen according to Liu et al., (2018) and Anusha et al., (2017), respectively.

Blood samples and liver tissues were collected at the end of the experimental period from all experimental animals. The serum was separated and liver samples were taken for the biochemical analysis and histological examination.
 
Biochemical analysis
 
Determination of liver function indices
 
Liver enzymes, including AST and ALT, were quantitatively estimated in serum using commercial kits, following the manufacturer’s instructions. Monoamine oxidase (MAO) was analyzed using a commercial kit.
 
Determination of oxidative stress markers
 
 Liver antioxidant CAT, SOD, GPx and TBARS as MDA levels were measured using standard assay kits, following the procedures of the kits’ manufacturer.
 
Determination of inflammatory markers
 The inflammatory cytokines interleukin-6 (IL-6), interleukin-4 (IL-4) and interleukin-10 (IL-10) levels were measured in serum using rat ELISA kits.The tumor necrosis factor-α (TNF-α) was analyzed using ELISA kit. The analyses were done according to the manufacturers’ instructions.
 
Apoptotic markers
 
According to the protocols described in the ELISA kits.
 
Histopathological analysis
 
Liver samples from each group were fixed in 4% formaldehyde for 24 h, dehydrated in ascending ethanol series, embedded in paraffin, sectioning (4 μm thick) and  stained with hematoxylin and eosin dye (H and E) and examined under light microscope. The histopathological alterations in tissue sections were scored and an average value was determined as follows: normal histostructure (0), mild (1), moderate (2) and severe (3) following extensive alterations according to their histopathological findings (Bancroft and Gamble 2002).
 
Statistical analysis
 
The SPSS software was used for data analysis and results presented as mean ± SEM. The one-way analysis of variance (ANOVA) was used to determine statistical differences among groups, followed by a post hoc “LSD test: the least significant difference.” A p<0.05 was considered statistically significant.

Effect of APG on liver function indices
 
ALT and AST are enzymes mainly confined within the hepatocytes, although they are also found in the heart, kidney, blood cells and pancreas (Aja et al., 2020) while monoamine oxidase is a mitochondrial enzyme with high activity in the brain, GIT and hepatic tissue (Jaka et al., 2021). CCl₄ significantly increased the serum activities of ALT, AST and MAO compared to normal control. However, the administration of APG prevented these toxic effects in a dose-dependent mannerreduction (Table 1).



Similar result was obtained by Liu et al., (2018) and Almatroodi et al., (2020). Elevations in activities of ALT and AST is due to cellular leakage and loss of functional integrity of hepatic cell membrane, where as elevated ALP activity is a marker of hepatic-cholestatic damage while increased hepatic MAO activity reveals mitochondrial injury (Abou Seif, 2016). Furthermore, hepatic necrosis which was observed in our histopathological analysis has been implicated as a contributory factor to ALT and AST release into the blood circulation. Contrarily, concomitant administration of APG (10 and 20 mg/kg body weight) to rats inhibited CCl₄-mediated hepatic damage dose-dependently in this study. This result agree with that study of Yue et al., (2020).
      
Effect of APG on oxidative stress markers
 
It is known that SOD, CAT and GPx are cellular antioxidant enzymes that scavenge ROS and thus promote antioxidant mechanism. CCl₄ sigmificantly reduced the activities of SOD, CAT and GPx and increased MDA level.

However, the administration of APG (at 10 and 20 mg/kg body weight) significantly increased the activities of these enzymes and decreased MDA level compared to CCl₄ group. APG exerted significant dose-dependent reduction on MDA alone (Table 2). These findings  can be confirm by previous reports (Ubhenin et al., 2016). The significant depression in the hepatic activities of SOD, CAT and GPx implies the overwhelming oxidative imbalance exerted by the CCl₄ metabolites leading to antioxidant imbalance and/or oxidative stress in the rat liver exposed to CCl₄. 



The chief mechanism underlying CCl₄ hepatotoxicity is oxidative stress arising from the hepatic metabolites of CCl₄ (trichloromethyl and trichloromethyl peroxyl radicals). These hepatic metabolites, are ROS generators and consumers of antioxidant balance (El-Hadary and Hassanien, 2016). Intriguingly, trichloromethyl can react with sulfhydryl groups present in glutathione, GPx and protein thiols to form an oxidative complex, exerting deleterious effects on SOD and CAT (El-Hadary and Hassanien, 2016).

Interestingly, the APG administration scavenged the ROS and enhanced hepatic antioxidant homeostasis. This was evident through prominently elevated hepatic activities of SOD, CAT and GPx  and decreased level of MDA compared to CCl₄ group, in consonance with earlier studies (Raskovic et al., 2017 and Ubhenin et al., 2016). Consequently, appreciably indicating ability of APG to  inhibit  oxidative stress.

It was noteworthy to observe that the two doses of APG failed to demonstrate dose-dependent increases in SOD, CAT and GPx activities but only in MDA level (Table 2). A robust body of literature indicates APG antioxidant property (DeRango-Adem and Blay, 2021). It is a natural polyphenolic flavonoid flavone with ROS-scavenging activity and other pharmacological properties (Salehi et al., 2014). By implication, therefore, APG demonstrates a hepatoprotective effect against CCl4 oxidative stress via its antioxidant property. The free hydroxyl groups present on the A/B rings of APG are responsible for the antioxidant effects of this flavone (Singh et al., 2014).
 
Effect of APG on inflammatory markers in CCl₄-intoxicated rats
 
In CCl₄ group, the levels of IL-6 and TNF-α significantly increased while the levels of anti-inflammatory markers, IL-4 and IL-10  is significantly reduced compared to normal control. This result indicates that CCl4 provokes pro-inflammation and depresses anti-inflammation (Yang et al., 2018). The oxidative stress observed herein might have enhanced the induction of cytokine expression. Oxidative stress status may trigger the nuclear translocation of nuclear factor-kappa B (NF-κB) to stimulate the expression of cytokine proteins Edeogu et al., (2020), which has been reported to occur during CCl₄ hepatotoxicity (Tsai et al., 2017).

However, APG administration prominently abrogated the effect of CCl₄ on these cytokines. Interestingly, the two doses of APG expressed a dose-dependent effect on IL-6 and TNF-α only (Table 3). Accumulating number of studies demonstrate that APG can suppresses inflammatory cascades (Salehi et al., 2019). 


 
Effect of APG on apoptosis markers in CCl4-intoxicated rats
 
Serum levels of M30 and M65 are indicator of apoptosis of cells undergoing necrosis and cell death de Haas et al. (2008). The release of TNF-α promotes cell apoptosis leading to hepatocyte cell death (Li et al., 2020). The CCl₄ significantly increased the level of M65 and M30 in comparison to the normal control (Fig 1). The induction of apoptosis by CCl₄ in this study corroborate the findings of previous studies (Li et al., 2020).



On the contrary, the APG doses significantly reduced the levels of M65 and M30 compared to CCl₄ group (Fig 1). Interestingly, the two doses of APG revealed a dose-dependent effect on M65 and M30, respectively.

Also, there was dose-dependent antiapoptotic effects of APG as mentioned  in previous studies (Mohamed et al., 2020 and Zhong et al., 2017).  
 
Histopathological analysis
 
Fig 2 showed that, the control group (1a), the liver architecture appears normal with hepatocytes, blood sinusoids and central vein. On the contrary, the liver histological analysis from CCl₄ group revealed inflammatory cells infiltration (star), cytoplasmic vacuolation and degeneration and severe hepatic necrosis (arrow) (2a). The oxidative milieu created by CCl₄ may be the cause of these histopathologic lesions. The observed infiltration of inflammatory cells in our histopathological analysis could also account for the increased IL-6 and TNF-α levels in this study (Yeh et al., 2013).



The administration of APG in the APG + CCl4 groups ameliorated the CCl₄-induced alterations to mild lesions (3a and 4a). The APG only did not alter the liver structures (5a and 6a).   

The present study demonstrated the hepatotoxic effect of CCl₄ and emphasized that APG possesses a mechanistic hepatoprotective effect against CCl₄ induced hepatotoxicity via abrogation of oxidative stress, pro-inflammation and apoptosis. Chiefly, these beneficial effects can be attributed to antioxidant and anti-inflammatory activities of APG .

Deanship of Scientific Research, King Faisal University, Saudi Arabia, with Grant Number: GRANT 2214.

The authors acknowledge the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research at King Faisal University, Saudi Arabia for financial support under the Student Researcher Funding track [GRANT 2214].

The authors declare no conflict of interests.