INTRODUCTION:

Peripheral neuropathic pain (NP) is a spontaneous pain characterized by foot mechanical/cold allodynia, paresthesia or dysesthesia, motor and cognitive deficits ^[1]. NP is commonly seen with diseases such as cancer chemotherapy^[2]and diabetes^[3]. The pathophysiology of NP is complex in nature with multiple events such as excitotoxicity, oxidative stress and inflammation in the synaptic cleft of neurons^[4].

N-methyl D-aspartate (NMDA) receptor activation is the major contributor for excitotoxicity NR2B subtype of NMDA receptor, is postulated in the molecular pathogenesis of various neurological disorders^[5,6]. It is treated with the conventional drugs such as opioids, antidepressants, antiepileptic drugs adverse effects are major limitations for their use^[7]. Therefore, alternative therapies especially with natural products have been gaining importance now-days for various diseases including neuropathic pain^[8].

Papain (PN), a cysteine protease found in Carica papaya reported with antioxidant activity^[9]. PN has proven efficient in maintaining the intact morphology of cortical rat neurons^[10]. Hence, we proposed to evaluate the potency of Papain for NR2B antagonism by in-silico docking studies and its neuroprotective action against PSNL induced oxidative neuronal damage in rats.

MATERIALS AND METHODS:

In-silico docking studies:

In silico analysis was performed in Dell Precision T7610 workstation (8 processors; 8 GB RAM; ZOTAC 3GB graphics; Maestro 9.8, Schrodinger, New York, U.S.A) workstation running on Redhat 6.1 Linux environment.

Ligand Preparation:

Molecular docking studies were performed against target protein NR2B (NMDA) receptor. The structure of ligand was downloaded from PubChem. The 3D coordinate file of target protein was retrieved from protein data bank (PDB ID: 5EWJ).

Protein Preparation:

The protein was prepared with the help of Protein Preparation Wizard of Schrödinger Suite 9.8. The prepared protein was optimized and minimized using algorithm OPLS_2005 (optimized potential for liquid simulations) force field and grid was generated using the Glide Grid Generation panel in Glide. Grid was generated around the co-crystal ligand of the target protein.

The structure of test ligand was downloaded from PubChem. The known inhibitor co-crystal ligand 4-[(1R, 2S)-2-(4-benzylpiperidin-1-yl)-1-hydroxypropyl] phenol ^[11] and test compound PN along with PREG were minimized using Lig Prep module. The minimized test compound PN were docked in to the receptor grid using Glide XP docking calculations. The molecular orientation and docking scores were compared with the co-crystal ligand and standard drug Pregabalin. The XP Glide scoring function was used to get the best ranked compounds and the specific interactions like H-bonds, and van der Waals were analyzed using XP visualizer in Glide module^[12,13].

In-vivo neuroprotective studies:

Drugs and Chemicals:

Papain (PN) was obtained from Enzyme Biosciences, Gujarat, India. All the chemicals used were of analytical grade and were obtained from Sigma and S.D. Fine Chemicals.

Animals:

Male albino rats weighing 150–200g were used for the study. Animals were acclimatized under laboratory conditions for 2 weeks prior to the experimentation after Institutional Animal Ethics Committee with No. BRWN/IAEC/2014 dated 20-3-2014. They were maintained under standard conditions of temperature (23±2°C) and humidity (50±10%) with an alternating 12 h light/dark cycles. The animals had free access to tap water and fed standard pellet diet (Agro Corporation Private Ltd., Bangalore, India).

Study protocol:

Rats were randomly divided into six groups of six animals in each group, group-I (Sham control), group -II (PSNL control) received vehicle only, group -III received Pregabalin 10mg/kg orally (standard control), group IV and V received PN 50 and 100mg/kg p.o respectively, group VI received PN 100mg/kg alone (without surgery). The animals underwent PSNL surgery except group I and group VI animals. All the animals were received their respective treatment for a period of 21 days.

Surgery:

Partial sciatic nerve ligation was done under general anesthesia with thiopental sodium 35mg/kg, i.p. Briefly, the rats were placed in prone position, an incision was made starting 0.5cm laterally from the animal’s midline and extended laterally for 3cm towards the tibio femoral articulation. The femoral biceps and gluteus muscles were separated using blunt dissection forceps to allow access to provide exposure to sciatic nerve and half of the left sciatic nerve was ligated at the upper thigh level using an 8-0 nylon suture. Sham surgery was done by exposing the sciatic nerve without ligation^[14].

Assessment of behavioral changes:

Behavioral changes were assessed on 0^th, 7^th, 14^th and 21^st day intervals. On 21^st day, after behavioral assessment, the animals were sacrificed by cervical decapitation, sciatic nerve was collected, homogenized and supernatant was used for biochemical studies.

Foot deformity score:

The foot deformation in ligated and drug treated groups were assessed by score. The rat was placed on a plate with a neutral temperature and the posture of the foot was observed. The foot deformation was scored as follows: Score 0 if the paw is in normal position with fanned toes, Score 1 if the toe is ventroflexed, Score 2 if the paw is averted so that only the internal edge of the paw touches the floor^[15].

Cold allodynia test:

Cold allodynia test was performed as per the method described by Naik et al.,^[16]. In this method, the left hind paw of the rat was gently submerged in ice cold water (4 ±1⁰C). The paw withdrawal latency was observed with a maximum cutoff time of 20 S.

Motor co-ordination test:

Motor co-ordination was evaluated by a Rota rod device as described by Jones and Roberts,^[17]. Briefly, rats were placed individually on the rotating rod (25rpm). The time taken by the rat to fall off from the rotating rod was recorded with a cut off time of 120 S.

Assessment of Biochemical Changes:

On 21^st day, animals were sacrificed by cervical dislocation and sciatic nerve was immediately isolated, homogenized with 0.1 M Tris–HCl buffer (pH 7.4,) and supernatant of homogenate was used for the estimation of levels of superoxide dismutase (SOD)^[18], catalase (CAT)^[19], malondialdehyde (MDA)^[20] and glutathione reductase (GR)^[21].

Histopathology:

Samples of sciatic nerve were kept in the fixative solution (10% formalin) and 4-μm thickness slices were performed and stained with hematoxylin and eosin as described by Yukari et al.,^[23]. Nerve sections were analyzed qualitatively under light microscope for axonal degeneration.

Statistical analysis:

All the data were expressed as mean ± standard error of mean (S.E.M) and analyzed using one-way ANOVA followed by post hoc analysis of Dunnett’s test. A value of p <0.05 was considered to be statistically significant.

RESULTS:

In-silico molecular docking studies:

In order to understand the probable binding mode of the compounds, molecular docking was performed against target protein NR2B (NMDA) receptor. The target enzyme was confirmed from X-ray having resolution of 2.77 Å and it was used for docking studies in reported literature. In-silico studies, test compound Papain showed good binding energy to the target compared to co-crystal ligand 4-[(1R,2S)-2-(4-benzylpiperidin-1-yl)-1-hydroxypropyl]phenol. The docking results were drawn based on the docking score, hydrogen bonding and van der Waals interactions of the ligand with the enzyme (Fig. 1). The co-crystal ligand is making hydrogen bonding with Ser132, Glu236 and Gln110, it is also involved in π-π interactions with Phe114, Arg115 and Phe176. Similarly, the test compound Papain is retained the hydrogen bonds with residues Ser132, Gln110 and π- π interaction with Phe176. Additionally it is also forming hydrogen bonding with Tyr109, Arg115 and Glu106.The standard drug pregabalin is able to form hydrogen bond with the Ile133 and Leu135 residues. Thus, the activity of test compounds could be due to the inhibition of NR2B receptor (Fig 1).

Effect of PN on foot deformity score:

Foot deformity was significantly (p<0.001) increased in PSNL group as compared with sham operated group. PREG 10mg/kg, PN 50 and 100 mg/kg treated rats showed significant (p<0.001, p<0.01, p<0.001) improve in foot deformity score when compared with PSNL control group (Fig 2A). There was no significant difference between PN 100 mg/kg alone treated group and sham group.

Fig 1. In-silico molecular docking studies of papain with NR2B ligand of NMDA receptors

Docking of a) co-crystal ligand, b) Papain and c) Pregabalin into the active site of NR2B receptor. Interactions between the inhibitor (Yellow color) and the protein (Wheat color) are signified by magenta dashed lines.

Fig 2. Effect of Papain on PSNL induced behavioral changes

Effect of Papain on PSNL induced changes in foot deformity score (2A), Cold allodynia (2B), Motor Coordination (2C). Values expressed as Mean ± SEM (n=6), Analyzed by one-way ANOVA followed by post hoc Dunnett’s test **, ***, (p<0.01), (p<0.001) Vs Sham Control group, ++, +++, (p<0.01), (p<0.001) Vs PSNL Control group

Effect of PN on cold allodynia:

Paw withdrawal latency time was significantly (p<0.001) decreased in PSNL group as compared with sham group, whereas PREG 10 mg/kg and PN 100 mg/kg treated rats showed significant (p<0.05) reversal of the values from 7^th day onwards when compared with PSNL control group (Fig 2B). We found no significant difference between PN 100 mg/kg alone treated group and sham group.

Effect of PN on motor co-ordination:

PSNL induced significantly (p<0.001) decreased motor performance when compared with sham control group. PREG 10 mg/kg, PN 50 and 100 mg/kg post treated groups showed significantly (p<0.01) restored motor performance when compared with PSNL control group (Fig 2C). We observed no significant difference between PN 100 mg/kg alone treated group and sham group.

Effect of PN on Biochemical levels:

SOD, CAT and GR levels were found significantly (p<0.001) decreased in the PSNL group as compared with the sham group. PREG 10 mg/kg PN 50 and 100 mg/kg treated groups showed significant (p<0.01) restoration in SOD levels (Fig 3A), CAT levels (Fig 3B), GR levels (Fig 3C) when compared with PSNL control group.

A significant increase (p<0.001) in the content of MDA was observed in PSNL group as compared with sham group. PREG 10 mg/kg, PN 50 mg/kg and 100 mg/kg treated groups has showed significantly (p<0.05) reversed PSNL induced increase in the MDA levels as compared with PSNL group (Fig 3D). We found no significant difference between sham control and PN 100 mg/kg alone treated group.

Fig 3. Effect of Papain on PSNL induced biochemical changes

Effect of PN on SOD Levels (3A), CAT levels (3B), GR levels (3C) and MDA levels (3D).

Values expressed as Mean ± SEM (n=6), Analyzed by one-way ANOVA followed by post hoc Dunnett’s test, *, ***, (p<0.05), (p<0.001) Vs Sham Control group; ++, +++, (p<0.01), (p<0.001) Vs PSNL Control group

Histological studies:

Sciatic nerve from PSNL group showed severe structural damage which indicate degeneration like necrotic changes (at magnification 40X). A regenerative change in sciatic nerve takes place and it shows similar to normal cyto-architecture of tissue in PN 50 and 100 mg/kg treated groups compared to PSNL group (Fig 4).

Fig 4. Effect of PN on histological changes in sciatic nerve

AX-Axon, C-Congestion, DC-Degenerative Changes, MY-Myelin Sheet, NC-Necrotic Change, NU-Perineurial Cell Nuclei, PE-Perineurium.

DISCUSSION:

The pathogenesis of neuropathic pain involves multiple pathological consequences such as excitotoxicity, oxidative stress, inflammation and apoptosis. The excitotoxicity is due to the over activation of glutaminergic mediated NMDA receptor activation, which is responsible for sensitization of nociceptive signaling pathway in neurons peripherally or centrally ^{[4, 24]}. In-silico molecular docking studies revealed that Papain perfectly fits into the inhibitory binding pocket of NR2B protein particularly of NMDA receptor which is involved in nociception and may be the contributing factor for the neuroprotective activity of PN against PSNL induced neuropathic pain in rats.

PSNL is one of the best model to study the neuropathic pain. PSNL causes neurodegeneration via excitotoxicity, oxidative stress and inflammation. It alters the neurovascular system leading to enhance the neuropathic pain symptoms ^[25,26]. In our study, we observed PSNL induced neurodegeneration as evident from foot deformity, cold allodynia and motor coordination which was significantly restored to normal condition with the post treatment of PN 100 mg/kg and PREG 10 mg/kg. Our results are inconsistent with the previous reports^{[27, 4]}.

Several studies reported that free radical mediated oxidative stress and inflammation together play a major role in the pathogenesis of neurodegenerative diseases including neuropathic pain^[28-30]. Evidence supports that increased extracellular glutamate levels activates several intracellular pathways including ROS formation; this change in redox status promotes a leukocyte mediated pro-inflammatory response that ultimately leads to the exacerbation of secondary damage^[31]. Abundant accumulation of cytosolic free radicals is known to activate lipid peroxidation of the neuronal membrane. In this sense, some studies have implicated the lipid peroxidation byproduct causing damage to biomolecules and altering several cellular processes in neurons, including mitochondria functionality in many neuropathological diseases^[32,33]. In our study, lipid peroxide levels were measured in terms of malondialdehyde which was significantly reversed in PSNL group and was observed reduced in PREG 10 mg/kg, PN 100mg/kg treated animals indicating that PN significantly attenuated PSNL induced change in MDA levels.

Physiological functions such as mitochondrial function and phagocytic activity produce excessive ROS, which are quenched by defensive antioxidant mechanism (SOD, CAT and GR) in the body. There is a direct relationship between increased levels of ROS and inflammatory hyperalgesia^[34]. Scientific evidence reported the use of antioxidant supplementation in several pathological conditions for preventive and therapeutic uses^[35]. Antioxidants have been reported to reduce not only oxidative stress but also inflammatory response and pain in several diseases. Our study revealed that PN 100 mg/kg and PREG 10 mg/kg post treatment significantly increased the levels of SOD, CAT and GR as compared with PSNL group which was inconsistent with previous reports^[29,30]. It has been demonstrated that conventional drugs used for the treatment of NP have significant adverse events which redirects the use of antioxidant supplementation as an alternative therapy clinically. Several natural compounds such as curcumin ^[36], α-tocopherol^[37], quercetin^[38] have exhibited antinociceptive effects in different models of neuropathic pain through antioxidant and anti-inflammatory mechanism. Interestingly, in our study PN exhibited significant protective effect through attenuation of oxidative stress parameters. The results were confirmed with the histological changes in which PN 100 mg/kg and PREG 10mg/kg rat nerve showed normal architecture and regenerative changes.

CONCLUSION:

From the results, it was concluded that Papain attenuated PSNL induced behavioral and biochemical changes as evident by histopathological studies. Further studies are warranted to study the in-depth mechanism in the neuroprotective effect of papain.

CONFLICTS OF INTEREST:

The authors declared that they don’t have any conflicts of interest.

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Received on 26.09.2019 Modified on 30.11.2019

Research J. Pharm. and Tech. 2020; 13(8):3807-3812.

DOI: 10.5958/0974-360X.2020.00674.5