Hepatoprotective potential of sciadopitysin against paraquat induced liver damage in rats

1 Introduction

Paraquat (PQ) is a potent synthetic herbicide that is extensively used in agricultural practices to prevent the growth of harmful weeds and undesirable vegetation (Peiró et al., 2007). PQ poses a substantial risk to human health due to its chemical characteristics such as high water solubility and volatility. It is reported that PQ is a hazardous toxicant due to lack of effective strategies against PQ induced toxicity (Ortiz-ortiz et al., 2011). PQ poisoning is a major concern in the developing countries, particularly in Asia. Human exposure to PQ occurs via skin contact, inhalation, and ingestion, while acute poisoning of PQ leads to death in three and a half hour. PQ exposure leads to mortality due to failure of multiple organs. PQ administration culminates in ROS synthesis, which impairs the normal functioning of biological system in the body (Liu et al., 2011).

According to previous study, PQ-induces pulmonary toxicity, reproductive toxicity, neurotoxicity and gastrointestinal toxicity, which are responsible for more than 50 % of pesticide-related fatality cases (Peiró et al., 2007). Liver diseases and environmental pollutants are strongly related, which is a grave concern related to human health. The liver is the primary organ for detoxification as it is involved in metabolism and is a major target of pesticides induced toxicity (Peiró et al., 2007). PQ induces hepatotoxicity due to over production of reactive species. PQ exposure induces oxidative stress (OS) in hepatic tissues, leading to a substantial rise in the levels of liver enzymes and up-regulation of genes expression that promote apoptosis. PQ exposure also leads to mitochondrial dysfunction, which causes mitochondrial membrane swelling in the liver of rats (Han et al., 2014). PQ-instigated liver damage results in centrilobular cholestasis, apoptosis in hepatocytes and macrophagic infiltration in the portal regions. The exposure to PQ leads to enlarged portal tracts due to an increase in collagen stroma and decreased lymphocyte and leukocyte infiltration (Bataller et al., 2000).

Flavonoids belong to the class of polyphenols that are widely used in nutraceutical, pharmacological and cosmetic industry. Biflavonoids are a type of flavonoid dimers, composed of two flavonoid units that are either similar or non-similar, connected symmetrically or asymmetrically by an alkyl-based linker (DeForest et al., 2014). Sciadopitysin (SCD) is a biflavonoid that found in the leaves of Ginko biloba and exhibits various therapeutic properties (Li et al., 2019). Hence, the current experiment was conducted to evaluate the therapeutic effects of SCD on PQ-instigated hepatotoxicity.

2 Materials and methods

3 Research design

48 Rats were split into four different groups: Control group, PQ (5 mgkg⁻¹) treated group, PQ (5 mgkg⁻¹) + SCD (2 mgkg⁻¹) co-treated groups and SCD (2 mgkg⁻¹) only treated group. The doses of PQ and SCD were selected based on previous studies by Kheiripour et al. (2021) and El-Aarag et al. (2019), respectively. The doses were administered via oral gavage. After 30 days of experiment, rats were anesthetized with 60 mgkg⁻¹ of ketamine and 6 mgkg⁻¹ of xylazine before decapitation. Liver was removed and half lobe of liver was homogenized at 11,000 g for 20 min using cold phosphate-buffered saline (25 mM; pH: 7.4), for the estimation of different biomarkers, while the other lobe was fixed in CH₂O (10 %) for histomorphological analysis

4 Results

4.1

4.1 Impact of SCD on Nrf-2/Keap-1 pathway

PQ administration induced a significant (p < 0.05) reduction in the expressions of Nrf-2 and antioxidant genes, whereas elevating the Keap-1 expression as compared to the control group. However, co-treatment with SCD upregulated the expressions Nrf-2 and antioxidant genes, while downregulating the expression of Keap-1 as compared to PQ treated group. Furthermore, in rats treated with SCD only the expressions of these parameters were close to those of control group rats (Figs. 1, 2).

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Fig. 1

Protective effect of SCD on A) Nrf-2 and B) Keap-1 expression. Bars are shown on the basis of mean ± SEM. Different superscripts on bars presenting significant variation.

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Fig. 2

Protective effect of SCD on A) CAT, B) SOD, C) GPX, D) GSR, E) GST and F) HO-1 expression. Bars are shown on the basis of mean ± SEM. Different superscripts on bars presenting significant variation.

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4.4

4.4 Impact of SCD on hepatic apoptotic markers

PQ poisoning led to a notable increase in the expressions of Caspase-3 and Bax, besides decreased the expression of Bcl-2 as compared to the control group. Nevertheless, SCD + PQ supplementation notably (p < 0.05) downregulated the expressions of Caspase-3 and Bax, while upregulating the expression of Bcl-2 as compared to the PQ administered group. Additionally, in SCD only administered group these expressions were comparable to control group (Fig. 3).

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Fig. 3

Protective effect of SCD on A) Bax, B) Bcl2, and C) Caspase-3, expressions. Bars are shown on the basis of mean ± SEM. Different superscripts on bars presenting significant variation.

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5 Discussion

PQ is a prominent synthetic herbicide used in agriculture practices worldwide for many years. It is known for its ability to undergo redox cycling, acting quickly and increasing the synthesis of free radicals, leading to oxidative stress (OS) and cell damage (Asghari et al., 2017). PQ exposure can leads to dysfunction of vital organs such as kidney, brain, heart, and gastrointestinal tract. The liver, being the main site for xenobiotics transformation, has a higher potential for producing ROS, and is thus susceptible to toxins damage. Antioxidants can help prevent health risks resulting from the exposure of pesticides (Ahmadian et al., 2018). Biflavonoids have effective anti-oxidative properties and can be used to protect cells from detrimental impacts of these free radicals (Ye et al., 2012). Sciadopitysin (SCD) is a biflavonoid found in the leaves of Ginko biloba that displays ROS scavenging capabilities due to its structural configuration (Li et al., 2019). Therefore, this research was planned to determine the ameliorative potential of SCD against PQ induced hepatic impairment by evaluating antioxidants activity, levels of OS markers, liver serum markers, inflammatory markers, and apoptotic markers in albino rats.

PQ treatment reduced the expressions of antioxidant genes and Nrf-2, while increasing the expressions of Keap-1. Vomund et al. (2017) stated that Nrf-2 plays a key role in the regulation of OS, while Pintard et al. (2004) explained that Keap-1 acts as the inhibitor of Nrf-2 and controlling its stability. During ROS production, Nrf-2 separates from its negative inhibitor, Keap-1, through some physical modifications, moves into the nucleus, and stimulate the expression of various cellular proteins. Hawkes et al. (2014) reaffirmed that Nrf-2 plays an effective role in regulating the expression of antioxidant genes. Similarly, Yang et al. (2022) documented that high OS resulted in decreased Nrf-2 expressions, while increasing the expressions of Keap-1. Consequently, reduced Nrf-2 expressions leads to lower antioxidant genes expression. Plant-based flavonoid has potential to increase the activities of antioxidant enzymes (Ijaz et al., 2022a). However, SCD administration elevated the expressions of antioxidant genes and Nrf-2, while lowering Keap-1 expression. Therefore, it is assumed that SCD has the ability to regulate the Nrf-2 and Keap-1 expressions.

PQ administration resulted in a notable decrease in the activities of SOD, GPx, CAT, GSR, GST, OH-1, and GSH, while increasing the concentrations of MDA and ROS. These findings align with the study by Latif and Faheem (2020), who reported that PQ elevates ROS levels while reducing the activities of antioxidant enzymes. Antioxidant enzymes serve as a primary defense against harmful free radicals. SOD converts superoxide ions into H₂O₂, which is generated as a byproduct of oxidative stress (Kheradmand et al., 2010). CAT facilitates the transformation of H₂O₂ into H₂O and O₂ (Han et al., 2014). GPx helps in reducing H₂O₂ and lipid peroxide levels, working alongside CAT to mitigate the harmful effects of OH radicals by limiting free radical production (Gharu, 2022). GSH donates electrons in these reactions, playing the role of a donor, while GSR maintains GSH activity. HO-1 is involved in the breakdown of heme and plays a crucial role in cellular homeostasis (Bai et al., 2017). Reduced antioxidant activities lead to elevated ROS levels in the body, ultimately causing oxidative stress (Huang et al., 2016). The excessive production of radicals results in lipid peroxidation (LP), which disrupts macromolecules such as lipids, DNA, and proteins. MDA, a marker of LP, is associated with several negative outcomes, including increased membrane stiffness, osmotic fragility, and decreased mitochondrial longevity (Aydin et al., 2004). Moreover, excessive production of free radicals decreases the activities of antioxidant enzymes which ultimately impairs the endogenous cellular defence system (Ahmad et al., 2023). However, supplementation with SCD restored the biochemical profile and reduced the levels of oxidative stress indicators due to its antioxidant properties. Additionally, Liu et al. (2021) demonstrated that the three methoxy groups in the structure of SCD are responsible for its antioxidant activity.

PQ intoxication upregulated Bax and Caspase-3 while downregulating the Bcl-2 expression. Apoptotic indicators are the members of Bcl-2 family. Bax induces apoptosis, while Bcl-2 prevents apoptosis (Frenzel et al., 2009). An increase in the expressions of pro-apoptotic markers (Bax and Caspase-3), while reduction in anti-apoptotic protein (Bcl-2) results in an alteration in the selectivity of mitochondrial membrane, which increases cytochrome C liberation into cytosol (Gu et al., 2017). The increase in cytochrome C causes activation of Caspase-3, which triggers apoptosis (Cain et al., 2002). Grippa et al. (2015) reaffirmed that apoptosis can be averted by blocking the activation of Caspase-3, which is a key molecule in the apoptotic pathway. However, SCD treatment lowered the expressions of Bax and Caspase-3 whereas increased the Bcl-2 expression possibly due to its anti-apoptotic property.

6 Conclusion

The outcomes of this study revealed that PQ administration culminated in a reduction in the enzymatic activity of antioxidants, increased the oxidative stress markers and apoptotic markers in the hepatic tissues. Nevertheless, SCD treatment abrogated all these PQ induced adverse impairments in liver on account of its anti-apoptotic, antioxidant and hepatoprotective nature. So, it may be concluded that SCD can be used to treat liver damage in humans and animals. However, in this study, rats are used as animal model, so further clinical trials on humans are required in the future.

CRediT authorship contribution statement

Ansa Javed: Writing – original draft, Methodology, Investigation, Conceptualization. Rabia Azmat: Writing – review & editing, Writing – original draft, Methodology, Investigation, Conceptualization. Moazama Batool: Visualization, Validation, Formal analysis, Data curation. Amjad Islam Aqib: Writing – review & editing, Software, Formal analysis. Shaik Althaf Hussain: Writing – review & editing, Resources, Funding acquisition. Ayesha Ishtiaq: Writing – original draft, Validation, Software.