Neuroprotective effect of naringin by modulation of endogenous biomarkers in streptozotocin induced painful diabetic neuropathy

Fitoterapia 83 (2012) 650–659

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Neuroprotective effect of naringin by modulation of endogenous biomarkers in streptozotocin induced painful diabetic neuropathy Amit D. Kandhare, Kiran S. Raygude, Pinaki Ghosh, Arvindkumar E. Ghule, Subhash L. Bodhankar ⁎ Centre of Advance Research in Pharmaceutical Sciences (CARPS), Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, Maharashtra, 411038, India

a r t i c l e

i n f o

Article history: Received 6 November 2011 Accepted in revised form 25 January 2012 Available online 9 February 2012 Keywords: Apoptosis Diabetic neuropathy Motor nerve conduction velocity Naringin Oxidative stress Tumor necrosis factor-α

a b s t r a c t Diabetes mellitus is a serious debilitating epidemic affecting all social strata in developing as well as developed countries. Diabetic neuropathy is most common of secondary complications associated with diabetes mellitus and is characterized by slowing of nerve conduction velocity, elevated pain, sensory loss and nerve fiber degeneration. The aim of the present investigation was to evaluate the neuroprotective effect of naringin against streptozotocin (STZ) induced diabetic neuropathic pain in laboratory rats. Four weeks after intraperitoneal injection of STZ resulted in significant decrease in mechano-tactile allodynia, mechanical hyperalgesia, thermal hyperalgesia and motor nerve conduction velocity. Activity of endogenous antioxidant like superoxide dismutase as well as membrane bound inorganic phosphate enzyme was also found to be significantly decreased. It not only caused neural cell apoptosis but also enhanced lipid peroxide, nitrite, and inflammatory mediators' (TNF-α) level. Chronic treatment with naringin (40 and 80 mg/kg) for 4 weeks significantly and dose dependently attenuated the decrease in level of nociceptive threshold, endogenous antioxidant and membrane bound inorganic phosphate enzyme. It also decreased the elevated levels of oxidative–nitrosative stress, inflammatory mediators as well as apoptosis in neural cells significantly and dose dependently. The important finding of the study is that, the naringin–insulin combination not only attenuated the diabetic condition but also reversed the neuropathic pain, whereas insulin or naringin alone only improved hyperglycemia but partially reversed the pain response in diabetic rats. Thus, naringin is a potential flavonone bearing antioxidant, antiapoptotic and disease modifying property acting via modulation of endogenous biomarker to inhibit diabetes induced neuropathic pain. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Diabetes has transformed into a metabolic epidemic projected to affect 366 million individuals by 2050. Diabetic retinopathy, neuropathy, nephropathy and cardiomyopathy are the most frequent pathological features of diabetic complications appearing in around 50% diabetics [1,2]. Diabetic neuropathy is precipitated due to an array of factors including elevated hexosamine shunt, aldose reductase activation,

⁎ Corresponding author at: Department of Pharmacology, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Erandwane, Pune411038, Maharashtra, India. Tel.: + 91 2025437237; fax: + 91 2025439383. E-mail address: [email protected] (S.L. Bodhankar). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.01.010

decrease in the nerve myoinositol content, an impaired neurotrophic support, activation of protein kinase C (PKC), activation of poly (ADP‐ribose) polymerase (PARP), impaired insulin/C peptide action, and formation of advanced glycation end products (AEG) which modulate various intertwining biochemical pathways to orchestrate autooxidative glycosylation and polyol pathways leading to structural and functional aberration of peripheral neurons, spinal glial cells and nerve fibers [3,4]. Various cytokines and excitatory neurotransmitters (NT) also contribute to downregulation of pain threshold of the neurons [5]. STZ, an antibiotic is a routinely used agent to induce experimental diabetes in laboratory animals. STZ induced diabetic neuropathy is a reproducible laboratory animal model

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for preclinical evaluation of potential drug for diabetic neuropathy [6,7]. The current treatment regimen of diabetic neuropathy including antioxidant, antidepressants, polyphenols, selective serotonin reuptake inhibitors (SSRIs), anti-arrhythmics, opioids and anticonvulsants has met with limited success in clinical trials [8–11]. The chief lines of therapy for treatment of diabetic neuropathy are alpha-lipoic acid, acetyl-L-carnitine, benfotiamine and methylcobalamin, etc. [12]. However, these therapies provide relief only to a fraction of patients and their side effect profiles limit their use. Isolated bioactive moieties from the class of flavonoids are being recognized as promising free radical scavengers playing pivotal role in amelioration of various diseases [13]. The hydrogen donating substituent (hydroxyl groups) attached to the aromatic ring structures of flavonoids, which enable the flavonoids to undergo a redox reaction enabling them to scavenge free radicals easily [14]. Naringin (4′,5,7-trihydroxy flavonone 7-rhamnoglucoside) is derived from grape fruit and related citrus species [15] which possess metal-chelating, antioxidant and free radical scavenging properties [16]. Naringin has been evaluated for a wide spectrum of activities including anticancer, antiinflammatory and cardioprotective activity [17]. It has been reported to lower glucose level and elevate plasma insulin in STZ induced diabetic rats [18,19]. It also lowered oxidative stress in-vitro [18]. However, the role of naringin in diabetic complications has not been investigated. The aim of the present investigation was to evaluate the neuroprotective effect of naringin against STZ induced diabetic neuropathic pain in laboratory rats by assessing various behavioral, and biochemical parameters and apoptotic changes. 2. Materials and methods 2.1. Animals

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chloride, sodium carbonate, sodium bicarbonate, potassium chloride, calcium chloride, disodium hydrogen orthophosphate, potassium dihydrogen orthophosphate, carbon tetrachloride, chloroform, ether, hydrochloric acid and conc. sulfuric acid were purchased from S.D. Fine Chemicals, Mumbai, India. Sulphanilamides, naphthalamine diamine HCl, and phosphoric acid were obtained from LobaChemi Pvt. Ltd., Mumbai, India. TNF-α ELISA kit was obtained from Thermo Scientific, USA. 2.3. Induction and assessment of diabetes A single dose of 55 mg/kg streptozotocin (STZ) prepared in citrate buffer (pH 4.4, 0.1 M) was injected intraperitoneally to induce diabetes [20]. The age‐matched control rats received an equal volume of citrate buffer and were used along with diabetic animals. Diabetes was confirmed 48 h after streptozotocin injection, the blood samples were collected via retro-orbital plexus technique using heparinized capillary glass tubes and plasma glucose levels were estimated by the enzymatic GOD‐POD (glucose oxidase peroxidase) diagnostic kit method. The rats having plasma glucose levels more than 250 mg/dL were selected and used for the present study. The body weight and plasma glucose levels were measured before and at the end of the experiment. 2.4. Experimental design After a basal recording of nociceptive reaction at week 4 after streptozotocin injection, the control and diabetic rats were randomly selected and divided into seven groups of 8–10 animals each as follows: [A] Non-diabetic animals Group 1 Normal non-diabetic (ND): animals received a single injection of citrate buffer (vehicle) and oral gavage of double distilled water. [B] Diabetic animals

Adult male Wistar rats (150–200 g) were obtained from the National Institute of Biosciences, Pune (India). They were maintained at 24 °C ± 1 °C, with relative humidity of 45–55% and 12:12 h dark/light cycle. The animals had free access to standard pellet chow (Pranav Agro industries Ltd., Sangli, India) and water throughout the experimental protocol. All experiments were carried out between 09:00 and 17:00 h. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Poona College of Pharmacy, Pune (CPCSEA/44/2010) and performed in accordance with the guidelines of Committee for Control and Supervision of Experimentation on Animals (CPCSEA), Government of India on animal experimentation. 2.2. Drugs and chemicals Naringin and streptozotocin were purchased from Sigma Chemical Co. (St Louis, MO, USA). 1,1′,3,3′-Tetraethoxypropane, crystalline beef liver catalase, reduced glutathione, 5,5′-dithiobis (2-nitrobenzoic acid), bovine serum albumin, thiobarbituric acid, Tris buffer, sucrose, trichloroacetic acid, citric acid monohydrate, sodium nitrate, copper sulfate, sodium potassium tartarate, ethylene diamine tetra acetic acid disodium salt, Folin's phenol reagent, sodium hydroxide, sodium carbonate, magnesium

Group 2 Diabetic (STZ) control: animals received oral gavage of double distilled water. Group 3 Diabetic (STZ) + N (20): animals received oral gavage of naringin (20 mg/kg) in double distilled water. Group 4 Diabetic (STZ) + N (40): animals received oral gavage of naringin (40 mg/kg) in double distilled water. Group 5 Diabetic (STZ) + N (80): animals received oral gavage of naringin (80 mg/kg) in double distilled water. Group 6 Diabetic (STZ) + N (80) + I (10): animals received oral gavage of naringin (80 mg/kg) in double distilled water along with subcutaneous injections of insulin (10 IU/kg, s.c.). Group 7 Diabetic (STZ) + I (10): animals received subcutaneous injection of insulin (10 IU/kg, s.c.) only. The naringin was freshly prepared in three different dosages (20, 40 and 80 mg/kg) and administered for 4 weeks starting from week 5 of streptozotocin injection [18]. After 8 weeks, rats were killed under deep anesthesia and sciatic nerves were immediately isolated and tissue homogenate

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was prepared in 0.1 M Tris–HCl buffer (pH 7.4) for the biochemical and molecular estimations. 2.5. Behavioral tests 2.5.1. Mechanical hyperalgesia (Randall–Selitto paw pressure test) Mechanical nociceptive threshold, an index of mechano hyperalgesia, was assessed by previously described method [21]. The nociceptive flexion reflex was quantified using the Randall–Selitto paw pressure device (UGO Basile, SRL Biological Research Apparatus, Italy). Withdrawal of hind paw was used to assess the nociceptive threshold. 2.5.2. Mechano-tactile allodynia (Von frey hair test) Mechano-tactile allodynia (non-noxious mechanical stimuli) was assessed by previously described method [22]. Von-frey hairs (IITC, Woodland Hills, USA) with calibrated bending forces (in g) of different intensities were used to deliver punctuate mechanical stimuli of varying intensities. The criterion for the threshold value, in grams, was equal to the filament evoking a withdrawal of the paw 5 times out of 10 trials, i.e., 50% response. 2.5.3. Thermal hyperalgesia (tail immersion test) Spinal thermal sensitivity was assessed by the tail immersion test according to previously described method [23]. The duration of the tail withdrawal reflex was recorded. 2.5.4. Motor nerve conduction velocity (MNCV) The recording of MNCV was performed in rats according to previously described method [24]. Briefly, rats were anesthetized using thiopental sodium (50 mg/kg, i.p.) for electrophysiological recording. The dorsal side of rats' paw was shaved with hair removal cream and cleaned using moist cotton plug. MNCV was recorded by stimulating the sciatic and tibial nerves at sciatic and tibial notch respectively by 200 μs square wave pulse delivered through a pair of monopolar needle electrodes (1.0–1.5 mA, 2.0 mV/D) using a stimulator (Weltronics, India). Responses were recorded from the plantar muscles using data acquisition system (AD Instrument Pvt. Ltd., LabChart 7.3, Australia). The MNCV was determined using the following formula:

MNCV ¼ V½distance between sciatic and tibial stimulation point ðin mÞ =½latency for sciatic ðin sÞ–latency for tibial ðin sÞg:

2.6.2. Estimation of TNF-α The quantifications of TNF-α were performed with the help and instructions provided by Thermo Scientific, USA Rat TNF-α immunoassay kit. It contains rat TNF-α immunoassay which is a 4.5 h solid phase ELISA designed to measure rat TNF-α levels. The assay employs the sandwich enzyme immunoassay technique. A monoclonal antibody specific for rat TNF-α had been pre-coated in the microplate. Briefly 50 μL of pretreated buffer was added to each well. Then, 50 μL of standards, control and test samples (aliquot of sciatic nerve homogenate) were added into each well and incubated at R.T. for 1 h. If any rat TNF-α is present it would have bound by the immobilized antibody. After having washed away any unbound substance, 50 μL of biotinylated antibody reagent was added to each well and incubated at R.T. for 1 h. After washing away any unbound substance, 100 μL of streptavidin-HPR reagent was added to each well which is an enzyme-linked polyclonal antibody specific for rat TNF-α. Then it was followed by washing to remove any unbound antibody–enzyme reagent. The 100 μL of TMB a substrate solution and consequently an enzyme reaction was added which made the blue product turn yellow. The intensity of the color was measured at 550 nm, in proportion to the amount of rat TNF-α bound in the initial steps. The sample values were then read off using the standard curve. Values were expressed as means ± S.E.M.

2.6.3. Preparation of single Schwann cell (SC) suspensions Preparation of single Schwann cell (SC) suspensions and determination of apoptotic cell populations were determined as previously described in [29]. At the end of treatment the sciatic nerves of rats were collected and mixed with 0.4% collagenase and 0.25% Trypsin at 37 °C for 30 min and dissociated, grinded and obtained homogenate was passed through a 70 μm nylon mesh. Single Schwann cell (SC) suspension was washed three times with phosphate-buffered saline (PBS).

2.6.4. Flow cytometry analysis In order to determine Schwann cell (SC) apoptosis, the isolated SCs were incubated with rabbit anti cow S-100 antibody and followed by staining with APC-goat anti rabbit IgG (both from BD) with FITC-Annexin V and PI (Sigma). The percentages of expression of Fas and Annexin-Von gated S-100 positive SC were analyzed by a FACSCalibur cytometer using CellQuest software (Becton & Dickinson, San Diego, USA).

2.7. Statistical analysis 2.6. Biochemical estimations 2.6.1. Sciatic nerve homogenate preparation All animals were sacrificed at the end of study i.e. 8th week and sciatic nerves were immediately isolated. Tissue homogenates were prepared with 0.1 M Tris–HCl buffer (pH 7.4) and supernatant of homogenates was employed to estimate superoxide dismutase (SOD) [25], lipid peroxidation (MDA content) [26], nitric oxide (NO content) [27], membrane bound inorganic phosphate (Na +K +ATPase) [28] and TNF-α.

Data was expressed as mean ± standard error mean (SEM). Data analysis was performed using Graph Pad Prism 5.0 software (Graph Pad, San Diego, USA). Data of behavioral tests were statistically analyzed using two-way repeated analysis of variance (ANOVA) and Bonferroni's multiple range test was applied for post hoc analysis, while data of biochemical parameters were analyzed using one-way analysis of variance (ANOVA) and Tukey's multiple range test was applied for post hoc analysis. A value of P b 0.05 was considered to be statistically significant.

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3. Result 3.1. Effect of naringin on body weight and plasma glucose level Before the induction of diabetic neuropathy by intraperitoneal injection of STZ there was no significant difference in the body weight and plasma glucose level of normal nondiabetic rats (183.61 ± 2.17 g and 161.22 ± 2.01 mg/dL respectively) and diabetic (STZ) control rats (185.12 ± 3.04 g and 159.47 ± 4.01 mg/dL respectively). Four weeks after intraperitoneal injection of STZ resulted in significant decrease of body weight (147.70 ± 2.89 g; P b 0.05) and increase of plasma glucose level (460.10 ± 5.63 mg/dL; P b 0.05) of diabetic (STZ) control rats as compared to normal non-diabetic rats. Chronic treatment with naringin (20, 40 and 80 mg/kg) for 4 weeks significantly and dose dependently ameliorated these decreased body weights (158.00 ± 5.00, 176.20 ± 4.73 and 221.50 ± 2.99 g respectively; P b 0.05) as compared to diabetic control rats. Also chronic treatment with naringin (40 and 80 mg/kg) for 4 weeks significantly and dose dependently ameliorated (345.70 ± 5.76 and 264.40 ± 5.71 mg/dL respectively, P b 0.05) these increased plasma glucose level as compared to diabetic control rats. Moreover, diabetic rats treated with insulin–naringin combination significantly attenuated this alteration in body weight (253.70 ± 2.60, P b 0.05) and plasma glucose level (206.80 ± 9.25, P b 0.05) as compared to insulin or naringin alone treated groups (Table 1).

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However, treatment with insulin–naringin combination significantly decreased food intake, water intake and urine output (28.65 ± 3.44 g, 52.98 ± 3.87 mL and 14.73 ± 1.50 mL respectively, P b 0.05) as compared to insulin or naringin alone treated groups (Table 1). 3.3. Effect of naringin on diabetes-induced mechano-tactile allodynia The mean paw withdrawal threshold in diabetic control rats (75.20 ± 2.31 g) before the induction of diabetic neuropathy was not significantly different than that of normal nondiabetic rats (75.75 ± 2.39 g). After 4 weeks of STZ injection, in response to Von-frey hair stimulation, a significant decrease (P b 0.05) in mean paw withdrawal threshold (i.e. mechanical allodynia) was produced in diabetic control rats (16.32 ± 1.73 g) as compared to normal non-diabetic rats (72.38 ± 3.28 g). Chronic treatment with naringin (20, 40 and 80 mg/kg) for 4 weeks significantly and dose dependently ameliorated (P b 0.05) this decrease in mean paw withdrawal threshold (29.91 ± 1.66, 47.14 ± 2.32 and 54.02 ± 2.10 g respectively) as compared to diabetic control rats. Moreover, diabetic rats treated with insulin–naringin combination significantly attenuated this decreased paw withdrawal threshold (58.57 ± 3.32 g, P b 0.05) as compared to naringin alone treated groups (Fig. 1A). 3.4. Effect of naringin on diabetes-induced mechanical hyperalgesia

3.2. Effect of naringin on food intake, water intake and urine output There was no significant difference in intake of food and water along with urine output of diabetic control rats (22.84 ± 1.74 g, 23.78 ± 2.15 mL and 23.78 ± 2.15 mL respectively) and normal non-diabetic rats (24.20 ± 3.04 g, 23.78 ± 2.15 mL and 23.78 ± 2.15 mL respectively) before induction of diabetic neuropathy. Four weeks after STZ injection resulted in significant increase of food intake, water intake and urine output (78.23 ± 3.89 g, 135.90 ± 5.11 mL and 49.85 ± 2.58 mL respectively, P b 0.05) of diabetic (STZ) control rats as compared to normal non-diabetic rats. Chronic treatment with naringin (40 and 80 mg/kg) for 4 weeks significantly and dose dependently decreased food intake (56.22 ± 2.64 and 39.57 ± 4.28 g respectively), water intake (101.10 ± 4.33 mL and 70.63 ± 5.02 mL respectively) and urine output (28.45 ± 2.40 mL and 21.05 ± 1.71 mL respectively) (P b 0.05) as compared to diabetic control rats.

On day 0 the mean paw withdrawal threshold in diabetic (STZ) control rats (280.00 ± 12.04 g) did not differ significantly than that in normal rats (272.50 ± 8.13 g). There was no significant change in the mean paw-withdrawal threshold of normal rats during the time period of 8 weeks. A significant decrease in (P b 0.05) mean paw withdrawal threshold was produced in the diabetic (STZ) control rats (50.00 ± 7.41 g) after 4 weeks of STZ injection as compared to normal non-diabetic rats (270.00 ± 13.96 g). In rats receiving treatment of naringin (40 and 80 mg/kg) mean paw withdrawal threshold was significantly and dose dependently increased (132.50 ± 11.88 and 185.00 ± 13.22 g respectively, P b 0.05) compared to diabetic control rats (50.00 ± 9.22 g). Moreover, attenuation of decreased mean paw withdrawal threshold by insulin–naringin combination (217.50 ± 11.45 g) was more significant (P b 0.05) compared to insulin (200.00± 17.60 g) or naringin alone treated groups (Fig. 1B).

Table 1 Effect of naringin and insulin treatment on body weight, plasma glucose, food intake, water intake and urine output of diabetic rats. Treatment

Body weight (g)

Plasma glucose (mg/dL)

Food intake (g)

Water intake (mL)

Urine output (mL)

Normal (ND) STZ control STZ + N (20) STZ + N (40) STZ + N (80) STZ + N (80) + I (10) STZ + I (10)

275.70 ± 3.18 147.70 ± 2.89# 158.00 ± 5.00⁎,$ 176.20 ± 4.73⁎,$ 221.50 ± 2.99⁎,$ 250.50 ± 2.68⁎,$ 253.70 ± 2.60⁎,$

169.10 ± 6.88 460.10 ± 5.63# 415.90 ± 5.16 345.70 ± 5.76⁎,$ 264.40 ± 5.71⁎,$ 197.30 ± 6.02⁎,$ 206.80 ± 9.25⁎,$

23.78 ± 2.15 78.23 ± 3.89# 72.60 ± 4.88 56.22 ± 2.64⁎,$ 39.57 ± 4.28⁎,$ 28.65 ± 3.44⁎,$ 27.63 ± 2.83⁎,$

43.83 ± 4.52 135.90 ± 5.11# 124.30 ± 4.98 101.10 ± 4.33⁎,$ 70.63 ± 5.02⁎,$ 52.98 ± 3.87⁎,$ 59.93 ± 3.70⁎,$

7.20 ± 1.01 49.85 ± 2.58# 45.53 ± 2.92 28.45 ± 2.40⁎,$ 21.05 ± 1.71⁎,$ 14.73 ± 1.50⁎,$ 16.48 ± 1.93⁎,$

Data are expressed as mean ± S.E.M. (n = 6) and analyzed by one way ANOVA followed by Tukey's multiple range test. ⁎P b 0.05 as compared to the diabetic control group, #P b 0.05 as compared to normal group and $P b 0.05 as compared to one another. ND: non-diabetic rats; STZ: diabetic (STZ) control rats; N (20): naringin (20 mg/kg, p.o.) treated rats; N (40): naringin (40 mg/kg, p.o.) treated rats; N (80): naringin (80 mg/kg, p.o.) treated rats; I (10): insulin (10 mg/kg, s.c.) treated rats.

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Fig. 1. Effect of chronic treatment of naringin and insulin on mechanical allodynia in Von-frey hair test (A), mechanical hyperalgesia in paw pressure test (B), thermal hyperalgesia in tail immersion test (C), and nerve conduction velocity (D).Data are expressed as mean± S.E.M. (n= 6) and analyzed by two way ANOVA followed by Bonferroni's test. *P b 0.05 as compared to the diabetic control group, #P b 0.05 as compared to the normal group and $P b 0.05 as compared to one another. ND: nondiabetic rats; STZ: diabetic (STZ) control rats; N (20): naringin (20 mg/kg, p.o.) treated rats; N (40): naringin (40 mg/kg, p.o.) treated rats; N (80): naringin (80 mg/kg, p.o.) treated rats; I (10): insulin (10 mg/kg, s.c.) treated rats.

3.5. Effect of naringin on diabetes-induced thermal hyperalgesia Tail withdrawal latency in diabetic (STZ) control rats (12.73 ± 0.78 s) before the induction of diabetic neuropathy was not significantly different than that in normal nondiabetic rats (13.00 ± 0.45 s). Significant decrease (P b 0.05) in mean tail withdrawal latency was produced in the diabetic control rats (2.50± 0.35 s) after 4 weeks of STZ injection as compared to normal non-diabetic rats (13.13 ± 0.55 s). Rat treated with naringin (40 and 80 mg/kg) for 4 weeks significantly (P b 0.05) and dose dependently attenuated this decreased mean tail withdrawal latency (6.08 ± 0.26 and 8.28 ± 0.46 s) as compared to diabetic control rats. However, insulin–naringin combination was more significant (9.90 ± 0.56 s, P b 0.05) as compared to insulin (9.45 ± 0.43 s) or naringin alone treated groups in attenuating reduction of mean paw withdrawal threshold (Fig. 1C). 3.6. Effect of naringin on diabetes-induced alterations in motor nerve conduction velocity Motor nerve conduction velocity in diabetic control rats was 54.96 ± 2.47 m/s on day 0 while it was 53.39 ± 3.36 m/s in normal rats. There was no significant change in the motor nerve conduction velocity of normal rats over the time period of 8 weeks. A significant decrease (P b 0.05) in motor nerve conduction velocity (17.62 ± 2.54 m/s) was recorded in the diabetic control rats after 4 weeks of

intraperitoneal injection of STZ as compared to normal rats. Chronic treatment with naringin (40 and 80 mg/kg) for 4 weeks significantly and dose dependently attenuated (P b 0.05) these decreased motor nerve conduction velocities (27.62 ± 2.00 and 35.05 ± 1.43 m/s respectively) as compared to diabetic control rats. However, treatment with insulin–naringin combination significantly increased motor nerve conduction velocity (40.57 ± 2.31 m/s, P b 0.05) as compared to insulin (38.55 ± 1.57 m/s) or naringin alone treated groups (Fig. 1D). 3.7. Effect of naringin on diabetes-induced alterations in the superoxide dismutase profile Intraperitoneal administration of STZ resulted in significant decrease (P b 0.05) in superoxide dismutase (SOD) level in sciatic nerve of diabetic (STZ) control rats (5.78 ± 0.89 U/mg of protein) compared to normal non-diabetic rats (29.46 ± 0.83 U/mg of protein). Superoxide dismutase (SOD) level in naringin treated rats (40 and 80 mg/kg) was increased (17.89 ± 0.94 and 22.85 ± 0.89 U/mg of protein, P b 0.05) significantly and dose dependently as compared to diabetic (STZ) control rats. However, rats treated with insulin–naringin combination significantly inhibited this decreased level of superoxide dismutase (27.41 ± 0.91 U/mg of protein, P b 0.05) as compared to insulin (26.38 ± 0.97 U/mg of protein) or naringin alone treated groups (Table 2).

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3.8. Effect of naringin on diabetes-induced alterations in lipid peroxide profile

P b 0.05) as compared to insulin (82.24 ± 6.18 pg/mL) or naringin alone treated groups (Table 2).

After 8 weeks of STZ injection, neural lipid peroxide (LPO) level in diabetic control rats was significantly increased (10.29 ± 0.49 nM/mg of protein, P b 0.05) as compared to normal non-diabetic rats (2.22 ± 0.34 nM/mg of protein). The lipid peroxide level in naringin treated rats (40 and 80 mg/kg) was decreased (6.85 ± 0.48 and 5.01 ± 0.42 nM/ mg of protein, P b 0.05) significantly and dose dependently compared to diabetic control rats. Moreover, attenuation of increased lipid peroxide level by insulin–naringin combination was more significant (2.82 ± 0.39 nM/mg of protein, P b 0.05) as compared to insulin (3.82 ± 0.43 nM/mg of protein) or naringin alone treated groups (Table 2).

3.11. Effect of naringin on diabetes-induced alterations in the membrane bound inorganic phosphate

3.9. Effect of naringin on diabetes-induced alterations in the nitrosative stress Neural nitrite level in sciatic nerve of diabetic control rats was significantly increased (310.30 ± 7.25 μg/mL, P b 0.05) after 8 weeks of intraperitoneal STZ injection as compared to normal non-diabetic rats (110.50 ± 9.20 μg/mL). The neural nitrite level in naringin treated rats (40 and 80 mg/kg) was significantly and dose dependently decreased (232.90 ± 12.61 and 201.20 ± 12.71 μg/mL, P b 0.05) as compared to diabetic control rats. Moreover, diabetic rats treated with insulin–naringin combination significantly attenuated these elevated neural nitrite level (132.70 ± 10.88 μg/mL, P b 0.05) as compared to insulin (165.00 ± 10.22 μg/mL) or naringin alone treated groups (Table 2). 3.10. Effect of naringin on diabetes-induced alterations in the TNF-α Rats treated with STZ resulted in significant increase (P b 0.05) in TNF-α level in sciatic nerve of the diabetic control rats (163.00 ± 6.52 pg/mL) as compared to normal nondiabetic rats (57.47 ± 4.49 pg/mL). Rats treated with naringin (40 and 80 mg/kg) significantly and dose dependently attenuated this increase (124.50 ± 7.19 and 93.02 ± 6.12 pg/mL, P b 0.05) in the level of inflammatory cytokine i.e. TNF-α as compared to diabetic control rats. However, diabetic rats treated with insulin–naringin combination significantly attenuated these elevated TNF-α level (70.62 ± 5.66 pg/mL,

After 8 weeks of intraperitoneal STZ injection membrane bound inorganic phosphate i.e. Na–K-ATPase level in diabetic control rats was significantly increased (2.31 ± 0.42 μmol/mg of protein P b 0.05) as compared to normal non-diabetic rats (10.87 ± 0.68 μmol/mg of protein). The Na–K-ATPase level in naringin treated rats (40 and 80 mg/kg) was decreased significantly and dose dependently (6.01 ± 0.47 and 7.94 ± 0.61 μmol/mg of protein, P b 0.05) as compared to diabetic control rats. However, insulin–naringin combination treatment more significantly attenuated this elevated membrane bound inorganic phosphate level (9.74 ± 0.48 μmol/mg of protein, P b 0.05) as compared to insulin (8.84 ± 0.37 μmol/mg of protein) or naringin alone treated groups (Table 2). 3.12. Effect of naringin on diabetes-induced alterations in SC apoptosis Percent apoptosis in sciatic nerve of diabetic (STZ) control rats was significantly increased (7.32 ± 0.24, P b 0.05) after 8 weeks of STZ injection as compared to normal non-diabetic rats (0.81 ± 0.22). Percent apoptosis in naringin treated rats (40 and 80 mg/kg) was significantly and dose dependently decreased (3.85 ± 0.34 and 2.43 ± 0.25, P b 0.05) as compared to diabetic (STZ) control rats. However, rats treated with insulin–naringin combination significantly inhibited this elevated level of apoptosis (1.16 ± 0.22, P b 0.05) as compared to insulin (1.67 ± 0.27) or naringin alone treated groups (Figs. 2 and 3). 4. Discussion Diabetic neuropathy is characterized by clinical features like allodynia, hyperalgesia due to elevated nociceptive response, reduced motor nerve condition velocity, neuronal hypoxia, reduced threshold to painful stimuli, etc. [30]. Similar symptoms are exhibited by STZ induced diabetic animals [31]. STZ injected rats exhibit clinicopathological features including biochemical, oxidative and metabolic changes which also presented in human [32].

Table 2 Effect of naringin and insulin treatment on various endogenous biomarkers of diabetic rats. Treatment

SOD (U/mg of protein)

LPO (nM/mg of protein)

NO (μg/mL)

TNF-α (pg/mL)

Na+K+ATPase (μmol/mg of protein)

Normal (ND) STZ control STZ + N (20) STZ + N (40) STZ + N (80) STZ + N (80) + I (10) STZ + I (10)

29.46 ± 0.83 5.78 ± 0.89# 9.53 ± 0.94 17.89 ± 0.94⁎,$ 22.85 ± 0.89⁎,$ 27.41 ± 0.91⁎,$ 26.38 ± 0.97⁎,$

2.22 ± 0.34 10.29 ± 0.49# 9.62 ± 0.45 6.85 ± 0.48⁎,$ 5.01 ± 0.42⁎,$ 2.82 ± 0.39⁎,$ 3.82 ± 0.43⁎,$

110.50 ± 9.20 310.30 ± 7.25# 282.00 ± 11.77 232.90 ± 12.61⁎,$ 201.20 ± 12.71⁎,$ 132.70 ± 10.88⁎,$ 165.00 ± 10.22⁎,$

57.47 ± 4.49 163.00 ± 6.52# 152.50 ± 5.60 124.50 ± 7.19⁎,$ 93.02 ± 6.12⁎,$ 70.62 ± 5.66⁎,$ 82.24 ± 6.18⁎,$

10.87 ± 0.68 2.31 ± 0.42# 3.04 ± 0.44 6.01 ± 0.47⁎,$ 7.94 ± 0.61⁎,$ 9.74 ± 0.48⁎,$ 8.84 ± 0.37⁎,$

Data are expressed as mean ± S.E.M. (n = 6) and analyzed by one way ANOVA followed by Tukey's multiple range test. ⁎P b 0.05 as compared to diabetic control group, #P b 0.05 as compared to normal group and $P b 0.05 as compared to one another. ND: non-diabetic rats; STZ: diabetic (STZ) control rats; N (20): naringin (20 mg/kg, p.o.) treated rats; N (40): naringin (40 mg/kg, p.o.) treated rats; N (80): naringin (80 mg/kg, p.o.) treated rats; I (10): insulin (10 mg/kg, s.c.) treated rats.

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Fig. 2. Effect of chronic treatment of naringin and insulin on representative image of apoptotic cell populations observed after FACS analysis using Annexin V/FITCPI stain.Four distinct cell distribution patterns are visible: normal viable cells (lower left quadrant), apoptotic cells (lower right quadrant), late apoptotic or necrotic cells (upper right quadrant) and necrotic cells (upper left quadrant). Normal non-diabetic rats (A); diabetic (STZ) control rats (B); insulin (10 mg/kg, s.c.) treated rats (C); naringin (40 mg/kg, p.o.) treated rats (D); naringin (80 mg/kg, p.o.) treated rats (E); naringin (80 mg/kg, p.o.)+insulin (10 mg/kg, s.c.) combination treated rats (F).

In the present investigation, loss in body weight was halted in naringin treated animals when compared with vehicle treated animals exhibiting observation similar to earlier reports [18,19]. Features like polydipsia, and polyphagia were lowered due to drug treatment and hence excess food and water intake was downregulated. These features have been investigated by other researchers and the phenomenon of decrease in the availability of glucose and amino acid to cell has been implicated [33,34]. Cellular biosynthesis and metabolism has been proven to be the underlying cause of diabetes induced polydipsia, polyphagia, polyuria and body weight loss. In diabetes, pain threshold of the neurons is reduced due to oxidative stress generated by free radicals such as super oxide dismutase, hydroxyl radical, and peroxynitrite which impair blood supply to the neurons leading to impaired neuronal function and hypoxia. These conditions were investigated by assessing the biochemical markers like SOD, MDA, and NO. Von frey hair, Randall Selitto and tail flick have been reported methods to record behavioral measures of mechano tactile allodynia, peripheral analgesia and central pain in laboratory animals [35,36]. Intraperitoneal administration of STZ results in central sensitization of neurons due to the damage of the motor as well as sensory fibers resulting in reduction in thermal withdrawal latency. The decreased pain threshold in the control animals and its dose dependant amelioration were seen in the naringin treated group. These findings are in accordance with the previous study [37]. In diabetic rats endoneural hypoxia is caused by chronic hyperglycemic state due to oxidation of proteins and lipid of

neurons leading to reduced pain threshold and motor nerve conduction velocity [5,24]. Conventional drugs provide symptomatic relief to neuropathic pain whereas herbal moieties like naringin

Fig. 3. Effect of chronic treatment of naringin and insulin on percent apoptosis.Data are expressed as mean ± S.E.M. (n = 6) and analyzed by one way ANOVA followed by Tukey's multiple range test. *P b 0.05 as compared to diabetic control group, #P b 0.05 as compared to normal group and $P b 0.05 as compared to one another. ND: non-diabetic rats; STZ: diabetic (STZ) control rats; N (20): naringin (20 mg/kg, p.o.) treated rats; N (40): naringin (40 mg/kg, p.o.) treated rats; N (80): naringin (80 mg/kg, p.o.) treated rats; I (10): insulin (10 mg/kg, s.c.) treated rats.

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have anti-lipoperoxidation and metal ion chelating properties leading to amelioration of neuropathy. SOD and MDA are endogenous enzymes closely intertwined with oxidative stress [38]. Superoxide and hydroxyl free radicals are mainly responsible for vascular endothelial damage [5]. Superoxide anions are also believed to cause increase in aldose reductase and protein kinase C activity which are further implicated in pain perception [39]. SOD provides antioxidant protection from superoxide anions by transforming them to H2O2. MDA is elevated in stress conditions. It is mainly responsible for destruction of lipid membrane via rearrangement of the double bond in the unsaturated fatty acids of the membrane caused tissue damage [40]. The enhanced lipid peroxidation provides an index of oxidative stress and its levels are reduced by drug treatment. Nitric oxide (NO) is an unconventional intracellular messenger playing a vital role in various pathological and physiological processes. NO reacts with reactive oxygen species and acts as an oxidant. But, this oxidation is not specific and affects any cell molecule [41]. A localized increase in NO level leads to formation of peroxynitrite by reacting with superoxide anions which causes rapid protein nitration or nitrosylation, lipid peroxidation, DNA damage and cell death and which intern contribute to elevated pain [42]. The peroxynitrite decomposition catalyst, Fe (III) tetrakis‐2‐(N‐triethylene glycol monomethyl ether) pyridyl porphyrin, counteracts sensory neuropathy in streptozotocin‐diabetic animals [43]. Results of present study are in accordance with previous reports [44]. It has been documented that flavonoids having both a C-4 carbonyl group and a C-3 or C-5 hydroxyl group in their structure form complex with metal ions like iron can retain their antioxidant activity [45]. Naringin also contains hydroxyl groups at C-5 of A ring and C-4′ of B ring and carbonyl group at C-4 of the C ring [18,46]. These structural properties of naringin may contribute to its antioxidant property thus showing inhibition of neurodegeneration. Diabetic neuropathy is characterized by accelerated endogenous TNF-α production in microvascular and neural tissues leading to micro-vascular permeability microangeopathy and nerve damage [47]. Uses of agents that suppress cytokine elevation have been advocated to be used to treat diabetic complication [48]. Hence dose dependant reduction of TNF-α by naringin demonstrates its crucial role in inhibiting the upsurge of cytokine. Our results provide credence to the previous studies carried out by Lee et al. and Tiwari et al. [49,50]. It has been reported that generation and transmission of bioelectricity is mediated via the uniform distribution of Na–KATPase, a membrane bound enzyme [51]. It has been proposed that reduction in the MNCV is associated with the disturbances in membrane bound enzyme activity in the sciatic nerve [52]. Chronic hyperglycemia leads to activation of polyol pathways and reduction in Na–K-ATPase activity leading to inactivation of phosphate [4,53]. Thus, membrane bound Na–K-ATPase enzymes are inhibited and its restoration is desired [54,55]. In present investigation naringin restored the decreased level of NaKATPase exhibiting its neuroprotective effect. Reduced nerve conduction velocity has been attributed to nerve dysfunction due to pathological changes of arterioles perfusing the sciatic nerve in diabetic neuropathy. These results are in accordance with the other reports, wherein similar reduction of motor nerve conduction velocity and nerve blood

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flow in streptozotocin-induced diabetic rats was reported [38,44,56]. MNCV was decreased in vehicle treated animals whereas elevated in a dose dependant manner in naringin treated animals. Apoptosis is a phenomenon characterized by programmed cell death and is a facet of various degenerative maladies including neuropathy [29]. The present study demonstrates that intraperitoneal injection of STZ results in a rapid production and release of proinflammatory cytokine including TNF-α which was responsible for tissue necrosis [57]. The present investigation demonstrates that naringin was able to dose dependently reduce the population of early apoptotic cells revealed by flow cytometric determination. The pharmacotherapy of neuropathic pain has not advanced and there are no approved therapies to improve the long-term prognosis of peripheral nerve injury. Even with intensive insulin therapy, incidence of diabetic neuropathy is as high as 7.0% [58]. Insulin is an anabolic hormone and it acts primarily through cyclic AMP (cAMP) and protein kinase A. Treatment with various agents including gamma-linolenic acid, protein kinase C-β inhibitor (LY333531), RAC-alpha-lipoic acid and methylcobalamin for 6 months has been found to protect against diabetic peripheral neuropathy [9–11]. In the present investigation various behavioral, biochemical and molecular effects of naringin with insulin alone and naringin–insulin combination were compared. Insulin alone restored the elevated levels of blood glucose level but partially reversed the neuropathic pain in STZ induced diabetic rats. However, naringin–insulin combination not only attenuated the diabetic condition but also reversed neuropathic pain through modulation of oxidative–nitrosative stress, inflammatory cytokine release and reduction in apoptosis in the STZ induced diabetic rats. Therefore, naringin may exhibit its neuroprotective effect by down regulation of free radical, cytokine including tumor necrosis factor-α (TNF-α), an important mediator of neuropathic pain, apoptosis and restoration of membrane bound inorganic phosphate activity. Thus, naringin is a potential flavonone bearing antioxidant, antiapoptotic and disease modifying property acting via modulation of endogenous biomarker to inhibit diabetes induced neuropathic pain.

Conflict of interest There is no conflict of interest between any of the authors.

Acknowledgments The authors would like acknowledge Dr. S. S. Kadam, ViceChancellor and Dr. K. R. Mahadik, Principal, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune, India, for providing the necessary facilities to carry out the study. The authors thank Dr. A. A. Khan, Dr. S.K. Tiwari, Dr. G. Shivaram, and Dr. Avinash Bardia from CLRD, Hyderabad for providing the infrastructure for flow cytometric determination. We are also thankful to the All India Council of Technical and Education (AICTE), India for the financial support by awarding GATE Scholarship to one of the authors (Mr. Kandhare Amit) for the research work.

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