http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–11 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.943247
ORIGINAL ARTICLE
Effect of curcumin in mice model of vincristine-induced neuropathy Anand Babu, K. G. Prasanth, and Bhaskar Balaji
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Department of Pharmacology, PSG College of Pharmacy, Coimbatore, Tamil Nadu, India
Abstract
Keywords
Context: Curcumin exhibits a wide spectrum of biological activities which include neuroprotective, antinociceptive, anti-inflammatory, and antioxidant activity. Objective: The present study evaluates the effect of curcumin in vincristine-induced neuropathy in a mice model. Materials and methods: Vincristine sulfate (0.1 mg/kg, i.p. for 10 consecutive days) was administered to mice to induce neuropathy. Pain behavior was assessed at different days, i.e., 0, 7, 10, and 14 d. Sciatic nerve total calcium, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), nitric oxide (NO), and lipid peroxidation (LPO) were also estimated after the 14th day of study. Pregabalin (10 mg/kg, p.o.) and curcumin (15, 30, and 60 mg/kg, p.o.) were administered for 14 consecutive days. Results: Curcumin at 60 mg/kg significantly attenuated the vincristine-induced neuropathic pain manifestations in terms of thermal hyperalgesia (p50.001) and allodynia (p50.001); mechanical hyperalgesia (p50.001); functional loss (p50.001); and in the delayed phase of formalin test (p50.001). Curcumin at 30 and 60 mg/kg exhibited significant changes (p50.001) in antioxidant levels and in total calcium levels in vincristine-injected mice. Conclusion: Curcumin at 30 and 60 mg/kg dose levels significantly attenuated vincristineinduced neuropathy which may be due to its multiple actions including antinociceptive, calcium inhibitory, and antioxidant effect.
Antinociceptive, chemotherapy, allodynia, formalin test, hyperalgesia, neuroprotective, pain, sciatic functional index
Introduction Chemotherapy-induced painful neuropathy (CIPN) is a common side effect of anticancer treatment with vinca alkaloids, platinum drugs, taxanes, and other chemotherapeutic drugs affecting up to 30–40% of patients (Kaley & Deangelis, 2009). Symptoms usually start when chemotherapy is in process and tend to improve after completing therapy. However, in 25–30% of patients, pain or unpleasant paresthesias remain or even increase after terminating the chemotherapeutic treatment (Verstappen et al., 2005). Vincristine is one of the most common anticancer drugs applied for the treatment of many types of cancers such as leukemias, lymphomas, and sarcomas and entails the side effect of a dose and duration-dependent peripheral neuropathy. The anticancer action of vincristine is due to its binding to b-tubulin, which leads to disorganization of the axonal microtubule cytoskeleton. This effect underlies its antimitotic property and also in part explains the neurotoxic effect (Tanner et al., 1998). Conventional analgesic agents like non-steroidal antiinflammatory drugs (NSAIDs) and opioids are clinically
Correspondence: Bhaskar Balaji, Assistant Professor, Department of Pharmacology, PSG College of Pharmacy, Coimbatore 641004, Tamil Nadu, India. Tel: +91 422 4345843. Fax: +91 422 2594400. E-mail: [email protected]
History Received 22 April 2014 Revised 9 June 2014 Accepted 7 July 2014 Published online 27 November 2014
ineffective for attenuation of CIPN. Usefulness of tricyclic antidepressants and anticonvulsants in management of neuropathic pain is indicated but these medications exhibit a wide spectrum of adverse effects which limit their full clinical exploitation (Dworkin et al., 2010). Several herbal medicines such as Phyllanthus emblica Linn. (Phyllanthaceae), Cannabis sativa Linn. (Cannabaceae), Nigella sativa Linn. (Ranunculaceae), Ocimum sanctum Linn. (Lamiaceae), and Ginkgo biloba Linn. (Ginkgoaceae) are shown to have potential in different types of experimentally induced neuropathic pain (Kim et al., 2009; Muthuraman et al., 2008; Wirth et al., 2005). Some clinical reports have also advocated beneficial effect of drugs from plant origin in neuropathic pain conditions (Ellis et al., 2009; Zareba, 2009). Curcuma longa L. (Zingiberaceae), a traditional herb, has been used to manage various disorders for thousands of years. Curcumin isolated from Curcuma longa has shown to be nontoxic and non-mutagenic and exhibits a wide spectrum of biological activities which include neuroprotective, antiinflammatory, antidepressant, and antioxidant activity (Fan et al., 2013; Gupta et al., 2013). Curcumin has also been reported to exert antinociceptive effects in different animal models of pain (Mittal et al., 2009; Tajik et al., 2008), which also includes diabetic neuropathic pain (Sharma et al., 2006). With the above background, the present study was undertaken to investigate the effect of curcuminin vincristine-induced neuropathic pain in a mice model. Pregabalin, a selective Cav
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2.2 (a2-d subunit) channel antagonist served as positive control in this study.
Materials and methods
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Chemicals and preparation of drug solutions Vincristine sulfate (Sun Pharma, Mumbai, India), pregabalin (Ranbaxy Research Laboratories, Gurgaon, India), curcumin (Himedia Laboratories, Mumbai, India), nitro blue tetrazolium (NBT), (Sigma Aldrich, St. Louis. MO), Folin– Ciocalteu’s phenol reagent (Merck Limited, Mumbai, India), 5,50 -dithiobis (2-nitrobenzoic acid) (DTNB), bovine serum albumin (BSA), (Sisco Research Laboratories Pvt. Ltd. Mumbai, India), reduced glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH) (Himedia Laboratories, Mumbai, India), thiobarbituric acid (TBA) (Sigma Aldrich, St. Louis. MO) were procured for the present study. All the reagents used in the present study were of analytical grade. Pregabalin and curcumin were suspended in 0.3% w/v carboxy methyl cellulose (CMC) solution and vincristine was diluted with normal saline. Experimental animals Thirty adult male Swiss albino mice with a body weight of 22–25 g were used for this study. Mice were group-housed (n ¼ 6 per cage) in a room with controlled temperature (21 ± 2 C), and 12 h light-dark cycle was maintained. All the animals had free access to food and water ad libitum. The experimental protocols were approved by the Institutional Animal Ethical Committee and experiments were performed in accordance to the committee for the purpose of control and supervision of experiments on animals (CPCSEA) guidelines for ethical use of animals (CPCA no. 158/99/CPCSEA/14/ 2011). Preselection of animals The rota-rod test was performed to investigate the motor coordination of the mice. Mice were placed on a rotating rod (10 rpm) and the latency to falling was measured for up to 2 min according to the method described previously by Egashira et al. (2004). Three trials per day for 2 d were enough for the mice to learn this task. On the day of the test, only the mice that were able to stay balanced on rotating rod for 2 min were selected for further study.
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Group III, IV, V, and VI mice received pregabalin (10 mg/kg/ d p.o.), curcumin (15 mg/kg/d p.o.), curcumin (30 mg/kg/d p.o.), and curcumin (60 mg/kg/d p.o.), respectively, 1 h before each vincristine injection for 14 consecutive days. The behavioral tests like hot plate, cold plate, pin prick test, rota-rod, and sciatic functional index (SFI) were performed on different days, i.e. days 0, 7, 10, and 14. On the 14th day, mice were subjected to the formalin test. Thereafter, all the animals were sacrificed under deep ether anesthesia and subjected to biochemical analysis for estimating the total protein, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), GSH, lipid peroxidation (LPO), nitric oxide (NO), and total calcium activity in sciatic nerve tissue sample. Behavioral assessment Hot plate test (thermal hyperalgesia) In this test, mice were individually placed on a hot plate (Eddy’s hot plate) with the temperature adjusted to 55 ± 1 C. The latency to the first sign of paw licking or jump response to avoid the heat was taken as an index of the pain threshold; the cut-off time was 10 s in order to avoid damage to the paw (Tiwari et al., 2011). Cold plate test (thermal allodynia) Mice were placed on an ice platform submerged approximately 1 cm below the surface of cold water (4 C), such that the hairy and glabrous skin of the mice feet were in contact with the cold water. The latency prior to the first reaction (licking, moving the paws, and leaps) was recorded with a cutoff time of 30 s (Beyreuther et al., 2006). Pin prick test (mechanical hyperalgesia) Mechanical hyperalgesia was evaluated by the pin prick test as described in the method by Erichsen & Blackburn-Munro (2002). Briefly, the plantar surface of the left hind paw was touched with the point of the bent 18 gauge needle (at 90 angle) at intensity sufficient to produce a reflex withdrawal response in normal mice, but at an intensity which was insufficient to penetrate the skin in all groups. The duration of the paw withdrawal was recorded in seconds. A cut-off time of 20 s was maintained. Rota-rod test
Induction of peripheral neuropathy by vincristine Peripheral painful neuropathy was induced in mice which stood on a rotating rod for 2 min by intraperitoneal administration of vincristine sulfate (0.1 mg/kg) once per day for 7 consecutive days as per the method adopted by Kiguchi et al. (2008). Experimental design Group I served as normal control in which mice received 0.3% w/v carboxy methyl cellulose (CMC) (p.o.) vehicle administration for 14 d. Group II served as vincristine control in which mice were administered 0.3% w/v CMC (p.o.) 1 h before each vincristine injection for 14 consecutive days.
Mice were subjected to the rota-rod test to assess the motor coordination as described earlier. Determination of SFI Mice were subjected to walking-track analysis and measurement of SFI using a method similar to that described by De Medinaceli et al. (1982). The trials were done in an 8.2 42 cm corridor darkened at one end and covered with a sheet of white paper. The hind paws of the mice were dipped in black India-ink and then each animal was allowed to walk freely in the corridor mentioned above. From the analysis of the footprints of the feet, the following lengths were obtained: (a) distance from the heel to the third toe, the print length
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(PL); (b) distance from the first to the fifth toe, the toe spread (TS); and (c) distance from the second to the fourth toe, the intermediary TS (ITS). All three measurements were taken from the experimental (E) and normal (N) sides. The factors were calculated as follows: (I) PL factor (PLF) ¼ (EPL NPL)/NPL; (II) TS factor (TSF) ¼ (ETS NTS)/ NTS; (III) intermediary TS factor (ITF) ¼ (EIT NIT)/NIT. These factors were then incorporated into the SFI formula derived by Bain et al. (1989): SFI ¼ 38.3 PLF + 109.5 TSF + 13.3 ITF 8.8. An SFI of 0 is normal. An SFI of 100 indicates total impairment, such as would result from a complete transaction of the sciatic nerve.
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Formalin test in mice At the end of the study, all the groups were subjected to the formalin test. Briefly, each mouse was acclimatized to the observation box before the formalin test. After an adaptation period of 15 min, the right hind footpaw was injected with 50 mL of 2.5% formalin in the intraplantar region. Nociception was evaluated by quantifying paw licking time and paw elevation time during the first 10 min (acute phase) and at 20– 40 min (delayed phase) (Khalilzadeh et al., 2008; Luiz et al., 2007). Biochemical estimation All the mice were sacrificed with chemical euthanasia on the 14th day after behavioral tests. The portions of the sciatic nerve were isolated immediately and processed for SOD, CAT, GPx, GSH, NO, and LPO. The sciatic nerve homogenate (10% w/v) was prepared with 0.1 M Tris-HCl buffer (pH 7.4) and deionised water, respectively, for total protein and total calcium estimation. Superoxide dismutase SOD was assayed in the nerve homogenate according to the method described by Kakkar et al. (1984). The reduction of NBT to blue formazan mediated by superoxide anions was measured at 560 nm under aerobic conditions. Addition of SOD inhibits the reduction of NBT and the extent of inhibition is taken as a measure of enzyme activity. The activity of enzyme was expressed as units/mg protein, where one unit is defined as the amount of enzyme inhibiting the rate of reaction by 50%. Catalase CAT activity was assayed in the nerve homogenate by the Beers and Sizer (1952) method. Hydrogen peroxide (H2O2) decomposition by CAT was monitored spectrophotometrically by following the decrease in absorbance at 240 nm. The activity of enzyme was expressed as mmoles of H2O2 decomposed/min/mg protein. Glutathione peroxidase GPx activity was measured in the nerve homogenate by the method of Paglia and Valentine (1967). The reaction measured the rate of reduced GSH oxidation by H2O2 catalyzed by the GPx. GSH is maintained at constant concentration by the addition of exogenous glutathione
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reductase and NADPH, which immediately converts any oxidized glutathione disulfide (GSSG) produced to GSH. The rate of GSSG formation is then measured by following the absorbance of NADPH at 340 nm for 5 min. The activity of enzyme was expressed as nmoles NADPH oxidized/min/mg protein. Reduced Glutathione Glutathione content was estimated by following the Jollow et al. (1974) method. In this assay, GSH reduced 5,50 -dithiobis (2-nitrobenzoic acid) (DTNB) to 5-thio-2-nitrobenzoic acid (TNB) and the GSSG. TNB is a yellow product which was measured spectrophotometrically at 412 nm. The amount of GSH was expressed in mM/mg protein. LPO levels The quantitative measurement of LPO was performed in the nerve homogenate according to the Ohkawa et al. (1979) method. The amount of malondialdehyde (MDA) and other thiobarbituric acid reactive substances (TBARS) are quantified by their reactivity with TBA in acidic conditions. The reaction generated a pink-colored chromophore which was measured in a UV spectrophotometer at 532 nm. The results were expressed as nmoles MDA/mg protein. Nitric oxide Nitrate/nitrite was assayed in the nerve homogenate according to the Green et al. (1982) method using the Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyl ethylenediamine dihydrochloric acid in water in the ratio of 1:1). The color intensity of chromogen was read at 540 nm. The results were expressed as mM/mg protein. Estimation of total protein content Protein concentration of nerve homogenate was estimated according to the Lowry et al. (1951) method, using bovine serum albumin (BSA) as a standard. The absorbance was determined spectrophotometrically at 750 nm. Estimation of total calcium Total calcium levels were estimated in sciatic nerve homogenate according to the Severinghaus and Ferrebee (1950) method. Briefly, homogenate was mixed with 1 ml of trichloroacetic acid (4%) in ice cold conditions and centrifuged at 2000 rpm for 10 min. The clear supernatant was used for the estimation of total calcium ion by atomic emission spectroscopy at 556 nm. Statistical analysis All the results were expressed as mean ± standard deviation. The data were statistically analyzed by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple comparison test for biochemical estimations and formalin test. Two-way ANOVA (treatment versus duration) followed by the Bonferroni post test was used for the hot plate test, cold plate test, pin prick test, and SFI. Probability values of less than 0.05 were considered as significant. The analysis was
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carried out using Graph pad prism software (version 4.03) (GraphPad Software, San Diego, CA).
Results
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Effect of curcumin on hot plate test Administration of vincristine resulted in a significant (p50.001) development of thermal hyperalgesia as reflected by an increase either in the hind paw licking, lifting, or jumping from the hot plate surface on days 7, 10, and 14 in comparison to day 0 (Figure 1). Different treatments (F (5, 120) ¼ 66.58; p50.001) and duration of treatment (F (3, 120) ¼ 36.66; p50.001) significantly improved the hyperalgesic response. The interaction data (treatment versus duration) was also found to be significant (F (15, 120) ¼ 8.36; p50.001) which indicates time-dependent improvement in the analgesic response. Curcumin (15 mg/kg) did not significantly increase the pain threshold in comparison with vincristine-treated mice on days 7, 10, and 14. Curcumin (30 mg/kg) significantly increased the pain threshold in comparison with vincristine-treated mice on day 7 (p50.05) and days 10 and 14 (p50.001). Treatment of pregabalin and
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curcumin (60 mg/kg) significantly increased (p50.001) the pain threshold in comparison with vincristine-treated mice on days 7, 10, and 14. In pregabalin- and curcumin (60 mg/kg)-treated mice, the duration of paw withdrawal latency was not significantly different on days 7, 10, and 14 in comparison with the baseline (day 0). This indicates that curcumin (60 mg/kg) was able to restore the physiological thermal pain perception (Figure 1). Effect of curcumin on the cold plate test Vincristine-administered mice exhibited a significant development (p50.001) in thermal allodynia which was reflected by a decrease in the paw withdrawal latency period on days 7, 10, and 14 in comparison with day 0. Different treatments [F (5, 120) ¼ 91.08; p50.001] and duration of treatment [F (3, 120) ¼ 6.39; p50.001] significantly improved the allodynic response. The interaction data (treatment versus duration) was also found to be significant [F (15, 120) ¼ 15.90; p50.001] which indicates time-dependent improvement in the allodynic response. Administration of pregabalin and curcumin at 15, 30, and 60 mg/kg significantly attenuated
Figure 1. Effect of curcumin (15, 30, and 60 mg/kg) on thermal hyperalgesia induced by the hot plate in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. Two-way ANOVA followed by post-hoc Bonferonni’s test. a – indicates significance at p50.001 in comparison to normal control mice at corresponding days. b,d – indicates significance at p50.001 and p50.05, respectively, in comparison to day 0. x,z – indicates significance at p50.001 and p50.05, respectively, in comparison to vincristine-treated mice at corresponding days.
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(p50.001) vincristine-induced thermal allodynia in comparison with vincristine-treated mice. Interestingly, curcumin (60 mg/kg)-treated mice exhibited significant increase (p50.001) in paw withdrawal latency on days 10 and 14 in comparison with the baseline. Pregabalin treatment also showed similar results on days 7, 10, and 14 in comparison with the baseline (Figure 2).
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Effect of curcumin on pin prick test Administration of vincristine resulted in a significant (p50.001) development of mechanical hyperalgesia as reflected by a significant increase in the left hind paw lifting duration in the pinprick test. Different treatments [F (5, 120) ¼ 140.74; p50.001] and duration of treatment [F (3, 120) ¼ 102.30; p50.001] significantly improved the hyperalgesic response. The interaction data (treatment versus duration) was also found to be significant [F (15, 120) ¼ 16.04; p50.001] and indicates time-dependent improvement in the analgesic response. Curcumin at 15 mg/kg significantly (p50.05) attenuated the mechanical hyperalgesia in comparison with vincristine-treated mice on day 14. Administration of pregabalin and curcumin (30 and 60 mg/kg) significantly attenuated
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(p50.001) the increase in the left hind paw lifting duration in comparison with vincristine-treated mice on days 7, 10, and 14. In curcumin (60 mg/kg)-treated mice, the duration of paw lifting was not significantly different on days 10 and 14 in comparison with the baseline (day 0). This indicates that curcumin (60 mg/kg) was able to restore the physiological mechanical pain perception. Pregabalintreated mice exhibited no significant difference in the paw withdrawal latency on days 7, 10, and 14 in comparison with the baseline (Figure 3). Effect of curcumin on motor performance Vincristine treatment did not show any significant difference in the performance of mice on the accelerating rota-rod from that of the normal control group on days 7, 10, and 14. Curcumin (15, 30 and 60 mg/kg) and pregabalin treatment did not show any significant difference in comparison with vincristine-treated mice (data not shown). Effect of curcumin in formalin test The effect of curcumin in formalin test was measured in two different phases: acute and delayed phase.
Figure 2. Effect of curcumin (15, 30, and 60 mg/kg) on thermal allodynia induced by cold plate in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. Two-way ANOVA followed by post-hoc Bonferonni’s test. a – indicates significance at p50.001 in comparison to normal control mice at corresponding days. b,c – indicates significance at p50.001 and p50.01, respectively, in comparison to day 0. x – indicates significance at p50.001 in comparison to vincristine-treated mice at corresponding days.
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Figure 3. Effect of curcumin (15, 30, and 60 mg/kg) on mechanical hyperalgesia by the pin prick test in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. Two-way ANOVA followed by post-hoc Bonferonni’s test. a – indicates significance at p50.001 in comparison to normal control mice at corresponding days. b,c – indicates significance at p50.001 and p50.01, respectively, in comparison to day 0. x,z – indicates significance at p50.001 and p50.05 in comparison to vincristine-treated mice at corresponding days.
Acute phase Formalin injection in vincristine-administered mice produced a significant increase in paw licking (F (5,30) ¼ 5.98; p50.001) and paw elevation (F (5,30) ¼ 3.33; p50.05) in comparison with normal control mice. Administration of pregabalin and curcumin (15, 30, and 60 mg/kg) did not produce any significant alterations in paw licking and paw elevation in comparison with vincristine-administered mice (Figure 4a). Delayed phase Formalin injection in vincristine-administered mice produced a significant increase in paw licking (F (5,30) ¼ 8.26; p50.001) and paw elevation (F (5,30) ¼ 26.02; p50.001) in comparison with normal control mice. Curcumin at 30 mg/ kg showed a significant decrease (p50.01) in paw elevation in comparison with vincristine-administered mice, while curcumin at 60 mg/kg also significantly decreased the paw elevation (p50.001) and paw licking (p50.01) in comparison with vincristine-administered mice. Pregabalin at 10 mg/ kg significantly decreased (p50.001) the paw licking time and paw elevation time in comparison with vincristineadministered mice (Figure 4b).
SFI level. Different treatments (F (5, 120) ¼ 728.73; p50.001) and duration of treatment (F (3, 120) ¼ 516.95; p50.001) significantly improved the SFI. The interaction data (treatment versus duration) was also found to be significant (F (15, 120) ¼ 86.83; p50.001) and indicated time-dependent improvement in SFI. Administration of curcumin (15, 30, and 60 mg/kg) significantly attenuated (p50.001) vincristine-induced rise in SFI in a dose-dependent manner. However, curcumin treatment at all the dose levels was unable to restore the SFI to baseline score. Similar effects were seen with pregabalin administration (Figure 5). Effect of curcumin in total calcium levels Administration of vincristine exhibited a significant increase (p50.001) in total calcium levels in comparison with normal control mice in the sciatic nerve homogenate (Figure 6). Curcumin at 15 mg/kg significantly decreased (p50.01) the calcium levels in comparison with vincristine-treated mice. Pregabalin and curcumin (30 and 60 mg/kg) significantly decreased (p50.001) the calcium levels in comparison with vincristine-treated mice (F (5,30) ¼ 132). Effect of curcumin on SOD activity
Effect of curcumin on SFI Vincristine-administered mice resulted in sciatic functional loss as reflected by a significant rise (p50.001) in the
In comparison with normal control mice, administration of vincristine significantly (p50.001) depleted the SOD activity in mice. Curcumin (15, 30, and 60 mg/kg) treated mice
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Figure 4. (a) Effect of curcumin (15, 30, and 60 mg/kg) on nociception induced by formalin (acute phase) in the vincristine-induced neuropathic pain mice model. (b) Effect of curcumin (15, 30, and 60 mg/kg) on nociception induced by formalin (delayed phase) in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. One-way ANOVA followed by post-hoc Tukey’s multiple comparison test. $$,$$$ Indicate significance at p50.01 and p50.001, respectively, in comparison to normal control group. **,***Indicate significance at p50.01.
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Figure 5. Effect of curcumin (15, 30, and 60 mg/kg) on sciatic functional index in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. Two-way ANOVA followed by post-hoc Bonferonni’s test. a – indicates significance at p50.001 in comparison to normal control mice at corresponding days. b,c,d – indicates significance at p50.001 and p50.01, and p50.05, in comparison to day 0. x – indicates significance at p50.001 in comparison to vincristine-treated mice at corresponding days.
Figure 6. Effect of curcumin (15, 30, and 60 mg/kg) on sciatic nerve total calcium levels in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. One-way ANOVA followed by post-hoc Tukey’s multiple comparison test. $$$Indicates significance at p50.001 in comparison to normal control group. **,***Indicate significance at p50.01. and p50.001 respectively, in comparison to vincristine-treated group.
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Table 1. Effect of curcumin (15, 30, and 60 mg/kg) on sciatic nerve homogenate oxidative markers in the vincristine-induced neuropathic pain mice model.
Treatment and dose
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Normal control Vincristine control Vincristine + pregabalin 10 mg/kg Vincristine + curcumin 15 mg/kg Vincristine + curcumin 30 mg/kg Vincristine + curcumin 60 mg/kg
SOD CAT (units/mg protein) (units/mg protein)
GPx (units/mg protein)
GSH (mM/mg protein)
LPO (nM/mg protein)
NO (mM/mg protein)
48.26 ± 6.40 22.19 ± 2.43$$$ 40.75 ± 4.07**
62.34 ± 3.61 32.74 ± 4.94$$$ 44.88 ± 8.76
151.94 ± 23.50 77.81 ± 16.87$$$ 101.60 ± 17.64
44.68 ± 6.65 17.99 ± 4.56$$$ 33.09 ± 4.69
591.92 ± 82.70 134.92 ± 21.70 1077.35 ± 190.82$$$ 266.37 ± 38.56$$$ 821.66 ± 80.11* 166.05 ± 36.75***
42.85 ± 4.31***
48.14 ± 3.98**
107.55 ± 13.66
33.22 ± 6.48
760.45 ± 112.94**
197.51± 26.30*
49.84 ± 7.59***
50.49 ± 6.46**
139.78 ± 20.31**
38.01 ± 6.86*
710.22 ± 57.51**
188.60 ± 28.67**
63.64 ± 7.37***
60.12 ± 8.00***
190.38 ± 12.36***
44.47 ± 11.63**
655.68 ± 49.74***
150.18 ± 39.23***
Results were expressed as mean ± S.D., n ¼ 6 mice per group. One-way ANOVA followed by post hoc Tukey’s multiple comparison test. $$$Significance at p50.001 in comparison with the normal control group. Significance at *p50.05, **p50.01, and ***p50.001 in comparison with the vincristine-treated group.
exhibited dose-dependent significant (F (5,30) ¼ 22.85; p50.001) increase in the SOD activity in the sciatic nerve in comparison with vincristine-treated mice. Pregabalintreated mice also exhibited significant (p50.01) increase in the SOD activity (Table 1). Effect of curcumin on CAT activity Vincristine-administered mice showed a significant decrease (p50.001) in CAT levels in comparison with normal control mice. Treatment with curcumin at 15, 30, and 60 mg/kg exhibited a significant dose-dependent increase (p50.01, p50.01, and p50.001) in CAT activity, respectively, in the sciatic nerve in comparison with vincristine-treated mice (F (5,30) ¼ 14.83). However, pregabalin-treated mice did not exhibit any significant alterations in the CAT activity in comparison with vincristine-treated mice (Table 1). Effect of curcumin on GPx activity GPx activity was significantly depleted (p50.001) in vincristine-administered mice in comparison with normal control mice. Treatment with curcumin (30 and 60 mg/kg) treated mice significantly elevated the GPx activity at p50.01 and p50.001, respectively, in comparison with vincristine-treated mice (F (5,30) ¼ 20.81). Pregabalin and curcumin (15 mg/kg) -treated mice did not show any significant alterations in the GPx enzyme activity in comparison with vincristine-treated mice (Table 1). Effect of curcumin on GSH levels Significant decrease (p50.001) in GSH levels was observed in vincristine-administered mice in comparison with normal control mice. Curcumin treatment at 30 and 60 mg/kg exhibited a significant dose-dependent increase in the GSH levels at p50.05 and p50.01, respectively, in comparison with vincristine-treated mice [F (5,30) ¼ 8.36]. No significant alterations in GSH levels were observed in pregabalin and curcumin (15 mg/kg) -treated mice (Table 1).
Effect of curcumin on LPO levels In comparison with control mice, vincristine-treated mice exhibited a significant elevation (p50.001) in LPO levels. Curcumin 15, 30, and 60 mg/kg treatment significantly decreased the LPO levels at p50.01, p50.01, and p50.001, respectively, in comparison with vincristine-treated mice [F (5,30) ¼ 10.24]. Pregabalin treatment also exhibited significant decrease (p50.05) in LPO levels in comparison with vincristine-treated mice (Table 1). Effect of curcumin on NO levels Vincristine-administered mice exhibited a significant increase in NO levels in comparison with normal control mice. Treatment with curcumin 15, 30, and 60 mg/kg significantly decreased the NO levels at p50.05, p50.01, and p50.001, respectively, in comparison with vincristine-treated mice [F (5,30) ¼ 12.29]. Pregabalin at 10 mg/kg significantly decreased the NO levels at p50.001 in comparison with vincristine-treated mice (Table 1).
Discussion Vincristine has been commonly used as an anticancer agent for the management of various cancer disorders. However, its utilization has been limited due to unavoidable CIPN. Till date there is no effective prevention or management for CIPN, hence several patients are forced to decrease the dose or discontinue their drugs like vincristine, paclitaxel, oxaliplatin, and cisplatin (Geis et al., 2011). Vincristine possesses neurotoxicity as well as anticancer action due to its binding towards neuronal cytoskeleton protein (b-tubulin) and distruptive action in polymerization of microtubules (Tanner et al., 1998). Vincristine is also known to modulate the cellular Ca2+ levels, generation of free radicals, and release of inflammatory mediators which leads to axonal degeneration (Muthuraman et al., 2008). Here, the protective potential of curcumin in the mouse model of vincristine-induced CIPN was evaluated. As demonstrated earlier, vincristine-injected mice exhibited CIPN which was evident from significant decrease in nociceptive threshold in hyperalgesic, allodynic, and formalin
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test models (Hansen et al., 2011; Saika et al., 2009). Vincristine-injected mice also displayed functional (SFI) loss and oxidative stress in sciatic nerve in the present study. Pregabalin, a selective Cav 2.2 (a2 -d subunit) channel antagonist, was used as a standard and protected the vincristine-induced hyperalgesic response in this study, which is in accord with previous report (Thiagarajan et al., 2012). In the present study, treatment with curcumin in vincristine-injected mice significantly increased the nociceptive threshold, decreased the oxidative stress and total calcium levels, and improved functional index in a dose-dependent manner. Pain behavior due to vincristine intoxication is associated with dysfunction and increase in spontaneous discharge of A-fibers and C-fibers (Xiao & Bennett, 2008). The antinociceptive effect of curcumin could be due to the inhibitory effect on cytokine production. Earlier, curcumin has been reported to show antinociceptive effect in normal mice and in diabetic neuropathic pain-induced conditions which support the present findings (Aggarwal & Harikumar, 2009; Sharma et al., 2006). The nociception induced by formalin is associated with injured tissue. It has been reported that formalin-induced nociception resembles clinical pain more closely in comparison with other tests (Lopes et al., 2009). The acute phase (0–10 min) of the formalin test is short-lived and is characterized by C-fiber activation due to peripheral stimuli. The delayed phase (20–40 min) is a longer, persistent period caused by local tissue inflammation and also by functional changes in the dorsal horn of the spinal cord. Substances that act primarily as central analgesics inhibit both phases while peripherally acting drugs inhibit only the delayed phase (Le Bars et al., 2001). Curcumin exhibited analgesia only in the delayed phase which suggests that curcumin would possibly prevent the inflammatory proteins-induced pain perception (peripheral action). However, curcumin produced antinociceptive activity in a hot plate model indicating possible central activity. Hence, it can be stated that the antinociceptive effect of curcumin can be attributed due to its metabolite (tetrahydrocurcumin) or due to the inhibitory effect on cytokine production or both. Tetrahydrocurcumin has been reported to possess better anti-inflammatory activity by decreasing the levels of inducible NOS and COX-2 through downregulation of ERK1/2 activation than curcumin (Lai et al., 2011). The descending monoamine pathway, chiefly noradrenergic and serotonergic transmission, is also a key factor of the endogenous pain modulatory system (Millan, 2002). Earlier, curcumin increased the release of monoamines in the brain of mice or rats (Kulkarni et al., 2008; Zhao et al., 2012), suggesting curcumin may activate the descending monoamine system to contribute to its analgesic actions. In the present study, curcumin also exhibited antinociceptive activity in the hot plate model indicating possible central activity. Hence, it can be stated that the antinociceptive effect of curcumin can be due to the multiple actions which include suppression of inflammatory proteins and by inhibitory actions of cytokines. The SFI is an assessment of functional loss using three variables that measure PL, intermediary TS, and total TS. Significant positive correlation between SFI and distal and proximal fiber diameter has been reported earlier
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(Martins et al., 2006). Thus, SFI provides a non-invasive and quantitative method to evaluate functional recovery of walking ability, the ultimate goal in the regeneration of injured sciatic nerve (Varejao et al., 2004). In the present study, curcumin significantly improved the SFI index which indicates its potential neuroprotective action against vincristine. Further, it has been recently reported that curcumin promotes nerve regeneration and functional recovery in the rat model of sciatic nerve crush injury (Noorafshan et al., 2011). Massive accumulation of intracellular calcium has been reported in various types of neuropathic disorders which include vincristine-induced neuropathy (Jaggi & Singh, 2010). Accumulation of calcium ion has been known to activate the secondary messengers (calpain and calmodulin) which can lead to axonal degeneration by altering the stability of axonal cytoskeleton protein (Glass et al., 2002). In the present study, curcumin significantly decreased the calcium ion accumulation in the sciatic nerve which could be a reason for protective effect of curcumin against vincristine-induced CIPN. Previous findings also indicate that curcumin possesses calcium inhibitory action (Wang et al., 2012). Oxidative stress is another major contributor in the development of neuropathy in vincristine-injected mice. In the present study, significant increase in LPO levels and reductions in endogenous antioxidant enzyme levels in vincristine-injected mice have been observed. Oxidative stress is also known to cause vascular impairment leading to endoneurial hypoxia which leads to impaired neural function and reduced nerve conduction velocity (Pop-Busui et al., 2002). In the present study, curcumin exhibited significant decrease in LPO and NO and increase in endogenous antioxidant enzymes better than pregabalin. This antioxidant activity of curcumin could also have influenced its activity in vincristine-induced neuropathic pain. In conclusion, the protective effect of curcumin in vincristine-induced neuropathy includes multiple actions, namely antinociceptive, calcium inhibitory, and antioxidative activity.
Declaration of interest The authors declare that they have no conflict of interest.
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ORIGINAL ARTICLE
Effect of curcumin in mice model of vincristine-induced neuropathy Anand Babu, K. G. Prasanth, and Bhaskar Balaji
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Department of Pharmacology, PSG College of Pharmacy, Coimbatore, Tamil Nadu, India
Abstract
Keywords
Context: Curcumin exhibits a wide spectrum of biological activities which include neuroprotective, antinociceptive, anti-inflammatory, and antioxidant activity. Objective: The present study evaluates the effect of curcumin in vincristine-induced neuropathy in a mice model. Materials and methods: Vincristine sulfate (0.1 mg/kg, i.p. for 10 consecutive days) was administered to mice to induce neuropathy. Pain behavior was assessed at different days, i.e., 0, 7, 10, and 14 d. Sciatic nerve total calcium, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), nitric oxide (NO), and lipid peroxidation (LPO) were also estimated after the 14th day of study. Pregabalin (10 mg/kg, p.o.) and curcumin (15, 30, and 60 mg/kg, p.o.) were administered for 14 consecutive days. Results: Curcumin at 60 mg/kg significantly attenuated the vincristine-induced neuropathic pain manifestations in terms of thermal hyperalgesia (p50.001) and allodynia (p50.001); mechanical hyperalgesia (p50.001); functional loss (p50.001); and in the delayed phase of formalin test (p50.001). Curcumin at 30 and 60 mg/kg exhibited significant changes (p50.001) in antioxidant levels and in total calcium levels in vincristine-injected mice. Conclusion: Curcumin at 30 and 60 mg/kg dose levels significantly attenuated vincristineinduced neuropathy which may be due to its multiple actions including antinociceptive, calcium inhibitory, and antioxidant effect.
Antinociceptive, chemotherapy, allodynia, formalin test, hyperalgesia, neuroprotective, pain, sciatic functional index
Introduction Chemotherapy-induced painful neuropathy (CIPN) is a common side effect of anticancer treatment with vinca alkaloids, platinum drugs, taxanes, and other chemotherapeutic drugs affecting up to 30–40% of patients (Kaley & Deangelis, 2009). Symptoms usually start when chemotherapy is in process and tend to improve after completing therapy. However, in 25–30% of patients, pain or unpleasant paresthesias remain or even increase after terminating the chemotherapeutic treatment (Verstappen et al., 2005). Vincristine is one of the most common anticancer drugs applied for the treatment of many types of cancers such as leukemias, lymphomas, and sarcomas and entails the side effect of a dose and duration-dependent peripheral neuropathy. The anticancer action of vincristine is due to its binding to b-tubulin, which leads to disorganization of the axonal microtubule cytoskeleton. This effect underlies its antimitotic property and also in part explains the neurotoxic effect (Tanner et al., 1998). Conventional analgesic agents like non-steroidal antiinflammatory drugs (NSAIDs) and opioids are clinically
Correspondence: Bhaskar Balaji, Assistant Professor, Department of Pharmacology, PSG College of Pharmacy, Coimbatore 641004, Tamil Nadu, India. Tel: +91 422 4345843. Fax: +91 422 2594400. E-mail: [email protected]
History Received 22 April 2014 Revised 9 June 2014 Accepted 7 July 2014 Published online 27 November 2014
ineffective for attenuation of CIPN. Usefulness of tricyclic antidepressants and anticonvulsants in management of neuropathic pain is indicated but these medications exhibit a wide spectrum of adverse effects which limit their full clinical exploitation (Dworkin et al., 2010). Several herbal medicines such as Phyllanthus emblica Linn. (Phyllanthaceae), Cannabis sativa Linn. (Cannabaceae), Nigella sativa Linn. (Ranunculaceae), Ocimum sanctum Linn. (Lamiaceae), and Ginkgo biloba Linn. (Ginkgoaceae) are shown to have potential in different types of experimentally induced neuropathic pain (Kim et al., 2009; Muthuraman et al., 2008; Wirth et al., 2005). Some clinical reports have also advocated beneficial effect of drugs from plant origin in neuropathic pain conditions (Ellis et al., 2009; Zareba, 2009). Curcuma longa L. (Zingiberaceae), a traditional herb, has been used to manage various disorders for thousands of years. Curcumin isolated from Curcuma longa has shown to be nontoxic and non-mutagenic and exhibits a wide spectrum of biological activities which include neuroprotective, antiinflammatory, antidepressant, and antioxidant activity (Fan et al., 2013; Gupta et al., 2013). Curcumin has also been reported to exert antinociceptive effects in different animal models of pain (Mittal et al., 2009; Tajik et al., 2008), which also includes diabetic neuropathic pain (Sharma et al., 2006). With the above background, the present study was undertaken to investigate the effect of curcuminin vincristine-induced neuropathic pain in a mice model. Pregabalin, a selective Cav
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2.2 (a2-d subunit) channel antagonist served as positive control in this study.
Materials and methods
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Chemicals and preparation of drug solutions Vincristine sulfate (Sun Pharma, Mumbai, India), pregabalin (Ranbaxy Research Laboratories, Gurgaon, India), curcumin (Himedia Laboratories, Mumbai, India), nitro blue tetrazolium (NBT), (Sigma Aldrich, St. Louis. MO), Folin– Ciocalteu’s phenol reagent (Merck Limited, Mumbai, India), 5,50 -dithiobis (2-nitrobenzoic acid) (DTNB), bovine serum albumin (BSA), (Sisco Research Laboratories Pvt. Ltd. Mumbai, India), reduced glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH) (Himedia Laboratories, Mumbai, India), thiobarbituric acid (TBA) (Sigma Aldrich, St. Louis. MO) were procured for the present study. All the reagents used in the present study were of analytical grade. Pregabalin and curcumin were suspended in 0.3% w/v carboxy methyl cellulose (CMC) solution and vincristine was diluted with normal saline. Experimental animals Thirty adult male Swiss albino mice with a body weight of 22–25 g were used for this study. Mice were group-housed (n ¼ 6 per cage) in a room with controlled temperature (21 ± 2 C), and 12 h light-dark cycle was maintained. All the animals had free access to food and water ad libitum. The experimental protocols were approved by the Institutional Animal Ethical Committee and experiments were performed in accordance to the committee for the purpose of control and supervision of experiments on animals (CPCSEA) guidelines for ethical use of animals (CPCA no. 158/99/CPCSEA/14/ 2011). Preselection of animals The rota-rod test was performed to investigate the motor coordination of the mice. Mice were placed on a rotating rod (10 rpm) and the latency to falling was measured for up to 2 min according to the method described previously by Egashira et al. (2004). Three trials per day for 2 d were enough for the mice to learn this task. On the day of the test, only the mice that were able to stay balanced on rotating rod for 2 min were selected for further study.
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Group III, IV, V, and VI mice received pregabalin (10 mg/kg/ d p.o.), curcumin (15 mg/kg/d p.o.), curcumin (30 mg/kg/d p.o.), and curcumin (60 mg/kg/d p.o.), respectively, 1 h before each vincristine injection for 14 consecutive days. The behavioral tests like hot plate, cold plate, pin prick test, rota-rod, and sciatic functional index (SFI) were performed on different days, i.e. days 0, 7, 10, and 14. On the 14th day, mice were subjected to the formalin test. Thereafter, all the animals were sacrificed under deep ether anesthesia and subjected to biochemical analysis for estimating the total protein, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), GSH, lipid peroxidation (LPO), nitric oxide (NO), and total calcium activity in sciatic nerve tissue sample. Behavioral assessment Hot plate test (thermal hyperalgesia) In this test, mice were individually placed on a hot plate (Eddy’s hot plate) with the temperature adjusted to 55 ± 1 C. The latency to the first sign of paw licking or jump response to avoid the heat was taken as an index of the pain threshold; the cut-off time was 10 s in order to avoid damage to the paw (Tiwari et al., 2011). Cold plate test (thermal allodynia) Mice were placed on an ice platform submerged approximately 1 cm below the surface of cold water (4 C), such that the hairy and glabrous skin of the mice feet were in contact with the cold water. The latency prior to the first reaction (licking, moving the paws, and leaps) was recorded with a cutoff time of 30 s (Beyreuther et al., 2006). Pin prick test (mechanical hyperalgesia) Mechanical hyperalgesia was evaluated by the pin prick test as described in the method by Erichsen & Blackburn-Munro (2002). Briefly, the plantar surface of the left hind paw was touched with the point of the bent 18 gauge needle (at 90 angle) at intensity sufficient to produce a reflex withdrawal response in normal mice, but at an intensity which was insufficient to penetrate the skin in all groups. The duration of the paw withdrawal was recorded in seconds. A cut-off time of 20 s was maintained. Rota-rod test
Induction of peripheral neuropathy by vincristine Peripheral painful neuropathy was induced in mice which stood on a rotating rod for 2 min by intraperitoneal administration of vincristine sulfate (0.1 mg/kg) once per day for 7 consecutive days as per the method adopted by Kiguchi et al. (2008). Experimental design Group I served as normal control in which mice received 0.3% w/v carboxy methyl cellulose (CMC) (p.o.) vehicle administration for 14 d. Group II served as vincristine control in which mice were administered 0.3% w/v CMC (p.o.) 1 h before each vincristine injection for 14 consecutive days.
Mice were subjected to the rota-rod test to assess the motor coordination as described earlier. Determination of SFI Mice were subjected to walking-track analysis and measurement of SFI using a method similar to that described by De Medinaceli et al. (1982). The trials were done in an 8.2 42 cm corridor darkened at one end and covered with a sheet of white paper. The hind paws of the mice were dipped in black India-ink and then each animal was allowed to walk freely in the corridor mentioned above. From the analysis of the footprints of the feet, the following lengths were obtained: (a) distance from the heel to the third toe, the print length
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(PL); (b) distance from the first to the fifth toe, the toe spread (TS); and (c) distance from the second to the fourth toe, the intermediary TS (ITS). All three measurements were taken from the experimental (E) and normal (N) sides. The factors were calculated as follows: (I) PL factor (PLF) ¼ (EPL NPL)/NPL; (II) TS factor (TSF) ¼ (ETS NTS)/ NTS; (III) intermediary TS factor (ITF) ¼ (EIT NIT)/NIT. These factors were then incorporated into the SFI formula derived by Bain et al. (1989): SFI ¼ 38.3 PLF + 109.5 TSF + 13.3 ITF 8.8. An SFI of 0 is normal. An SFI of 100 indicates total impairment, such as would result from a complete transaction of the sciatic nerve.
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Formalin test in mice At the end of the study, all the groups were subjected to the formalin test. Briefly, each mouse was acclimatized to the observation box before the formalin test. After an adaptation period of 15 min, the right hind footpaw was injected with 50 mL of 2.5% formalin in the intraplantar region. Nociception was evaluated by quantifying paw licking time and paw elevation time during the first 10 min (acute phase) and at 20– 40 min (delayed phase) (Khalilzadeh et al., 2008; Luiz et al., 2007). Biochemical estimation All the mice were sacrificed with chemical euthanasia on the 14th day after behavioral tests. The portions of the sciatic nerve were isolated immediately and processed for SOD, CAT, GPx, GSH, NO, and LPO. The sciatic nerve homogenate (10% w/v) was prepared with 0.1 M Tris-HCl buffer (pH 7.4) and deionised water, respectively, for total protein and total calcium estimation. Superoxide dismutase SOD was assayed in the nerve homogenate according to the method described by Kakkar et al. (1984). The reduction of NBT to blue formazan mediated by superoxide anions was measured at 560 nm under aerobic conditions. Addition of SOD inhibits the reduction of NBT and the extent of inhibition is taken as a measure of enzyme activity. The activity of enzyme was expressed as units/mg protein, where one unit is defined as the amount of enzyme inhibiting the rate of reaction by 50%. Catalase CAT activity was assayed in the nerve homogenate by the Beers and Sizer (1952) method. Hydrogen peroxide (H2O2) decomposition by CAT was monitored spectrophotometrically by following the decrease in absorbance at 240 nm. The activity of enzyme was expressed as mmoles of H2O2 decomposed/min/mg protein. Glutathione peroxidase GPx activity was measured in the nerve homogenate by the method of Paglia and Valentine (1967). The reaction measured the rate of reduced GSH oxidation by H2O2 catalyzed by the GPx. GSH is maintained at constant concentration by the addition of exogenous glutathione
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reductase and NADPH, which immediately converts any oxidized glutathione disulfide (GSSG) produced to GSH. The rate of GSSG formation is then measured by following the absorbance of NADPH at 340 nm for 5 min. The activity of enzyme was expressed as nmoles NADPH oxidized/min/mg protein. Reduced Glutathione Glutathione content was estimated by following the Jollow et al. (1974) method. In this assay, GSH reduced 5,50 -dithiobis (2-nitrobenzoic acid) (DTNB) to 5-thio-2-nitrobenzoic acid (TNB) and the GSSG. TNB is a yellow product which was measured spectrophotometrically at 412 nm. The amount of GSH was expressed in mM/mg protein. LPO levels The quantitative measurement of LPO was performed in the nerve homogenate according to the Ohkawa et al. (1979) method. The amount of malondialdehyde (MDA) and other thiobarbituric acid reactive substances (TBARS) are quantified by their reactivity with TBA in acidic conditions. The reaction generated a pink-colored chromophore which was measured in a UV spectrophotometer at 532 nm. The results were expressed as nmoles MDA/mg protein. Nitric oxide Nitrate/nitrite was assayed in the nerve homogenate according to the Green et al. (1982) method using the Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyl ethylenediamine dihydrochloric acid in water in the ratio of 1:1). The color intensity of chromogen was read at 540 nm. The results were expressed as mM/mg protein. Estimation of total protein content Protein concentration of nerve homogenate was estimated according to the Lowry et al. (1951) method, using bovine serum albumin (BSA) as a standard. The absorbance was determined spectrophotometrically at 750 nm. Estimation of total calcium Total calcium levels were estimated in sciatic nerve homogenate according to the Severinghaus and Ferrebee (1950) method. Briefly, homogenate was mixed with 1 ml of trichloroacetic acid (4%) in ice cold conditions and centrifuged at 2000 rpm for 10 min. The clear supernatant was used for the estimation of total calcium ion by atomic emission spectroscopy at 556 nm. Statistical analysis All the results were expressed as mean ± standard deviation. The data were statistically analyzed by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple comparison test for biochemical estimations and formalin test. Two-way ANOVA (treatment versus duration) followed by the Bonferroni post test was used for the hot plate test, cold plate test, pin prick test, and SFI. Probability values of less than 0.05 were considered as significant. The analysis was
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carried out using Graph pad prism software (version 4.03) (GraphPad Software, San Diego, CA).
Results
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Effect of curcumin on hot plate test Administration of vincristine resulted in a significant (p50.001) development of thermal hyperalgesia as reflected by an increase either in the hind paw licking, lifting, or jumping from the hot plate surface on days 7, 10, and 14 in comparison to day 0 (Figure 1). Different treatments (F (5, 120) ¼ 66.58; p50.001) and duration of treatment (F (3, 120) ¼ 36.66; p50.001) significantly improved the hyperalgesic response. The interaction data (treatment versus duration) was also found to be significant (F (15, 120) ¼ 8.36; p50.001) which indicates time-dependent improvement in the analgesic response. Curcumin (15 mg/kg) did not significantly increase the pain threshold in comparison with vincristine-treated mice on days 7, 10, and 14. Curcumin (30 mg/kg) significantly increased the pain threshold in comparison with vincristine-treated mice on day 7 (p50.05) and days 10 and 14 (p50.001). Treatment of pregabalin and
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curcumin (60 mg/kg) significantly increased (p50.001) the pain threshold in comparison with vincristine-treated mice on days 7, 10, and 14. In pregabalin- and curcumin (60 mg/kg)-treated mice, the duration of paw withdrawal latency was not significantly different on days 7, 10, and 14 in comparison with the baseline (day 0). This indicates that curcumin (60 mg/kg) was able to restore the physiological thermal pain perception (Figure 1). Effect of curcumin on the cold plate test Vincristine-administered mice exhibited a significant development (p50.001) in thermal allodynia which was reflected by a decrease in the paw withdrawal latency period on days 7, 10, and 14 in comparison with day 0. Different treatments [F (5, 120) ¼ 91.08; p50.001] and duration of treatment [F (3, 120) ¼ 6.39; p50.001] significantly improved the allodynic response. The interaction data (treatment versus duration) was also found to be significant [F (15, 120) ¼ 15.90; p50.001] which indicates time-dependent improvement in the allodynic response. Administration of pregabalin and curcumin at 15, 30, and 60 mg/kg significantly attenuated
Figure 1. Effect of curcumin (15, 30, and 60 mg/kg) on thermal hyperalgesia induced by the hot plate in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. Two-way ANOVA followed by post-hoc Bonferonni’s test. a – indicates significance at p50.001 in comparison to normal control mice at corresponding days. b,d – indicates significance at p50.001 and p50.05, respectively, in comparison to day 0. x,z – indicates significance at p50.001 and p50.05, respectively, in comparison to vincristine-treated mice at corresponding days.
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(p50.001) vincristine-induced thermal allodynia in comparison with vincristine-treated mice. Interestingly, curcumin (60 mg/kg)-treated mice exhibited significant increase (p50.001) in paw withdrawal latency on days 10 and 14 in comparison with the baseline. Pregabalin treatment also showed similar results on days 7, 10, and 14 in comparison with the baseline (Figure 2).
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Effect of curcumin on pin prick test Administration of vincristine resulted in a significant (p50.001) development of mechanical hyperalgesia as reflected by a significant increase in the left hind paw lifting duration in the pinprick test. Different treatments [F (5, 120) ¼ 140.74; p50.001] and duration of treatment [F (3, 120) ¼ 102.30; p50.001] significantly improved the hyperalgesic response. The interaction data (treatment versus duration) was also found to be significant [F (15, 120) ¼ 16.04; p50.001] and indicates time-dependent improvement in the analgesic response. Curcumin at 15 mg/kg significantly (p50.05) attenuated the mechanical hyperalgesia in comparison with vincristine-treated mice on day 14. Administration of pregabalin and curcumin (30 and 60 mg/kg) significantly attenuated
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(p50.001) the increase in the left hind paw lifting duration in comparison with vincristine-treated mice on days 7, 10, and 14. In curcumin (60 mg/kg)-treated mice, the duration of paw lifting was not significantly different on days 10 and 14 in comparison with the baseline (day 0). This indicates that curcumin (60 mg/kg) was able to restore the physiological mechanical pain perception. Pregabalintreated mice exhibited no significant difference in the paw withdrawal latency on days 7, 10, and 14 in comparison with the baseline (Figure 3). Effect of curcumin on motor performance Vincristine treatment did not show any significant difference in the performance of mice on the accelerating rota-rod from that of the normal control group on days 7, 10, and 14. Curcumin (15, 30 and 60 mg/kg) and pregabalin treatment did not show any significant difference in comparison with vincristine-treated mice (data not shown). Effect of curcumin in formalin test The effect of curcumin in formalin test was measured in two different phases: acute and delayed phase.
Figure 2. Effect of curcumin (15, 30, and 60 mg/kg) on thermal allodynia induced by cold plate in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. Two-way ANOVA followed by post-hoc Bonferonni’s test. a – indicates significance at p50.001 in comparison to normal control mice at corresponding days. b,c – indicates significance at p50.001 and p50.01, respectively, in comparison to day 0. x – indicates significance at p50.001 in comparison to vincristine-treated mice at corresponding days.
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Figure 3. Effect of curcumin (15, 30, and 60 mg/kg) on mechanical hyperalgesia by the pin prick test in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. Two-way ANOVA followed by post-hoc Bonferonni’s test. a – indicates significance at p50.001 in comparison to normal control mice at corresponding days. b,c – indicates significance at p50.001 and p50.01, respectively, in comparison to day 0. x,z – indicates significance at p50.001 and p50.05 in comparison to vincristine-treated mice at corresponding days.
Acute phase Formalin injection in vincristine-administered mice produced a significant increase in paw licking (F (5,30) ¼ 5.98; p50.001) and paw elevation (F (5,30) ¼ 3.33; p50.05) in comparison with normal control mice. Administration of pregabalin and curcumin (15, 30, and 60 mg/kg) did not produce any significant alterations in paw licking and paw elevation in comparison with vincristine-administered mice (Figure 4a). Delayed phase Formalin injection in vincristine-administered mice produced a significant increase in paw licking (F (5,30) ¼ 8.26; p50.001) and paw elevation (F (5,30) ¼ 26.02; p50.001) in comparison with normal control mice. Curcumin at 30 mg/ kg showed a significant decrease (p50.01) in paw elevation in comparison with vincristine-administered mice, while curcumin at 60 mg/kg also significantly decreased the paw elevation (p50.001) and paw licking (p50.01) in comparison with vincristine-administered mice. Pregabalin at 10 mg/ kg significantly decreased (p50.001) the paw licking time and paw elevation time in comparison with vincristineadministered mice (Figure 4b).
SFI level. Different treatments (F (5, 120) ¼ 728.73; p50.001) and duration of treatment (F (3, 120) ¼ 516.95; p50.001) significantly improved the SFI. The interaction data (treatment versus duration) was also found to be significant (F (15, 120) ¼ 86.83; p50.001) and indicated time-dependent improvement in SFI. Administration of curcumin (15, 30, and 60 mg/kg) significantly attenuated (p50.001) vincristine-induced rise in SFI in a dose-dependent manner. However, curcumin treatment at all the dose levels was unable to restore the SFI to baseline score. Similar effects were seen with pregabalin administration (Figure 5). Effect of curcumin in total calcium levels Administration of vincristine exhibited a significant increase (p50.001) in total calcium levels in comparison with normal control mice in the sciatic nerve homogenate (Figure 6). Curcumin at 15 mg/kg significantly decreased (p50.01) the calcium levels in comparison with vincristine-treated mice. Pregabalin and curcumin (30 and 60 mg/kg) significantly decreased (p50.001) the calcium levels in comparison with vincristine-treated mice (F (5,30) ¼ 132). Effect of curcumin on SOD activity
Effect of curcumin on SFI Vincristine-administered mice resulted in sciatic functional loss as reflected by a significant rise (p50.001) in the
In comparison with normal control mice, administration of vincristine significantly (p50.001) depleted the SOD activity in mice. Curcumin (15, 30, and 60 mg/kg) treated mice
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Figure 4. (a) Effect of curcumin (15, 30, and 60 mg/kg) on nociception induced by formalin (acute phase) in the vincristine-induced neuropathic pain mice model. (b) Effect of curcumin (15, 30, and 60 mg/kg) on nociception induced by formalin (delayed phase) in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. One-way ANOVA followed by post-hoc Tukey’s multiple comparison test. $$,$$$ Indicate significance at p50.01 and p50.001, respectively, in comparison to normal control group. **,***Indicate significance at p50.01.
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Figure 5. Effect of curcumin (15, 30, and 60 mg/kg) on sciatic functional index in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. Two-way ANOVA followed by post-hoc Bonferonni’s test. a – indicates significance at p50.001 in comparison to normal control mice at corresponding days. b,c,d – indicates significance at p50.001 and p50.01, and p50.05, in comparison to day 0. x – indicates significance at p50.001 in comparison to vincristine-treated mice at corresponding days.
Figure 6. Effect of curcumin (15, 30, and 60 mg/kg) on sciatic nerve total calcium levels in the vincristine-induced neuropathic pain mice model. Results are expressed as mean ± SD, n = 6 mice per group. One-way ANOVA followed by post-hoc Tukey’s multiple comparison test. $$$Indicates significance at p50.001 in comparison to normal control group. **,***Indicate significance at p50.01. and p50.001 respectively, in comparison to vincristine-treated group.
Curcumin against vincristine-induced neuropathy
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Table 1. Effect of curcumin (15, 30, and 60 mg/kg) on sciatic nerve homogenate oxidative markers in the vincristine-induced neuropathic pain mice model.
Treatment and dose
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Normal control Vincristine control Vincristine + pregabalin 10 mg/kg Vincristine + curcumin 15 mg/kg Vincristine + curcumin 30 mg/kg Vincristine + curcumin 60 mg/kg
SOD CAT (units/mg protein) (units/mg protein)
GPx (units/mg protein)
GSH (mM/mg protein)
LPO (nM/mg protein)
NO (mM/mg protein)
48.26 ± 6.40 22.19 ± 2.43$$$ 40.75 ± 4.07**
62.34 ± 3.61 32.74 ± 4.94$$$ 44.88 ± 8.76
151.94 ± 23.50 77.81 ± 16.87$$$ 101.60 ± 17.64
44.68 ± 6.65 17.99 ± 4.56$$$ 33.09 ± 4.69
591.92 ± 82.70 134.92 ± 21.70 1077.35 ± 190.82$$$ 266.37 ± 38.56$$$ 821.66 ± 80.11* 166.05 ± 36.75***
42.85 ± 4.31***
48.14 ± 3.98**
107.55 ± 13.66
33.22 ± 6.48
760.45 ± 112.94**
197.51± 26.30*
49.84 ± 7.59***
50.49 ± 6.46**
139.78 ± 20.31**
38.01 ± 6.86*
710.22 ± 57.51**
188.60 ± 28.67**
63.64 ± 7.37***
60.12 ± 8.00***
190.38 ± 12.36***
44.47 ± 11.63**
655.68 ± 49.74***
150.18 ± 39.23***
Results were expressed as mean ± S.D., n ¼ 6 mice per group. One-way ANOVA followed by post hoc Tukey’s multiple comparison test. $$$Significance at p50.001 in comparison with the normal control group. Significance at *p50.05, **p50.01, and ***p50.001 in comparison with the vincristine-treated group.
exhibited dose-dependent significant (F (5,30) ¼ 22.85; p50.001) increase in the SOD activity in the sciatic nerve in comparison with vincristine-treated mice. Pregabalintreated mice also exhibited significant (p50.01) increase in the SOD activity (Table 1). Effect of curcumin on CAT activity Vincristine-administered mice showed a significant decrease (p50.001) in CAT levels in comparison with normal control mice. Treatment with curcumin at 15, 30, and 60 mg/kg exhibited a significant dose-dependent increase (p50.01, p50.01, and p50.001) in CAT activity, respectively, in the sciatic nerve in comparison with vincristine-treated mice (F (5,30) ¼ 14.83). However, pregabalin-treated mice did not exhibit any significant alterations in the CAT activity in comparison with vincristine-treated mice (Table 1). Effect of curcumin on GPx activity GPx activity was significantly depleted (p50.001) in vincristine-administered mice in comparison with normal control mice. Treatment with curcumin (30 and 60 mg/kg) treated mice significantly elevated the GPx activity at p50.01 and p50.001, respectively, in comparison with vincristine-treated mice (F (5,30) ¼ 20.81). Pregabalin and curcumin (15 mg/kg) -treated mice did not show any significant alterations in the GPx enzyme activity in comparison with vincristine-treated mice (Table 1). Effect of curcumin on GSH levels Significant decrease (p50.001) in GSH levels was observed in vincristine-administered mice in comparison with normal control mice. Curcumin treatment at 30 and 60 mg/kg exhibited a significant dose-dependent increase in the GSH levels at p50.05 and p50.01, respectively, in comparison with vincristine-treated mice [F (5,30) ¼ 8.36]. No significant alterations in GSH levels were observed in pregabalin and curcumin (15 mg/kg) -treated mice (Table 1).
Effect of curcumin on LPO levels In comparison with control mice, vincristine-treated mice exhibited a significant elevation (p50.001) in LPO levels. Curcumin 15, 30, and 60 mg/kg treatment significantly decreased the LPO levels at p50.01, p50.01, and p50.001, respectively, in comparison with vincristine-treated mice [F (5,30) ¼ 10.24]. Pregabalin treatment also exhibited significant decrease (p50.05) in LPO levels in comparison with vincristine-treated mice (Table 1). Effect of curcumin on NO levels Vincristine-administered mice exhibited a significant increase in NO levels in comparison with normal control mice. Treatment with curcumin 15, 30, and 60 mg/kg significantly decreased the NO levels at p50.05, p50.01, and p50.001, respectively, in comparison with vincristine-treated mice [F (5,30) ¼ 12.29]. Pregabalin at 10 mg/kg significantly decreased the NO levels at p50.001 in comparison with vincristine-treated mice (Table 1).
Discussion Vincristine has been commonly used as an anticancer agent for the management of various cancer disorders. However, its utilization has been limited due to unavoidable CIPN. Till date there is no effective prevention or management for CIPN, hence several patients are forced to decrease the dose or discontinue their drugs like vincristine, paclitaxel, oxaliplatin, and cisplatin (Geis et al., 2011). Vincristine possesses neurotoxicity as well as anticancer action due to its binding towards neuronal cytoskeleton protein (b-tubulin) and distruptive action in polymerization of microtubules (Tanner et al., 1998). Vincristine is also known to modulate the cellular Ca2+ levels, generation of free radicals, and release of inflammatory mediators which leads to axonal degeneration (Muthuraman et al., 2008). Here, the protective potential of curcumin in the mouse model of vincristine-induced CIPN was evaluated. As demonstrated earlier, vincristine-injected mice exhibited CIPN which was evident from significant decrease in nociceptive threshold in hyperalgesic, allodynic, and formalin
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test models (Hansen et al., 2011; Saika et al., 2009). Vincristine-injected mice also displayed functional (SFI) loss and oxidative stress in sciatic nerve in the present study. Pregabalin, a selective Cav 2.2 (a2 -d subunit) channel antagonist, was used as a standard and protected the vincristine-induced hyperalgesic response in this study, which is in accord with previous report (Thiagarajan et al., 2012). In the present study, treatment with curcumin in vincristine-injected mice significantly increased the nociceptive threshold, decreased the oxidative stress and total calcium levels, and improved functional index in a dose-dependent manner. Pain behavior due to vincristine intoxication is associated with dysfunction and increase in spontaneous discharge of A-fibers and C-fibers (Xiao & Bennett, 2008). The antinociceptive effect of curcumin could be due to the inhibitory effect on cytokine production. Earlier, curcumin has been reported to show antinociceptive effect in normal mice and in diabetic neuropathic pain-induced conditions which support the present findings (Aggarwal & Harikumar, 2009; Sharma et al., 2006). The nociception induced by formalin is associated with injured tissue. It has been reported that formalin-induced nociception resembles clinical pain more closely in comparison with other tests (Lopes et al., 2009). The acute phase (0–10 min) of the formalin test is short-lived and is characterized by C-fiber activation due to peripheral stimuli. The delayed phase (20–40 min) is a longer, persistent period caused by local tissue inflammation and also by functional changes in the dorsal horn of the spinal cord. Substances that act primarily as central analgesics inhibit both phases while peripherally acting drugs inhibit only the delayed phase (Le Bars et al., 2001). Curcumin exhibited analgesia only in the delayed phase which suggests that curcumin would possibly prevent the inflammatory proteins-induced pain perception (peripheral action). However, curcumin produced antinociceptive activity in a hot plate model indicating possible central activity. Hence, it can be stated that the antinociceptive effect of curcumin can be attributed due to its metabolite (tetrahydrocurcumin) or due to the inhibitory effect on cytokine production or both. Tetrahydrocurcumin has been reported to possess better anti-inflammatory activity by decreasing the levels of inducible NOS and COX-2 through downregulation of ERK1/2 activation than curcumin (Lai et al., 2011). The descending monoamine pathway, chiefly noradrenergic and serotonergic transmission, is also a key factor of the endogenous pain modulatory system (Millan, 2002). Earlier, curcumin increased the release of monoamines in the brain of mice or rats (Kulkarni et al., 2008; Zhao et al., 2012), suggesting curcumin may activate the descending monoamine system to contribute to its analgesic actions. In the present study, curcumin also exhibited antinociceptive activity in the hot plate model indicating possible central activity. Hence, it can be stated that the antinociceptive effect of curcumin can be due to the multiple actions which include suppression of inflammatory proteins and by inhibitory actions of cytokines. The SFI is an assessment of functional loss using three variables that measure PL, intermediary TS, and total TS. Significant positive correlation between SFI and distal and proximal fiber diameter has been reported earlier
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(Martins et al., 2006). Thus, SFI provides a non-invasive and quantitative method to evaluate functional recovery of walking ability, the ultimate goal in the regeneration of injured sciatic nerve (Varejao et al., 2004). In the present study, curcumin significantly improved the SFI index which indicates its potential neuroprotective action against vincristine. Further, it has been recently reported that curcumin promotes nerve regeneration and functional recovery in the rat model of sciatic nerve crush injury (Noorafshan et al., 2011). Massive accumulation of intracellular calcium has been reported in various types of neuropathic disorders which include vincristine-induced neuropathy (Jaggi & Singh, 2010). Accumulation of calcium ion has been known to activate the secondary messengers (calpain and calmodulin) which can lead to axonal degeneration by altering the stability of axonal cytoskeleton protein (Glass et al., 2002). In the present study, curcumin significantly decreased the calcium ion accumulation in the sciatic nerve which could be a reason for protective effect of curcumin against vincristine-induced CIPN. Previous findings also indicate that curcumin possesses calcium inhibitory action (Wang et al., 2012). Oxidative stress is another major contributor in the development of neuropathy in vincristine-injected mice. In the present study, significant increase in LPO levels and reductions in endogenous antioxidant enzyme levels in vincristine-injected mice have been observed. Oxidative stress is also known to cause vascular impairment leading to endoneurial hypoxia which leads to impaired neural function and reduced nerve conduction velocity (Pop-Busui et al., 2002). In the present study, curcumin exhibited significant decrease in LPO and NO and increase in endogenous antioxidant enzymes better than pregabalin. This antioxidant activity of curcumin could also have influenced its activity in vincristine-induced neuropathic pain. In conclusion, the protective effect of curcumin in vincristine-induced neuropathy includes multiple actions, namely antinociceptive, calcium inhibitory, and antioxidative activity.
Declaration of interest The authors declare that they have no conflict of interest.
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