Neuro-protective effect of rutin against Cisplatin-induced neurotoxic rat model

Platinum-based compounds, such as cisplatin, are part of standard treatment for various cancers [1]. Cisplatin is an old drug approved by the Food and Drug Administration in 1978 [2, 3], then it becomes one of the most commonly prescribed anti-cancer drugs. Cisplatin causes cell-cycle arrest leading to apoptosis [4], but the core mechanism is not only its ability to covalently bind to DNA but also to a broad range of essential RNA molecules. Recent near atomic resolution study showed that cisplatin interacts with various RNA sites in the ribosome [5].

Cisplatin-related side effects (ototoxicity, nephrotoxicity, neurotoxicity and cerebral disorders) limits its clinical use at the desired dosage [6, 7]. Several studied have investigated the mechanisms of cisplatin toxicity but the mechanisms for induction of peripheral neuropathies is poorly understood [8‐10]. One study showed the ability of cisplatin to penetrate into the brain where it inhibits neuronal stem cell proliferation [11]. Cisplatin-induced neurotoxicity leads to dose reduction or early termination of chemotherapy that can affect patient life [12, 13]. Cisplatin-induced neurotoxicity via oxidative damage, inflammation, mitochondrial dysfunction, DNA damage, and apoptosis in the nervous system [11, 12]. Cisplatin-induced neurotoxicity through the formation of nucleoli abnormalities in the spinal root ganglion cells of rat embryo [14, 15]. The cisplatin side effects on both human and animal nervous systems can be proven with electrophysiological and histopathological experiments [5, 7, 9]. Chronic cisplatin administration leads to severe damage in spinal ganglia neurons and decreases cell size [16] via interference of platinum with DNA synthesis [4].

Paraoxonase (PONs) is a multigene family composed of three members (PON1, PON2, PON3) coded for enzymes capable of hydrolyzing organophosphate compounds; and plays a role in inflammation and oxidative stress [17]. The enzymes of PONs have anti-atherogenic role through its ability to retard the oxidation of LDL [18]. PON1 is paraoxonase/arylesterase that hydrolyses a broad range of substrates and is a lactonase with lipophilic lactones substrates [19]. PON2 hydrolyses and inactivates N-acyl-homoserine lactones. PON1 gene is expressed in brain [20] and PON2 gene is expressed in lungs, heart, liver and brain, but is not detected in blood [21]. PON3 can hydrolyse bulky drug substrates, such as lovastatin and spironolactone [19]. PON1 and PON3 are synthesized in liver and are attached to high-density lipoproteins (HDL) in blood [22, 23].

Oxidative stress has an important role in toxicity produced by different drugs such as doxorubicin and cisplatin [24, 25]. Cisplatin produces oxidative stress through reduction of plasma antioxidant enzymes levels such as catalase, glutathione peroxidase and superoxide dismutase leading to a failure of the antioxidant defense against free radical damage generated by antitumor drugs [26]. DNA damage and inflammatory cytokines are major players in cisplatin-induced cytotoxicity [27]. The increased reactive oxygen species (ROS) react with DNA to permit the formation of 8-hydroxy guanine causing damage to DNA [28]. The excess generation of ROS increases the damage of biomolecules resulting in lipid peroxidation.

Antioxidants play a vital role in inhibiting the generation of free radicals subsequently preventing the oxidative damage. The antioxidants are naturally present in the body, while others have to be provided as supplements. Several antioxidant agent can reduce the cisplatin-induced cytotoxicity. Parsley oil, with its antioxidant activity, used in the treatment of cisplatin-induced hepatic and cardiac injuries [29]. Other study found that ceftriaxone displayed protective efficacy against cisplatin-induced renal tubule-interstitial fibrosis, possibly via anti-fibrotic potential [30]. Other natural product such as honey bee and royal jelly could be used as dietary preventive natural products against subchronic cisplatin-induced renal injury [31]. Flavonoids are poly-phenolic compounds with anti-inflammatory, antiviral, antibacterial, and neuroprotective properties [32].

Rutin, a flavonoid glycoside, found in vegetables, fruits, tea and herbs [33]. Moreover, rutin possess different protective effects including antioxidant, anti-cancer and anti-inflammatory properties [34]. Interestingly, several studies showed that rutin significantly reduced the cisplatin-induced oxidative stress via decreasing lipid peroxidation and increasing antioxidant activity [35‐37]. Also, rutin has a protective effect against doxorubicin-induced memory deficits and has neuroprotective effects in streptozotocin-induced diabetes in rats [38, 39]. In addition, it has a protective function in ischemic organs including the heart and brain [40]. Rats are used as models of human disease because the rats provide many advantages over other organisms, including the size of their body and substructures in organs. In addition, the ability to measure drug effects at specific anatomical areas [41]. Therefore, this study aimed to investigate the possible protective effect of the rutin via studying some genes of the antioxidant pathway in the brain tissues of cisplatin-induced neurotoxic rat model.

Cisplatin is a wildly used anticancer drug but its toxicities have limited its uses in cancer treatment at effective doses. Cisplatin causes lipid membrane peroxidation by increasing free oxygen radicals and reducing antioxidant production, finally resulting in extensive tissue damage [49]. Several mechanisms are proposed for the cisplatin-neurotoxicity in which oxidative damage is one of the important mechanism in cisplatin and other chemotherapeutic agents’ toxicity. The oxidative stress alters the cell structure and function, and reduces the antioxidant mechanisms resulting in DNA damage in biological systems [50]. Combining the drug with another protective agent is one of the methods used to decrease the severity of the drug-related toxicity. Several studies conclude that the antioxidant agent such as rutin, L-acetayle carnitine, Parsley oil, ceftriaxone, honey bee and royal jelly have antioxidant activity against chemotherapy [29‐31, 51, 52]. The current study investigated the protective effect of rutin on the brain of the rat against cisplatin-induced neurotoxicity via studying the gene expression level of some genes related to the antioxidant pathway.

The neurotoxic effect was determined by measuring the TBAR, GSH and antioxidant genes levels in the brain tissue of the rat. In this study, administration of cisplatin significantly increase the TBAR and decline the GSH levels. Similarly, Turan and coworker found that cisplatin-induced oxidative stress in the brain tissue via significantly increased the TBAR and reduced the GSH levels [53]. The elevated TBARS levels in tissues indicate the increase in the free oxygen radical that results in cells destruction [54]. Glutathione provides the first line of defense against oxidative damage and toxic compounds and has role in several metabolic processes [55]. The decrease in the glutathione levels leads to reduction in the NADPH or GSH utilization in exclusion of peroxides [56].

Previous studies demonstrated that antioxidant agents could prevent cisplatin-induced neurotoxicity [43, 44]. Rutin is a potent bioflavonoid with powerful antioxidant, anti-cancer and anti-inflammatory properties [34]. In the current study, rutin co-administration with cisplatin reversed the changes in TBAR and GSH to their normal levels as in control group. Therefore, rutin may prevent lipid peroxidation on the cell membrane by scavenging the free oxygen radicals.

The oxidative stress can cause cell damage when losing the imbalance between ROS production and antioxidant defense [57]. In the brain, PONs are important in nerves myelination due to their protective function against lipid oxidation. PON-1 and PON3 are expressed in liver, and their protein products are associated with high-density lipoproteins in plasma. PON-1 and PON3 can protect LDL from oxidation by scavenging free radicals [46]. The antioxidant activity of PON1 is via its association with its –SH group that can affect its activity [58]. The inhibition of PON-1 expression and activity plays a role in neurotoxicity and oxidative stress [59]. In the brain, PON1 polymorphisms rs662 and rs854560 is involved in Alzheimer’s disease neuropathology [60]. The decrease in PON1 and PON3 expression levels is associated with toxicity induced by oxidative stress. Similarly, in our previous study, the decrease in PON1 and PON3 expression levels is associated with hepatotoxicity induced by carbon tetrachloride [61]. In the present study, rutin co-administration with cisplatin reverses the alteration in PON1 and PON3 expression levels and increases its antioxidant activity. Rutin reduces neurotoxicity via antioxidant activity. Previous study found that the neuroprotective effect of rutin in the rat brain ischemia was through its ability to reduce TBARS, H₂O₂ and GSH in the hippocampus and frontal cortex in the middle cerebral artery occlusion. In addition to its ability to reduce the expression of p53 and increasing of antioxidant enzymatic activities [40].

PON2 is a member of paraoxonase family [62] and is a ubiquitously expressed intracellular enzyme [63, 64]. PON2 mRNA and protein are detected in the brain [64, 65]. PON2 exerts an antioxidant effect and play a major role in neuroprotection [66, 67]. PON2 is localized primarily in the mitochondria [20, 68] and this support its role in protecting cells from oxidative damage. In the current study, cisplatin significantly increased the PON2 expression levels. Rutin administration decreases the expression levels of PON2 as in control group. Similarly, PON2 high expression is accompanying with resistance to oxidative stress-induced toxicity and may be one of its neuroprotective mechanisms [69]. The previous study showed that rutin has a neuroprotective effect in the brain ischemia in rats [40]. It also ameliorated morphological damage and attenuated ischemic neural apoptosis by reducing the p53 expression and increasing of antioxidant enzymatic activities [40].

The PON2 over-expression by cisplatin might be associated with increased the cells’ ability to scavenge ROS and to antagonize oxidant-induced toxicity. Other study found that the macrophage PON2 expression and activity were increased under oxidative stress and suggested that this increase might be a compensatory mechanism against oxidative stress [18].

Among the PPAR isoforms, PPARδ expression is abundant in the neural cell types and might play a role in the brain physiological functions [70] but its exact roles needs more clarification. The activation of PPARδ induced by a neurotransmitter involved in neurological disorders such as Alzheimer’s disease [71] and reduced the intracellular ROS accumulation. In the present study, cisplatin-induced reduction in the PPARδ expression level and this alteration was reversed by administering rutin. Similarly, previous study showed that PPARδ activation could induce antioxidant systems [72] as well as provide neuroprotection [73, 74].

In conclusion, rutin showed neuroprotective effect on the brain of rat cisplatin- neurotoxic model with a possible mechanism via the antioxidant pathway.