|Year : 2014 | Volume
| Issue : 3 | Page : 131-138
Effect of Ashwagandha (Withania somnifera) against chronic constriction injury induced behavioral and biochemical alterations: Possible involvement of nitric oxide mechanism
Anil Kumar, Seema Meena, Raghavender Pottabathini
Department of Pharmacology, Neuropharmacology Division, University Institute of Pharmaceutical Sciences, UGC Centre for Advance Studies, Punjab University, Chandigarh, India
|Date of Submission||27-Nov-2013|
|Date of Acceptance||25-Jan-2014|
|Date of Web Publication||16-May-2014|
Neuropharmacology Division, Department of Pharmacology, University Institute of Pharmaceutical Sciences, UGC Centre for Advance Studies, Punjab University, Chandigarh 160 014
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: Neuropathic pain (NP) arises due to lesion or disease in somatosensory nervous system. Recent reports indicate the role of Withania somnifera (WS) in various inflammatory pain conditions. The objective of the present study was to explore the possible protective effect of WS against chronic constriction injury (CCI) induced NP in rats. Materials and Methods: CCI of sciatic nerve was performed in male Wistar rats. Various behavioral parameters (thermal hyperalgesia, cold allodynia) followed by biochemical parameters (lipid peroxidation, reduced glutathione, catalase and nitrite) were assessed in sciatic nerves. Drugs were administered consecutively for 21 days from the day of surgery. CCI to sciatic nerve significantly caused thermal hyperalgesia, cold allodynia and oxidative damage in the sciatic nerves when compared with naive and sham control. Results: Chronic administration of WS (100 and 200 mg/kg, p.o.) significantly attenuated hyperalgesia, cold allodynia and oxidative damage (as indicated by reduction in lipid peroxidation, nitrite concentration, restoration of reduced glutathione and catalase activity). Further, L-NAME (5 mg/kg, i.p.) pre-treatment with WS (100 mg/kg, p.o.) significantly potentiated the protective effect of WS which was significant when compared with their effect per se However, L-arginine (100 mg/kg, i.p.) pre-treatment with WS (100 mg/kg, p.o.) significantly reversed the protective effects of WS in sciatic nerve. Conclusion: Result of present study suggests that nitric oxide mechanism could be involved in the protective effect of WS against CCI induced behavior alterations and oxidative damage in rats.
Keywords: Chronic constriction injury, neuropathic pain, oxidative stress, Withania somnifera
|How to cite this article:|
Kumar A, Meena S, Pottabathini R. Effect of Ashwagandha (Withania somnifera) against chronic constriction injury induced behavioral and biochemical alterations: Possible involvement of nitric oxide mechanism. Int J Nutr Pharmacol Neurol Dis 2014;4:131-8
|How to cite this URL:|
Kumar A, Meena S, Pottabathini R. Effect of Ashwagandha (Withania somnifera) against chronic constriction injury induced behavioral and biochemical alterations: Possible involvement of nitric oxide mechanism. Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2019 Feb 19];4:131-8. Available from: http://www.ijnpnd.com/text.asp?2014/4/3/131/132664
| Introduction|| |
Neuropathic pain (NP) is one of the chronic painful complications of the nervous system. NP represents a pathological change at both peripheral and central level and involves damage to nerves by a variety of insults such as traumatic nerve injury, metabolic diseases, viral infections, or stroke etc. , Despite the extensive research progressed, pathophysiology of NP is still not completely understood. Based on various experimental models several hypotheses have been suggested such as oxidative stress, nitric oxide (NO) and neuro-inflammatory cascades to explain the pathogenesis of NP. ,,
The main source of reactive oxygen species (ROS) in-vivo is aerobic respiration, although ROS can also be produced by paroxysmal-beta oxidation of fatty acids, or stimulation of phagocytosis and arginine metabolism.  Under normal physiological conditions, excessive ROS neutralized and cleared off by the action of superoxide dismutase (SOD), catalase or glutathione, as well as antioxidant (vitamin C and E). , However, under pathological conditions increased intracellular levels of ROS can cause severe cell damage, produce malonaldehyde through peroxidation of membrane lipids and even cell death, sometimes. Obviously, repair of oxidative stress is often important and essential in restoring normal conditions.
NO, a free radical synthesized from L-arginine by calcium-dependent constitutive NO synthase (NOS) isoforms, including neuronal NOS (nNOS) and endothelial NOS, or the calcium-independent inducible NOS that require an activation by endotoxin or cytokines.  Evidence has shown the involvement of both central and peripheral NO in nociceptive processing.  It is now well accepted that NO plays a vital role in neural transmission and acts as second messenger at both central and peripheral level.  However, when NO react with superoxide radical it forms peroxynitrite which is several times more potent than their parents (superoxide and NO) in terms of tissue toxicity. It is now believed that mechanism responsible for hyperalgesia in chronic pain may involve not only NO itself, but also the product of its reaction with superoxide radicals, peroxynitrite.  However, the functional role of NO-Guanosine 3':5'- cyclic monophosphate (NO-cGMP) pathway in the spinal mechanism of chronic NP remains to be clarified.
Currently, NP has been treated symptomatically and demands urgently for effective strategies for its successful treatment and management. Withania somnifera (WS) (Dunal), popularly known as Indian Ginseng or Ashwagandha, belongs to the family Solanaceae. Earlier, it has been reported that glycowithanolides alter cortical and striatal antioxidant enzyme activities (SOD, catalase and glutathione peroxidase) in rats.  A 21 day treatment with WS root extract significantly decreased lipid peroxidation and restored both SOD and catalase , indicating its free radical scavenging activity. A number of earlier investigations have also indicated that WS exhibits a potent antioxidant, neuroprotective actions. ,, However, its role in NP conditions is not been explored yet.
Therefore, the present study has been designed to elucidate the possible role of WS and its interaction with NO modulators against chronic constriction injury (CCI) induced NP in rats.
| Materials and methods|| |
Male Wistar rats (180-200 g) bred in Central Animal House facility of the Panjab University, Chandigarh were used. Animals were acclimatized to laboratory conditions prior to experimentation. Animals were housed under standard laboratory conditions, maintained on a natural light-dark cycle and had free access to food (Ashirwad Industries, Mohali, India) and water. During the entire study period, animals were kept under 12 h light/dark cycle in a group of two rats in one plastic cage with soft bedding. All the experiments were carried out between 09:00 and 15:00 h. The protocol was approved by the Institutional Animal Ethics Committee and carried out in accordance with the Indian National Science Academy Guidelines for the use and care of experimental animals.
Induction of NP by CCI
The unilateral mononeuropathy was induced according to the modified method of Bennett and Xie.  Briefly, the rats were anesthetized using thiopental sodium (40 mg/kg, i.p.). The hair of the rat's lower back and thigh were shaved. The skin of the lateral surface of the right thigh was incised and the common sciatic nerve was exposed at the middle of the thigh by blunt dissection through the biceps femoris. Proximal to the sciatic trifurcation, approximately 7 mm of nerve was freed and four ligatures of 4-0 chromic gut were placed around the sciatic nerve with 1 mm intervals. Extra care has been given not to interrupt epineural blood flow while tying the ligatures. After performing the ligation, muscular and skin layer were sutured in two layers with thread and topical antibiotic was applied. Nociceptive threshold was assessed at weekly intervals on day 7 th , 14 th and 21 st after the surgery. In sham operated rats, the same surgical procedure was followed, the connective tissue was freed and no ligatures were applied.
Drugs and treatment schedule
Study protocol includes 11 treatment groups, consisting of 6 animals in each group. Group-1: Naive (without treatment), Group-2: Sham (sciatic nerve was exposed but not ligated, vehicle administered), Group-3: CCI group, Group-4: CCI + gabapentin (50 mg/kg, i.p.), Group-5: CCI + gabapentin (100 mg/kg, i.p.), Group-6: CCI + WS (100 mg/kg, p.o.), Group-7: CCI + WS (200 mg/kg, p.o.). Group-8: L-arginine (100 mg/kg, i.p.) + CCI, Group-9: L-arginine (100 mg/kg, i.p.) + WS (100 mg/kg, p.o.) + CCI, Group-10: L-NAME (5 mg/kg, i.p.) + CCI, Group-11: L-NAME (5 mg/kg, i.p.) + WS (100 mg/kg, p.o.) + CCI.
WS powder was purchased from Dabur India Limited, gabapentin; L-arginine and L-NAME were purchased from Sigma-Aldrich (Sigma Chemicals, St. Louis, MO, USA). All drugs were freshly dissolved in saline and were administered for respective groups, once daily for the duration of 3 weeks. However, L-arginine and L-NAME pretreatment were given 30 min before WS treatment. Drug doses selection was made on the basis of earlier reports. , All the reagents used in the present study were of analytical grade.
Hot plate test
The hyperalgesic response using hotplate is considered to result from a combination of central and peripheral mechanisms.  Thermal hyperalgesia was assessed by placing individually each animal on a hot plate (Eddy's Hot Plate), maintained at 55 ± 1°C on weekly intervals on (7 th , 14 th and 21 st day) after CCI. The latency to first sign of paw licking or jumping response to avoid thermal pain was taken as an index of pain threshold. A cut off time of 15 s was maintained throughout the experimental protocol.
Cold allodynia was assessed after 2 h of assessment of hyperalgesia by measuring ipsilateral paw withdrawal latency (PWL), ice-cold water (4°C ± 2°C) was taken in a beaker. The paws of animals were submerged gently in water and the withdrawal time was measured, on weekly intervals on (7 th , 14 th and 21 st days) after CCI. A cut off time of 15 s was maintained throughout the experimental protocol. 
Dissection and homogenization
On 21 st day, animals were sacrificed by decapitation immediately after behavioral assessments. A segment of sciatic nerve, approximately 1.5 cm in length, 5 mm proximal and 5 mm distal to the injured site was used for preparing the homogenate for biochemical estimation 10% (w/v). Tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates were centrifuged at 10000 × g for 15 min at 4°C and supernatant was used for estimation of various biochemical parameters.
Measurement of oxidative stress parameters
Lipid peroxidation assay
The quantitative measurement of lipid peroxidation was performed according to the method of Wills (1966).  The amount of malondialdehyde was measured by reaction with thiobarbituric acid 532 nm using Shimadzu spectrophotometer. The values were calculated using molar extinction coefficient of chromophore (1.56 × 10 5 /M/cm) and expressed as percentage of sham.
Estimation of reduced glutathione
Reduced glutathione was estimated according to the method described by Ellman (1959).  Briefly, 1 ml supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 4°C for 1 h. The samples were centrifuged at 1200 × g for 15 min at 4°C. To 1 ml of this supernatant, 2.7 ml of phosphate buffer (0.1 M, pH 8) and 0.2 ml of 5,5-dithiobis (2-nitrobenzoic acid) were added. The yellow color developed was read immediately at 412 nm using Shimadzu spectrophotometer. Results were calculated using molar extinction coefficient of chromophore (1.36 × 10 4 /M/cm) and expressed as percentage of sham.
Estimation of nitrite
The accumulation of nitrite in the supernatant, an indicator of the production of NO was determined with a colorimetric assay using Griess reagent (0.1% N-(1-Napthyl) ethylenediamine dihydrochloride, 1% sulfanilamide and 2.5% phosphoric acid).  Equal volumes of supernatant and Griess reagent were mixed, the mixture was incubated for 10 min at room temperature and the absorbance was measured at 540 nm using Shimadzu spectrophotometer. The concentration of nitrite in the supernatant was determined from a standard curve and expressed as percentage of sham.
Protein estimation was done by biuret method using bovine serum albumin as standard. 
Estimation of catalase
Catalase activity was assayed by method of Luck (1971),  where in the breakdown of H 2 O 2 was measured at 240 nm. Briefly, the assay mixture consisted of 3 ml of H 2 O 2 phosphate buffer (1.25 × 10 − 2 M H 2 O 2 ) and 0.05 ml of supernatant of the tissue homogenate (10%) and the change in absorbance was recorded at 240 nm using the Shimadzu spectrophotometer. Enzyme activity was calculated using the mill molar extinction coefficient of H 2 O 2 ( 0.07). The result was expressed as micromoles of H 2 O 2 decomposed/min/mg of protein.
All the values are expressed as mean ± standard error of the mean (SEM) data were analyzed by two way of analysis variance for thermal hyperalgesia and cold allodynia and one way of analysis variance followed by Tukey's test for oxidative stress measurement. In the entire tests criterion for significance was P < 0.05.
| Results|| |
Effects of gabapentin and WS on thermal hyperalgesia and cold allodynia in CCI rats
In the present study, CCI of sciatic nerve significantly (P < 0.001) caused thermal hyperalgesia (as reflected by percentage decrease in pain threshold) and cold allodynia (reduced PWL) when compared with sham or naive group. These behavioral changes were persisted up to 3 weeks. Treatment with gabapentin (50 and 100 mg/kg), significantly attenuated CCI induced thermal hyperalgesia (increased pain threshold) and cold allodynia (increased PWL) when compared with control CCI after 1 st , 2 nd and 3 rd weeks. WS (200 mg/kg) treatment significantly (P < 0.001) attenuated thermal hyperalgesia (increased pain threshold) and cold allodynia (increased PWL) when compared with control CCI on 2 nd and 3 rd week's interval [Figure 1] and [Figure 2]. However, lower dose of WS (100 mg/kg) has significant effect (P < 0.05) on 3 rd week only and did not show any significant effect on thermal hyperalgesia and cold allodynia effect when compared with control group CCI [Figure 1] and [Figure 2].
|Figure 1: Effects of nitric oxide (NO) modulators (L-NAME and L-arginine) on the protective effect of WS on thermal hyperalgesia in Chronic Constriction Injury (CCI) rats. The values are expressed as mean ± SEM. aP < 0.05 as compared to naive, bP < 0.05 as compared to sham group, cP < 0.05 as compared to control CCI group, dP < 0.05 as compared to gabapentin (GP) (50), eP < 0.05 as compared to WS (100), fP < 0.05 as compared to L-NAME (5) (Two way ANOVA followed by Bonferroni posttest)|
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|Figure 2: Effects of NO modulators (L-NAME and L-arginine) on the protective effect of WS on cold allodynia in Chronic Constriction Injury (CCI) rats. The values are expressed as mean ± SEM. aP < 0.05 as compared to naive, bP < 0.05 as compared to sham group, cP < 0.05 as compared to control CCI group, dP < 0.05 as compared to gabapentin (GP) (50), eP < 0.05 as compared to WS (100), fP < 0.05 as compared to L-NAME (5) (Two way ANOVA followed by Bonferroni posttest)|
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Effects of NO modulators (L-NAME and L-arginine) on the protective effect of WS on thermal hyperalgesia
L-NAME (5 mg/kg) or L-arginine (100 mg/kg) treatment did not influence pain threshold (jump or licking time latency) on 1 st , 2 nd and 3 rd week when compared with control CCI. However, L-NAME (5 mg/kg) pretreatment with lower dose of WS (100 mg/kg) significantly potentiated their protective effect (increased pain threshold) on 2 nd and 3 rd week when compared with their effect per se (P < 0.05) [Figure 1]. Further, pretreatment of L-arginine (100 mg/kg) with lower dose of WS (100 mg/kg) significantly reversed its protective effect (reduced pain threshold) which was significant when compared with WS (100 mg/kg) alone on 3 rd week (P < 0.05) [Figure 1].
Effects of NO modulators (L-NAME and L-arginine) on the protective WS on cold allodynia
Similarly, L-NAME (5 mg/kg) and L-arginine (100 mg/kg) treatment did not produce any significant effect on PWL on 1 st , 2 nd and 3 rd week when compared with control CCI. However L-NAME (5 mg/kg) pretreatment with lower dose of WS (100 mg/kg) significantly delayed PWL on 3 rd week, which was significant when compared with their effect per se (P < 0.05) [Figure 2]. Further, L-arginine pretreatment with lower dose of WS (100 mg/kg) significantly reversed its anti-allodynic effect on 3 rd week when compared with WS (100 mg/kg) treatment alone (P < 0.01) [Figure 2].
Effects of gabapentin and WS on oxidative damage in CCI rats
Sham treatment did not produce any significant effect on oxidative stress parameters when compared with naive animals. CCI of sciatic nerve significantly caused an oxidative damage as indicated by increase in lipid peroxidation, nitrite concentration, depletion of reduced glutathione level and catalase activity when compared with naive or sham treatment. 3 weeks treatment with gabapentin (50 mg/kg and 100 mg/kg) and WS (100 and 200 mg/kg) significantly attenuated oxidative damage (reduced lipid peroxidation, nitrite concentration and caused restoration of reduced glutathione and catalase activity) when compared with the control CCI [Table 1].
|Table 1: Effect of WS on oxidative damage and its interaction with NO modulators in CCI rats |
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Effects of NO modulators (L-NAME and L-arginine) on the protective effect of WS on oxidative damage in CCI rats
L-NAME (5 mg/kg) and L-arginine (100 mg/kg) treatment did not shown any significant effect on oxidative stress parameters when compared with control CCI. However, L-NAME (5 mg/kg) pretreatment with lower dose of WS (100 mg/kg) significantly potentiated their protective effect when compared with their effect per se [Table 1]. Further, L-arginine (100 mg/kg) pretreatment with WS (100 mg/kg) significantly reversed the antioxidant effects of WS treatment in CCI subjected animals [Table 1].
| Discussion|| |
Injury to peripheral nerve produces a persistent neuropathic painful state that is characterized by spontaneous pain, allodynia and hyperalgesia. , Various experimental animal models involving injury to sciatic nerve have been developed in recent years to study the mechanism of development and maintenance of NP. Among these partial sciatic nerve ligation, CCI, L5/L6-spinal nerve ligation and tibial-sural nerve transection etc., have been tried to simulate NP in humans. However, CCI in rats has been widely used as it simulates the clinical condition of chronic nerve compression such as that occurring in nerve entrapment neuropathy or spinal root irritation by lumbar disk herniation. This model also shows many of the pathophysiological properties of chronic NP in human subjects, such as allodynia and hyperalgesia.  In the present study, we employed CCI involving four loose ligations around the sciatic nerve caused significant thermal hyperalgesia as well as cold allodynia in response to hotplate and non-noxious cold stimulus respectively. These responses were more significant on 2 nd and 3 rd week post-surgery, when compared with sham or naive animals. Gabapentin is standard drug used in NP and was used to compare the effects of WS in the present study.
Earlier, it has also been reported that behavioral symptoms induced by CCI of sciatic nerve in rats, involves at least in part various independent neural pathways in their progression. , Studies have suggested that hyperalgesic like behavior may be partly due to sensitization of the primary afferent nerves and/or an oxidative/nitrosative stress caused by interaction of ROS or NO with superoxide radicals. ,,
In the present study, CCI caused thermal hyperalgesia and cold allodynia in animals which were significant on 2 nd and 3 rd week post-CCI. Treatment with higher dose of WS (200 mg/kg) significantly improved the threshold for thermal stimuli and increased the PWL to non-noxious cold stimuli. This result is noteworthy since the WS administration from day 1 following the CCI, therefore suggesting its ability to improve the established disease may have therapeutic potential against NP states. However, lower dose of WS failed to show any significant effect on thermal hyperalgesia and cold allodynia in 1 st and 2 nd week, which might be due to its insufficient dose level to counteract the symptoms.
CCI significantly caused oxidative damage as indicated by rise in lipid peroxidation, nitrite concentration and decreased cellular antioxidant defense system such as glutathione and catalase activity. ROS such as superoxide, NO and peroxynitrile plays an important role in neuroinflammatory and immune responses, including defense mechanism against invading microbes.  In an earlier related study in rat, 2 h ischemia followed by 3 h reperfusion of sciatic nerve resulted in significant increase in lipid peroxidation, nitrite levels.  There are also other studies suggesting the increase in lipid peroxidation as a consequence of CCI of sciatic nerve. , Treatment with free radical scavengers resulted in decrease in lipid peroxidation suggesting the involvement of ROS in the central sensitization and initial induction of allodynia. , Thus, evidence suggests that role of oxidative stress in NP and other neurological diseases, but the precise mechanism underlying the pain and oxidative stress still remains unclear. Increased production of ROS leads to direct damage to cellular proteins, lipids and DNA or indirectly by affecting cellular signaling causing impaired neuronal function.  The present study examines the role of WS against CCI induced oxidative stress. Chronic treatment with WS significantly attenuated hyperalgesia and allodynia response as well as caused antioxidant like effect against CCI induced NP. Further, these effects were comparable to the standard drug gabapentin.  In support, various flavonoids have also been claimed to be beneficial against oxidative stress and being investigated against NP and related conditions. ,,
WS is a well-known antioxidant, act by free radical scavenging action. , There is considerable evidence indicating its protective effect against lipid peroxidation.  Our data suggests that oxidative stress plays a critical role in pathogenesis of NP which is in line with the earlier studies indicating oxidative and nitrosative stress is critically involved in the development and maintenance of NP. ,, Even though studies have reported antioxidant actions of WS in various pathological events, its exact mechanism in NP is not been evaluated. So, in our study we have explored interaction of WS with NO modulators to evaluate at least in part the mechanism of WS in the treatment of NP.
Role of NO in spinal pain processing is mainly based on work with inhibitors of NOSs.  Many recent experiments with selective NOS inhibitors and NOS-deficient mice revealed the nNOS isoform to be the most important NO-producing enzyme in the spinal cord during development and maintenance of inflammatory and NP. ,,, Tetrahydrobiopterin (BH4) is an essential cofactor for NO production by NOS and inhibiting BH4 synthesis de novo attenuated inflammatory and NP in rodents further confirming the role of NO in NP.  At rest, nNOS is expressed in few inhibitory interneurons and <5% of dorsal root ganglia (DRG) neurons. During NP, number of nNOS positive DRG neurons is increased and its content is up regulated in central terminals of primary afferent neurons. , The increased NO production leads to activation of NO-guanylyl cyclase (NO-GC) and subsequent cGMP production in NO-GC expressing neurons.  This NO-cGMP pathway modifies several intracellular processes, including activation of protein kinases, ion channels and phosphodiesterases. 
Thus, inhibitors of NO and cGMP production in the spinal cord reduces pain and activators of NO and cGMP with various NO donors increases the excitability of nociceptive neurons leading to enhancement of pain sensation. In the present study, L-NAME pretreatment with lower dose of WS, caused potentiation in their protective effect (antihyperalgesic, antiallodynic and antioxidant like effect). Further, L-arginine (NO precursor) treated group caused hyperalgesia and allodynia like behaviors, indicating that NO may be a pain producing substance. However, their per se effect was not significant when compared with CCI group. L-arginine pretreatment with lower dose of WS reversed their protective effect, suggesting the involvement of NO pathway in their action. Various NOS inhibitors have also been used for the treatment of NP in animal models of NP.  It is known that NO activates soluble guanylate cyclase, which further convert guanosine triphosphate to cGMP. Both L-arginine (a precursor of NO) and N (G)-nitro-L-arginine (L-NNA, a NOS inhibitor) displays anti-nociceptive activity in NP models.  It is further hope that NO modulators and its various downstream processes that are responsible for NP could be the future drugs for the treatment of NP.
| Conclusion|| |
The present study attempts to highlights the protective effect of WS against CCI induced behavioral and biochemical alterations. The behavioral and biochemical alterations clearly implicated the role of free radicals in NP and highlight the involvement of NO mechanism in WS action. However, further studies with various specific inhibitors of different isoforms of NOS are required to confirm this hypothesis.
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