The protective effect of chrysin on oxidative stress, inflammation, and apoptosis in lithium-induced hepatotoxicity in rats

Raghda A E Elsayed; Marwa M Atef; Noha M Shafik; Samia A H El-Dardiry

doi:10.4103/tmj.tmj_57_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 51 | Issue : 2 | Page : 139-143

The protective effect of chrysin on oxidative stress, inflammation, and apoptosis in lithium-induced hepatotoxicity in rats

Raghda A E Elsayed, Marwa M Atef, Noha M Shafik, Samia A H El-Dardiry
Department of Medical Biochemistry, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission	25-Sep-2022
Date of Acceptance	01-Dec-2022
Date of Web Publication	14-Sep-2023

Correspondence Address:
Raghda A E Elsayed
Faculty of Medicine, Tanta University, Ahmed Ali Street, Gharbia 31527
Egypt

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/tmj.tmj_57_22

Abstract

Background Drugs are an important cause of liver injury. Chrysin is a flavonoid and the main constituent of Oroxylum indicum. Many pharmacological and biological benefits have been attributed to it. These include anti-apoptotic, anti-inflammatory, and antioxidant effects. Aim We intended investigating the anti-apoptotic, anti-inflammatory, in addition antioxidant influence of chrysin on lithium-induced hepatotoxicity in rats. Materials and methods The present research was conducted at the Medical Biochemistry Department, Faculty of Medicine, Tanta University, Egypt. A total of 40 albino male rats weighing between 150 and 200 g were included in this experiment and were split randomly into 4 equal groups (each group involved 10 rats): group I (control), group II (lithium treated), group III (lithium+chrysin), and group IV (chrysin treated). All rats that were acquired originated in the laboratory’s animal colony at Tanta University. Liver function tests, total and direct bilirubin, malondialdehyde, superoxide dismutase, reduced glutathione, caspase-3, and nuclear factor-kappa B levels were measured. Results Chrysin treatment resulted in significant decrease in liver functions, total and direct bilirubin, caspase-3, nuclear factor-kappa B, and malondialdehyde levels and significant increase in superoxide dismutase activity, with reduced glutathione intensity. Conclusion Based on these correlating findings, it is possible to conclude that chrysin has protective potential on lithium-induced hepatotoxicity; therefore, it represents a promising therapeutic strategy in its management.

Keywords: anti-inflammatory, anti-apoptotic, chrysin, drug-induced hepatotoxicity, lithium, oxidative stress

How to cite this article:
Elsayed RA, Atef MM, Shafik NM, El-Dardiry SA. The protective effect of chrysin on oxidative stress, inflammation, and apoptosis in lithium-induced hepatotoxicity in rats. Tanta Med J 2023;51:139-43

How to cite this URL:
Elsayed RA, Atef MM, Shafik NM, El-Dardiry SA. The protective effect of chrysin on oxidative stress, inflammation, and apoptosis in lithium-induced hepatotoxicity in rats. Tanta Med J [serial online] 2023 [cited 2023 Nov 30];51:139-43. Available from: http://www.tdj.eg.net/text.asp?2023/51/2/139/385705

Introduction

Drug-induced hepatotoxicity causes liver damage, as neutrophils and Kupffer cells in the liver produce inflammatory cytokines and reactive free radicals, leading to oxidative stress [1].

Lithium’s dominant cytotoxic mechanism is oxidative stress and reactive oxygen species (ROS) generation [2].

Besides ROS, lithium cytotoxicity was also associated with mitochondrial membrane collapse and cytochrome C release into the cytoplasm of hepatocytes [3].

Nuclear factor-kappa B (NF-KB) is a dictating factor that is present in each cell’s cytoplasm, and after being triggered, it translocates to the nucleus [4].

Reduced glutathione (GSH) plays a vital role in protection from reactive metabolites [5].

Materials and methods

Materials

Study design and animal grouping

The Medical Research Ethics Committee at Tanta University’s School of Medicine in Egypt approved the conduct of the present research at the Medical Biochemistry Department (Approval code: 34393/1/21). A total of 40 albino male rats weighing between 150 and 200 g were included in this investigation. These rats were obtained from Tanta University’s experimental animal colony. Animals were provided free access to water and kept in wire mesh cages for the duration of the trial. They were subjected to a controlled environment consisting of a constant temperature (25°C) and a strict lighting schedule (12 h of darkness followed by 12 h of light).

Experimental design

Once adapted to environment during a period of 1 week, 40 rats were equally split among four test groups in a random manner:

Group I (control group): it received vehicle (0.5% sodium carboxymethylcellulose) for 7 weeks.

Group II (lithium-treated group): this group received vehicle for 3 weeks and then received oral dose of 30 mg/kg lithium carbonate dosage daily (dissolved in distilled water) for 4 weeks [6,7].

Group III (chrysin and lithium-treated group): it received chrysin dosage of 100 mg/kg weight of the body dissolved in 0.5% sodium carboxymethylcellulose for 3 weeks and then received oral dose of 30 mg/kg of lithium carbonate daily for 4 weeks [8].

Group IV (chrysin-treated group): it received a measured dosage of chrysin 100 mg/kg dissolved in 0.5% sodium carboxymethylcellulose for 3 weeks and then received vehicle daily for 4 weeks.

After decapitation

(1)Serum samples were assessed for alkaline phosphatase, alanine aminotransferase, total and direct bilirubin, and aspartate aminotransferase.

(2)Liver tissue samples were homogenized using a Potter-Elvehjem tissue homogenizer and assessed for reduced GSH, superoxide dismutase, and malondialdehyde (MDA) activity by spectrophotometry utilizing an accessible kits supplied by BIOMED and for protein caspase-3 and NF-KB levels by an ELISA kit purchased from Innova Biotech Co. Ltd, Beijing, China.

Statistical analysis

Analysis of statistics and presentation of the results of the current research was performed, and the data were presented as number and percentage or mean±SD. The analysis of variance (ANOVA) and Tukey tests were calculated using the computer program SPSS V.20 (SPSS inc. IBM, Chicago, USA). P value less than 0.05 was judged significant.

Results

[Table 1] shows a difference among the studied groups regarding NF-ΚB level in the liver tissue homogenate using ANOVA test and then by Tukey’s test. Group II, which received lithium treatment, showed a statistically significant rise in NF-KB compared with the other groups. In addition, the NF-KB level was significantly reduced in both lithium and chrysin-exposed groups (group III) and chrysin cured (group IV) when compared with lithium-treated group (group II) (P<0.001).

Table 1: Comparison among the studied groups regarding nuclear factor-kappa B level in liver tissue homogenate (ng/mg tissue protein)

Click here to view

[Table 2] shows a difference among the groups analyzed regarding caspase-3 level in the liver tissue homogenate using Tukey’s test after ANOVA test. Statistical analysis shows a significant increase in caspase-3 level in lithium-treated group (group II) compared with other studied group. In addition, there was a significant decrease in caspase-3 level in both chrysin and lithium-treated group (group III) and chrysin-treated group (group IV) when compared with lithium-treated group (group II) (P<0.001).

Table 2: Comparison among the studied groups regarding caspase-3 level in liver tissue homogenate (pg/mg tissue protein)

Click here to view

[Table 3] shows a difference among the groups researched regarding MDA level in liver tissue homogenate using ANOVA test followed by Tukey’s test. There was a statistically significant increase in MDA level in lithium-treated group (group II) versus other groups. In addition, there was a significant decrease in MDA levels in both chrysin and lithium-treated group (group III) and chrysin-treated group (group IV) when compared with lithium-exposed group (group II) (P<0.001).

Table 3: Comparison among the studied groups regarding malondialdehyde level in liver tissue homogenate (nmol/mg tissue protein)

Click here to view

[Table 4] shows a difference among the studied groups regarding reduced GSH level in liver tissue homogenate using Tukey’s test after ANOVA test. There was a significant statistical decrease in reduced GSH level in lithium-treated group (group II) compared with other groups in the study. In addition, there was a significant increase in reduced GSH level in both chrysin and lithium-treated group (group III) and chrysin-treated group (group IV) versus lithium-treated group (group II) (P<0.001).

Table 4: Comparison among the studied groups regarding reduced glutathione level in liver tissue homogenate (mg/gm. tissue)

Click here to view

Discussion

Overall, we aimed to monitor an essential regulator of many cellular processes, NF-KB gene, which encodes for products implicated in tissue damage and inflammation. This may be an initial biomarker regarding toxicity of chemical. The current research shows NF-KB was formed to be increase significantly in lithium-intoxicated group (group II), which is explained to occur, during conditions of oxidative stress. It represents one of the main molecules triggered by inflammatory cues involving hydrogen peroxide (H₂O₂). In harmony, in an early report concerning treatment of human T lymphocytes with the N-acetyl cysteine, an antioxidant prevents the activation of NF-KB in response to micromolar doses of H₂O₂. Recent evidence suggests that H₂O₂ may not serve as a direct inducer but rather as a modulator of the NF-KB pathway [9]. This explains also the reason of decreased NF-KB levels in groups treated by chrysin (groups III and IV).

In agreement with the results obtained here, Marwa et al. [10] revealed that bisphenol, which is a drug that causes hepatotoxicity, results in significant increase in liver NF-KB. This was evident in comparison with the control group. Moreover, when treated with thymoquinone, which has antioxidant and anti-inflammatory effects as chrysin, there was a significant decrease in liver NF-KB level.

Matching with these findings, Mustafa et al. [11] revealed that chrysin was found to be able to reduce inflammation by suppressing COX-2 activity, PGE-2, and NF-KB production in dependence on the amount of drug used.

In relevance, previous reports noted that apoptosis and autophagy include two types of programmed cell death with important role in organism homeostasis. Apoptosis extrinsic and intrinsic pathways meet at the caspase-3 level. Intrinsic pathway is activated by cellular stresses. Hence, oxidative stress and caspase-3 represent the key executioner of apoptosis present at the cross road of both apoptotic pathways [12]. In fact, a hallmark of fulminant hepatic failure is usually the rapid induction of apoptosis to hepatocyte. Accordingly, chronic treatment with lithium induces features of apoptosis, such as caspase activation and the discharge of mitochondrial cytochrome C.

Confirmatively, specifically, here, treatment with lithium revealed significant elevation in the hepatic caspase-3, compared with other studied groups. This finding can be explained by induction of caspase-3 activation via several mechanisms, including the activation of intracellular ROSs production, cytochrome C, and the increase in intracellular calcium level.

However, another study by Justin et al. [13] revealed that caspase-3 activation caused by rotenone and H₂O₂ is suppressed by continuous lithium treatment. To avoid caspase-3 activation, lithium may inhibit GSK3, an intermediary in several apoptotic signaling pathways [14] and a shared target of lithium and valproate. In addition, lithium may have a role in the mitochondrial death pathway before the activation of caspase-3.

The effective role of chrysin-treated groups (groups III and IV) had exhibited a significant decreased in expression of caspase-3, revealing chrysin’s anti-apoptotic protective function. In consistent with our data, Mustafa et al. [11] stated that chrysin was able to reduce testicular tissue apoptosis as there was no manifestation of TNF-α and caspase-3 in the CHR-50-cured group. This was explained by suppressing the expression in the research of PbAc-activated caspase-3.

Nonetheless, it may agree with Neha et al. [15], who have revealed that chrysin decelerates apoptosis by decreasing expression of caspase-3 in rats with diabetes. These findings prove that chrysin can alleviate liver toxicity caused by lithium with its anti-apoptotic properties.

In reference, impairment in membrane function and structural integrity [16] are both effects of lipid peroxidation, resulting from oxidative destruction of polyunsaturated fatty acids. Lithium was associated with an elevation in hepatic MDA in the current study, when compared with other studied groups. The observed increase in MDA is associated with a significant reduction in animals with lithium intoxication and their GSH levels. This confirms the report that lithium induces lipid peroxidation as a result of the magnitude in the form of oxidative stress that promotes the development of hepatotoxicity.

Conceivably, chrysin pretreatment revealed significant reduction of the MDA concentration, most likely owing to its free radical scavenging properties. This means that when chrysin is used alone or in combination with lithium treatment, it might inhibit hepatotoxic effects of lithium. In harmony with this finding, Ganesan et al. [17] reported that silymarin with chrysin treatment was significantly decrease in lipid peroxidation which might be owing to their antioxidant ability. Free radical scavenging properties and antioxidant have been attributed to chrysin.

A selected antioxidant biomarker target, which was monitored in here, was presented by reduced GSH level, which is a tri-peptide synthesized from three amino acids glycine, cysteine, and glutamate in the liver. The role of GSH is associated with cellular protection against the multifaceted toxic effects [18]. GSH serves as a redox buffer under circumstances of oxidative stress, as it is a cofactor for the enzyme peroxidase, thus acting as an indirect antioxidant. This is induced by donating the electrons necessary for decomposition of H₂O₂, and these reactions generate oxidized GSH. Cellular levels of GSH are an oxidative stress significant indicator [19].

In harmony, loss of tissue GSH (an intracellular antioxidant) causes membrane lipid peroxidation, which in turn compromises the integrity of these structures and, in extreme cases, may be lethal [20]. Cell mortality, increased protein breakdown rates, and changes in membrane fluidity and permeability have all been linked to lipid peroxidation in membranes [21]. Therefore, determination of membrane integrity and the severity of toxicant-induced liver cell damage is mostly dependent on intracellular GSH content [20].

Notably, our results revealed that GSH depletion could have occurred in lithium-treated group because of ROS formation when compared with the control group. Explicably and in line with the aforementioned data, Vijayta et al. [22] documented that reduced GSH, catalase, and glutathione S-transferase levels were significantly reduced in lithium-treated rats, following daily lithium treatment for 2–4 months.

On the contrary, Alfonso et al. [19] have emphasized the role of GSH in oxidative reactions on the basis that it participates in metal ion-mediated processes that generate free radicals and ROS.

However, GSH levels were significantly raised after pretreatment with the chrysin [23]. Hence, chrysin (C15H10O4) is a flavonoid and has remarkably compelling effects; thus, a hydroxyl group is present on carbon 5. Polyphenols’ ability to act as antioxidants is dependent on the ring B being hydroxylated. However, chrysin’s beneficial antioxidant activity is only moderately influenced by the meta hydroxyl groups in the A ring at positions 5 and 7.

Conclusion

Chrysin’s modulatory effect in maintaining an appropriate intracellular redox state was associated with a number of beneficial activities as confirmed by decreasing lipid peroxidation, oxidative stress makers, and caspase-3 levels and increasing antioxidants such as superoxide dismutase and reduced GSH levels with improvement of the histopathological abnormalities of hepatic tissues. It implies that chrysin has prophylactic potential by maintaining intracellular redox homeostasis in the treatment of hepatotoxicity and represents a promising therapeutic strategy in its management.

Financial support and sponsorship

Nil.

Conflicts of interest

No Conflict of Interest.

References

1.	Priyankar D, Manas R, Arnab S An overview on drug-induced hepatotoxicity. Asian J Pharm Clin Res 2013; 6:1–4.
2.	Mukherjee S, Gogoi BJ Free radicals in disease and potential role of phytoconstituents – a cause with emerging importance. Curr Chem Bio 2011; 5:197–212.
3.	Haussmann R, Bauer M, Von B, Grof P, Lewitzka U Treatment of lithium intoxication: in facing the need for evidence. Int J Bipolar Disord 2015; 3:1–5.
4.	Serasanambati M, Shanmuga R Function of nuclear factor kappa B (NF-kB) in human disease. South Indian J Bio Sci 2016; 2:368–387.
5.	Ganesaratnam K, Rajesh D, Deborah F The role of glutathione in cancer. Cell Biochem Funct 2004; 22:343–352.
6.	Mohammad A, Yasser E, Muhammad F, Muhammad W Lithium induced toxicity in rat: blood serum chemistry, anti-oxidative enzymes in red blood cells and histopathological studies. Bio Pharm Bull 2011; 34:272–277.
7.	Abeer E, Nahla E Impact of resveratrol versus platelet-rich plasma for treatment of experimental lithium-induced thyroid follicular cell toxicity in rats. A histology and immunohistochemical study. Ultrastruct Pathol 2019; 43:80–93.
8.	Anca HT, Mariasiu I, Navarro G Hepato-protective action of chrysin is mediated through TNF-α in chemically induced acute liver damage: In vivo study and molecular modeling. Exp Therap Med 2017; 13:1671–1680.
9.	Virgínia OM, Susana M, Luísa C, fernando A Role of hydrogen peroxide in NF-κB activation: from inducer to modulator. Antioxi Redox Signal 2009; 11:2223–2243.
10.	Marwa M, Randa M, Mona S, Afnan E, Hend E Protective effect of thymoquinone on bisphenol induced hepatotoxicity in male rats, targeting the role of associated pro-inflammatory cytokines and NF-kB. Benha Med J 2021; 38(Academic issue): 61–72.
11.	Mustafa I, Fulya B, Emrah H, Yildirim S, Kandemir FM, Dogan T, Dortbudak MB, Genc A Chrysin protects against testicular toxicity caused by lead acetate in rats with its anti-oxidant, anti-inflammatory, and anti-apoptotic properties. J Food Biochem 2021; 45:e13593.
12.	Alfons L Apoptosis—an introduction. Bioessays 2003; 25:888–896.
13.	Justin SL, Chunnian Z, Jerry JW, Peter PL Cyto-protection by lithium and valproate varies between cell types and cellular stresses. Eur J Pharm 2006; 539:18–26.
14.	Carol AG, Richard SJ The multifaceted roles of glycogen synthase kinase 3β in cellular signaling. Progr Neurobio 2001; 65:391–426.
15.	Neha R, Saurabh B, Jagriti B, Nag TC, Ruma R, Dharamvir SA Chrysin, a PPAR-γ agonist improves myocardial injury in diabetic rats through inhibiting AGE-RAGE mediated inflammation and oxidative stress. Chemico-biol Interact 2016; 250:59–67.
16.	Barry H, John G The importance of free radicals and catalytic metals in human diseases. Mol Aspects Med 1985; 8:89–193.
17.	Ganesan P, Panneerselvam K, Chinnadurai V, Pugalendi KV Effect of chrysin on hepato-protective and anti-oxidant status in D-galactosamine-induced hepatitis in rats. Eur J Pharm 2010; 631:36–41.
18.	Wen XW, Wen LJ, Gau JM, Tan M, Fei J, Li Y, Li CY Monitoring the fluctuation of hydrogen peroxide (H₂O₂) in diabetes and its complications with a novel near infrared fluorescent probe. Anal Chem 2021; 93:3301–3307.
19.	Alfonso P, Athanase V, Aldo P, Vincenzo DT, Alessandro FC The changing faces of glutathione, a cellular protagonist. Biochem Pharm 2003; 66:1499–1503.
20.	Güven A, Güven A, Gülmez M The effect of kefir on activities of GSH-Px, GST, CAT, GSH and LPO levels in carbon tetrachloride induced toxicity in mice tissues. J Vet Med B 2003; 50:412–416.
21.	Joaquı́n J, Russel J, Juan M, Escames G, Yu BP, Oh CS, Munoz-Hoyos A Melatonin prevents changes in microsomal membrane fluidity during lipid peroxidation. FEBS Lett 1997; 408:297–300.
22.	Vijayta DC, Bhalla P, Devinder KD Zinc modulates lithium-induced hepatotoxicity in rats. Liver Int 2008; 28:558–565.
23.	Mir T, Sarwat S Chrysin modulates ethanol metabolism in Wistar rats: the promising role against organ toxicities. Alcohol Alcohol 2011; 46:383–392.