Hesperidin, a flavanoglycone attenuates experimental diabetic neuropathy via modulation of cellular and biochemical marker to improve nerve functions

Abstract

Aim: Diabetic neuropathy (DN) is one of the most common long-term complications of diabetes mellitus and clinically can be characterized by an elevated nociceptive response with electrophysiological conduction abnormalities. The present investigation was designed to evaluate the neuroprotective effect of hesperidin against STZ induced diabetic neuropathic pain in laboratory rats.

Materials and methods: DN was induced in Sprague–Dawley rats (150–200 g) by intraperitoneal administration of streptozotocin (STZ) (55 mg/kg, p.o.). Rats were divided into various groups, namely, STZ control (vehicle), hesperidin (25, 50, and 100 mg/kg, p.o.), insulin (10 IU/kg, s.c.), and combination of hesperidin (100 mg/kg, p.o.) with insulin (10 IU/kg, s.c.) for 4 weeks. Various behavioral (allodynia and hyperalgesia), biochemical parameters [oxido-nitosative stress, Na–K–ATPase, aldose reductase (AR)], and molecular changes (TNF-α and IL-1β) along with hemodynamic changes were determined.

Results: Rats treated with hesperidin (50 and 100 mg/kg, p.o., 4 weeks) significantly reduced (p < 0.05) hyperglycemia and its metabolic abnormalities induced by intraperitoneal administration of STZ. The decreased nociceptive threshold, motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV), serum insulin as well as Na–K–ATPase activity were significantly increase (p < 0.05) by hesperidin (50 and 100 mg/kg, p.o.) treatment. It significantly attenuated (p < 0.05) elevated glycated hemoglobin, AR activity, oxido-nitrosative stress, neural calcium, and pro-inflammatory cytokines (TNF-α and IL-1β) levels. Histological aberration induced after STZ administration was restored by administration of hesperidin (50 and 100 mg/kg, p.o.)

Conclusion: In combination with insulin, hesperidin not only attenuated the diabetic condition but also reversed neuropathic pain via control over hyperglycemia as well as hyperlipidemia to down-regulate generation of free radical, release of pro-inflammatory cytokines as well as elevation in membrane bound enzyme.

• IL-1β
• insulin
• nerve conduction velocity
• oxido-nitrosative stress
• TNF-α

Introduction

Diabetes mellitus is a major global health problem caused due to imbalanced metabolic pathways. According to the World Health Organization (WHO), the prevalence of diabetes is predicted to increase by 366 million people worldwide by the year 2025 (Wild et al., 2004). Despite appropriate treatment, long-standing diabetes mellitus leads to microvascular and neurologic complications such as cardiomyopathy, neuropathy, nephropathy, and retinopathy (Ghosh et al., 2012; Said, 2007). Among various microvascular complications, diabetic neuropathy (DN) is the most common complication affecting almost 50% of diabetic patients who suffer from severe and incessant pain (Kandhare et al., 2012b; Visnagri et al., 2012). Clinically, diabetic neuropathic pain can be recognized by persistent burning or tingling sensation in legs and feet. Besides this, other symptoms include inability to detect heat and cold, loss of vibration sensation and, paradoxically, and the loss of pain perception (Dyck et al., 1988).

Although the pathophysiology of DN is not fully understood, it has been reported that an array of neuroanatomical, neurophysiologic, and neurochemical mechanisms are responsible for the development and maintenance of DN. DN is associated with demyelination of pheripheral neurons, myelinated as well as unmyelinated sensory fibers degeneration results in decreased nerve conduction (Dyck et al., 1988). Increased blood glucose level leads to activation of polyol pathway, increased oxidative stress via generation of reactive oxygen species (superoxide, hydroxyl radical, and hydrogen peroxide), nerve hypoxia/ischemia, formation of advanced glycation end product, hexosamine flux, and activation of protein kinase C (Obrosova et al., 2005; Vincent et al., 2004). Besides this, fatty acid metabolic changes and ischemic-hypoxic factors also played an important role in the development of DN (Friedmann et al., 1966).

Hyperglycemia caused generation of reactive oxygen species via glucose autooxidation and formation of advanced glycation end products leads to diminished activity of endogenous antioxidant enzyme defense system (superoxide dismutase (SOD) and glutathione peroxidase) causing damage to cell protein, lipid, and DNA via abnormal cell signaling and gene regulation (Kandhare et al., 2012e; Vincent et al., 2004). It causes endoneurial hypoxia and impaired nerve conduction velocity with neural function leading to the development of DN (Stevens et al., 2000).

Over the last two decades, in vivo as well as in vitro studies have provided important information about biochemical pathways that play a role in the development of DN. Neuropathy induced by streptozotocin (STZ) is widely used, well-established, and reproducible animal model used to screen potential therapeutic moieties in the field of DN (Kandhare et al., 2011b; Sharma et al., 1977). Intraperitoneal administration STZ exerts its diabetogenic effect via destruction of pancreatic β-cells which leads to down-regulation of intracellular nicotinamide adenine dinucleotide. This depletion caused methylation of islet cells of pancreas via DNA damage leads to hyperglycemia (Bhattacharya, 1955; Matkovics et al., 1997) and the development of DN.

Progressive development of DN is associated with various problems in the daily life of diabetic patients. Therefore, strict control over elevated blood sugar level may provide some relief from onset and progression of DN (Group, 1994). But adjuvant treatment based on pathogenic mechanisms is important along with hyperglycemic control. Current treatment regimens for painful DN include lidocaine patches 5%, duloxetine, gabapentin, and pregabalin (Gidal & Billington, 2006; Gosavi et al., 2011). Besides that, α-lipoic acid, acetyl-l-carnitine, benfotiamine, and methylcobalamin are the chief lines of therapy for treatment of DN (Davidson, 2004; Group, 1994). However, relief to only fraction of patients and debilitating side effect profiles limit their use in clinical settings for the management of DN.

As previously described, reactive oxygen/nitrogen species and inflammatory cytokines have played a vital role in the development of DN. Hence, use of potential antioxidant treatment (α-lipoic acid, β-carotene, vitamin C, and vitamin E) has been carried out clinically as well as preclinically both to combat elevated levels of oxidative stress (Sayyed et al., 2006; Sima, 2006). Various scientific communities, including the National Center for Complementary and Alternative Medicine, United States, have shown interest in the development of herbal medicine for management of DN (Yoon et al., 2004). In the Indian traditional system of medicine, various isolated bioactive moieties are being recognized for disease treatment. Flavonoids are a class of compound that possesses hydrogen donating substituent (hydroxyl groups) present at aromatic ring enabling them to undergo a redox reaction and thus act as an antioxidant (Rice-Evans et al., 1997).

Hesperidin (hesperetin-7-rhamnoglucoside) is abundant and inexpensive major plant flavonoid derived from citrus species including sweet orange and lemon. It has been reported that immature orange species of genus Citrus aurantium L., C. sinensis, C. unshiu (Rutaceae) contain large amounts of hesperidin (Emim et al., 1994; Kawaguchi et al., 1997). Hesperidin has been reported to have a battery of pharmacological properties, including antihyperlipidemic, anti-inflammatory, analgesic, antifungal, anticarcinogenic, antioxidant, antiallergic, antidiabetic, anti-hypertensive, and anti-atherogenic potential (Akiyama et al., 2010; Jin et al., 2008; Kaur et al., 2006; Kawaguchi et al., 1997; Lee et al., 2004; Shah & Patel, 2010; Shi et al., 2012). Further, hesperidin was found to be effective in diabetic nephropathy and cardiomyopathy in type 2 diabetic rats (Shah & Patel, 2010). It has been shown to attenuate various diseases including sodium arsenite induced toxicity (Pires Das Neves et al., 2004), diabetes induced brain damage (Ibrahim, 2008) and breast cancer in rats (Nandakumar et al., 2011). However, very little is known about the possible attenuating effect in STZ-induced DN. Hence, the objective of the present investigation was to evaluate the neuroprotective effect of hesperidin against STZ induced diabetic neuropathic pain in laboratory rats by assessing various behavioral, biochemical parameters, and molecular changes.

Materials and methods

Animals

Adult Sprague Dawley rats (150–200 g) were obtained from the National Institute of Biosciences, Pune, India. They were maintained at 24 ± 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/25/2012) and performed in accordance with the guidelines of Committee for Control and Supervision of Experimentation on Animals (CPCSEA).

Drugs and chemicals

Hesperidin and STZ were purchased from Sigma Chemical Co. (St Louis, MO). 1,1′,3,3′-Tetraethoxypropane, crystalline beef liver catalase, reduced glutathione (GSH), 5,5′-dithiobis (2-nitrobenzoic acid), bovine serum albumin (BSA), thiobarbituric acid, Tris buffer, sucrose, trichloroacetic acid, citric acid monohydrate, sodium nitrate, copper sulfate, sodium potassium tartarate, and ethylene diamine tetra acetic acid disodium salt were purchased from S.D. Fine Chemicals, Mumbai, India. Sulphanilamides, naphthalamine diamine HCl, and phosphoric acid were obtained from LobaChemi Pvt. Ltd., Mumbai, India. GOD‐POD, cholesterol, and triglyceride diagnostic kits were purchased from Accurex Biomedical Pvt. Ltd., Mumbai, India. Rat serum insulin ELISA kit was purchased from Mercodia AB, Sweden.

Induction and assessment of diabetes

A single dose of 55 mg/kg STZ prepared in citrate buffer (pH 4.4, 0.1 M) was injected intraperitoneally to induce diabetes (Bhatt & Veeranjaneyulu, 2010). 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 STZ 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. Food intake and water intake were measured with by placing the animals in metabolic cage (Metabolic cage, Techniplast, Varese, Italy) before and at the end of the experiment.

Experimental design

After a basal recording of nociceptive reaction at week 4 after STZ injection, the control and diabetic rats were randomly selected and divided into seven groups of 8–10 animals each as follows:

1. 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.

1. Diabetic animals

• Group 2: diabetic (STZ) control: animals received double distilled water by oral gavage.

• Group 3: diabetic (STZ) + H (25): animals received hesperidin (25 mg/kg) in double distilled water by oral gavage.

• Group 4: diabetic (STZ) + H (50): animals received hesperidin (50 mg/kg) in double distilled water by oral gavage.

• Group 5: diabetic (STZ) + H (100): animals received hesperidin (100 mg/kg) in double distilled water by oral gavage.

• Group 6: diabetic (STZ) + I (10): animals received subcutaneous injection of insulin (10 IU/kg, s.c.) only.

• Group 7: diabetic (STZ) + H (100) + I (10): animals received hesperidin (100 mg/kg) in double distilled water by oral gavage along with subcutaneous injections of insulin (10 IU/kg, s.c.).

Freshly prepared hesperidin was administered daily in three different dosages (25, 50, and 100 mg/kg) and administered for 4 weeks starting from week 5 of STZ injection (Pires Das Neves et al., 2004).

Behavioral tests

Thermal hyperalgesia (radiant heat test): radiant heat hyperalgesia of the left hind paw was assessed using the radiant heat lamp source according to the method described elsewhere (Hargreaves et al., 1988) for assessing the reactivity to noxious thermal stimuli.

Mechanical hyperalgesia (Randall–Selitto paw pressure test): mechanical nociceptive threshold, an index of mechanohyperalgesia, was assessed according to the method described elsewhere (Randall & Selitto, 1957).

Mechano-tactile allodynia (von Frey hair test): mechano-tactile allodynia (non-noxious mechanical stimuli) was assessed according to the method described elsewhere (Chaplan et al., 1994). von-Frey hairs (IITC, Woodland Hills, CA) with calibrated bending forces (in g) of different intensities were used to deliver punctuates mechanical stimuli of varying intensities.

Thermal hyperalgesia (tail immersion test): spinal thermal sensitivity was assessed by the tail immersion test according to the method described elsewhere (Necker & Hellon, 1978).

Nerve conduction velocity: the recording of motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) was performed in rats according to previously described method (Kandhare et al., 2012c; Raygude et al., 2012b).

Invasive measurement of hemodynamic changes

Blood pressure was measured by means of a polyethylene cannula (PE 50) filled with heparinized saline (100 IU/ml) inserted into the left femoral artery. The cannula was connected to a transducer and the signal was amplified by means of a bioamplifier. Left ventricular systolic pressure was measured using PowerLab 8/35 Data Acquisition Systems (AD Instrument Pvt. Ltd., Bella Vista, Australia). Heart rate, SBP, DBP, and MABP were obtained by means of an acquisition data system (LabChart 7.3; AD Instrument Pvt. Ltd., Bella Vista, Australia).

Biochemical estimations

At the end of study, i.e., 8th week, blood was withdrawn from rat by retro orbital puncture under light ether anesthesia. The serum was used for the estimation of cholesterol, triglyceride, glycated hemoglobin, and serum insulin. All animals were sacrificed after blood collection and sciatic nerves were immediately isolated for biochemical and molecular estimation.

Determination of serum cholesterol and triglyceride levels

Serum levels of cholesterol and triglyceride were measured by spectroflurimetrically using reagent kits (Accurex Biomedical Pvt. Ltd., Mumbai, India).

Determination of serum insulin

The quantifications of serum insulin were performed with the help and instructions provided by Mercodia AB, Uppsala, Sweden, with a rat insulin ELISA kit, which is a 4.5-h solid-phase ELISA. The assay employs the sandwich enzyme immunoassay technique. A monoclonal antibody specific for rat insulin had been precoated in the microplate. Briefly, 10 μL of standards, control, and test samples (serum) were added into each well followed by addition of 100 μL of enzyme conjugate to it and incubated at RT for 2 h. If any rat insulin is present, it would have bound by the immobilized antibody. After having washed away any unbound substance, 200 μL TMB, a substrate solution and consequently an enzyme reaction, was added to each well and incubated at RT for 15 min. Then, it was followed by addition of 50 μL stop solution, which causes blue product to turn yellow. The intensity of the color was measured at 450 nm, in proportion to the amount of rat insulin bound in the initial steps. The sample values were then read off using the standard curve. Values were expressed as mean ± SEM.

Sciatic nerve homogenate preparation

Tissue homogenates were prepared with 0.1 M Tris–HCl buffer (pH 7.4) and supernatant of homogenates was employed to estimate SOD, GSH, lipid peroxidation (malondialdehyde (MDA) content), nitric oxide (NO content), total calcium content, and membrane bound enzyme (Na⁺K⁺ATPase).

Determination of total protein in sciatic nerve

Protein concentration was estimated according to the method according to the method described elsewhere (Lowry et al., 1951), using BSA as a standard.

Determination of SOD contents in sciatic nerve

The neural pathological alteration occurs due to the overproduction of reactive oxygen species (ROS). SOD assay were performed according to the method described elsewhere (Misra & Fridovich, 1972). SOD activity was expressed as U/mg of protein.

Determination of GSH contents in sciatic nerve

The GSH assay was performed according to the method described elsewhere (Moron et al., 1979). The amount of GSH was expressed as µg/mg of protein.

Determination of MDA content in sciatic nerve

MDA level in the neural tissue was performed according to the method described elsewhere (Slater & Sawyer, 1971). The values were expressed in nanomoles/mg of protein.

Determination of nitrite level in sciatic nerve

The NO level was estimated as nitrite by the acidic Griess reaction after reduction in nitrate to nitrite by vanadium trichloride according to the method described elsewhere (Miranda et al., 2001).

Determination of total calcium in sciatic nerve

Total calcium levels were estimated in sciatic nerve according to the method described elsewhere (Severenghaus & Ferrebee, 1950).

Determination of membrane-bound inorganic phosphate (Na⁺K⁺ATPase) in sciatic nerve

Membrane-bound inorganic phosphate (Na⁺K⁺ATPase) was estimated in sciatic nerve according to the method described elsewhere (Bonting, 1970).

Determination of TNF-α and IL-1β by reverse transcriptase PCR in sciatic nerve

RNA isolation: the nerve tissue was chopped and minced. The specimens were disrupted using mortar pestle in liquid nitrogen. Total cytoplasmic RNA was extracted from nerve samples using a guanidium isothiocyanate/phenol chloroform/trizol method (Thermo Fischer, Mumbai, India). Following an isopropanol precipitation, the RNA was washed with 70% ethanol and treated with RNAse Inhibitor (Thermo Fischer) for 45 min. Following re-suspension of the RNA at 65 °C for 15 min, RNA preparations were further purified using the Qiagen RNA isolation kit and were treated with RNAse-free DNase as directed by the manufacturer (Qiagen, Foster City, CA). Following precipitation, RNA was resuspended in RNAse-free water and its concentration was quantified by absorbance at 260 nm wavelength. RNA samples were stored at −80 °C until analysis.

cDNA preparation: single-stranded cDNA was synthesized from 5 µg of total cellular RNA using reverse transcriptase and oligo-(dT)-primers (Takara, Berkeley, CA) according to the method described elsewhere (Kandhare et al., 2013a). The polymerase chain reaction mixture was amplified in a DNA thermal cycler (Eppendorf, Hauppauge, NY). The primer sequence for TNF-α, IL-1β, and β-actin were synthesized by Ocimum Biosolutions, Hyderabad, India, and is in Table 1. Polymerase chain reaction products were detected by electrophoresis on a 1.5% agarose gel containing ethidium bromide. The size of amplicons was confirmed by using 100-bp ladder (Takara) as a standard size marker. The amplicons were visualized and images were captured using gel documentation system (Alpha Innotech Inc., San Leandro CA). The expression of all the genes was assessed by generating densitometry data for band intensities in different sets of experiments was generated by analyzing the gel images on the Image J program (Version 1.33, National Institutes of Health (NIH), Bethesda, MD) semi-quantitatively. The band intensities were compared with constitutively expressed β actin. The intensity of mRNAs was standardized against that of the β-actin mRNA from each sample, and the results were expressed as PCR-product/β-actin mRNA ratio.

Table 1. Sequences of primers used in experiments and product size.

Primer	Sequence	bp
β-Actin	5′-ACGGTCAGGTCATCACTATCG-3′	746
	5′-GCTCAAGCAGCAAAGGATCT-3′
TNF-α	5′-GACCCTCACACTCAGATCATCTTCT-3′	480
	5′-TCACAGAGCAATGACTCCAAAG-3′
IL-1β	5′-ATAGCAGCTTTCGACAGTGAG-3′	748
	5′-GTCAACTATGTCCCGACCATT-3′

Histopathological analysis of sciatic nerve: samples of sciatic nerve were placed in the fixative solution (10% formalin), cut into 5 mL thickness and stained with hematoxylin and eosin (Sudoh et al., 2004). Nerve sections were analyzed qualitatively under light microscope (400× for axonal degeneration and various histopathological alterations.

Statistical analysis

Data were expressed as mean ± standard error mean (SEM). Data analysis was performed using Graph Pad Prism 5.0 software (Graph Pad, San Diego, CA). Data of behavioral tests were statistically analyzed using a 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 a one-way analysis of variance (ANOVA) and Tukey's multiple range test was applied for post hoc analysis. A value of p < 0.05 was considered to be statistically significant.

Results

Effect of hesperidin on body weight and plasma glucose level

STZ (diabetic) control rats showed a significant decrease (p < 0.05) in body weight and an increase in plasma glucose level as compared to normal non-diabetic rats. Diabetic rats treated with hesperidin (50 and 100 mg/kg) showed significant (p < 0.05) and dose-dependent prevention in body weight reduction and increase in plasma glucose level as compared to STZ control rats. There was a significant increase (p < 0.05) in body weight and decrease in plasma glucose level of insulin (10 IU/kg)-treated rats when compared with STZ control rats. Moreover, there was significant inhibition (p < 0.05) in decreased body weight and increased plasma glucose level in hesperidin (100 mg/kg) and insulin (10 IU/kg) combination treated rats as compared to hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treated rats (Table 2).

Table 2. Effect of chronic treatment of hesperidin and insulin on body weight, plasma glucose, food intake, water intake, and urine output in STZ-induced diabetic neuropathic rats.

Treatment	Body weight (g)	Plasma glucose (mg/dL)	Food intake (g)	Water intake (mL)	Urine output (mL)
Normal (ND)	261.6 ± 3.18	171.9 ± 2.51	23.20 ± 1.07	42.16 ± 1.18	7.74 ± 0.62
STZ control	155.6 ± 4.94#	458.6 ± 10.82#	70.44 ± 2.09#	137.1 ± 2.12#	53.22 ± 1.81#
STZ + H (25)	163.4 ± 3.50	421.4 ± 13.58	65.22 ± 2.08	114.8 ± 4.03	49.90 ± 1.05
STZ + H (50)	186.8 ± 2.47*,$	321.4 ± 13.70*,$	47.24 ± 2.73*,$	81.80 ± 3.34*,$	34.38 ± 1.72*,$
STZ + H (100)	211.8 ± 3.99*,$	248.0 ± 11.57*,$	35.00 ± 1.06*,$	72.46 ± 1.35*,$	21.26 ± 1.43*,$
STZ + I (10)	237.8 ± 9.85*,$	174.7 ± 4.47*,$	26.70 ± 0.96*,$	60.72 ± 1.71*,$	16.28 ± 0.67*,$
STZ + H (100) + I (10)	256.4 ± 6.84*,$	173.4 ± 4.98*,$	27.54 ± 2.08*,$	55.34 ± 2.42*,$	11.28 ± 1.22*,$

Data are expressed as mean ± SEM from six rats and analyzed by a two-way ANOVA followed by Bonferroni’s test. ND: non-diabetic rats; STZ: diabetic (STZ) control rats; H (25): hesperidin (25 mg/kg, p.o.) treated rats; H (50): hesperidin (50 mg/kg, p.o.) treated rats; H (100): hesperidin (100 mg/kg, p.o.) treated rats; I (10): insulin (10 IU/kg, s.c.) treated rats; H (100) + I (10): hesperidin (100 mg/kg, p.o.) and insulin (10 IU/kg, s.c.) combination treated rats.

*p < 0.05 as compared to the STZ diabetic control group.

#p < 0.05 as compared to normal non-diabetic control animals.

$p < 0.05 as compared to one another group.

Effect of hesperidin food intake, water intake, and urine output

The food intake, water intake, and urine output in STZ control rats significantly increased (p < 0.05) after 4 weeks of STZ administration as compared to normal non-diabetic rats. When compared with STZ control rats, hesperidin (50 and 100 mg/kg) 4 week treatment significantly and dose dependently (p < 0.05) ameliorated the elevated food intake, water intake, and urine output. Moreover, insulin (10 IU/kg) treatment also showed the significant reduction (p < 0.05) in food intake, water intake, and urine output as compared to STZ control rats. But this attenuation in food intake, water intake, and urine output was more significant (p < 0.05) in hesperidin (100 mg/kg) and insulin (10 IU/kg) combination treated rats as compared to hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treated rats (Table 2).

Effect of hesperidin on diabetes-induced mechano-tactile allodynia and hyperalgesia

There was no significant difference in mechano-tactile allodynia and hyperalgesia in STZ control rats and normal non-diabetic rats before induction of DN. Intraperitoneal administration of STZ resulted in significant decrease (p < 0.05) in mechano-tactile allodynia and increased hyperalgesia in STZ control rats as compared to normal non-diabetic rats. There was significant (p < 0.05) and dose dependent increase in mechano-tactile allodynia and reduction in hyperalgesia in hesperidin (25, 50, and 100 mg/kg) treated rats as compared to STZ control rats. Treatment with insulin (10 IU/kg) also showed significant increase (p < 0.05) in mechano-tactile allodynia and hyperalgesia as compared to STZ control rats. Moreover, treatment with combination of hesperidin (100 mg/kg) and insulin (10 IU/kg) also showed significant increase (p < 0.05) in mechano-tactile allodynia and reduction in hyperalgesia as compared to hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treated diabetic rats (Figure 1A and B).

Figure 1. Effect of chronic treatment of hesperidin and insulin on (A) mechanical allodynia in von Frey hair test, (B) mechanical hyperalgesia in paw pressure test, (C) thermal hyperalgesia in plantar test, and (D) thermal hyperalgesia in tail immersion test. Data are expressed as mean ± SEM from six rats and analyzed by a two-way ANOVA followed by Bonferroni’s test. *p < 0.05 as compared to the STZ diabetic control group, #p < 0.05 as compared to normal non-diabetic control animals, and $p < 0.05 as compared to one another group. STZ: diabetic (STZ) control rats; H (25): hesperidin (25 mg/kg, p.o.) treated rats; H (50): hesperidin (50 mg/kg, p.o.) treated rats; H (100): hesperidin (100 mg/kg, p.o.) treated rats; I (10): insulin (10 IU/kg, s.c.) treated rats; H (100) + I (10): hesperidin (100 mg/kg, p.o.), and insulin (10 IU/kg, s.c.) combination treated rats.

Effect of hesperidin on diabetes-induced thermal hyperalgesia

There was no significant difference in thermal hyperalgesia of STZ-treated rats as compared to normal rats before STZ administration. Intraperitoneal STZ administration resulted in a significant decrease (p < 0.05) in thermal hyperalgesia of STZ control rats as compared to normal non-diabetic rats. Hesperidin (50 and 100 mg/kg) treatment for 4 weeks significantly and dose dependently (p < 0.05) increased thermal hyperalgesia as compared to STZ control rats. This decreased thermal hyperalgesia was also significantly (p < 0.05) elevated by insulin (10 IU/kg) treatment. Combination treatment of hesperidin (100 mg/kg) and insulin (10 IU/kg) showed significant amelioration in decreased (p < 0.05) thermal hyperalgesia when compared with hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treated rats.

Effect of hesperidin on nerve conduction velocity

There was a significant decrease (p < 0.05) in MNCV as well as SNCV of STZ-treated rats as compared to normal rats. Treatment with hesperidin (50 and 100 mg/kg) for 4 weeks significantly and dose dependently (p < 0.05) inhibited this decreased MNCV and SNCV as compared to STZ control rats. Insulin (10 IU/kg) treated rats also showed significant (p < 0.05) increase in MNCV and SNCV as compared to STZ control rats. When compared with hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treated rats, MNCV and SNCV were more significantly increased (p < 0.05) in hesperidin (100 mg/kg) and insulin (10 IU/kg) combination treated rats (Figure 2).

Figure 2. Effect of chronic treatment of hesperidin and insulin on (A) motor nerve conduction velocity and (B) sensory nerve conduction velocity. Data are expressed as mean ± SEM from six rats and analyzed by a two-way ANOVA followed by Bonferroni’s test. *p < 0.05 as compared to the STZ diabetic control group, #p < 0.05 as compared to normal non-diabetic control animals, and $p < 0.05 as compared to one another group. STZ: diabetic (STZ) control rats; H (25): hesperidin (25 mg/kg, p.o.) treated rats; H (50): hesperidin (50 mg/kg, p.o.) treated rats; H (100): hesperidin (100 mg/kg, p.o.) treated rats; I (10): insulin (10 IU/kg, s.c.) treated rats; H (100) + I (10): hesperidin (100 mg/kg, p.o.) and insulin (10 IU/kg, s.c.) combination treated rats.

Effect of hesperidin on levels of serum glucosuria, cholesterol, and triglyceride

There was a significant increase (p < 0.05) in glucosuria, cholesterol, and triglyceride level of STZ control rats as compared to normal rats after 4 weeks of intraperitoneal STZ administration. When compared with STZ control rats, elevated levels of glucosuria, cholesterol, and triglyceride were significantly and dose dependently (p < 0.05) attenuated by the treatment of hesperidin (50 and 100 mg/kg). Rats treated with insulin (10 IU/kg) also showed significant decrease (p < 0.05) in the elevated levels of glucosuria, cholesterol, and triglyceride as compared to STZ control rats. However, hesperidin (100 mg/kg) and insulin (10 IU/kg) combination treatment showed more significant decrease (p < 0.05) in glucosuria, cholesterol, and triglyceride levels as compared to hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treatment (Table 3).

Table 3. Effect of chronic treatment of hesperidin and insulin on glucosuria, serum cholesterol, serum triglyceride, serum glycated hemoglobin, serum insulin, and neural aldose reductase in STZ-induced diabetic neuropathic rats.

Treatment	Normal (ND)	STZ control	STZ + H (25)	STZ + H (50)	STZ + H (100)	STZ + I (10)	STZ + H (100) + I (10)
Glucosuria (mg/dL)	29.40 ± 1.69	959.4 ± 16.48#	851.4 ± 35.02	595.0 ± 10.57*,$	162.9 ± 21.58*,$	35.58 ± 1.48*,$	29.52 ± 5.47*,$
Cholesterol (mg/dL)	64.82 ± 4.09	219.1 ± 5.04#	199.2 ± 3.95	166.0 ± 4.03*,$	134.7 ± 5.64*,$	104.0 ± 4.29*,$	87.54 ± 5.06*,$
Triglyceride (mg/dL)	88.76 ± 4.32	354.1 ± 14.03#	304.6 ± 13.64	244.5 ± 8.13*,$	165.3 ± 10.73*,$	135.2 ± 9.21*,$	105.1 ± 4.73*,$
Glycated Hb (%)	3.88 ± 0.36	9.22 ± 0.44#	8.10 ± 0.37	6.64 ± 0.55*,$	5.18 ± 0.35*,$	4.78 ± 0.40*,$	4.58 ± 0.53*,$
Insulin (µg/L)	2.29 ± 0.22	0.46 ± 0.10#	0.61 ± 0.12	1.29 ± 0.10*,$	1.88 ± 0.28*,$	2.11 ± 0.15*,$	2.12 ± 0.18*,$
Aldose reductase  (nM of NADPH  oxidized/min/mg of protein)	2.42 ± 0.26	7.90 ± 0.31#	7.36 ± 0.33	5.84 ± 0.55*,$	4.56 ± 0.37*,$	5.25 ± 0.70*,$	3.54 ± 0.31*,$

Data are expressed as mean ± SEM from six rats and analyzed by a two-way ANOVA followed by Bonferroni’s test. ND: non-diabetic rats; STZ: diabetic (STZ) control rats; H (25): hesperidin (25 mg/kg, p.o.) treated rats; H (50): hesperidin (50 mg/kg, p.o.) treated rats; H (100): hesperidin (100 mg/kg, p.o.) treated rats; I (10): insulin (10 IU/kg, s.c.) treated rats; H (100) + I (10): hesperidin (100 mg/kg, p.o.) and insulin (10 IU/kg, s.c.) combination treated rats.

*p < 0.05 as compared to the STZ diabetic control group.

#p < 0.05 as compared to normal non-diabetic control animals.

$p < 0.05 as compared to one another group.

Effect of hesperidin on glycated hemoglobin and neural aldose reductase (AR) levels

There was a significant increase (p < 0.05) in glycated hemoglobin and AR level in STZ control rats as compared to normal non-diabetic rats. Treatment with hesperidin (50 and 100 mg/kg) showed significant and dose-dependant reduction (p < 0.05) in elevated glycated hemoglobin and AR levels as compared to STZ control rats. When compared with STZ control rats, the level of glycated hemoglobin and AR was significantly reduced (p < 0.05) in the insulin (10 IU/kg) treated rats. But when compared with hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treatment, the hesperidin (100 mg/kg) and insulin (10 IU/kg) combination treatment showed more significant reduction (p < 0.05) in glycated hemoglobin and AR level (Table 3).

Effect of hesperidin on insulin level

Intraperitoneal administration of STZ caused a significant reduction (p < 0.05) in serum insulin level as compared to normal non-diabetic rats. Hesperidin (50 and 100 mg/kg) treated rats showed significant (p < 0.05) and dose-dependent increased in serum insulin level as compared to STZ control rats. Whereas insulin (10 IU/kg) treated rats also showed a significant increase (p < 0.05) in serum insulin level as compared to STZ control rats. Moreover, hesperidin (100 mg/kg) and insulin (10 IU/kg) combination treated rats showed a more significant increase (p < 0.05) in serum insulin level as compared to hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treated rats (Table 3).

Effect of hesperidin on hemodynamic parameters

A significant increase (p < 0.05) in SBP, DBP, and MABP was recorded in STZ control rats as compared to normal non-diabetic rats. STZ induced elevated SBP, DBP, and MABP were significantly and dose dependently (p < 0.05) attenuated by the hesperidin (50 and 100 mg/kg) treatment as compared to STZ control rats. Insulin (10 IU/kg) also showed significant reduction (p < 0.05) in these elevated hemodynamic parameters, i.e. SBP, DBP, and MABP. The treatment with hesperidin (100 mg/kg) and insulin (10 IU/kg) combination showed a more significant decrease (p < 0.05) in elevated SBP, DBP, and MABP as compared to hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treated rats. Intraperitoneal administration of STZ did not produce any significant change in heart rate as compared to normal- as well as hesperidin-treated rats (Table 4).

Table 4. Effect of chronic treatment of hesperidin and insulin on hemodynamic parameters in STZ-induced diabetic neuropathic rats.

Treatment	Normal (ND)	STZ control	STZ + H (25)	STZ + H (50)	STZ + H (100)	STZ + I (10)	STZ + H (100) + I (10)
SBP (mmHg)	112.6 ± 4.12	152.9 ± 3.79#	145.6 ± 4.02	135.5 ± 3.38*,$	128.4 ± 2.84*,$	123.5 ± 3.55*,$	119.2 ± 3.34*,$
DBP (mmHg)	76.60 ± 2.33	115.7 ± 2.96#	105.1 ± 6.18	96.98 ± 4.49*,$	90.32 ± 2.79*,$	81.40 ± 1.24*,$	83.70 ± 3.15*,$
MABP (mmHg)	76.86 ± 1.97	126.6 ± 2.17#	116.7 ± 2.76	107.1 ± 4.28*,$	99.46 ± 2.22*,$	87.66 ± 2.50*,$	86.40 ± 1.72*,$
HR (BPM)	373.1 ± 9.23	345.3 ± 8.67	337.8 ± 10.2	363.0 ± 8.39	358.0 ± 13.00	367.2 ± 5.93	368.0 ± 12.1

Data are expressed as mean ± SEM from six rats and analyzed by a two-way ANOVA followed by Bonferroni’s test. ND: non-diabetic rats; STZ: diabetic (STZ) control rats; H (25): hesperidin (25 mg/kg, p.o.) treated rats; H (50): hesperidin (50 mg/kg, p.o.) treated rats; H (100): hesperidin (100 mg/kg, p.o.) treated rats; I (10): insulin (10 IU/kg, s.c.) treated rats; H (100) + I (10): hesperidin (100 mg/kg, p.o.) and insulin (10 IU/kg, s.c.) combination treated rats.

*p < 0.05 as compared to the STZ diabetic control group.

#p < 0.05 as compared to normal non-diabetic control animals.

$p < 0.05 as compared to one another group.

Effect of hesperidin on neural SOD and glutathione level

There was a significant decrease (p < 0.05) in SOD and glutathione level in STZ control rats as compared to non-diabetic rats after 8 weeks of STZ administration. This decreased SOD and GSH levels were significantly and dose dependently (p < 0.05) increased by the 4-week hesperidin (50 and 100 mg/kg) treatment as compared to STZ control rats. Insulin (10 IU/kg) treatment also showed significant increase (p < 0.05) in SOD and GSH level as compared to STZ control rats. When compared with hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treated rats, SOD and GSH levels were more significantly increased (p < 0.05) by the treatment of hesperidin (100 mg/kg) and insulin (10 IU/kg) combination (Table 5).

Table 5. Effect of chronic treatment of hesperidin and insulin on neural levels of endogenous biomarkers, i.e., SOD, GSH, LPO, nitrite, calcium, and Na–K–ATPase in STZ-induced diabetic neuropathic rats.

Treatment	SOD (U/mg of protein)	GSH (μg/mg protein)	LPO (nM/mg of protein)	NO (μg/ml)	Calcium (ppm/mg of protein)	Na^+K^+ATPase (μmoles/mg of protein)
Normal (ND)	24.12 ± 1.53	1.28 ± 0.11	2.60 ± 0.35	97.64 ± 4.94	5.26 ± 0.32	9.26 ± 0.45
STZ control	6.14 ± 0.58#	0.32 ± 0.06#	9.56 ± 0.31#	315.5 ± 6.57#	20.90 ± 0.92#	2.78 ± 0.46#
STZ + H (25)	5.98 ± 0.68	0.39 ± 0.04	8.60 ± 0.66	296.3 ± 5.72	20.06 ± 1.00	4.30 ± 0.38
STZ + H (50)	10.30 ± 0.63*,$	0.82 ± 0.08*,$	6.94 ± 0.57*,$	261.3 ± 9.60	14.86 ± 1.03*,$	5.72 ± 0.31*,$
STZ + H (100)	17.36 ± 0.98*,$	1.05 ± 0.08*,$	4.30 ± 0.23*,$	193.5 ± 6.01*,$	10.44 ± 0.61*,$	7.62 ± 0.31*,$
STZ + I (10)	16.14 ± 0.57*,$	0.80 ± 0.08*,$	3.87 ± 0.42*,$	156.7 ± 10.91*,$	13.26 ±0.70*,$	8.28 ± 0.27*,$
STZ + H (100) + I (10)	21.51 ± 0.50*,$	1.06 ± 0.13*,$	3.16 ± 0.27*,$	161.0 ± 4.38*,$	7.28 ± 0.58*,$	9.02 ± 0.45*,$

Data are expressed as mean ± SEM from six rats and analyzed by a two-way ANOVA followed by Bonferroni’s test. ND: non-diabetic rats; STZ: diabetic (STZ) control rats; H (25): hesperidin (25 mg/kg, p.o.) treated rats; H (50): hesperidin (50 mg/kg, p.o.) treated rats; H (100): hesperidin (100 mg/kg, p.o.) treated rats; I (10): insulin (10 IU/kg, s.c.) treated rats; H (100) + I (10): hesperidin (100 mg/kg, p.o.) and insulin (10 IU/kg, s.c.) combination treated rats.

*p < 0.05 as compared to the STZ diabetic control group.

#p < 0.05 as compared to normal non-diabetic control animals.

$p < 0.05 as compared to one another group.

Effect of hesperidin on neural lipid peroxidase, NO, and total calcium level

Intraperitoneal administration of STZ caused significant elevation (p < 0.05) in lipid peroxidase, NO, and total calcium levels in STZ control rats as compared to normal rats. This elevated lipid peroxidase and total calcium levels were significantly and dose dependently (p < 0.05) attenuated by hesperidin (50 and 100 mg/kg) treatment whereas rats treated with hesperidin (100 mg/kg) showed significant reduction (p < 0.05) in NO level as compared to STZ control rats. Treatment with insulin (10 IU/kg) also significantly reduced (p < 0.05) lipid peroxidase, NO, and total calcium level as compared to STZ control rats. Rats treated with combination of hesperidin (100 mg/kg) and insulin (10 IU/kg) showed more significant attenuation (p < 0.05) in lipid peroxidase, NO, and total calcium as compared to hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treated rats (Table 5).

Effect of hesperidin on neural Na⁺K⁺ATPase level

There was a significant reduction (p < 0.05) in Na⁺K⁺ATPase level in STZ control rats as compared to normal rats. There was a significant increase (p < 0.05) in Na⁺K⁺ATPase level of hesperidin (50 and 100 mg/kg) treated rats as compared to STZ control rats. Also, insulin (10 IU/kg) treatment significantly increased (p < 0.05) Na⁺K⁺ATPase level when compared with STZ control rats. There was a significant elevation (p < 0.05) in the Na⁺K⁺ATPase level of hesperidin (100 mg/kg) and insulin (10 IU/kg) combination treated rats as compared to hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treated rats (Table 5).

Effect of hesperidin on mRNA expression levels of neural TNF-α and IL-1β

As shown in Figure 3, mRNA expression of TNF-α and IL-1β were significantly increased (p < 0.05) in STZ control rats as compared to normal non-diabetic rats. Treatment with hesperidin (50 and 100 mg/kg) significantly and dose dependently (p < 0.05) down-regulated TNF-α and IL-1β mRNA expression as compared to STZ control rats. When compared with STZ control rats, insulin (10 IU/kg) treatment also significantly inhibited (p < 0.05) up-regulated mRNA expression of TNF-α and IL-1β. Moreover, these elevated TNF-α and IL-1β expressions were more significantly attenuated (p < 0.05) by hesperidin (100 mg/kg) and insulin (10 IU/kg) combination treatment as compared to hesperidin (100 mg/kg) or insulin (10 IU/kg) alone treatment.

Figure 3. Effect of chronic treatment of hesperidin and insulin on (A) reverse transcriptase analysis of protein expression of TNF-α and IL-1β. (B) Quantitative representation of TNF-α and IL-1β protein expression. Data are expressed as mean ± SEM from six rats and analyzed by a one-way ANOVA followed by Tukey's multiple range test. *p < 0.05 as compared to the STZ diabetic control group, #p < 0.05 as compared to normal non-diabetic control animals, and $p < 0.05 as compared to one another group. STZ: diabetic (STZ) control rats; H (50): hesperidin (50 mg/kg, p.o.) treated rats; H (100): hesperidin (100 mg/kg, p.o.) treated rats; I (10): insulin (10 IU/kg, s.c.) treated rats; H (100) + I (10): hesperidin (100 mg/kg, p.o.), and insulin (10 IU/kg, s.c.) combination treated rats.

Effect of hesperidin on histological alteration of sciatic nerve

Figure 4(A) represents the normal architecture of the sciatic nerve reflected by the absence of inflammatory cell infiltration, necrosis, edema, and congestion. Intraperitoneal administration of STZ caused histological alteration in the sciatic nerve of the STZ control rats reflected by the presence of the neutrophiles and macrophages infiltration (yellow arrow), congestion (red arrow) as well as edema (black arrow) (Figure 4B). The necrosis (blue arrow) and nerve cell vacuolization resulted in swelling of nerve fibers and decreased the number of myelinated fibers. A histological section of sciatic nerve from rats treated with hesperidin (50 and 100 mg/kg) showed reduction in infiltration of neutrophilic as well as macrophages (yellow arrow) (Figure 4D and E). But it showed the presence of the mild congestion (red arrow) along with edema (black arrow). Swelling of nonmyelinated and myelinated nerve fibers as well axonal degeneration was inhibited by the treatment of insulin (10 IU/kg) (Figure 4C). STZ induced infiltration of neutrophiles, and macrophages (yellow arrow), congestion (red arrow), and edema (black arrow) were reduced by the combination of hesperidin (100 mg/kg) and insulin (10 IU/kg) treatment thus reducing the swelling of nonmyelinated and myelinated nerve fibers (Figure 4F).

Figure 4. Effect of chronic treatment of hesperidin and insulin on histopathological analysis of sciatic nerve in STZ-induced diabetic neuropathy. Photomicrographs of sections of sciatic nerve from rats stained with H&E. Sciatic nerve microscopic image of (A) normal rat, (B) STZ control rat, (C) insulin (10 IU/kg, s.c.), (D) hesperidin (50 mg/kg, p.o.), (E) hesperidin (100 mg/kg, p.o.), and (F) hesperidin (100 mg/kg, p.o.) and insulin (10 IU/kg, s.c.) combination-treated rats (microscopic examination under 400 × light microscopy).

Discussion

DN, a consequence of metabolic disorder, results from hyperglycemia, alterations in glucose-sensitive signal transduction pathways, and increased oxidative stress that further causes neurochemical and neurovascular defects in the peripheral nervous system (Group, 1994). An elevated nociceptive response (allodynia and hyperalgesia) with electrophysiological conduction abnormalities is a characteristic clinical feature observed in diabetic patients suffering from neuropathy (Kandhare et al., 2012b). In the present investigation, STZ-induced diabetic rat exhibits neuronal complications like peripheral sensory nerve injury assessed by allodynia, hyperalgesia, and reduced conduction velocities after 8 weeks of intraperitoneal STZ administration. These preclinical features of DN are in line with the clinicopathology that is present in humans (Courteix et al., 1993).

In the present investigation, STZ-induced DN was associated with decreased body weight with increased food intake, water intake, and urine output as compared to normal non-diabetic rats. These features of cellular metabolic abnormalities are caused due to decreased availability of glucose and amino acids to cells which create shortage of cellular biosynthesis substrates (Ar'Rajab & AhréN, 1993). Rats treated with hesperidin significantly inhibited features of cachexia, polydipsia, and polyphagia which are in line with findings of previous workers who showed improvement in body weight, food intake, and water intake after treatment with hesperidin in STZ-induced nephropathic rats (Shah & Patel, 2010).

Hyperglycemia is one of the most important pathogenic factors responsible for the development of diabetic peripheral neuropathy. Sustained increased blood glucose level as well as STZ induced damage to pancreatic β-cells caused release of insulin and its resistance in peripheral tissues (Davidson, 2004; Rossetti et al., 1990). It is also associated with elevated glycated hemoglobin (HbA₁c) levels and it has been clinically proven that HbA₁c concentration is closely related with the incidence of diabetic complications (Davidson, 2004). Hyperglycemia produces formation as well as accumulation of advanced glycosylation end-products (AGE) in neural tissue via glycosylation and auto-oxidation of cellular protein. These increased levels of glycated hemoglobin as well AGE contribute to free radical generation and tissue damage in DN. Treatment with hesperidin significantly elevated levels of insulin and decreased levels of HbA₁c thus exerting its neuroprotective effect. Results of the present investigation are in accordance with the findings of previous researchers which showed HbA₁c lowering potential of hesperidin in serum of diabetes induced rat (Shah & Patel, 2010).

Elevated blood glucose levels are a major factor that has been reported to play a vital role in the development of DN via activation of bundle of pathways including aldol reductase, non-enzymatic glycation, protein kinase C (PKC), mitogen-activated protein kinases, poly ADP ribose polymerase (PARP), etc. (Sima, 2006; Soriano et al., 2001). Type IV collagen, laminin, and fibronectin are the extracellular matrix (ECM) molecules which is a multifunctional complex made up of proteins and proteoglycans that provide structural integrity to the vasculature (Yurchenco & Schittny, 1990). The activation of various pathways like PKC, PARP, and AR modulated the structural integrity to the vasculature via ECM results in tissue damaged (Matrisian, 2005). In the present investigation, hyperglycemia was induced after 48 h of intraperitoneal STZ administration. This may have activated the various pathways that produced structural abnormalities in sciatic nerve via DNA (Kumar et al., 2007). Treatment with hesperidin significantly attenuated hyperglycemia that might inhibit the activation of cytotoxicity pathways to exert its neuroprotective potential. Antidiabetic potential of hesperidin also has been reported by Jung et al. (2004) and Lonchampt et al. (1989) via inhibition of hepatic gluconeogenesis and scavenging of active oxygen species, respectively.

Nociception is encoding and processing of noxious stimuli by the nervous system. DN is associated with abnormal sensation and pain which can be determined by behavioral responses to external stimuli (Newby, 2005). In inflamed tissue, nociceptive pain appears as spontaneous pain, as hyperalgesia, or allodynia. It has been reported that von Frey hair, Randall selitto, and tail flick are the well-established methods for measuring mechano-tactile allodynia, peripheral analgesia, and central pain in laboratory animals (Kandhare et al., 2013a,b; Raygude et al., 2012b; Visnagri et al., 2012). Intraperitoneal administration of STZ is associated with two phases of thermal pain sensitivity composed of transient hyperalgesia phase followed by a hypoalgesia phase (Pabbidi et al., 2008). In the present investigation, hyperalgesia or allodynia was developed after 4 weeks of STZ administration which is in accordance with several other reports (Kapur, 2003; Wuarin-Bierman et al., 1987). It has been documented that various mechanisms such as peripheral receptors sensitization, ectopic activity in sprouting fibers, and alterations in dorsal root ganglia cells played a vital role in the development of nociception (Jensen & Baron, 2003; Quattrini & Tesfaye, 2003). STZ-induced DN is a symmetric type of neuropathy which involves distal sensory and motor nerves. As diabetes progresses, the sensation of distal extremities decreases leading to loss of pain sensation. Hence, tail withdrawal threshold is an important parameter to assess the response of rats to thermal noxious stimuli. It has been reported that STZ-induced diabetic rat exhibits an elevated tail-flick threshold response to the noxious thermal stimuli (Calcutt et al., 2004). Treatment with hesperidin has been shown to inhibit diabetes-induced elevation in hyperalgesia or allodynia.

Diabetic microangiopathy and macroangiopathy is developed due to alterations in fatty acid metabolism and elevated levels of serum lipoprotein (Cameron et al., 1991). In diabetic condition, erythrocyte dysfunction occurred due to alteration in the lipid composition of the erythrocyte membrane. Clinically it has been proven that high cholesterol levels and hyperlipidemia are associated with decreased oxygen release leading to vascular complications (Ditzel & Dyerberg, 1977). Cell membrane dysfunction of nerve occurred due to inhibition of β-oxidation of fatty acids leading to accumulation of long chain fatty acid ester caused alteration in PKC and Na⁺K⁺ATPase activity (Brecher, 1983; Kandhare et al., 2012a; Raygude et al., 2012a). Treatment with hesperidin showed significant attenuation in elevated levels of cholesterol and triglyceride to inhibit accumulation of long chain fatty acid ester thus improved conduction via Na⁺K⁺ATPase activity. Effectiveness of hesperidin in hyperlipidemic condition is further supported by the study of Akiyama et al. (2010) wherein hypolipidemic potential of dietary hesperidin on diabetic rats is reported.

Membrane bound inorganic phosphate enzyme, i.e., Na⁺K⁺ATPase is composed of catalytic α-subunit (Mr 112 kd) with three different isoforms of protein kinases substrate for binding of ATP and ouabain. One of the isoform of Na⁺K⁺ATPase α-subunit is located in Schwann cell in peripheral nerve which plays an important role in nerve condition velocity. It has been documented that increased level of blood glucose results in the activation of polyol pathway as well as PKC activity with the inhibition of myoinositol and Na⁺K⁺ATPase activity (Greene et al., 1992). This decreased activity of Na⁺K⁺ATPase associated with down-regulation of NADPH activity leads to energy-dependent slowing of nerve conduction. In this investigation, significant decrease in the motor as well as sensory nerve conduction velocity was recorded after 4 weeks of STZ administration. Moreover, Hirata and Okada (1990), Goto et al. (1995), and Garner and Spector (1987) reported that many therapeutic agents like AR inhibitors (Epalrestat) exert its neuroprotective effect via inhibition of AR activity to improve Na⁺K⁺ATPase and thus corrected delayed MNCV in the clinical setting (Garner & Spector, 1987, Goto et al., 1995). Rats treated with hesperidin showed significant reduction in elevated levels of AR thus improved activity of membrane bound inorganic phosphate enzymes which in turn improved MNCV as well as SNCV. This notion of reduction in AR activity is further supported by earlier reports of Shi et al. (2012), which showed AR inhibitory potential of hesperidin.

An array of studies carried out by various researchers reported that hyperglycemia is associated with uncontrolled generation of free radical which gives rise to oxidative stress in STZ-induced diabetic rats (Callaghan et al., 2005; Niedowicz & Daleke, 2005). Generation of free radical via increased glucose levels involves non-enzymatic and enzymatic protein glycation mechanisms with a complex series of chemical and cellular intermediates (Greene et al., 1992). The elevated level of oxidative stress may attribute to vascular impairment and neuronal damage via oxidation of lipoprotein of cellular membrane leading to impaired neural function and decreased nerve conduction velocity with increased sensitivity to painful stimuli (Baydas et al., 2005; Serpell, 2006). Therefore, the use of potent antioxidant like α-lipoic acid, acetyl-l-carnitine, benfotiamine, methylcobalamin, and topical capsaicin with free radical scavenging potential can be effective in the treatment of DN (Head, 2006).

SOD is an essential antioxidant enzyme that provides protection against highly reactive superoxide anions () and converts them to H₂O₂ in turn reduced oxidative stress (Gosavi et al., 2012; Halliwell, 1991; Kandhare et al., 2011a). SOD is responsible for maintenance of redox balance in neuron as well as vascular endothelial damage. An elevated glucose level caused oxidation of NADP⁺/NADPH thus brings out reduction in SOD activity (Low & Nickander, 1991; Nagamatsu et al., 1995) in turn activates AR and protein kinase C thus resulted in pain perception. Formation of sorbitol and fructose occurred via activation of NADPH, but competitive antagonism in between NADPH with GSSG–reductase reduced the availability of GSH which is an important enzyme that provides protection to sulfahydryl group of cysteine in proteins and thus cell damage (Kandhare et al., 2013b, 2012d). In hyperglycemic condition, the activity of neural SOD as well as GSH was significantly decreased in diabetic animals (Arai et al., 1987). Similar findings were observed in our study where the activity of SOD and GSH was significantly decreased after 8 weeks of STZ. Hence, diminished activity of SOD and GSH makes sciatic nerve more prone towards oxidative stress. Treatment with hesperidin significantly elevated decreased activity of SOD and GSH in sciatic nerve of rats. The result of the present investigation are in accordance with the findings of previous workers who showed that hesperidin treatment improved GSH levels in liver and kidney of diabetic rats. Jean and Bodinier (1994) reported the antioxidant potential of hesperidin in zymosan-stimulated human polymorphonuclear neutrophils in vitro.

It has been reported that increased activity of MDA is responsible for destruction of cell membranes and nerve damage via changing the arrangement of double bonds of unsaturated fatty acid of lipid membrane (Patil et al., 2012b,c). In the diabetic condition, post-translational modifications of various antioxidants plays a vital role in its potential. As the duration of diabetes is increased, the activity of antioxidant enzymes like SOD and GSH is decreased whereas the activity of MDA is increased in the sciatic nerve (Kishi et al., 2000). Similar findings were observed in the present investigation where STZ control rats exhibit decreased SOD as well as GSH activity and elevated MDA activity in peripheral nerve tissue. In the present study, treatment with hesperidin significantly attenuated elevated levels of MDA which might be due to its potential to provide more substrate for ROS that are responsible for cellular toxicity.

Cook and Samman (1996) reported that flavonoids with the molecular structure of a C-4 carbonyl group and a C-3 or C-5 hydroxyl group have more antioxidant potential due to its ability to form complexes with metal ions. Hesperidin, (2 S)-5-hydroxy-2 -(3-hydroxy-4-methoxyphenyl)-7 -{(2 S,3 R,4 S,5 S,6 R)-3,4,5-trihydroxy-6 -(((2 R,3 R,4 R,5 R,6 S)-3,4,5-trihydroxy-6-methyloxan-2-yl) oxymethyl)oxan-2-yl)oxy-2,3-dihydrochromen-4-one is a plant flavonoid with a C-5 hydroxyl group at A ring and a C-3′ hydroxyl group at B ring (Figure 5). This structural property of hesperidin may contribute to free radical scavenging activity and exert its neuroprotective potential.

Figure 5. Structure of the flavonoid hesperidin.

NO is an unconventional intracellular messenger playing a vital role in various pathological and physiological processes (Patil et al., 2012a,d). But, increased levels of NO may lead to neuronal damage (Zochodne & Levy, 2005). Peroxynitrite is formed when NO reacts with free radicals including superoxides and hydroxyl radicals that leads to protein nitration, lipid peroxidation, DNA damage, and cell death. It also is responsible for impaired vascular relaxation of epineural arterioles of sciatic nerve in diabetic rats (Coppey et al., 2001; Nishikawa et al., 2000). Together, this leads to the development of neuropathic pain (Kim et al., 2003). In STZ-induced DN, elevated levels of NO are a hallmark of nitrosative stress which was increased after 8 weeks of STZ administration (Kuhad & Chopra, 2009). However, treatment with hesperidin significantly restored elevated level of NO. Recent studies by Kaur et al. (2006) and Lee et al. (2004) reported protective effect of hesperidin hepatic as well as intestinal dysfunction possibly through down-regulation of NO; this investigation agrees with previous studies.

Increased levels of tissue calcium results in exitotoxicity accompanying unfettered oxidative stress (Kandhare et al., 2012c). Elevated calcium along with released free radicals cause a vicious cycle of biochemical changes resulting in the degeneration of axonal cytoskeleton and neuronal dysfunction. The results of the present investigation are in accordance with the previous study carried out in our laboratory (Kandhare et al., 2012b,c,e). Rats treated with hesperidin significantly attenuated elevated levels of neural calcium levels to prevent neural damage.

DN is associated with decreased nerve blood flow leading to endoneurial hypoxia and impaired nerve conduction velocity (Coppey et al., 2001; Low & Nickander, 1991). Hypertension is a hallmark of metabolic and insulin resistance syndrome that has been reported to contribute in morbidity and mortality during cardiovascular disease. Hence, in the present study, we investigate the cardiovascular effects of hesperidin in systolic, diastolic, mean arterial blood pressure, and heart rate after 4 weeks of treatment. Treatment with hesperidin significantly attenuated elevated levels of SBP, DBP, and MABP. This correction in hemodynamic parameters reflected in improved nerve conduction velocity as well as increased insulin level. The result of the present investigation is in accordance with the findings of previous researchers who reported efficacy of hesperidin in myocardial improvement in diabetic rats (Shah & Patel, 2010).

TNF-α and IL-1β are two important pro-inflammatory cytokines that have been reported to produce and release via glial cells by activation of the innate immune response. Release of TNF-α after tissue injury in turn triggers a cascade of interleukin like IL-1β, IL-6, and IL-8 (Dobretsov et al., 2007). Research carried out by various authors has shown the induction of thermal hyperalgesia and mechanical allodynia via release of TNF-α (Sorkin & Doom, 2001). In STZ-induced DN, TNF-α and IL-1β played a pivotal role in elevated hyperalgesia via central sensitization (Gustafson-Vickers et al., 2008). IL-1β which is synthesized as 31 kDa precursor peptides converted into mature cytokine of 17 kDa triggers inflammatory response resulted in tissue necrosis (Dinarello, 2009; Gabay et al., 2010). It has been also reported that over-expression of cyclo-oxygenase-2 caused release of IL-1β that is responsible for inflammatory pain hypersensitivity (Samad et al., 2001). Increased production of TNF-α and IL-1β is responsible for the development and maintenance of diabetic polyneuropathy via increased microvascular permeability and nerve damage. Hence, the use of agents including ghrelin, quercetin, curcumin, etc., that suppress cytokine elevation, have been advocated to be used to treat diabetic complications (Guneli et al., 2007). In the present study, rats treated with hesperidin significantly attenuated up-regulation of TNF-α and IL-1β mRNA expression in sciatic nerve of diabetic rats. Findings of the present investigation are in line with previous research carried out by Shi et al. (2012) which showed that levels of TNF-α as well as IL-1β were significantly ameliorated in the retina of STZ -induced diabetic rats.

Rapid production and release of pro-inflammatory cytokines caused infiltration of inflammatory cells including neutrophils and monocytes into sciatic nerve of diabetic rats. The inflammatory cells are responsible for reduction in nociceptive hypersensitivity and alleviate neuropathic pain (Perkins & Tracey, 2000). The results suggest an important role of TNF-α and IL-1β in the development of neuropathic pain in animal models (Scholz & Woolf, 2007; Verri et al., 2006). Hence, many therapeutic strategies have been implicated for the reduction of neutrophil recruitment in peripheral nerve to provide better functional outcomes in DN. Histological finding indicated that sciatic nerve of hesperidin-treated rats showed attenuation of recruitment of inflammatory cells after peripheral nerve injury that may inhibit development of DN.

Current therapeutic regimens for the treatment of DN have not advanced, and there are no approved therapies to improve the long-term prognosis of peripheral nerve injury. Clinically, it has been proven that oxidative stress is a major mediator which plays an important role in the development and maintenance of DN. Many similar studies involving individual or mixtures of antioxidant therapies with trace elements and vitamins have been carried out (Opara, 2002). Huang and Appel (2003) also reported the antioxidant potential of α-tocopherol in clinical settings. An array of antioxidants including quercetin and α-lipoic acid has been successfully used in clinical trials for treatment of DN (Valensi et al., 2005; Ziegler et al., 1999). Hesperidin has now successfully entered into clinical trials for vascular-protective effects (Milenkovic et al., 2011). It has been proven that 500 mg daily oral administration of hesperidin for 3 weeks improved endothelial function to exert its vasculoprotective actions via reducing inflammatory markers including eNOs, AMPK, and TNF-α (Rizza et al., 2011).

In the present study, individual treatment with hesperidin showed partial attenuation in neuropathic pain; however, in combination with insulin, hesperidin not only attenuated the diabetic condition but also reversed neuropathic pain. Therefore, from the results of the present investigation, it can be concluded that hesperidin, a plant flavonoid, exerts its neuroprotective effect via control over hyperglycemia as well as hyperlipidemia and thus down-regulates generation of free radical, release of cytokines (TNF-α and IL-1β) as well as elevation in membrane bound enzyme to correct allodynia and hyperalgesia.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Acknowledgements

The authors acknowledge Dr. S.S. Kadam, Vice chancellor, Bharati Vidyapeeth University and Dr. K.R. Mahadik, Principal, Poona College of Pharmacy for keen interest and providing the necessary facilities to carry out the study.