Protective and restorative potency of diosmin natural flavonoid compound against tramadol-induced testicular damage and infertility in male rats

Abstract Flavonoids are polyphenolic natural compounds with various biological actions and limited toxicity including diosmin (DM) which is considered a safe flavonoid natural type with anti-inflammatory and antioxidant activities. Tramadol (TM) is a centrally long-acting analgesic class of opioids extensively being used among the population. It was reported that long-term exposure to TM triggers the releases of oxidative stress, inflammatory factors, and nitric oxides resulting in organs damage. This study aimed to investigate the possible ameliorative and restorative actions of DM against tramadol-induced testicular damage. Rats were divided into: GI: control; GII: Rats received TM, GIII: Rats received DM, GIV: Rats received TM + DM; GV: Rats received DM + TM. Rat's testicular tissue and blood samples were collected. A relevant improvement in all examined parameters was observed among GIV and GV groups. Thereby, it was highlighted that diosmin has beneficial natural actions against tramadol-induced testicular injury via suppressing triggered oxidative stress, and inflammatory factors. Graphical Abstract


Introduction
Flavonoids are polyphenolic secondary metabolites that are abundantly expressed in plants. Diosmin (DM) is a flavone polyphenolic glycoside type found to be present mainly in citrus fruits (Rashid et al. 2019;Ali et al. 2021). It has been characterized by having various biological activities including anti-inflammatory (Tekeli et al., 2021), and antioxidant effects (Feldo et al. 2018). Several studies previously indicated the preventive actions of diosmin against venous insufficiency, liver and kidney, myocardial infarction, and neuropathic pain (Feldo et al. 2018). Diosmin was also found to inhibit severe alloxan-induced diabetic neuropathy via impressing the activation of nuclear factor kappa B (NF-kB) signaling pathways resulting in the suppressed release of oxidative stress (Ahmed et al. 2016).
Tramadol is a centrally acting opioid class with rapid bioavailability and instant analgesic actions (Ahmed and Kurkar 2014). It is characterized by being a l-opioid receptor agonist in addition to inhabiting the reuptake of serotonin and norepinephrine, therefore enhancing pain transmission inhibitory actions in the spinal cord (Ahmed and Kurkar 2014). Tramadol was reported to induce oxidative stress by suppressing body antioxidant intensity resulting in an imbalanced level of reactive oxygen species (ROS) and antioxidants with a major shift towards ROS release leading to severe apoptosis and inflammation (Garc ıa-S anchez et al. 2020). Several studies have shown that tramadol induces severe testicular damage and lesions associated with elevated oxidative stress, nitric oxide (NO), and inflammatory factors (Ahmed and Kurkar 2014).
The ameliorative and prophylactic actions of diosmin against tramadol-induced testicular damage haven't been previously demonstrated before in a sequential descriptive manner. Hence; this study aimed to evaluate diosmin natural flavonoid compound efficiency to alleviate tramadol-mediated testes injury by highlighting its natural biological actions including antioxidant, anti-inflammatory, and antiapoptotic effects.

Results and discussion
Tramadol is the most extensively used opioid drug type for its analgesic actions and rapid onset. Several drawbacks have been reported among long-term users which can be suggested to be due to triggered oxidative stress and inflammatory cytokines storms (Mohamed and Mahmoud 2019). Our results revealed that the daily administration of tramadol resulted in severe testicular damage associated with increased oxidative stress and inflammatory factors. Figure (S1) and Table (S1) showed that the protective administration of (DM þ TM) (GV) effectively improved the body and organ weight in addition to a relevant mitigating effect on Hb, RBCs count, and HCT due to its flavonoids' protective actions. Meanwhile, the administration of (TM þ DM) (GIV) resulted also in marked improvement in organ weight and hematologic parameters. Additionally, data of the present study Table (S1) demonstrated a suppressive action of tramadol on sperm count, viability, and motility when compared to control GI (p < 0.05). On the other hand, the administration of (DM þ TM) (GV) and (TM þ DM) (GIV) revealed that DM attenuated the undue effect of tramadol on sperm effectively. Testicular Leydig cells defined as the main androgen source in males were also observed to be affected by tramadol administration resulting in severe damage in Leydig cells leading to an observed decrease in testosterone level. It was previously reported that elevated NO may affect sperm functionality by forming toxic peroxynitrite nitrite (O'Bryan et al. 2000). This finding may justify our observed results regarding decreased sperm motility and damaged testicular Leydig cells in tramadolinduced rats.
A significant increase in lipid peroxidation, ROS, 8-OHdG, PC, TNF-a, and NO was highly observed following tramadol administration as shown in Figure S2 (a & c) and Table (S2). Meanwhile, TAC, and catalase were found to be suppressed following tramadol administration as shown in Figure S2 (b & d). All these tramadol damageable effects were restored effectively following the administration of (DM þ TM) (GV) and (TM þ DM) (GIV). Additionally, it was observed that tramadol-induced rats suffered from suppressed FSH, LH, and testosterone levels indicating increased hormones degradation levels as shown in Figure S3 (a, b, & c). The chronic long time effects of tramadol on the endocrine system include decreased testosterone level associated with a central reduction in FSH and LH release via affecting the hypothalamic-pituitarygonadal axis (Katz and Mazer 2009). The administration of (DM þ TM) (GV) and (TM þ DM) (GIV) attenuated the undue hormonal effects of tramadol as shown in Figure S3 (a, b, & c). These findings justify our results regarding the increased DNA fragme-ntation and damage in tramadol-induced rats along with the increased ROS and oxidative stress in testes tissue activating instant immune responses via triggering TNF-a, NF-kB pathways which results in apoptotic and inflammatory damage (Anwar 2022) as shown in Figure S3 (d) and Table (S2). On the other hand, groups (DM þ TM) (GV) and (TM þ DM) (GIV) revealed a significant change in DNA damage (tail length) observed in comet test as shown in Table (S2) and Figure S4 (a) with normal histopathological findings observed in group (GIV) as shown in Figure S4 (b).
The main aim of our study was to evaluate the antioxidant and inflammatory actions associated with the protective efficiency of diosmin against tramadol-induced toxicity on testicular tissues. Interestingly, all the above-mentioned tramadol mediated defects were all reversed and improved on the administration of diosmin whether after and/or before tramadol administration acting as a potent natural flavonoid compound. Diosmin treatment effects can be related to the very potential mechanism of flavonoids by acting as an active anti-inflammatory and antioxidant agent. Most importantly, its relevant radical scavenger abilities are due to breaking free radicle chain reaction thereby inhibiting inflammatory cytokines and testicular damage. It can also be related to the ability of polyphenols to chelate metal ions preventing the release of toxic hydroxyl factors and thereby TNF-a (Bozda g and Eraslan 2020). Meanwhile, the protective efficiency of diosmin against tramadol-induced testicular damage is suggested to be related to its bioactive natural constituents leading to relevant protective actions.

Conclusion
The main aim of the current study was to address the drawbacks of prolonged or misuse of tramadol which is considered as one of the most used analgesics among the population. It was observed that on accumulative administration, triggered inflammatory cytokines and oxidative stress were observed resulting in systemic organ damage including testes. The results obtained from this conducted study proved the efficiency of diosmin as a potential natural flavonoid compound against tramadol-mediated testicular Injury. This likely is related to mitigating oxidative stress, inflammatory and apoptotic factors leading to various ameliorative and protective actions.

Supplementary material (S)
Experimental details related to this article are available online.

Disclosure statement
No potential conflict of interest was reported by authors

Funding
The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.