Azadirachta indica attenuates cisplatin-induced neurotoxicity in rats.

OBJECTIVE: The objective of this study is to investigate the neuroprotective effects of Azadirachta indica leaves against cisplatin (CP)-induced neurotoxicity.
MATERIALS AND METHODS: Female Wistar rats were treated with vehicle (control); a single intraperitoneal 5 mg/kg CP (CP group); neem leaves (orally 500 mg/kg) for 5 and 10 days, N5 and N10 groups, respectively; neem leaves (500 mg/kg) for 5 days after CP injection, collagenous protein nitrogen (CPN) group; neem leaves (500 mg/kg) for 5 days before CP injection, noncollagenous protein group and neem leaves in a dose of 500 mg/kg for 5 days before and after CP injection, noncollagenous protein nitrogen group. Rats were sacrificed 5 days after CP injection to determine neural lipid peroxidation (LPO), nitric oxide (NO), and glutathione (GSH) levels. The neuronal antioxidant enzymes were evaluated in brain homogenates.
RESULTS: CP injection increased LPO, NO levels and decreased GSH level, whereas neem reversed these effects. Morphological brain damage and apoptosis induction were apparent in the CP group. In the CPN group, the histological damage and apoptosis induction caused by CP was improved, whereas morphological findings of neem before and after CP injection implied a well preserved brain tissue. No changes, in biochemical parameters were observed with neem treated groups.
CONCLUSION: This study suggests that methanolic extract of neem leaves may be of therapeutic benefit when used with CP.

Introduction

Plants with diverse medicinal properties have come under extensive study in the light of their antioxidant, antimutagenic, and anticarcinogenic effects.[1] Azadirachta indica (A. indica; neem) used as food and folklore medicine offers promise as an antioxidative agent due to its beneficial effects on health. Extracts of neem leaf have been found to possess immunomodulatory, antiinflammatory and anticarcinogenic properties.[2] Studies have demonstrated antigenotoxic and chemopreventive potential of ethanol extract of neem leaves against oral and fore stomach tumors are mediated by up-regulation of antioxidant defenses and induction of differentiation and apoptosis.[3] Although a number of therapeutically useful compounds have been identified from neem leaf, most of the pharmacological properties have been reported only with crude extracts.[34]

Cisplatin [cis-diamminedichloroplatinum (II)] (CP) is one of the most effective chemotherapeutics used in the treatment of testicular, ovarian, bladder, and neck. Endometrial cancer, nonsmall lung cancer, malignant melanoma, penile cancer and adrenocorticol carcinoma.[5] However, its therapeutic use is limited by its severe toxicity such as nephrotoxicity, ototoxicity, and neurotoxicity and liver toxicity.[6]

Recently, Ezz-Din et al.[7] and Dkhil et al.[8] have reported that methanolic extract of neem leaves (MENL) attenuated CP-induced hepatotoxicity and nephrotoxicity. However, the efficacy of neem extract to protect against CP-induced neurotoxicity has not yet been evaluated. Therefore, the purpose of this study was to examine the protective effects of MENL on CP-induced neurotoxicity.

Materials and Methods

Chemicals and Experimental Animals

Cisplatin was purchased from Sigma (St. Louis, MO, USA). All other chemicals and reagents used in this study were of analytical grade. Double-distilled water was used as the solvent.

Adult female Wistar albino rats weighing 180-200 g were obtained from the holding company for biological products and vaccines (VACSERA, Cairo, Egypt). After an acclimatization period of 1 week, the animals were divided into seven groups of six rats each and housed in wire bottomed cages in a room under standard condition of illumination with a 12 h light-dark cycle at 25 ± 1°C. Rats were provided with water and balanced diet ad libitum. All animals received care in compliance with the Egyptian rules for animal protection. The protocol approved by the Research Ethics Committee “Approval Number: 132/03/04/2012”.

Preparation of the Neem Leaf Extract

Fresh matured leaves of neem were collected in August from the garden in Obour City, Cairo, Egypt. The plant material was authenticated in the Botany Department, Faculty of Science, Helwan University, Cairo-Egypt on the basis of taxonomic characters. Neem leaf extract was prepared according to the method described by Manikandan et al.[9] with some modifications. Air-dried powder (100 g) of neem leaves was extracted by percolation with 70% methanol and kept at 4°C for 24 h. The obtained extract was concentrated and dried under reduced pressure (100 mmHg, 50°C). The residue was dissolved in distilled water, filtered, and used in the experiment.

Experimental Protocol

Forty-two adult female Wistar albino rats were randomly divided into seven groups of six rats each. Group I served as untreated control (Con group). Group II (CP group) received single intraperitoneal (i.p.) injection of CP (5 mg/kg body weight [bwt]) and left for 5 days. Groups III and IV (N5 and N10 groups) were treated with MENL (500 mg/kg bwt orally) for 5 and 10 consecutive days, respectively. Groups V and VI (collagenous protein nitrogen and noncollagenous protein groups) received MENL for 5 days pretreatment and posttreatment with CP (5 mg/kg bwt i.p.), respectively. Group VII (noncollagenous protein nitrogen group) received MENL (500 mg/kg bwt orally) for 5 days pre- and postCP treatment.

The dosage of MENL (500 mg/kg bwt) was based on the previous work of Ezz-Din et al.[7] The i.p. injection of CP (5 mg/kg bwt) was chosen according to Uchino et al.[10] and Kamisli et al.[11] where CP causes neurotoxicity in rats at doses ranged from 2 mg/kg to 7 mg/kg, respectively.

After 24 h of last drug administration, rats were euthanized under mild ether anesthesia. Brain was promptly excised, washed in chilled saline, blotted and processed for biochemical and histological studies.

Oxidative Stress Measurement

Lipid peroxidation (LPO) in the brain was determined using 1 ml of trichloroacetic acid 10% and 1 ml of thiobarbituric acid 0.67% and were then heated in a boiling water bath for 30 min. Thiobarbituric acid reactive substances were determined by the absorbance at 535 nm and expressed as malondialdehyde formed. Nitric oxide (NO) was determined by optimized acid reduction method at 540 nm.

In addition, the neuronal glutathione (GSH) was determined by the reduction of Elman's reagent (5,5′-dithiobis[2-nitrobenzoic acid] “DTNB”) and measured at 405 nm.

Enzymatic Antioxidant Status

The neuronal antioxidant enzymes as superoxide dismutase (SOD) were determined by nitroblue tetrazolium method.

Brain catalase (CAT) was assayed by adding 50 μl of brain homogenates to 30 mM H2O2 in 50 mM of potassium phosphate buffer (pH 7.8), and the consumption of H2O2 was measured at 340 nm for 120 s at 20 s intervals.

The GSH-S-transferase (GST) activity was determined by measuring the conjugation of 1-chloro-2,4-dinitrobenzene with reduced GSH. In addition, GSH reductase (GRd) assayed indirectly by GRd catalyses the reduction of GSH in the presence of nicotinamide adenine dinucleotide phosphate (NADPH), which is oxidized to NADPH+. The decrease in absorbance at 340 nm is measured. Finally, GSH peroxidase (GPx) activity in the brain homogenates was measured using the method of Paglia and Valentine.[12]

Histopathological Examination

Tissue samples were fixed in 10% neutral formalin for 24 h and paraffin blocks were obtained and routinely processed for light microscopy. Slices of 4-5 μm were obtained from the prepared blocks and stained with H and E. The preparations obtained were visualized using a Nikon microscopy at a magnification of ×400.

Immunohistochemical Analyses of Nuclear Factor Kappa B

For immunohistochemistry, brain sections (4 μm) were deparaffined and then boiled in Declere (Cell Marque, Hot Springs, AR, USA) to unmask antigen sites; the endogenous activity of peroxidase was quenched with 0.03% H2O2 in absolute methanol. Brain sections were incubated overnight at 4°C with a 1:200 dilution of antinuclear factor kappa B (NF-κB) antibodies in phosphate buffered saline (PBS). Following removal of the primary antibodies and repetitive rinsing with PBS, slides were incubated with a 1:500 dilution of biotinylated goat antiIgG secondary antibody. Bound antibodies were detected with avidin biotinylated peroxidase complex ABC-kit Vectastain and diaminobenzidine substrate. After appropriate washing in PBS, slides were counterstained with hematoxylin. All sections were incubated under the same conditions with the same concentration of antibodies and at the same time; hence, the immunostaining was comparable among the different experimental groups.

Detection of Apoptosis by Propidium Iodide Staining

Sections were incubated with 4 mg/ml propidium iodide (PI) (Sigma) and 100 mg/ml RNase (Sigma; DNase-free) in PBS for 60 min at 37°C. The slides were washed in PBS, mounted and examined with a Zeiss fluorescence microscope.

Statistical Analysis

Results were expressed as the mean ± standard error of the mean. Data for multiple variable comparisons were analyzed by one-way analysis of variance. For the comparison of significance between groups, Duncan's test was used as posthoc test according to SPSS statistical package software, version 17 (SPSS Inc., Chicago, Illinois).

Results

Table 1 summarizes the potential role of MENL on LPO in neural tissue homogenates. MENL was able to reduce the increase in LPO-induced by CP injection. The maximum protective effect of MENL was observed following neem administration after or concurrently with CP. There was a significant reduction (P < 0.05) in LPO of the brain homogenates in animals treated with neem extract as compared to CP injected rats.

Table 1

Effects of MENL on CP induced alternation in oxidative stress markers and enzymatic antioxidant molecules in rats brain

Cisplatin significantly (P < 0.05) induced in vivo production of NO in the neural tissues of rats [Table 1]. However, MENL treatment before CP failed to decrease the production of NO in brain tissues when compared with control rats. Moreover, MENL treatment either delivered after or concurrently with CP, significantly (P < 0.05) lowered the production of NO in brain tissue, when compared to the CP group, where the level of NO was returned to the control value.

Treatment with MENL alone for 10 days caused a significant elevation (P < 0.05) in GSH content of the brain homogenate [Table 1], while CP injection induced a significant (P < 0.05) reduction in GSH levels when compared to the control group. Moreover, when rats were treated with MENL before or after their treatment of CP, there were a significant (P < 0.05) reduction in GSH levels in the neural tissues, when compared to the group treated only with CP. Furthermore, the concurrent treatment of MENL with CP reversed the neuronal GSH depletion-induced by CP. Where, the GSH content of the brain was returned toward the control value.

Furthermore, GPx, GRd, and GST activities in the brain were significantly (P < 0.05) decreased in CP-treated rats with respect to nontreated rats [Table 1]. MENL treatment also significantly increased GRd, GST, and GPx activities as compared with CP alone and the activity of neuronal GPx was greatly improved.

The SOD activity in the brain significantly decreased (44% of control) in CP-treated rats [Table 1]. The treatment with MENL induced reversion of SOD activity up to values significantly higher to those of the nontreated control group. Thus, neem treatment exerted a stimulating effect in neuronal SOD activity as compared with CP alone. This effect was in time dependent manner. In addition, CP injection did not cause any effect in CAT activity as compared to the control group.

Histopathological study of brain sections showed that CP caused degeneration in the most examined regions characterized by the presence of necrotic and apoptotic nuclei [Figure 1]. However, MENL pre-, post- and co-administration showed a striking level of protection, with no signs of the adverse effects of the CP injection in the brain sections of the rats [Figure 1].

Figure 1

Control group showing normal histological features of different brain regions with well-formed neurons. Sections of cisplatin (CP) injected rats with damaged hippocampus, striatum, and cerebral cortex regions. Sections of methanolic extract of neem leaves (MENL) posttreated for 5 days showing brain regions with some signs of the damage tissues like CP group. Sections of MENL pretreated for 5 days showing brain regions with less damage tissues than CP group. Sections of brain regions of MENL administered 5 days before and after CP injection showed with better recovery in tissues and well-formed nuclei without vacuolation and irregular features (×400)

Immunohistochemical investigation for NF-κB showed that there was some immunoreactivity in the neurons of the different brain regions in the control group, indicating the normal life cycle of cells [Figure 2]. The number of NF-κB positive immunostaining neurons was increased slightly when MENL administered for 10 days. The immunostaining activity for NF-κB was increased strongly in the CP group. The beneficial effect of neem was shown when rats were treated after or concurrently with MENL during CP injection. In this case, the numbers of NF-κB immunostaining neurons were decreased. These effects of MENL were also shown when neem was administered before CP treatment.

Figure 2

Immunohistochemical localizations of nuclear factor kappa B (NF-κB) in the different brain regions of rats. Control and methanolic extract of neem leaves (MENL) administered for 5 and 10 days sections showing low distribution of NF-κB. Sections from rats treated with CP with high affinity to NF-κB. Sections from rats treated with cisplatin (CP) then MENL showing low distribution of NF-κB. Sections from rats treated with MENL then CP showing medium distribution of NF-κB, and Sections from rats treated with MENL and CP showing low distribution of NF-κB (×400)

Furthermore, PI analysis showed that neem treatment reduces the effects of CP-mediated neuronal cell damage [Figure 3]. The maximum protective effect of MENL against CP-induced apoptosis was shown when neem was administered either after or concurrently with CP treatment, while, the number of the apoptotic neurons still higher when MENL was administered before CP.

Figure 3

Morphological changes visualized under fluorescence microscope with propidium iodide staining in different brain regions of female rats treated with methanolic extract of neem leaves and cisplatin (×400)

Discussion

Cisplatin is a potent antitumor drug against various types of malignant tumors. It is known to produce toxicity and several studies suggest that supplemental antioxidants can reduce CP-induced neurotoxicity.[1314] Compared to the other tissues, brain has a higher probability to be challenged by reactive oxygen species (ROS), because it consumes more than 20% of all the oxygen utilized by the other organs during mitochondrial respiration; moreover specific reactions, such those catalyzed by monoamine oxidases, produce H2O2 and neurotransmitters themselves autoxidize and generate ROS.[15]

Deoxyribonucleic acid (DNA) was the primary target of CP. The formation of CP-DNA adducts structurally distorted the DNA and restrained the DNA replications, which was established as main events responsible for its antitumor property. During the formation of CP-DNA adducts, oxygen free radicals were generated. Overproduction of the oxygen free radicals induced oxidative stress, which was responsible for the CP-related tissue toxicity.[816] These DNA adducts are thought to mediate their cytotoxic effects by interfering with transcription and replication, ultimately leading to the induction of apoptosis. CP adducts distort the DNA duplex, resulting in the exposure of the DNA minor groove, to which several classes of proteins can bind, including high-mobility group proteins and transcription factors that contribute to CP-induced toxicity.[17]

There are many studies, which have demonstrated the involvement of oxidative stress, LPO and mitochondria dysfunction in CP-induced neurotoxicity.[1819] A mechanism by which CP exerts its cytotoxicity is through the generation of ROS. The administration of CP caused an increase in LPO and NO levels and a decrease in the activity of antioxidant defense enzymes, as well as in the concentrations of nonenzymatic components of GSH that prevent, or protect against, LPO in the brain tissues. It is accepted that both correlate to oxidative stress and cause an imbalance between the generation of oxygen derived radicals and the organism's antioxidant potential.[15]

The results of this study show that neuronal SOD, GPx, GRd, and GST activities as well as GSH level significantly decreased in the CP-treated animals compared to the normal group. These observations support the hypothesis that the mechanism of neurotoxicity in CP-treated animals is related to depletion of antioxidant defense system. Treatment with MENL (500 mg/kg bwt, orally) after, before or concurrently with CP-treatment prevents the depletion of brain antioxidants.

The decrease in SOD activity after CP injection might be due to the loss of copper and zinc, which are essential for enzyme activity.[20] CP has been demonstrated to induce the loss of copper and zinc in the brain. The decreased SOD activity is insufficient to scavenge the superoxide anion produced during the normal metabolic process. The superoxide anion can cause initiation and progression of LPO.

The activity of GPx is also found to decrease after CP injection. This resulted in the decreased ability of the brain to scavenge toxic H2O2 and lipid peroxides. Restoration of neuronal SOD and GPx activities by MENL suggests that the extract is capable of protecting the enzymes even 5 days before or after CP injection. GSH depletion can markedly increase the toxicity of CP. The increased GSH levels render protection, which is evident from the extract plus CP-treated group of animals.

Free radicals are known to play an important role in CP-induced neurotoxicity. The free radicals and ROS induce oxidative stress in brain.[15] Due to CP injection, platinumsulphydryl group complexes formed are taken up by neuronal cells and stabilized by intracellular GSH for several hours. In case of intracellular GSH depletion, the complexes undergo rapid transformation to reactive metabolites.[21] Thus, GSH depletion results in increased toxicity of CP. GSH depletion also results in LPO and this seems to be the prime factor that permits LPO and impaired antioxidant enzyme activities. These observations support the conclusion that the mechanism of neurotoxicity in CP-treated rats is related to depletion of antioxidant systems. Thus, neuroprotection by the MENL might be directly related to its antioxidant activity. The tested dose of methanolic extract of A. indica shows no signs of toxicity in rats. Survival rate of animals treated with CP and A. indica extract supports the neuroprotective effect of this tree.

The contribution of NO in the cytotoxicity and organ toxicity of anticancer drugs have been previously reported.[22] Many authors reported that increased NO production after CP injection was a secondary event following an increase in inducible NO synthase.[23] Furthermore, NO reacts spontaneously with the available superoxide radical to form the more potent and versatile oxidant peroxynitrite. This highly toxic species reacts with GSH, lipids, proteins and DNA. Moreover, NO production inactivates GPx activity via modification of a cysteine-like essential residue on GPx.[15]

The activation of NF-κB contributes to drug resistance in different carcinoma and it was demonstrated that pretreatment of carcinoma cells with genistein down-regulates NF-κB activity and contributes toward enhancing the apoptosis-inducing effect of CP, leading to greater antitumor activity in vivo.[24] Thus, high NF-κB activity indicates decreased apoptosis so that inhibition of NF-κB enhances the effect of CP. In the present study, MENL was able to down-regulate NF-κB in different brain regions.

Alcoholic neem leaf extract contains a number of potent antioxidants and anticarcinogens including β-carotene, nimbin, azadirachtin, nimbidiol, quercetin, nimbidin, and nimbatiktam.[3] β-Carotene is known to inhibit LPO by trapping peroxyl radicals. Azadirachtin has inhibitory effects on cell proliferation. Quercetin, a highly ethanol soluble neem bioflavonoid and potent antioxidant, has been reported to decrease N-methyl-N′-nitro-N-nitrosoguanidine-induced DNA damage and induce phase II enzymes.[25]

Neem leaf is reported to decrease the extent of LPO. Neem flowers are known to induce phase II enzymes such as GST in rats. Tepsuwan et al.[26] demonstrated the chemopreventive effects of neem flowers on 7,12-dimethylbenz[a] anthracene-induced mammary carcinogenesis and aflatoxin B-induced liver carcinogenesis in rats. The inhibitory action of neem stem bark on superoxide anion production has been reported.[3]

Dkhil et al.[27] have reported that one of the major protective functions of neem is to decrease the oxidative damage in mice. Indeed, neem prevents the infection-induced loss of GSH and increased production of NO and LPO. In accordance, Balasenthil et al.[28] have found that neem significantly decreased LPO and increased GSH. The antioxidative property of neem has been previously ascribed mainly to its major chemical component, azadirachtin.[9]

Although quantitation and characterization of individual components was not carried out, the results of the present study demonstrate that MENL may mediate its neuroprotective effects by modulation of LPO, apoptosis induction and enhancing antioxidant enzymes. These findings validate the hypothesis that medicinal plants rich in antioxidants are potential neuroprotective agents.