Rhamnetin abrogates polystyrene microplastics prompted hepatic damage by regulating Nrf-2/Keap-1 pathway

1 Introduction

Environmental pollution emerging from plastic is a prevailing issue due to its extensive manufacturing and application in industry and agricultural sector (Hwang et al., 2020). Plastic manufacture has accelerated exponentially reaching to 368 million metric tons in 2019, moreover within 20 years it is expected to be two folds (Al Mamun et al., 2023). Plastics production has surpassed the production of all other synthetic products worldwide. This shows that billions of kilograms of plastics are produced per day, of which only 9 % are recycled, 12 % are incinerated, whereas remaining 79 % are added in the natural environment (Geyer et al., 2017; Almadhi et al., 2023). The remaining plastics (79 %) undergo weathering and breaking down into minute particles that are known as microplastics (MPs) (<5 mm in diameter). Previous literature have described the presence of MPs in facial skin, hair, placenta, saliva, testis and liver. Additionally, MPs pollution contributes to global issues such as ocean acidification, global warming, and ozone depletion (Abbasi and Turner, 2021).

Polystyrene (PS) is one of the most renowned and frequently used type of plastics, which is valued for its persistent physical characteristics and inexpensive price (Cincinelli et al., 2017; Wang et al., 2022). Polystyrene microplastics (PS-MPs) is frequently used to manufacture disposable cups, paper clips, toys cosmetics, pharmaceuticals and trays. PS-MPs exposure to human occur directly through the drinking water, inhalation, skin and ingestion. PS-MPs can enter easily into the organisms owing to low density, smaller size and relatively large surface area; thus, they assemble in organic food chains (Campbell et al., 2017).

Mounting evidences have revealed that PS-MPs intoxication leads to gastrointestinal toxicity, nephrotoxicity, neurotoxicity, testicular toxicity and hepatotoxicity (Ge et al., 2023; Hamza et al., 2023a). PS-MPs treatment provokes ROS generation, which in turn enhances the oxidative and immunological processes. Excessive ROS production leads to DNA damage, impaired protein expression at biochemical as well as cellular level (Yu et al., 2018).

Flavonoids, is a group of polyphenolic compounds that exhibit remarkable pharmacological potentials (Alvi et al., 2022; Surien et al., 2023). Rhamnetin (C₁₆H₁₂O₇) is a potent dietary bioflavonoid that is naturally extracted from diverse plant species i.e., halophytes (Halimione portulacoides, Salicornia europaea and Arthrocnemum macrostachyum) vegetables (Thymus nummularius, Ziziphus mistol) and fruits (clove, apple, sour cherries). RHM exhibits excellent therapeutic properties including neuro-protective, anti-tumor, anti-cancer and anti-bacterial (Mondal et al., 2013). Keeping these attributes under consideration, the present study was planned to determine the protective role of RHM against PS-MPs induced hepatic damage in rats.

2 Material and methods

3 Results

3.1

3.1 Protective role of RHM on the expressions of Nrf-2/Keap-1

PS-MPs treatment induced a significant (P<0.05) reduction in the expressions of Nrf-2 and antioxidant genes, on the other hand elevated Keap-1 expression in PS-MPs administered rats in contrast to the control rats. Nevertheless, treatment of RHM increased the Nrf-2 and anti-oxidant genes expression and down-regulated the expression of Keap-1. Additionally, RHM alone administered rats presented these expressions near to the control rats (Figs. 1, 2 presenting the findings).

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Fig. 1

Impact of RHM and PS-MPs on a) Nrf-2 and b) Keap-1 expressions. The data were interpreted with the help of One-way ANOVA and Tukey's test. Different superscripts on the graphs are presenting significant difference.

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Fig. 2

Impact of RHM and PS-MPs on a) CAT, b) SOD, c) GPx, d) GSR, e) GST and f) HO-1 expressions. The data were interpreted with the help of One-way ANOVA and Tukey's test. Different superscripts on the graphs are presenting significant difference.

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3.4

3.4 Protective role of RHM on the expressions of apoptotic markers

PS-MPs intoxication markedly (P<0.05) increased Bax and Caspase-3 expressions. Although, Bcl-2 expression was lowered in rats treated with PS-MPs in contrast to the control rats. Nevertheless, RHM+PS-MPs supplementation considerably restored these expressions in contrast to the PS-MPs rats. RHM alone treated rats these expression were same as in the control rats (Fig. 3 presenting the findings).

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Fig. 3

Impact of RHM and PS-MPs on a) Bax, b) Bcl-2 and c) Caspase-3 expressions. The data were interpreted with the help of One-way ANOVA and Tukey's test. Different superscripts on the graphs are presenting significant difference.

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4 Discussion

The pollution of plastic is one of the most prevalent issues, due to its massive production and detrimental effects on organisms (Zhang et al., 2020). PS-MPs are ubiquitous environmental contaminants that pose adverse effect on human health such as cancer, infertility, respiratory disease, brain damage, heart disease, gastrointestinal blockage, impairment in placental tissues and blood as well as remarkable dysfunctions in intestine, excretory systems and liver (Zolotova et al., 2022). PS-MPs have been reported in beer, salt and honey. According to the study of Hwang et al. (2019) humans are exposed to PS-MPs via skin, ingestion, drinking water and inhalation. PS-MPs exposure leads to OS and inflammatory responses (Kim et al., 2021). Moreover, PS-MPs administration disturb the apoptotic markers, anti-oxidants activities as well as liver serum marker enzymes in rats. RHM is a flavone that is abundantly reported in halophytes plants (Halimione portulacoides, Arthrocnemum macrostachyum), vegetables (Moringa oleifera), Haplopappus multifolius and fruits i.e. grapes, oranges, mangoes (Ahmad et al., 2023). RHM exhibits various protective activities i.e., anti-tumor, anti-arthritic, anti-tumor and anti-oxidant (Jia et al., 2016). Therefore, the present study was planned to assess the mitigative role of RHM against PS-MPs induced hepatotoxicity.

PS-MPs treatment reduced anti-oxidant genes and Nrf-2 expressions, while increasing the expression of Keap-1. Vomund et al. (2017), stated that Nrf-2 performs crucial function in the regulation of OS. While Keap-1 acts as the inhibitor of Nrf-2 and controls its stability. Keap-1 detaches from Nrf-2 during ROS production via some physical modifications, then travels into the nucleus, where it attaches to small MAF proteins (Pintard et al. 2004). Nrf-2 along with MAF proteins attach to the antioxidant responsive elements and induces anti-oxidants genes expressions. Therefore, Nrf-2 has a critical part in monitoring the stimulation of anti-oxidant genes (Hawkes et al. 2014). Nevertheless, during extreme OS Nrf-2 expression is decreased, whereas increasing the Keap-1. Consequently, reduced Nrf-2 lowers the genes expressions of anti-oxidants, as Nrf-2 has a crucial role in regulating the genes expressions anti-oxidants (Yang et al., 2022). Nevertheless, RHM administration elevated anti-oxidant genes and Nrf-2 expressions, while lowering Keap-1 expression. Therefore, it is assumed that RHM has the ability to regulate Nrf-2 and Keap-1 expressions.

PS-MPs administration reduced the anti-oxidants activities such as GPx, GSH, GST, SOD, GSR, HO-1, CAT enzymes, besides increased ROS and MDA level. These anti-oxidants are the first wall of protection that shield DNA, lipids and proteins from oxidative damage (Ighodaro et al., 2018). CAT is referred as primary enzyme, which has important role in the catabolism of hydrogen peroxide (H₂O₂) (Selamoglu Talas et al., 2008). SOD splits superoxide radicals into oxygen (O₂) and H₂O₂. According to the study of Ulhaq and Tse (2023), GSR maintains reduced GSH levels in cells, via reducing ROS levels. HO-1 is an essential enzyme, which helps in the breakdown of heme and performs a crucial function in homeostasis. MDA is a marker of LP and directly indicates the level of LP and ROS (Samad et al., 2019). The body's anti-oxidant defense mechanism is overwhelmed when excessive level of ROS are created that results in OS. Besides natural anti-oxidants, these anti-oxidants from different plants may be used to treat OS (Nahid et al., 2017). Nevertheless, RHM+PS-MPs supplementation attenuated the PS-MPs damages by escalating anti-oxidants activity and lowering ROS and MDA contents owing to its ROS salvaging property. Moreover, according to previous study flavonoids have the anti-oxidant potential due to the presence of phenolic rings and OH groups in their structural formula (Rodríguez De Luna et al., 2020).

The intoxication of PS-MPs substantially escalated the levels of hepatic function enzymes. Hepatic function enzymes are suitable indicators to evaluate the hepatic function. When the plasma membrane of hepatocytes is disrupted, these enzymes are released from the cytosol and enter into the blood, resulting in hepatic dysfunction. Our outcomes are also evinced by the study of Cheng et al. (2022) who reported that PS-MPs intoxication increased the concentration of liver function enzymes, indicating the adverse condition of liver tissues that consequently leads to liver dysfunction (Kandemir et al., 2020). However, RHM administration dramatically reduced the levels of these enzymes due to hepato-protective and ROS scavenging properties.

PS-MPs-intoxication increased the expressions of Bax and Caspase-3, while decreasing the expression of Bcl-2. Caspase and Bcl-2 family play a major role in apoptosis (Hou et al., 2021). Bcl-2 protects the cells from apoptotic cell death. Contrarily, pro-apoptotic marker (Bax) triggers the cell death (Ehsan et al., 2023). A decrease in in Bcl-2 and escalation in Bax prompts cytochrome-C eviction in cytosol that instigates Caspase-3 (Yen et al., 2012). Caspase-3 break down the proteins and alters their structural makeup, leading to cell death. However, RHM+PS-MPs administration led to substantial reduction in Caspase-3 and Bax expression, besides a notable escalation in Bcl-2 expression was observed. This protective role might be ascribed to the anti-apoptotic property of RHM. Our results are further endorsed by the study conducted by Hamza et al. (2023b), who reported that RHM has to potential to inhibit apoptosis in the testicular tissues of rats.

The intoxication of PS-MPs increased the levels of inflammatory markers including NFκB, TNFα-, IL6, IL1β, and COX-2. The instigation of NF-κB performs a pivotal function in the expressions of other inflammatory indices (TNFα-, IL6, IL1β, and COX-2) that shows severe inflammation. The stimulation of NF-κB boosts inflammatory cytokines (TNFα-, IL6, IL1β, and COX-2) production that results in inflammation. COX-2 is an additional biomarker that also contributes significantly to inflammation (Kim et al., 2019). However, RHM supplementation significantly decreased the levels of inflammatory markers due to its anti-inflammatory nature.

5 Conclusion

Our research revealed that RHM shows potential mitigative role against PS-MPs-instigated hepatic damage. The administration of RHM regulated the anti-oxidants activities, MDA and ROS levels and hepatic function enzymes disturbed by PS-MPs intoxication. Moreover, it also restored apoptotic markers expression as well as inflammatory markers level, owing to its hepato-protective, anti-inflammatory, anti-oxidant and anti-apoptotic activities. Under the light of these findings, it can be deduced that RHM could be used to treat PS-MPs induced liver toxicity.

6 Limitations and future directions

The study was performed by using rats as an animal model and clinical trials are recommend in future to check the relevancy of these findings in human. The testing of rhamnetin with other natural flavonoids or compounds is also recommended in future to check its efficacy. The histopathological examination was not performed in this study.

CRediT authorship contribution statement

Saba Yaqoob: Conceptualization, Investigation, Methodology, Writing – original draft. Ali Hamza: Conceptualization, Investigation, Methodology, Writing – original draft. Moazama Batool: Data curation, Formal analysis, Software, Validation. Aisha Khatoon: Validation, Visualization, Writing – review & editing. Shaik Althaf Hussain: Funding acquisition, Resources, Validation, Writing – review & editing. Mian Nadeem Riaz: Software, Validation, Visualization, Writing – review & editing.