Juglanin cures polyethylene microplastics-induced testicular damage in rats

1 Introduction

Plastics is extensively used in healthcare, agriculture and many other industries due to its excellent physical and chemical properties (Jambeck et al., 2015; Rochman and Hoellein, 2020). It is reported that by the year 2050, there will be 33 billion tons of plastic products on the planet (Phillips and Bonner, 2015). Microplastics (MPs) are ubiquitous environmental pollutants that are produced by the fragmentation of large plastic products into tiny plastic particles (diameter < 5 mm). MPs are recognized as a fourth major environmental problem along with ozone depletion, acidification of ocean and climate change (Jambeck et al., 2015; Galloway and Lewis, 2016)). MPs can infiltrate the human body through many routes. They can penetrate in the body via food chain, drinking water (Kosuth et al., 2018) and by consuming sea salt (Yang et al., 2015). Several reports have illustrated that MPs induce a significant detrimental effects on the growth rate and the survival of different animals. MPs move via bloodstream into various organs to induce damage to human health by stimulating the inflammatory responses (Vethaak and Legler, 2021).

Polystyrene (PS), polyethylene (PE), polypropylene (PP) and polyester are main types of MPs. PEMP is the prevalent type of MPs, abundantly present in terrestrial and aquatic habitats. PEMP accounts for 93 % of all MPs used and it is also present in personal care and cosmetic products to enhance its cleaning properties (Gouin et al., 2015). Recently a study has been reported that illustrates the damaging impacts of PEMP on the testicular tissues. PEMP disturbs the antioxidant enzymatic profile as well as promotes the production of ROS (Ijaz et al., 2022a). PEMP significantly induces morphological damage to sperm head, tail and mid-piece while, it also reduces sperm motility and epididymal sperm count. Moreover, exposure to PEMP also lowers the hormonal level (testosterone, LH and FSH) (Ijaz et al., 2022a).

Flavonoids are plant-based chemicals that are widely used in medicine. They are found in fruits, nuts, vegetables, grains and legumes (Ahmad et al., 2023). Juglanin (Jug) is a flavonoid extracted from raw Polygonum aviculare. Jug shows multiple therapeutical potentials including anti-inflammatory, anti-cancer, anti-apoptotic, neuroprotective and anti-oxidant (Wei et al., 2022). According to the previous literature plant based anti-oxidant named as rhamnetin can be used to treat PS-MPs induced testicular damage in rats (Hamza et al., 2023a). However, literature is yet unavailable that demonstrates the curative effects of Jug against PEMP induced testicular damage. Therefore, keeping the therapeutical potentials of Jug under consideration the present research was performed to evaluate the curative effects of Jug against PEMP induced testicular damage in rats.

2 Material and methods

3 Results

3.1

3.1 Jug and PEMP effects on Nrf-2/Keap-1 pathway

PEMP intoxication induced a significant (P<0.05) decrease in Nrf-2 and anti-oxidants expressions, while increasing Keap-1 expression as matched with the control rats. Nevertheless, the administration of PEMP+Jug escalated Nrf-2 and anti-oxidants genes expression and down-regulated the expression of Keap-1. Additionally, Jug alone administered animals presented these expressions close to control animals (Fig. 1).

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Fig. 1

Effect of PEMP and Jug on the expression of Nrf-2 and Keap-1. Bars are are shown on the basis of Mean ± SEM. Bars with different superscripts are significantly (P<0.05) different from others.

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3.5

3.5 Jug and PEMP effects on apoptotic proteins

The exposure to PEMP increased (P<0.05) the Bax and Caspase-3 expressions, whereas substantially lowered Bcl-2 expression in contrast with the control group. Jug + PEMP co-administration lowered the augmented Bax and Caspase-3 expressions, while increasing the Bcl-2 expression relative to PEMP intoxicated rats. Jug-only treatment showed the same expression of the abovementioned proteins as in the control rats (Fig. 2).

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Fig. 2

Effect of PEMP and Jug on the expression of Bax, Bcl-2 and Caspase-3. Bars are are shown on the basis of Mean ± SEM. Bars with different superscripts are significantly (P<0.05) different from others.

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Fig. 2

Effect of PEMP and Jug on the expression of Bax, Bcl-2 and Caspase-3. Bars are are shown on the basis of Mean ± SEM. Bars with different superscripts are significantly (P<0.05) different from others.

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3.6

3.6 Jug and PE-MP effects on steroidogenic enzymes

PEMP intoxicated considerably (P<0.05) decreased the expression of 3β-HSD, StAR as well as 17β-HSD as compared to the control group. However, Jug + PEMP treatment increased these expression as compared to PEMP administrated group. However, Jug only treatment depicted similar profile of these enzymes as in the control group (Fig. 3).

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Fig. 3

Effect of PEMP and Jug on the expression of 3β-HSD, 17β-HSD and StAR. Bars are are shown on the basis of Mean ± SEM. Bars with different superscripts are significantly (P<0.05) different from others.

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4 Discussion

In the current study, PEMP exposure downregulated the Nrf-2 and anti-oxidants expression (GSR, CAT, SOD, HO-1 and GPx), whereas increased the Keap-1 expressions. The Nrf-2/Keap-1 signaling pathway is an important antioxidant defense system that promotes an adaptive cellular response to oxidative stress while also play a role in toxicant metabolism and detoxification. The transcription factor Nrf-2 has a key function in regulating both electrophilic stress and oxidative stress (OS). Conversely, Keap-1 interacts with Nrf-2 and inhibits it, and thus controlling its stability (Pintard et al., 2004). OS causes an increase in ROS production, which changes the cysteine residues on Keap-1. This causes Keap-1 to undergo a conformational shift, preventing it from efficiently binding to Nrf-2. Subsequently, Keap-1 liberates Nrf-2, rendering it to undergoing degradation. Because of this, Nrf-2 can build up in the cytosol. When Nrf-2 accumulates, it moves into the nucleus of the cell and attaches itself to anti-oxidant response element, a particular DNA sequence. Various anti-oxidant and detoxifying genes are activated when Nrf-2 binds to ARE (Bellezza et al., 2018). According to Hawkes et al. (2014), Nrf-2 is therefore essential for controlling the induction of antioxidant enzymes. This study found that exposure to PEMP suppressed Nrf-2 expression, while escalating Keap-1 expression, which in turn decreased cytoprotective anti-oxidants expressions. Conversely, the co-treatment of Jug significantly regulated anti-oxidant gene expression by regulating the Nrf-2/Keap-1 signaling pathway.

The oral administration of PEMP significantly dysregulated the antioxidant defense system by decreasing enzymatic activities such as GSR, CAT, SOD, GPx as well as HO-1. Whereas, MDA and ROS concentration was escalated. Antioxidant enzymes are considered a fundamental defense line that shields the DNA, protein and lipids (biomolecules of life) from OS by lowering the formation of ROS (Alvi et al., 2022). Hydrogen peroxide, nitric oxide and hydroxyl radical are the most important oxygen and nitrogen reactive species involved in body impairment (Mijatović et al., 2020). The imbalance between antioxidants and ROS concentration leads to the degradation of polyunsaturated fatty acids (PUFAs) present in the membrane of sperm (Fois et al., 2018), and it also initiates a self-perpetuating chemical reaction referred as lipid peroxidation (LP). SOD, a free radical scavenger turns oxygen anions (O^-) into hydrogen peroxide (H₂O₂), whereas CAT and GPx transformed H₂O₂ into hydrogen oxide (H₂O) (Aslani and Ghobadi, 2016; Ighodaro and Akinloye, 2018). HO-1 is responsible for the breakdown of heme and regulates cellular homeostasis (Bai et al., 2017). Apart from the intracellular antioxidant defense line, some plant-based remedies such as flavonoids hold hydroxy phenolic groups responsible for their antioxidant activity (Rodríguez De Luna et al., 2020). Jug co-administration with PEMP significantly attenuated the severe damages induced by OS in testes. Our findings coincided with the examination of Wei et al. (2022), who demonstrated that Jug administration alleviated the augmented MDA content and restored the normal anti-oxidant system (CAT, GPx, SOD, GSR and HO-1) activities in the brain cells. Moreover, the ability of Jug to salvage free radicals may be responsible for its antioxidant capacity. Furthermore, Proch́azkov́a et al. (2011) have demonstrated that flavonoids have multiple hydroxyl groups in their chemical structure that contribute to the antioxidant property.

PEMP administration significantly escalated the levels of inflammatory markers. Any cellular instigation activates the inflammatory mediator i.e., NF-κB, which eventually increases IL-6, IL-1β, TNF-α level and COX-2 activity (Taniguchi and Karin, 2018). Moreover, up-regulated expression of TNF-α initiates inflammatory responses and apoptosis (Fischer and Maier, 2015). Our outcomes elucidated the remedial role of the Jug responsible for the suppression of inflammatory responses by alleviating the level of inflammatory mediators. Zhang and Xu (2018), illustrated that inactivation of the NF-κB signaling might be the underlying reason associated with the anti-inflammatory potential of Jug. Furthermore our finding are further endorsed by the study of Wei et al. (2022) who reported that Jug supplementation alleviate doxorubicin-induced cognitive impairment in rats by regulating inflammatory responses.

According to our findings, PEMP administration reduced the levels of gonadotropin and androgen hormones (LH, FSH and testosterone respectively). Spermatogenesis is a vital phenomenon that is responsible for the generation of spermatozoa. The indication of a healthy male reproductive tract is associated with the normal production of these hormones. Hypothalamic gonadotrophin-releasing hormones control the liberation of LH and FSH (Khanehzad et al., 2021). LH encourages the activation of Leydig cells found in interstitial space. Activation of Leydig cells triggers testosterone production (O'Shaughnessy, 2014). FSH triggers the proliferation of spermatogonia as well as the Sertoli cells that are present inside the seminiferous tubules (Ramaswamy and Weinbauer, 2014). Ijaz et al. (2022a) evinced that PEMP exposure disrupts the hypothalamus-pituitary–gonadal axis (HPGA) that primarily regulates reproductive functions. However, Jug treatment considerably normalized the hormonal levels. Jug supplementation may avert these hazardous changes in hormonal levels by protecting any damage in HPGA.

PEMP treatment augmented the expressions of apoptotic proteins (Bax and Caspase-3), whereas considerably lowered the Bcl-2 expression. Apoptotic processes or events are regarded as indicator of OS (Ishtiaq et al., 2022). Bax and caspase-3 encourage apoptotic events. Whereas, Bcl-2 (antiapoptotic protein) plays an important role to restrain programmed cell death (Opferman and Kothari, 2018). Thus, the imbalance between these apoptotic and anti-apoptotic proteins is linked to OS produced in rats exposed to PEMP. Moreover, the permeability of the mitochondrial membrane is negatively altered due to a decline in Bcl-2 and an augmentation in Bax, leading to an increase in cytochrome c liberation in cytosol. Elevation in cytochrome c within cytoplasm triggers the stimulation of Caspase-3, the protein that promotes apoptosis (Kaur et al., 2020). The findings of our study predicted that Jug co-administration with PEMP reversed all the alternations in the apoptotic and anti-apoptotic proteins expression. Furthermore, our results are further endorsed by previous research reported that Caspase-3 expression was alleviated in rat’s brain cells supplemented with Jug (Wei et al., 2022).

The treatment of PEMP lowered the expressions of 3β-HSD and 17β-HSD. These enzymes play a vital role to maintain the steroidogenic processes and androgenesis. In steroidogenic events, StAR serves as a transport protein that helps in the shifting of cholesterol to the inner side of mitochondrial membrane (Das et al., 2012). While, steroidogenic (17β-HSD and 3β-HSD) enzymes trigger cholesterol conversion into testosterone hormone (Couture et al., 2020). Thus, downregulation in these enzymes expression in PEMP-administrated animals may cease cholesterol channeling leading to a reduction in the process of steroidogenesis. However, Jug + PEMP co-administration improved steroidogenesis by bringing an elevation in the expression of steroidogenic enzymes. It was also directed from a previous study that the molecular structure of flavonoids is identical to the cholesterol as well as other steroids which might have an impact on the generation of androgens (Martin and Touaibia, 2020). Consequently, it might be the main reason to detect increment in the steroidogenic enzymes followed by Jug + PEMP co-administration.

5 Conclusion

PEMP exposure induced damage in the testes by changing the profile of anti-oxidants and inflammatory indices. The level of hormones, expressions of steroidogenic enzymes and apoptotic/anti-apoptotic proteins were significantly disturbed in PEMP exposed rats. The disturbance in these parameters induced deterioration in the functioning of the reproductive system. While, Jug treatment repaired the above mentioned damages due to its anti-oxidant, anti-apoptotic and anti-inflammatory properties. Collectively, Jug exhibits pharmacological potential to normalize the testicular damage induced by PEMP exposure.

CRediT authorship contribution statement

Kaynat Alvi: Writing – original draft, Methodology, Investigation, Conceptualization. Ali Hamza: Writing – review & editing, Validation, Methodology, Investigation. Nazia Ehsan: Writing – original draft, Visualization, Methodology, Formal analysis, Conceptualization. Moazma Batool: Visualization, Software, Formal analysis, Data curation. Muhammad Zaid Salar: Software, Methodology, Formal analysis, Data curation. Zubair Ahmed: Writing – review & editing, Resources, Funding acquisition. Uman Atique: Writing – original draft, Formal analysis, Data curation.