Effect of curcumin (Curcuma longa, Zingiberaceae) against sciatic nerve ligation induced behavioral and biochemical alterations: Possible involvement of nitric oxide mechanism

About Authors: Seema Meena, Anil Kumar and Shikha Chauhan
a) Amity Institute of Pharmacy, Amity University, Noida,-201301, India.
b) Neuropharmacology division, University Institute of Pharmaceutical Sciences, UGC Centre for Advance Studies, Panjab University, chandigarh-160014, India.

Abstract:
Neuropathic pain represents a real clinical challenge because of its severity, chronic nature, and inadequate drug therapy. Recently, nitric oxide pathway has been proposed in the pathogenesis neuropathic pain like conditions. Curcumin is a well known for its antioxidant and medicinal values. The objective of the present study was to explore possible nitric oxide mechanism in the protective effect of curcumin against sciatic nerve ligation induced behavioral and biochemical alterations in rats.Sciatic nerve ligation was performed in Wistar rats. Various behavioral parameters (thermal hyperalgesia, cold allodynia) followed by assessment of biochemical parameters (lipid peroxidation, reduced glutathione, catalase, and nitrite) both on sciatic nerves and brain. Sciatic nerve ligation significantly caused thermal hyeralgesia, cold allodynia and oxidative damage as compared to sham (Sciatic nerve without ligation) and naïve animals. Chronic administration of curcumin (20 mg/kg, po) significantly reversed behavioral alteration and attenuated oxidative damage in both sciatic nerve and brain as compared to control.  Further, L-NAME (5 mg/kg) pretreatment with curcumin (10 mg/kg, po) potentiated protective effect of curcumin as compared to their effect per se. However, L-arginine (100 mg/kg) pretreatment with curcumin (10 mg/kg, po) significantly reversed the protective effects of curcumin. Result of present study suggests that nitric oxide mechanism could be involved in the protective effect of curcumin against sciatic nerve ligation induced behavior and biochemical alterations in rats.

INTRODUCTION:
Pain is a fundamental alarm system and is crucial for survival¹. In contrast, neuropathic pain is a severe pathological condition of the nervous system that offers no adaptive advantage². Neuropathic pain results from damage or abnormal function of the central or peripheral nervous system³. Neuropathic pain is caused by lesion or inflammation of the nervous system and is relatively common with an incidence estimated at 0.6% to 1.5% in the Indian population^4. It is often severely debilitating and largely resistant to treatment mainly because the underlying mechanisms are still poorly understood⁵. Patients with neuropathic pain frequently report sensory abnormalities including burning sensations, exaggerated responses to noxious stimuli (hyperalgesia), pain sensations resulting from innocuous stimuli (allodynia) and spontaneous pain episodes (dysesthesia)⁶. Neuropathic pain is probably multifactorial pathophysiological process) ^7. It is often refractory to the conventional treatment with analgesics such as opiates and nonsteroidal anti-inflammatory drugs⁸. Use of opioids in neuropathic pain is controversial because of their limited efficacy as compared to other pain states⁹.
Recently, significant scientific progress has been made to understand or explain the pathogenesis and cellular cascades underlying neuropathic pain, several treatments are being tried with inadequate therapeutic effect¹⁰. Oxidative damage, neuroinflammation and nitric oxide cascade involvement has been reported in its pathogenesis¹¹. Curcumin, one of active components of Curcuma longa, has been used since time immemorial. Curcumin is known for its diverse biological actions such as free radical scavenging action¹², anticarcinogenic, antimicrobial¹³, hepatoprotective¹⁴, hypoglycemic¹⁴, thrombosuppressive¹⁵, antiarthritic^16, anti-inflammatory properties¹⁷ including the production of interleukin- 8 (IL-8), interleukin-1β (IL-1β) and TNF-α by lipopolysaccharide stimulated monocytes and alveolar macrophages¹⁸.  Curcumin is a very potent free radical scavenger than vitamin E¹⁹.Studies have shown that curcumin is a powerful scavenger of the superoxide anion, the hydroxyl radical, and nitrogen dioxide²⁰ and protects DNA against singlet-oxygen-induced strand breaks²¹. Curcumin have also been found to inhibit nitric oxide production as measured by the amount of nitrite released from the culture medium²². Together with the ability of curcumin to inhibit NO and TNF-α release which cause nitrosative stress in neuropathic pain and promote inflammatory signals respectively,  the present study was designed to evaluate the effect of curcumin in neuropathic pain and an attempt was made to look for the participation of nitric oxide in curcumin's protective effect.
Oxidative stress and reactive oxygen species (ROS) have been implicated in neuropathic pain²³. Oxidative stress is imposed on the cells as a result of one of the three factors (i)an increase in oxidants generation (ii) decrease in antioxidant defense and (iii) a failure to repair oxidative damage²⁴. Cell damage is induced by ROS which are either free radicals, reactive anions containing oxygen atoms or molecules containing oxygen atoms that can either produce free radicals or chemically activated by them. Examples are hydroxyl radicals, superoxide, hydrogen peroxide and peroxinitrite²⁵. The main source of ROS in vivo is aerobic respiration, although ROS is also produced by peroxisomal-b oxidation of fatty acids, stimulation of phagocytosis by pathogens or lipopolysaccharides, arginine metabolism and tissue specific enzymes²⁶. Unchecked ROS attacks many key molecules including enzymes, membranes, lipids and DNA. Under normal conditions, ROS is cleared from cells by the action of superoxide dismutase (SOD), catalase or glutathione, as well as antioxidant vitamin C and E. In spite of much work on oxidative stress and ROS, studies involving ROS in chronic pain are limited. In chronic constriction injury (CCI) model of rat neuropathic pain, heat hyperalgesia was reduced by systemically injected antioxidants²⁶. Recently, in spinal nerve ligation (SNL) model of neuropathic pain, systemic injection of ROS scavenger phenyl-N-tert-butylnitrone (PBN) relieved mechanical allodynia²⁷. Although, these studies suggest the involvement of free radicals/oxidative stress in neuropathic pain, little attention has been paid to critical role of antioxidant enzymes in neuropathic pain.
Studies indicate the potential role for nitric oxide (NO) in maintaining chronic painful states in animals after lesions of peripheral nervous system and CNS²⁸. NO plays an important role in the maintenance of the behavioral signs of neuropathic pain and is involved in common steps in the maintenance of the different modalities of pain such as mechanical allodynia and cold allodynia^{29, 30, 31, 32}. It has been reported that intrathecal administration of NOS inhibitors reverses thermal hyperalgesic responses in rats after sciatic nerve ligation³³. There are also some reports that intracerebroventricular injection of L-arginine produces a biphasic effect on nociception, and NOS inhibitors have antinociceptive effects^{34, 35}. Relatively, little has been known about the role of NO in the mediation of nociception at supraspinal level³⁶. Sensory stimulation induces the release of L-arginine in the thalamus, which facilitate sensory transmission³⁷. Therefore, it appears that physiological role for NO at supraspinal a site is likely to be pronociceptive, although the site(s) and mechanisms for such action remain to be clarified³⁷. Together with the ability of curcumin to inhibit NO and TNF-α release which cause nitrosative stress in diabetes, promote pain and inflammatory signals respectively³⁸. Therefore, present study was designed to evaluate the effect of curcumin in sciatic nerve ligation induced behavioral and biochemical alterations and an attempt was made to look for the participation of NO in curcumin's antinociceptive effect.

Materials and Methods:
Animals
Wistar rats (Central Animal House of the facility of the Panjab University, Chandigarh) weighing 180–200 g were used at the start of the surgery. Animals were acclimatized to laboratory conditions prior to experimentation. Following surgery, the animals were kept under standard conditions of light and dark cycle with food and water ad libitum in groups of single in plastic cages 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 animals.

Induction of peripheral neuropathy by sciatic nerve ligation
Peripheral neuropathy was induced by sciatic nerve ligation (SNL) in rats. In brief, rats were deeply anesthetized with thiopental sodium (40mg/kg i.p). The hair of rat’s lower back and thigh were shaved. The skin of the lateral surface of the right thigh was incised and a cut was made directly through the biceps.  Expose the sciatic nerve and two ligatures (silk 4-0), were placed around the nerve. After performing the ligation, muscular and skin layer was immediately sutured with thread, and topical antibiotic was applied.  Nociceptive threshold was assessed at weekly intervals on 7, 14, and 21^{st (39, 40)}.

Drugs and treatment schedule
Animals were divided into eleven groups (5 animals in each). First, second and third group were treated as naïve (vehicle treated), sham group (expose the sciatic nerve without ligation) and sciatic nerve ligation (SNL ligated animals) respectively.  Curcumin (10 and 20 mg/kg, po), L-arginine (100 mg/kg, ip) and L-NAME (5 mg/kg, ip) were treated as groups 4–7, respectively. L-arginine (100 mg/kg, ip) or L-NAME (5 mg/kg) pretreatments with curcumin (10 and 20 mg/kg, po) were treated as groups 8–11, respectively. L-arginine and L-NAME were freshly prepared in distilled water and administered intraperitoneally 15 minutes before curcumin treatment.  Curcumin was suspended freshly in 0.5% w/v (sodium carboxy-methyl-cellulose) before administration in a constant volume of 5 ml/kg body weight. Curcumin treatment was given, once daily for the duration of 3 weeks by oral route. However, L-arginine and NAME treatment were given 10 minutes before. Doses of Curcumin, L-arginine and L-NAME were selected based on reported literature and laboratory reports ⁴¹.

Behavioral Examinations:
Hot plate test
Thermal hyperalgesia was assessed by placing animals individual on a hot plate (Eddy’s Hot Plate) maintained at 55 ⁰C on weekly interval on 7^th,  14 and  21^st day after SNL. 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 sec was maintained⁴².

Cold allodynia
Cold allodynia was assessed by measuring paw (both ipsilateral and collateral) withdrawal latency (PWL), when dipped in water bath maintained at 4⁰C  2⁰C on weekly interval on 7th, 14 and 21st days after SNL. A cut off 15 sec was maintained during the experiment⁴³.

Biochemical parameters:
Dissection and homogenization
On 21^st day, animals were sacrificed by decapitation immediately after behavioral assessments. The complete sciatic nerve and brains were removed and 10% (w / v^-1) tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). Homogenate were centrifuged for 20 minutes at 15000 rpm and supernatant was used for estimation of lipid peroxidation and reduced glutathione levels. The post nuclear fractions for catalase assay are obtained by centrifugation of the homogenate at 1000 × g for 20 min, at 4°C and for other enzyme assays centrifuged at 12,000 × g for 60 min at 4°C.

Lipid peroxidation assay
The quantitative measurement of lipid peroxidation in the whole brain was measured according to the method of Wills⁴⁴. The amount of malondialdehyde formed was measured by the reaction with thiobarbituric acid at 532 nm using Perkin Elmer lambda 20 spectrophotometer. The results were expressed as nanomole of malondialdehyde per milligram protein using the molar extinction coefficient of chromophore (1.56×10M¯^¹ cm¯^¹). 

Estimation of reduced glutathione
Reduced glutathione in the brain was estimated according to the method of Ellman⁴⁵. A 1.0 ml of homogenate was precipitated with 1.0 ml of 4% sulfosalicylic acid by keeping the mixture at 4°C for 1h and the samples were immediately centrifuged at 1200 ×g for 15 min at 4°C. The assay mixture contains 0.1ml of supernatant, 2.7 ml of phosphate buffer of pH 8.0 and 0.2 ml of 0.01M-dithiobisnitrobenzoic acid (DTNB). The yellow color developed was read immediately at 412nm using Perkin Elmer lambda 20 spectrophotometer. The results were expressed as nanomole GSH per milligram protein.

Nitrite estimation
Nitrite is the stable end product of nitric oxide (NO) in living system. Accumulation of nitrite was measured in cell free supernatants from brain homogenates by spectrophotometer assay based on Greiss reagent 15 (1% sulphanilamide/ 0.1% naphthylethylenediamine dihydrochloride/ 2.5% phosphoric acid) and incubated at room temperature for 10 min to yield a chromophore. Absorbance was read at 543 nm spectrophotometrically. The nitrite concentration was calculated from a standard curve using sodium nitrite as standard and expressed as micro molar nitrite per milliliter homogenate⁴⁶.

Protein estimation
The protein content was measured according to the method of Lowry using bovine serum albumin as standard⁴⁷.

Catalase estimation
Catalase activity was assayed by the method of Luck⁴⁸, wherein the breakdown of hydrogen peroxide (H₂O₂) is measured at 240 nm. Briefly, the assay mixture consisted of 3 ml of H₂O₂ phosphate buffer and 0.05 ml of supernatant of tissue homogenate (10%), and the change in absorbance was recorded at 240 nm. The results were expressed as micromole H₂O₂ decomposed per milligram of protein/min.

Statistical Analysis
All the values are expressed as mean ± SEM. The data were analyzed by using one way analysis of variance followed by Tukey’s test. P< 0.05 was considered statistically significant.

Results:

Figure 1.  L-NAME and L-arginine modulation on the protective effects of curcuminagainst thermal hyperalgesia Values are expressed as mean ± SEM, n=5 rats per group,  ^aP<0.05 as compared to naive group,^bP<0.05 as compared to sham and naive,^cP<0.05 as compared to Control (SNL), ^dP<0.05 as compared to Curcumin (10), ^eP<0.05 as compare to L-NAME (5).

Effects of Curcumin and its modulation by L-NAME and L-arginine on thermal hyperalgesia in SNL rats- Sciatic nerve ligation (SNL) significantly caused hyperalgesiaas reflected by shorten pain threshold (Jump or licking time latency) on weekly intervals as compared to naïve and sham group. Curcumin (20 mg/kg, po) treatment significantly reversed SNL induced hyperalgesia on weekly intervals as compared to control (SNL) (Figure 1).  However, lower dose of curcumin (10mg/kg, po) did not show any significant effect as compare to control group (SNL). Further, L-NAME (5mg/kg) pretreatment with curcumin (10 mg/kg, po) significantly potentiated the protective effect of curcumin on 2^nd and 3^rd week which was significant as compared to their effect per se (Figure 1). Similarly, L-arginine (100 mg/kg) pretreatment with curcumin (10mg/kg, po) significantly caused hyperalgesic response on 2^nd and 3^rd week as compared to control (Figure 1). L-arginine (100 mg/kg, per se) treatment did not produce any significant effect on hyperalgesia even after 3weeks.

Figure 2.  Effects of Curcumin and its interaction with L-NAME and L-arginine on cold allodynia Values are expressed as mean ± SEM, n=5 rats per group,  ^aP<0.05 as compared to naive group,^bP<0.05 as compared to sham and naive,^cP<0.05 as compared to Control (SNL), ^dP<0.05 as compared to Curcumin (10), ^eP<0.05 as compare to L-NAME (5).

Effects of curcuminand its modulation by L-NAME and L-arginine on cold allodynia in SNL rats- Sciatic nerve ligation (SNL) significantly caused cold allodynia as reflected by decreased paw withdrawal latency on weekly intervals as compared to naïve and sham group. Curcumin (20mg/kg, po) treatment significantly delayed withdrawal latency on 3^rd week as compared to control (SNL) (Figure 2). But lower dose of curcumin (10mg/kg, po) did not show any significance difference as compare to control (SNL).  L-NAME (5mg/kg) pretreatment with curcumin (10 mg/kg, po) caused potentiation in antiallodynic effect of curcumin on 2^nd and 3^rd week which was significant as compared their effect per se. Further, L-arginine (100mg/kg,per se) treatmentdid not produce any significant antiallodynic like behavior as compared to control (SNL) (P < 0.05). However, L-arginine (100 mg/kg, ip) pretreatment with curcumin (10 mg/kg, po) significantly reversed its antiallodynic effect on 2^nd and 3^rd weeks as compared to their effect per se (Figure 2).

Table 1. Effects of Curcuminand its interaction with L-NAME and L-arginine on oxidative damage on sciatic nerves

Effects of curcumin and its modulation by L-NAME and L-arginine on oxidative damage in SNL rats- Sciatic nerve ligation significantly caused oxidative damage as indicated by increased lipid peroxidation, nitrite concentration, depletion of reduced glutathione level and catalase activity in both sciatic nerve as well as whole brain respectively as compared to naïve or sham treated animals. Treatment with curcumin (10 and 20 mg/kg, po) significantly caused attenuation of lipid peroxidation, nitrite concentration and restoration of depleted reduced glutathione and catalase activity as compared to the control (SNL) both in brain and sciatic nerves respectively (P<0.05) (Table 1 and Table 2).

Table 2.  Effects of Curcuminand its interaction with L-NAME and L-arginine on oxidative damage in SNL brain

L-NAME (5mg/kg) pretreatment with lower dose of curcumin (10 mg/kg, po) caused potentiation in the antioxidant effect of curcumin both in brain and sciatic nerves which was significant as compared their effect per se ( Table 1 and Table 2). However, L-NAME (5mg/kg, per se) treatment did not influence significantly these oxidative stress parameters as compared to SNL group in both brain and sciatic nerves. Further, L-arginine (100 mg/kg/ip) pretreatment with curcumin (10 mg/kg, po) significantly reversed the protective effect of curcumin in both brain and sciatic nerve (Table 1 and Table 2). L-arginine (100 mg/kg, per se) did not produce any significant effect on oxidative stress parameters as compared to SNL group in both brain and sciatic nerves.

Discussion:
Neuropathic pain is a complexdebilitating and chronic disease that usually is accompanied by tissue injury⁴⁹. Peripheral nerve injury produces a persistent neuropathic pain state characterized by spontaneous pain, allodynia and hyperalgesia⁵⁰. With neuropathic pain, the nerve fibers themselves may be damaged, dysfunctional or injured. These damaged nerve fibers send incorrect signals to other pain centers⁵¹. In present study, unilateral sciatic nerve ligation induced significant behavioral and biochemical alterations resulting ipsilateral cold allodynia, thermal hyperalgesia and oxidative damage both in sciatic nerves as well as brain.
In the present study, partial sciatic nerve ligation (SNL) caused thermal hyperalgesia as well as cold allodynia in response to non-noxious cold stimulus which was more significant in 2^nd and 3^rd week of SNL. Studies have also reported that partial ligation of sciatic nerve induced hyperalgesia and cold allodynia like behavior in animals⁵².Reports further suggest the mechanism of hyperalgesia action could be due to sensitization of primary afferent nerves⁵³. In the present study, partial sciatic nerve ligation significantly raised lipid peroxidation, nitric concentration and depletion of reduced glutathione and catalase activity suggesting the involvement of oxidative damage during neuropathic pain.
In the present study, chronic treatment with curcumin significantly reversed thermal hyperalgesia, cold allodynia in sciatic nerve ligated animals indicating its therapeutic potential against neuropathic pain. The results of our study coincided with the earlier works which proved the analgesic activity of curcumin in neuropathic pain⁵⁴. These findings suggest that curcumin plays an important role in pain regulation at central and peripheral level.  Studies also reported the therapeutic potentials of curcumin against hyperalgesia and related states⁵⁵.
Curcumin exhibit differential antioxidant activity in several in vitro and in vivo models⁵⁶. The significance of Curcuma longa Linn (Turmeric) in health and nutrition has changed considerably since the discovery of the antioxidant property of naturally occurring phenolic compounds, curcuminoids. Earlier studies have shown that curcumin inhibits reactive oxygen species (ROS) production⁵⁷ as well as calcium entry⁵⁸ It can also affect different other cellular processes such as activation of apoptosis, inhibition of platelet aggregation, inhibition of inflammatory cytokine production, inhibition of cyclooxygenase and lipoxygenase isoenzymes. It may also affect the activity of different key enzymes such as PKC, protein tyrosine kinases and calcium dependent endonuclease⁵⁹.  In present study, curcumin treatment significantly attenuated oxidative damage in both sciatic nerves as well as in brain. Curcumintreatment significantly attenuated rise in lipid peroxidation, nitrite level and restored the depleted reduced glutatathione and catalase activity, suggesting its antioxidant like action against SNL. Although, pathogenesis of neuropathic pain is not clear so far.  Recent evidences suggested that antioxidant agents could be used to reverse peripheral nerve injuries⁶⁰.
Further, nitric oxide is an unconventional intercellular messenger in the nervous system. NO reacts with reactive oxygen species and acts as an oxidant⁶¹. The oxidation is not specific and affects any cell molecule. In the present study, pretreatment with L-arginine, nitric oxide precursor produced hyperalgesia, allodynic like conditions and oxidative damage suggesting the involvement of nitric oxide pathway in sciatic nerve ligation induced behavioral.  Studies reported the involvement of nitric oxide cascade in the pathogenesis of neuropathic pain⁶². However, an exact molecular and cellular cascade in the pathogenesis of neuropathic pain is still unclear. Besides, role of reactive oxygen species–reactive nitrogen species (ROS–RNS) and its interaction with NO in the regulation of stress responses are not clearly understood so far. Further, L-NAME pretreatment with curcumin caused potentiation of antihyperalgesic, antiallodynic and antioxidant like effect of curcumin, suggesting the possible involvement of NO pathway in its protective action. It seems nitric oxide mechanism is involved in the protective action of curcumin against sciatic nerve ligation induced neuropathic pain like states and oxidative damage.
In summary, nitric oxide mechanism could be involved in the protective action of curcumin against SNL induced neuropathic pain and oxidative damage. Study further suggests the potential of curcumin in effect management of neuropathic pain and related conditions.

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Reference ID: PHARMATUTOR-ART-1026