Escin

Escin alleviates peripheral neuropathy in streptozotocin induced diabetes in rats

Sachin V. Suryavanshi, Yogesh A. Kulkarni

Abstract

Aim: Inflammatory cascade and oxidative stress play a central role in diabetic peripheral neuropathy via activation of inflammatory cytokines. Escin has potent antioxidant and anti- inflammatory properties. Hence, the present study was conducted to evaluate the effect of escin on diabetic peripheral neuropathy in streptozotocin (STZ) induced diabetes in rats.
Main methods: Diabetes was induced in rats with streptozotocin (55 mg/kg). The animals with blood glucose above 250 mg/dl were randomized in different groups. Animals were treated with escin at a dose of 5, 10 and 20 mg/kg after six weeks of diabetes induction for the next four weeks. After completion of treatment, various parameters like glucose, thermal hyperalgesia, mechanical hyperalgesia, mechanical allodynia and nerve conduction velocities were evaluated. Oxidative stress parameters like malonaldehyde, catalase, reduced glutathione and superoxide dismutase were performed in sciatic nerves. Histopathology study of sciatic nerves was also studied.
Key findings: Escin treatment significantly reduced plasma glucose, thermal hyperalgesia, mechanical hyperalgesia and mechanical allodynia as compared to diabetic animals. The motor nerve conduction velocity and sensory nerve conduction velocities were significantly improved

in diabetic animals treated with escin. Escin significantly normalized oxidative stress parameters. Escin treatment also prevented progression of neuronal damage by reducing demyelination, leukocytic infiltration in sciatic nerves as compared to diabetic animals.
Significance: From the results of study it can be concluded that escin can be a useful option for management of diabetic peripheral neuropathy.
Keywords: Escin, Pentacyclic triterpenoids, diabetic neuropathy, nuclear factor – kappa beta, Aesculus
Introduction

Prolonged uncontrolled hyperglycemic condition results in microvascular and macro vascular complications involving kidney, eyes, nerves and heart. Diabetic neuropathy (DN) is attributed to an imbalance between nerve fiber damage and repair. The damage to autonomic and distal sensory fibers, results in progressive loss of sensation [1]. Diabetic neuropathy is one of the important complications of diabetes and major cause of mortality. Autonomic neuropathy and peripheral neuropathy are the most common types of neuropathies in diabetic patients [2]. Diabetic peripheral neuropathy (DPN) is one of the most common complications in diabetic patients. The global prevalence of diabetic peripheral neuropathy ranges from 16% to 66% [3]. The foot amputation which is severe form of peripheral neuropathy is 10-20 times more common in diabetic people compared to nondiabetic [3]. It is characterized by loss of sensory and motor nerves. At cellular level, pathological changes like axonal thickening, narrowing of neuronal capillary demyelination of nerves and loss of nerve fibers are observed [4]. Increased advanced glycation end products (AGEs), glycated hemoglobin (HbAc1), matrix metalloproteinase (MMP) 9 and oxidative stress in peripheral nerves overexpresses NF-κβ and causes neuronal damage [5].
Recent studies have revealed that triterpenes possess antidiabetic activity with several mechanisms. Triterpenes inhibit progression of insulin resistance and regulate glucose metabolism [6]. They possess potent antioxidant activity and inhibit AGEs [6]. Escin is a natural mixture of pentacyclic triterpenoid saponins present in plants like Aesculus hippocastanum and Aesculus indica [7]. Escin has been studied for its beneficial effects in treatment of varicose vein [8], anti-edematous property [9], NF-κβ inhibitory [10], anti-cancer [11], anti-inflammatory [12],and anti-apoptotic activity [13]. Hence, the present study was designed to study the effect of escin in diabetic neuropathy in rats.
Materials and methods

Drugs and chemicals
Escin, streptozotocin, 2-thiobarbituric acid, reduced glutathione, 1,1,3,3-tetramethoxypropane, and nitrobluetetrazolium were procured from Sigma Aldrich (St. Louis, USA).
Experimental Animals
The study was carried out on Male Sprague Dawley (SD) rats. The rats weighing between 180– 230 g were procured from the National Institute of Biosciences, Pune, India. Animals were housed under standard conditions (Temperature 22±2˚C, Humidity 75±5%, 12 h light and 12 h dark cycle) in animal facility throughout the study. A basal nutritional diet (Nutrivet life sciences, Pune, India) and purified water were provided ad libitum. The animals were allowed to acclimatize for one week to environment before starting the experiment. The experimental protocol was approved by the Institutional Animal Ethics Committee which was formed in accordance with norms of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. All animal experimentation procedures were performed as per NIH guidelines for handling of experimental animals [14].
Induction of diabetes and treatment
Diabetes was induced with a single intraperitoneal injection of STZ (55 mg/kg) dissolved in ice- cold citrate buffer (0.1 M, pH- 4.4) [4,15]. Plasma glucose level was measured 48 h after STZ administration to confirm induction of diabetes. Animals with plasma glucose levels above 250 mg/dl were considered as diabetic and were selected for further study. After six weeks, diabetic animals were randomized in different groups according to their plasma glucose level and body weight. The diabetic control group also received vehicle. One group of diabetic animals was kept as standard control and orally received pregabalin (30 mg/kg) with gavage once a day. Other groups of diabetic animals orally received escin at dose 5, 10 and 20 mg/kg once a day. One group of non-diabetic animals was kept as normal control and received vehicle. Distilled water was used as vehicle for administration of dose in all groups (10 ml/kg of body weight). Treatment was given for four weeks to all groups.

Evaluation parameters

Body weight and plasma glucose level
The body weight was measured at the end of study. One ml blood was collected by retro-orbital puncture method in centrifuge tubes containing 15 μl of 10% w/v disodium salt of ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Whole blood was centrifuged at 2500 rpm for 15 minutes at 4°C to separate the plasma and stored at temperature -20°C till further study. Plasma glucose level was measured using commercially available kits (Transasia Biomedicals Ltd. India)
Hot plate test
Eddy’s hot plate test was carried out to study the effect of treatment on thermal nociceptive threshold in diabetic rats. After 28 days of treatment, animals were placed on a hot plate (IITC Life Sciences, USA) maintained at 55±0.5°C. The time required for the first response (Flickering, licking of hind paw or jumping) was recorded. The cut off time was set to 15 seconds to avoid tissue damage [16].
Tail immersion test
The tail immersion test was carried out on 28th day of treatment to determine thermal nociceptive threshold. Animals were acclimatized to restrainer 30 minutes before experiment. The tail of animals was completely immersed in hot water maintained at 55±0.5°C. The time required to remove tail from hot water was considered as response time. The cut-off time for tail to hot water was set to 15 seconds to avoid tissue damage [16].
Mechanical hyperalgesia
Mechanical hyperalgesia was determined using Randall-Selitto test to determine mechanical nociceptive threshold. At the end of study, animals were restrained in cloth slings and subjected to constantly increasing pressure stimulus with pressure applicator (IITC Life Sciences, USA) to the dorsal surface of hind paw. The maximum pressure at which an animal showed response (paw withdrawal, squeaking or struggling) was recorded with digital meter (IITC Life Sciences, USA). The cut-off pressure was set to 400 g. Readings were recorded in triplicate separated by at least 20 minutes interval for each rat and mean values were considered as final readings [17].
Mechanical allodynia

Mechanical allodynia was recorded using the Von Frey test to determine nociceptive threshold to innocuous stimuli. At the end of study, animals were transferred to elevated cage with wire mesh bottom. Animals were allowed to acclimatize with cage for 20 minutes. A constantly increasing pressure stimulus was applied with electronic Von Frey pressure applicator attached with rigid tip (IITC Life Science, USA) to the ventral surface of hind paw. The maximum pressure at which animal showed response (paw withdrawal or brisk lifting) was recorded with digital meter (IITC Life Science, USA). The cut-off pressure was set to 400 g. Readings were recorded in triplicate separated by at least 20 minutes interval for each rat and mean values were considered as final readings [18].
Nerve conduction velocities
The nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) were recorded using data acquisition system (AD Instruments, Australia) in sciatica-posterior tibial conducting system. After completion of treatment, animals were anesthetized with ketamine (70 mg/kg, i.p.) and xylazine (10 mg/kg, i.m.). The body temperature was maintained at 37±0.5°C with homeothermic blanket with flexible probe (Harvard Apparatus, USA). The sciatic nerve was stimulated at sciatic notch and tibial nerve (proximal to ankle) with single stimulus (5V) using bipolar needle electrodes (24 gauge). The receiving electrodes were placed in the muscles of foot. The M-wave and H-wave reflexes were digitally recorded using data acquisition system (AD Instruments, Australia). The distance between two stimulation points was recorded manually. The MNCV was calculated from M-wave latencies and SNCV was calculated from H- wave latencies [19].
Oxidative stress parameters

At the end of study, sciatic nerves were carefully isolated and homogenized in ice-cold phosphate buffer (0.1M, pH – 7.4) by using probe homogenizer (Polytron PT 2500E, Kinematica, Switzerland). Homogenate was further divided into different aliquots to get post mitochondrial fraction and post-nuclear fraction [20]. The protein concentration was determined in tissue homogenate with method described earlier [21]. The lipid peroxidation was determined by assessing malondialdehyde (MDA) level [22]. The tissue sulfhydryl groups were determined by accessing reduced glutathione (GSH) levels [23]. Catalase activity was determined by

accessing moles of hydrogen peroxide decomposed per minute [24]. The superoxide dismutase (SOD) activity was determined as per method described earlier [25].
Histopathology Study

The sciatic nerves were carefully isolated and fixed in neutral formalin buffer and embedded in paraffin wax. Thin sections of 5 μm were taken with microtome (Leica Biosystems, Germany) and sections were stained with Mayer’s hematoxylin and eosin (HE) stain. The slides were observed for pathological changes. The slides were observed for the lesions, chromatolysis, leukocytic infiltration, neuronal swelling and degeneration, fragmentation of myelin and axon, inflammation, proliferation of Schwann cells and glial cells and neural edema. The severity lesions were scored as no abnormality detected (0), minimal changes (1), mild changes (2), moderate changes (3) and distribution was recorded as focal multifocal and diffuse.
Statistical analysis

All the data (n=6) was analyzed by one-way ANOVA following post hoc Dunnett’s multiple comparisons using graph pad prism software (version 5).
Results

Effect of escin treatment on body weight and plasma glucose levels

The diabetic animals showed significant weight loss which was decreased by treatment with escin at dose of 10 and 20 mg/kg. Pregabalin (30 mg/kg) treatment also significantly increased body weight as compared to diabetic animals (Figure 1). The diabetic animals showed increased plasma glucose levels as compared to normal animals (477.0±23.68 mg/dl v/s 81.71±6.18 mg/dl, p<0.001). Escin treatment for 28 days significantly reduced plasma glucose levels at dose of 10 mg/kg (382.7±11.72 mg/dl, p<0.01) and 20 mg/kg (350.4±23.46 mg/dl, p<0.001) when compared with diabetic animals. Escin at dose of 5 mg/kg non-significantly reduced the plasma glucose level (426.7±22.57 mg/dl) (Figure 1) Figure 1: Effect of treatment escin on body weight and plasma glucose levels All data are expressed as Mean ± SEM (n=6). ### p<0.001 when compared with normal control, **p<0.01, ***p<0.001 when compared with diabetic control. Effect of escin treatment on behavioral parameters The response latency and tail withdrawal latency was significantly decreased (p<0.001) in diabetic group in both hot plate test and tail immersion test when compared with normal group animals. This decrease in latency was significantly reversed by the four-week treatment of escin at all selected dose levels when compared with diabetic group. Paw withdrawal response was determined with Von Frey and Randall Selitto test. Diabetic animals showed significant reduction in paw withdrawal latency (64.74±3.95 g v/s 107.4±43.54 g, p<0.001) and paw withdrawal pressure (85.24±2.44 g v/s 311.8±11.50, p<0.001) when compared with normal animals. The treatment with escin significantly increased the paw withdrawal response at dose of 5 mg/kg (77.68±1.45 g, p<0.05), 10 mg/kg (97.41±3.54 g, p<0.001) and 20 mg/kg (98.95±2.54 g, p<0.001) when compared with diabetic animals. The paw withdrawal pressure was also significantly increased with treatment of escin at dose of 5 mg/kg (124.3±6.81 g, p<0.01), 10 mg/kg (154.8±6.85 g, p<0.001) and 20 mg/kg (201.8±5.37 g, p<0.001) when compared with diabetic animals. Treatment of pregabalin also showed significant increase in paw withdrawal latency and pressure (92.22±3.22 g and 190.8±.68 g respectively, p<0.001) as compared to diabetic animals. Escin treated groups at dose 10 and 20 mg/kg showed almost similar effect to that of pregabalin (30 mg/kg) treated group (Figure 2). Figure 2: Effect of escin treatment on a) Hot plate test b) tail immersion test c) Von Frey test and d) Randall Selitto Test All data are expressed as Mean ± SEM (n=6). ### p<0.001 when compared with normal control, *p<0.05, **p<0.01, ***p<0.001 when compared with diabetic control Effect of escin treatment on functional parameters The diabetic animals showed significant reduction in motor and sensory nerve conduction velocity (p<0.001) when compared with normal animals. Escin treatment for 28 days significantly increased the SNCV and MNCV in diabetic animals at dose of 10 mg/kg (p<0.01) and 20 mg/kg (p<0.001) when compared with diabetic control group. Escin at dose of 5 mg/kg did not show any significant increase in MNCV and SNCV. The standard drug showed significant increase in nerve conduction velocity (p<0.001). The high dose of escin showed almost similar effect as that of standard drug pregabalin (Figure 3). The representative graphs have been given in figure 4. Figure 3: Effect of escin treatment on nerve conduction velocity All data are expressed as Mean ± SEM (n=6). ### p<0.001 when compared with normal control, **p<0.01, ***p<0.001 when compared with diabetic control. Figure 4: Representative graphs of nerve conduction velocity. a) Normal control, b) Diabetic control, c) Diabetic+Pregabalin (30 mg/kg), d) Diabetic+Escin (5 mg/kg), e) Diabetic+Escin (10 mg/kg), f) Diabetic+Escin (20 mg/kg) Effect of escin treatment on oxidative stress parameters The oxidative stress in the sciatic nerves was significantly increased diabetic group which was indicated by significant increase in MDA and decrease in levels of GSH, SOD and catalase in diabetic animals when compared with normal animals. The MDA level was significantly reduced in escin treated animals at dose of 10 mg/kg (p<0.01) and 20 mg/kg (p<0.001) when compared to diabetic animals. Similarly, the GSH level was also significantly increased with escin treatment at dose of 10 mg/kg (p<0.05) and 20 mg/kg (p<0.01). The catalase and SOD levels were also significantly increased with escin treatment when compared with diabetic control. Table 1: Effect of escin treatment on oxidative stress parameters in sciatic nerve Normal Control Diabetic Control Diabetic + Pregabalin (30 mg/kg) Diabetic + Escin (5 mg/kg) Diabetic + Escin (10 mg/kg) Diabetic + Escin (20 mg/kg) MDA (nmol/mg 0.57±0.02 0.84±0.05# ## 0.66±0.04* 0.72±0.03 0.64±0.04** 0.61±0.04** protein) GSH (μmole/m 86.42±3.79 53.82±4.0 4### 71.15±2.90 ** 64.95±3.75 70.72±3.46* 72.92±3.51** g protein) Catalase (nmole of 59.02±2.68 28.22±2.3 1### 51.18±3.11 *** 34.83±1.92 48.20±3.91* * 56.58±5.29*** H2O2 decompos ed/ min/mg protein) SOD (unit/mg 4.88±0.39 2.25±0.16# ## 4.03±0.23* ** 3.27±0.21* 3.63±0.20** 4.13±0.30*** protein) All data are expressed as Mean ± SEM (n=6). ### p<0.001 when compared with normal control, *p<0.05, **p<0.01, ***p<0.001 when compared with diabetic control Effect of escin treatment on histopathology of sciatic nerves Histopathological study of sciatic nerve of different treated groups showed various lesions such as focal minimal to mild lymphocytic infiltration (Normal control: 0/3, Diabetic control: 2/3, Pregabalin 30 mg/kg: 0/3, escin 5 mg/kg: 2/3, Escin 10 mg/kg: 1/3, escin 20 mg/kg: 0/3), focal to multifocal and minimal to moderate axonal degeneration (Normal control: 0/3, Diabetic control: 3/3, Pregabalin 30 mg/kg: 0/3, escin 5 mg/kg: 3/3, Escin 10 mg/kg: 1/3, escin 20 mg/kg: 0/3), focal minimal to mild axonal swelling (Normal control: 0/3, Diabetic control: 2/3, Pregabalin 30 mg/kg: 0/3, escin 5 mg/kg: 2/3, Escin 10 mg/kg: 0/3, escin 20 mg/kg: 0/3). The diabetic animals showed minimal to moderate neuropathic lesions. Treatment with pregabalin and escin at dose of 10 and 20 mg/kg prevented the neuropathic lesions in sciatic nerves. However, escin at dose of 5 mg/kg did not alter the neuropathic lesions (Figure 5). Figure 5: Effect of escin treatment on sciatic nerves- H&E staining (400X) a) Normal Control: showing normal histology, myelin sheath (MS), schwann cells (SC), axon (A); b) Diabetic Control: showing degeneration of axon (AD), axonal swelling (AS), lymphocytic infiltration (L); c) Pregabalin (30 mg/kg): showing normal histology, myelin sheath (MS), schwann cells (SC), axon (A); d) Escin (5 mg/kg): showing degeneration of axon (AD), axonal swelling (AS), lymphocytic infiltration (L); e) Escin (10 mg/kg): showing degeneration of axon (AD), lymphocytic infiltration (L); f) Escin (20 mg/kg): showing normal histology, myelin sheath (MS), schwann cells (SC), axon (A). Discussion Diabetic complications like neuropathy, nephropathy, retinopathy and cardiomyopathy are the consequences of uncontrolled glycemia, increased advanced glycation end products (AGEs) and receptors for AGEs (RAGE) and stress-induced inflammatory cascade. Diabetic peripheral neuropathy is stubborn microvascular complication characterized by reduction in vibration perception and nerve conduction velocity [26]. Though the exact mechanism of diabetic peripheral neuropathy is poorly understood, it is majorly linked with increased oxidative stress and polyols [27]. Other risk factors involved in peripheral nerve injury are accumulation of glycated hemoglobin (HbAc1), and collagen in peripheral nerves [28]. This further causes neuronal injury by initiating apoptotic and inflammatory cascade through activation of inflammatory and apoptotic mediators like NF-κβ, Tumor necrosis factor (TNF-α) [26]. On other hand, high polyol flux triggers accumulation of sorbitol and reactive oxygen species (ROS) and affects nerve conduction velocities [5]. Overexpression of NF -κβ plays central role in neuronal injury by activating inflammatory mediators like tumor growth factor (TGF-β), TNF-α, interleukins (IL) -6. The arachidonic acid pathway is also activated in peripheral nerves and COX-2 in peripheral nerves are increased in response to NF –κβ activation [5]. In diabetes, insufficient insulin secretion of increased insulin resistance alters transport of glucose across the cell and increases blood glucose levels. This depletion in glucose transport tends to use burn reserved fats for energy leading to drastic weight loss. Hence, general body weight loss is observed in type 1 diabetic patients. Escin has property to increase the insulin secretion and thereby it helps in glucose transport across the cell membrane and reduction in plasma glucose level [29]. The reduction in the plasma glucose level with treatment of escin may due to its insulin secretory activity. Escin treatment also significantly increased body weight. Uncontrolled diabetes causes neuronal damage which result in the neuropathic pain. Neuronal dysfunction causes imbalance between non-painful and painful stimuli by damaging inhibitory pathway or overstimulation of nociceptive pathway which causes pain without involving nociceptors [30]. The hyperglycemic condition causes overexpression of NF-κβ and mitogen- activated protein kinase (MAPK) pathway leading to inflammatory cascade. Furthermore, it releases inflammatory cytokines like TGF-β, COX-2, TNF-α in sciatic nerves which causes mechanical allodynia and hyperalgesia [5,31]. Escin has potent anti-inflammatory effect without affecting immune system [32]. Escin also inhibits nociceptive pain via inhibition of inflammatory cascade [33]. Escin treatment significantly prevented increased in thermal hyperalgesia, mechanical hyperalgesia and mechanical allodynia. This reduction in neuropathic pain in escin treated diabetic animals may be through inhibition of NF-κβ pathway and thereby inflammatory cascade. Accumulation of ROS and altered polyol flux increases aldose reductase and sorbitol dehydrogenase activity in the peripheral nerves [34]. This rise in ROS and sorbitol in sciatic nerves interfere in the nerve conduction velocity by inactivating Na+/K+ ATPase activity [5]. Additionally, overexpression of NF-κβ due to increased phosphokinase C (PKC), ROS, and AGEs causes degeneration of peripheral neurons via leukocytic infiltration and decreased neuronal growth factor (NGF), TNF-α, IL6 and IL1β [35]. Escin has been proved to have anti- inflammatory and analgesic effect by inhibition of TNF-α and NF-κβ pathways [36]. Escin treatment significantly improved the motor and sensory nerve conduction velocities in diabetic rats. Escin has neuroprotective effect via inhibition of oxidative stress [37]. This may be the reason for increased nerve conduction velocities in escin treated diabetic rats. Generation of ROS in neuronal cells initiate pro-apoptotic cascade and inflammatory mediators leading to depletion in the antioxidant enzymes like malondialdehyde, glutathione, catalase and superoxide dismutase. Alteration in antioxidant enzymes causes mitochondrial dysfunction and causes demyelination of neurons and accumulation of extracellular matrix [5]. Escin treatment normalized the antioxidant enzyme levels and reduced the oxidative stress in the sciatic nerves. The normalization of oxidative enzymes prevents demyelination of nerves and provides neuroprotection. 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