双氯芬酸钠对大鼠神经病理性疼痛的疗效及机制
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摘要
目的观察不同剂量双氯芬酸钠灌胃对坐骨神经结扎(CCI)大鼠机械痛阈,背根神经节(DRG)及脊髓背角P物质(SP)、降钙素基因相关肽(CGRP)表达及胃肠粘膜的影响。探讨口服双氯芬酸钠对治疗神经病理性疼痛的疗效和可能机制,及其对胃肠道的副作用,为临床应用该药时提供参考。
方法雄性SD大鼠40只(体重240-260g),随机分为坐骨神经结扎(CCI)组(32只)和假手术组(n = 8)。CCI组又分为: a :双氯芬酸钠组(2mg/kg组, n = 8;4mg /kg组,n = 8;10mg/kg组,n = 8);b对照组(生理盐水1ml,n =8)。自术后第8天分别给予不同剂量双氯芬酸钠及生理盐水灌胃,每日两次共7天。采用自制的Von-Frey纤毛机械刺激器测定灌胃前及灌胃第1、3、5、7天时大鼠右后肢机械刺激缩足反射阈值(PWMT)及用药7天后免疫组化法测定L4-5背根神经节细胞及腰膨大处(L4-5节段)脊髓背角SP和CGRP表达,并在光镜下观察胃肠粘膜结构变化。
结果
(1)对CCI大鼠机械痛阈的影响:对照组、2mg/kg组、4mg/kg组、10mg/kg组在用药前及用药后各时间点的右后肢PWMT与假手术组相比显著降低(P<0.01)。4mg/kg组在用药第5、7天时右后肢PWMT与用药前及对照组相比显著增加(P<0.01或P<0.05);10mg/kg组用药后各时间点右后肢PWMT与用药前及对照组相比显著增加(P<0.01)。且呈现出剂量依赖效应(P<0.05)。
(2)对大鼠胃肠粘膜结构的影响:肉眼观各组大鼠胃窦部、腺体部及肠粘膜均未见溃疡和出血。假手术组、对照组病理切片观胃、空肠粘膜结构完整,未见明显的炎性细胞浸润。2mg/k组病理切片观胃粘膜腺体略有增厚;空肠腺体未见明确变化。4mg/kg组观胃粘膜腺体变少,腺壁变薄;肠绒毛变少、变短,杯状细胞减少。10mg/kg组观胃粘膜腺体增生、变厚、染色变深;肠绒毛变少、变短,肠嗜酸性细胞多,有炎症改变。
(3)对大鼠背根神经节(DRG) SP、CGRP表达的影响:对照组、2mg/kg组、4mg/kg组、10mg/kg组SP及CGRP表达的平均光密度值(OD)均明显高于假手术组(P <0.01)。4mg/kg组、10mg/kg组SP及CGRP表达的平均光密度值(OD)明显低于对照组(P<0.01或P<0.05)。
(4)对大鼠脊髓背角I、II层SP、CGRP表达的影响:对照组、2mg/kg组、4mg/kg组、10mg/kg组SP及CGRP表达的平均光密度值(OD)均明显高于假手术组(P <0.01)。4mg/kg组、10mg/kg组SP及CGRP表达的平均光密度值(OD)明显低于对照组(P <0.01或P<0.05)。
结论
1、一定剂量(4 mg/kg、10 mg/kg)双氯芬酸钠灌胃能够减轻CCI大鼠机械性痛敏反应,且具有剂量依赖效应。
2、双氯芬酸钠减轻机械痛敏的作用机制可能是使背根神经节和脊髓背角SP、CGRP的表达减少。
3、镇痛效果虽有剂量依赖效应,但随之副作用也增加,提示临床应用应严格限制用药剂量。说明双氯芬酸钠治疗神经病理性疼痛的有效剂量同时也会引起胃肠粘膜损害,因而还不是治疗神经病理性疼痛的理想药物。
Objective
To obseve the effects of diclofenac sodium on mechanical hyperalgia, substance P (SP) and calcitonin gene-related peptide (CGRP) expression in dorsal root ganglion (DRG) neurons and dorsal horn, as well as the structure of gastrointestinal mucosa in chronic constriction injury (CCI) rats.
Materials and Methods
Forty male SD rats weighing 240-260g were randomly divided into 5 groups : 1) Sham operation group; 2) Control group ( treated with CCI) ; 3) diclofenac sodium 2mg/kg group; 4) diclofenac sodium 4mg/kg group; 5) diclofenac sodium 10mg/kg group . From 8th day post-ligation. Nature saline, different dose of diclofenac sodium were administered by injecting into gastric cavity twice per day for 7 days according to different groups, respectively. Before and after drug injection(at 1~(st)、3~(rd)、5~(th)、7~(th) day), the posterior Paw Withdrawal Mechanical Threshold (PWMT) was measured by Von Frey hair, and the expressions of SP and CGRP in L4-5 dorsal root ganglion (DRG) neurons and dorsal horn were detected by immunohistochemistry method. Meanwhile, the structure of gastrointestinal mucosa was observed in light microscope.
Results
(1) The effects of diclofenac sodium on PWMT
At pre-drug , different time post-drug, the right posterior PWMTs of Control group, diclofenac sodium 2mg/kg group, 4mg/kg group, 10mg/kg group were significantly decreased compared with that of Sham operation group (p<0.01). At 5~(th) and 7~(th) day post-drug, the PWMTs of 4mg/kg group were significantly increased compared with that of pre-durg and control group(P<0.01或P<0.05). At different time post-drug, the PWMTs of10mg/kg group were significantly increased compared with that of pre-durg and control group(P<0.01). and the effect of diclofenac sodium was dose-dependent (p<0.05).
(2) The effects of diclofenac sodium on the structure of gastrointestinal mucosa.
At 7~(th) day post-drug, there were no any ulcer and bleeding in gastrointestinal mucosa of all five groups macroscopically. Under microscope, in Sham operation and Control group, the structure of gastrointestinal mucosa was intact without obvious inflammatory cells soakage. In 2mg/kg group, the structure of gastric mucosa was slightly incrassate, but the intestinal mucosa was intact. In 4mg/kg group, gastric mucosa gland became minor, the wall of the gland became tenuis, intestinal villi became minor and short, beaker cell decreased. In 10mg/kg group, gastric mucosa gland was hyperplastic and incrassate, intestinal villi became minor and short, acidophilic cell increased with inflammatory cells soakage.
(3) The effects of diclofenac sodium on SP and CGRP expressions in DRG neurons
At 7~(th) day post-drug, in Control group, diclofenac sodium 2mg/kg group, 4mg/kg group, 10mg/kg group, the average optical density (OD) of SP and CGRP in the right L4-5 DRG neurons were significantly increased compared with that of sham operation group(P <0.01).In 4mg/kg group, 10mg/kg group, the average optical density (OD) of SP and CGRP in the right L4-5 DRG neurons were significantly decreased compared with that of control group(P<0.01或P<0.05).
(4) The effects of diclofenac sodium on SP and CGRP expressions in the I, II layerof dorsal horn.
At 7~(th) day post-drug, in Control group, diclofenac sodium 2mg/kg group, 4mg/kg group, 10mg/kg group, the average optical density (OD) of SP and CGRP in the dorsal horn were significantly increased compared with that of sham operation group(P <0.01).In 4mg/kg group, 10mg/kg group, the average optical density (OD) of SP and CGRP in the dorsal horn were significantly decreased compared with that of control group(P<0.01或P<0.05).
Conclusions
Diclofenac sodium can dose-dependently (4mg/kg,10mg/kg) decrease the mechanical hyperalgia. The possible mechanism might be reducing SP and CGRP expressions in DRG neurons and dorsal horn in CCI rats; When administerd in large dose, it can damage gastrointestinal mucosa obviously, thus this dose shoud be cautious in rats and we should constrain dose in clinic.
方法雄性SD大鼠40只(体重240-260g),随机分为坐骨神经结扎(CCI)组(32只)和假手术组(n = 8)。CCI组又分为: a :双氯芬酸钠组(2mg/kg组, n = 8;4mg /kg组,n = 8;10mg/kg组,n = 8);b对照组(生理盐水1ml,n =8)。自术后第8天分别给予不同剂量双氯芬酸钠及生理盐水灌胃,每日两次共7天。采用自制的Von-Frey纤毛机械刺激器测定灌胃前及灌胃第1、3、5、7天时大鼠右后肢机械刺激缩足反射阈值(PWMT)及用药7天后免疫组化法测定L4-5背根神经节细胞及腰膨大处(L4-5节段)脊髓背角SP和CGRP表达,并在光镜下观察胃肠粘膜结构变化。
结果
(1)对CCI大鼠机械痛阈的影响:对照组、2mg/kg组、4mg/kg组、10mg/kg组在用药前及用药后各时间点的右后肢PWMT与假手术组相比显著降低(P<0.01)。4mg/kg组在用药第5、7天时右后肢PWMT与用药前及对照组相比显著增加(P<0.01或P<0.05);10mg/kg组用药后各时间点右后肢PWMT与用药前及对照组相比显著增加(P<0.01)。且呈现出剂量依赖效应(P<0.05)。
(2)对大鼠胃肠粘膜结构的影响:肉眼观各组大鼠胃窦部、腺体部及肠粘膜均未见溃疡和出血。假手术组、对照组病理切片观胃、空肠粘膜结构完整,未见明显的炎性细胞浸润。2mg/k组病理切片观胃粘膜腺体略有增厚;空肠腺体未见明确变化。4mg/kg组观胃粘膜腺体变少,腺壁变薄;肠绒毛变少、变短,杯状细胞减少。10mg/kg组观胃粘膜腺体增生、变厚、染色变深;肠绒毛变少、变短,肠嗜酸性细胞多,有炎症改变。
(3)对大鼠背根神经节(DRG) SP、CGRP表达的影响:对照组、2mg/kg组、4mg/kg组、10mg/kg组SP及CGRP表达的平均光密度值(OD)均明显高于假手术组(P <0.01)。4mg/kg组、10mg/kg组SP及CGRP表达的平均光密度值(OD)明显低于对照组(P<0.01或P<0.05)。
(4)对大鼠脊髓背角I、II层SP、CGRP表达的影响:对照组、2mg/kg组、4mg/kg组、10mg/kg组SP及CGRP表达的平均光密度值(OD)均明显高于假手术组(P <0.01)。4mg/kg组、10mg/kg组SP及CGRP表达的平均光密度值(OD)明显低于对照组(P <0.01或P<0.05)。
结论
1、一定剂量(4 mg/kg、10 mg/kg)双氯芬酸钠灌胃能够减轻CCI大鼠机械性痛敏反应,且具有剂量依赖效应。
2、双氯芬酸钠减轻机械痛敏的作用机制可能是使背根神经节和脊髓背角SP、CGRP的表达减少。
3、镇痛效果虽有剂量依赖效应,但随之副作用也增加,提示临床应用应严格限制用药剂量。说明双氯芬酸钠治疗神经病理性疼痛的有效剂量同时也会引起胃肠粘膜损害,因而还不是治疗神经病理性疼痛的理想药物。
Objective
To obseve the effects of diclofenac sodium on mechanical hyperalgia, substance P (SP) and calcitonin gene-related peptide (CGRP) expression in dorsal root ganglion (DRG) neurons and dorsal horn, as well as the structure of gastrointestinal mucosa in chronic constriction injury (CCI) rats.
Materials and Methods
Forty male SD rats weighing 240-260g were randomly divided into 5 groups : 1) Sham operation group; 2) Control group ( treated with CCI) ; 3) diclofenac sodium 2mg/kg group; 4) diclofenac sodium 4mg/kg group; 5) diclofenac sodium 10mg/kg group . From 8th day post-ligation. Nature saline, different dose of diclofenac sodium were administered by injecting into gastric cavity twice per day for 7 days according to different groups, respectively. Before and after drug injection(at 1~(st)、3~(rd)、5~(th)、7~(th) day), the posterior Paw Withdrawal Mechanical Threshold (PWMT) was measured by Von Frey hair, and the expressions of SP and CGRP in L4-5 dorsal root ganglion (DRG) neurons and dorsal horn were detected by immunohistochemistry method. Meanwhile, the structure of gastrointestinal mucosa was observed in light microscope.
Results
(1) The effects of diclofenac sodium on PWMT
At pre-drug , different time post-drug, the right posterior PWMTs of Control group, diclofenac sodium 2mg/kg group, 4mg/kg group, 10mg/kg group were significantly decreased compared with that of Sham operation group (p<0.01). At 5~(th) and 7~(th) day post-drug, the PWMTs of 4mg/kg group were significantly increased compared with that of pre-durg and control group(P<0.01或P<0.05). At different time post-drug, the PWMTs of10mg/kg group were significantly increased compared with that of pre-durg and control group(P<0.01). and the effect of diclofenac sodium was dose-dependent (p<0.05).
(2) The effects of diclofenac sodium on the structure of gastrointestinal mucosa.
At 7~(th) day post-drug, there were no any ulcer and bleeding in gastrointestinal mucosa of all five groups macroscopically. Under microscope, in Sham operation and Control group, the structure of gastrointestinal mucosa was intact without obvious inflammatory cells soakage. In 2mg/kg group, the structure of gastric mucosa was slightly incrassate, but the intestinal mucosa was intact. In 4mg/kg group, gastric mucosa gland became minor, the wall of the gland became tenuis, intestinal villi became minor and short, beaker cell decreased. In 10mg/kg group, gastric mucosa gland was hyperplastic and incrassate, intestinal villi became minor and short, acidophilic cell increased with inflammatory cells soakage.
(3) The effects of diclofenac sodium on SP and CGRP expressions in DRG neurons
At 7~(th) day post-drug, in Control group, diclofenac sodium 2mg/kg group, 4mg/kg group, 10mg/kg group, the average optical density (OD) of SP and CGRP in the right L4-5 DRG neurons were significantly increased compared with that of sham operation group(P <0.01).In 4mg/kg group, 10mg/kg group, the average optical density (OD) of SP and CGRP in the right L4-5 DRG neurons were significantly decreased compared with that of control group(P<0.01或P<0.05).
(4) The effects of diclofenac sodium on SP and CGRP expressions in the I, II layerof dorsal horn.
At 7~(th) day post-drug, in Control group, diclofenac sodium 2mg/kg group, 4mg/kg group, 10mg/kg group, the average optical density (OD) of SP and CGRP in the dorsal horn were significantly increased compared with that of sham operation group(P <0.01).In 4mg/kg group, 10mg/kg group, the average optical density (OD) of SP and CGRP in the dorsal horn were significantly decreased compared with that of control group(P<0.01或P<0.05).
Conclusions
Diclofenac sodium can dose-dependently (4mg/kg,10mg/kg) decrease the mechanical hyperalgia. The possible mechanism might be reducing SP and CGRP expressions in DRG neurons and dorsal horn in CCI rats; When administerd in large dose, it can damage gastrointestinal mucosa obviously, thus this dose shoud be cautious in rats and we should constrain dose in clinic.
引文
[1] Bennett GJ , Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man .Pain , 1988 ,33 : 87-107.
[2] Woolf CJ , Mannion RJ . Neuropathic pain: aetiology , symptoms ,mechanisms , and management . L ancet , 1999 ,353 : 1959-1964.
[3] Tracey DJ, Walker JS .Pain due to nerve damage: are inflammatory mediators involved? Inflamm Res, 1995 ,44:407–411.
[4] Taiwo YO, Levine JD. Effect of cyclooxygenase products of arachidonic acid metabolism on cutaneous nociceptive threshold in the rat. Brain Res, 1990,5(37): 372-374.
[5] Smith JA , Amagasu SM, Eglen RM,et al . Characterization of prostanoid receptor-evoked responses in rat sensory neurones . Pharmacol , 1998 ,124 (3) :513 - 523.
[6] Hingtgen CM, Waite KJ, Vasko MR..Prostaglandins facilitate peptide release from rat sensory neurons by activating the adenosine 3’,5’ -cyclic monophosphate transduction cascade. J Neurosci ,1995,15:5411–5419.
[7] Vasko MR Prostaglandin-induced neuropeptide release from spinal cord. Prog Brain Res ,1995,104:367–380.
[8] David J, John E, Michael A. Peripheral hyperalgesia in experiment neuropathy: mediation by α2-adrenoreceptors on post-ganglionic sympathetic terminals.Pain, 1995,60:317-327.
[9] Omana-Zapata I, Bley KR. A stable prostacyclin analog enhances ectopic activity in rat sensory neurons followingneuropathic injury. Brain Res,2001,904:85-92.
[10] Kulkarni SK, Satyanarayana SV. Differential effects of naproxen and rofecoxib on the development of hypersensitivity following nerve injury in rats. Pharmacology, Biochemistry and Behavior , 2004, 79:349–358
[11] Syriatowicz JP, Hu D, Walker JS, et al. Hyperalgesia due to nerve injury: role of prostaglandins. Neuroscience ,1999,94:587–594.
[12] Schafers M, Marziniak M. Cyclooxygenase inhibition in nerve-injury and TNF-induced hyperalgesia in the rat. Experimental Neurology, 2004, 185:160– 168
[13] Ma W, Eisenach JC. Morphological and pharmacological evidence for the role of peripheral prostaglandins in the pathogenesis of neuropathic pain. Eur. J.Neurosci, 2002, 15:1037– 1047.
[14] Chen J , Luo C, Li HL , et al. Primary hyperalgesia to mechanical and heat stimuli following subcutaneous bee venom injection into the plantar surface of hindpaw in the conscious rat:A comparative study with the formalin test. Pain, 1999,83 (1) : 67- 76.
[15] Chen J , Chen HS. Pivotal role of casaicin sensitive primary afferents in development of both heat and mechanical hyperalgesia induced by intraplantar bee venom injection. P ain, 2001, 91: 367- 376.
[16] 刘国凯,黄宇光,罗爱伦. 神经病理性疼痛动物模型及其评价.中国临床药理学与治疗学,2005,10(6) :601 – 603.
[17]Attal N, Jazat F, Kayser V, et al. Further evidence for pain-related behaviours in a model of unilateral peripheralmononeuropathy. Pain, 1990,41∶235-251.
[18] 路志红,梅其炳. 环氧合酶在神经病理性疼痛中作用的研究进展. 国外医学药学分册,2004 , 31( 5 ):274-278
[19] Ma W, Bisby MA. Increase of preprotachykinin mRNA and substance P immunoreactivity in spared dorsal root ganglion neurons following partial sciatic nerve injury. Eur J Neurosci,1998,10(7):2388-99.
[20] Harper AA, Lawson SN. Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurons. J Physiol , 1985 , 359:31-46.
[21] MA. W, Eisenach. JC. Intraplantar injection of a cyclooxygenase inhibitor ketorolac reduces immunoreactivities of substance p,Calcitonin gene - related peptide,and dynoprphin in the dorsal horn of rats with nerve injury or inflamimation. Neuroscience , 2003, 121:681–690.
[22] Salt TE, Hill RG. Nerotransmitter candidates of somatosensory primary afferent fiber. Neuroscience, 1983, 10:1083-1103.
[23] Skofitsch G, Jacobowitz DM. Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptide, 1985a, 6:721-745s.
[24] NasuF.Analysis of Calcitonin gene-related peptide containing nerve fibres in rat spinal cord using light and electron microscopy. J ElectronMicrosc, 1999 , 98:267-275
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[27] Ma W, Eisenach JC. Cyclooxygenase- 2 in infiltrating cells in injured nerve is universally up-regulated following various types of peripheral nerve injury. Neuroscience, 2003,121:691–704.
[28] Broom DC, Samad TA, Kohno T, et al.Cyclooxygenase-2 expression in the spared nerve injury model of neuropathic pain. Neuroscience, 2004,124:891– 900.
[29] Zhu X, Eisenach JC. Cyclooxygenase-1 in the spinal cord is altered after peripheral nerve injury. Anesthesiology ,2003,99:1175– 1179.
[30 ] Herrero JF , Romero2Sandoval EA , Gaitan G, et al .Antinociception and the new COX inhibitors : research approaches and clinical perspectives . CNS Drug Rev ,2003 ,9(3) :227 – 2.
[2] Woolf CJ , Mannion RJ . Neuropathic pain: aetiology , symptoms ,mechanisms , and management . L ancet , 1999 ,353 : 1959-1964.
[3] Tracey DJ, Walker JS .Pain due to nerve damage: are inflammatory mediators involved? Inflamm Res, 1995 ,44:407–411.
[4] Taiwo YO, Levine JD. Effect of cyclooxygenase products of arachidonic acid metabolism on cutaneous nociceptive threshold in the rat. Brain Res, 1990,5(37): 372-374.
[5] Smith JA , Amagasu SM, Eglen RM,et al . Characterization of prostanoid receptor-evoked responses in rat sensory neurones . Pharmacol , 1998 ,124 (3) :513 - 523.
[6] Hingtgen CM, Waite KJ, Vasko MR..Prostaglandins facilitate peptide release from rat sensory neurons by activating the adenosine 3’,5’ -cyclic monophosphate transduction cascade. J Neurosci ,1995,15:5411–5419.
[7] Vasko MR Prostaglandin-induced neuropeptide release from spinal cord. Prog Brain Res ,1995,104:367–380.
[8] David J, John E, Michael A. Peripheral hyperalgesia in experiment neuropathy: mediation by α2-adrenoreceptors on post-ganglionic sympathetic terminals.Pain, 1995,60:317-327.
[9] Omana-Zapata I, Bley KR. A stable prostacyclin analog enhances ectopic activity in rat sensory neurons followingneuropathic injury. Brain Res,2001,904:85-92.
[10] Kulkarni SK, Satyanarayana SV. Differential effects of naproxen and rofecoxib on the development of hypersensitivity following nerve injury in rats. Pharmacology, Biochemistry and Behavior , 2004, 79:349–358
[11] Syriatowicz JP, Hu D, Walker JS, et al. Hyperalgesia due to nerve injury: role of prostaglandins. Neuroscience ,1999,94:587–594.
[12] Schafers M, Marziniak M. Cyclooxygenase inhibition in nerve-injury and TNF-induced hyperalgesia in the rat. Experimental Neurology, 2004, 185:160– 168
[13] Ma W, Eisenach JC. Morphological and pharmacological evidence for the role of peripheral prostaglandins in the pathogenesis of neuropathic pain. Eur. J.Neurosci, 2002, 15:1037– 1047.
[14] Chen J , Luo C, Li HL , et al. Primary hyperalgesia to mechanical and heat stimuli following subcutaneous bee venom injection into the plantar surface of hindpaw in the conscious rat:A comparative study with the formalin test. Pain, 1999,83 (1) : 67- 76.
[15] Chen J , Chen HS. Pivotal role of casaicin sensitive primary afferents in development of both heat and mechanical hyperalgesia induced by intraplantar bee venom injection. P ain, 2001, 91: 367- 376.
[16] 刘国凯,黄宇光,罗爱伦. 神经病理性疼痛动物模型及其评价.中国临床药理学与治疗学,2005,10(6) :601 – 603.
[17]Attal N, Jazat F, Kayser V, et al. Further evidence for pain-related behaviours in a model of unilateral peripheralmononeuropathy. Pain, 1990,41∶235-251.
[18] 路志红,梅其炳. 环氧合酶在神经病理性疼痛中作用的研究进展. 国外医学药学分册,2004 , 31( 5 ):274-278
[19] Ma W, Bisby MA. Increase of preprotachykinin mRNA and substance P immunoreactivity in spared dorsal root ganglion neurons following partial sciatic nerve injury. Eur J Neurosci,1998,10(7):2388-99.
[20] Harper AA, Lawson SN. Conduction velocity is related to morphological cell type in rat dorsal root ganglion neurons. J Physiol , 1985 , 359:31-46.
[21] MA. W, Eisenach. JC. Intraplantar injection of a cyclooxygenase inhibitor ketorolac reduces immunoreactivities of substance p,Calcitonin gene - related peptide,and dynoprphin in the dorsal horn of rats with nerve injury or inflamimation. Neuroscience , 2003, 121:681–690.
[22] Salt TE, Hill RG. Nerotransmitter candidates of somatosensory primary afferent fiber. Neuroscience, 1983, 10:1083-1103.
[23] Skofitsch G, Jacobowitz DM. Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptide, 1985a, 6:721-745s.
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