【作者】 王军；

【导师】 张励才；

【作者基本信息】 中国医科大学 ， 麻醉学， 2009， 博士

【摘要】 目的乳铁蛋白(lactoferrin,LF)是转铁蛋白家族中的一种分子量约80,000的糖蛋白,广泛存在于哺乳动物的各种体液中,如乳汁、血液、唾液、泪液,胆汁、精液及胰液等。既往研究表明,乳铁蛋白除了抗炎、抗感染、抗肿瘤及免疫调节等多种作用外,还在炎性痛过程中产生镇痛作用。但乳铁蛋白是否在神经病理性痛发挥镇痛作用,如果有镇痛作用,其镇痛作用的分子机制如何,尚需进一步探讨。本研究采用坐骨神经慢性束缚损伤模型(chronic constriction injury of the sciaticnerve,CCI),从行为学、细胞化学和分子生物学等多层面探讨:(1)乳铁蛋白在神经病理性痛是否能产生镇痛作用;(2)乳铁蛋白在神经病理性痛的镇痛作用是否与NO-cGMP-PKG信号通路的激活有关;(3)乳铁蛋白在神经病理性痛的镇痛作用是否与阿片受体的激活有关;(4)乳铁蛋白产生镇痛作用的不同阶段信号分子蛋白激酶G(protein kinase G,PKG)的表达的变化。目的在于证实乳铁蛋白在神经病理性痛过程中的镇痛作用,阐明其发挥镇痛作用的分子机制,为临床治疗神经病理性痛提供理论参考和实践指导的依据。材料与方法1.大鼠坐骨神经慢性束缚损伤神经病理性痛模型(CCI)的制作雄性Sprague-Dawley(SD)大鼠腹腔内注射苯巴比妥钠(50mg/kg)进行麻醉,消毒后,沿大鼠右侧后肢大腿外侧切皮,暴露坐骨神经干,用4号铬制羊肠线环绕神经干分别作4个轻度结扎,结扎间距为1.0-1.5mm,结扎强度以引起同侧小腿肌肉轻度颤动为宜。最后缝合伤口。2.乳铁蛋白对神经病理性痛的镇痛作用将CCI大鼠分为乳铁蛋白腹腔内注射组和鞘内注射组两个大组,腹腔内注射组进一步分为30mg/kg(IP30组)、100mg/kg(IP100组)、300mg/kg(IP300组)3个剂量组;鞘内注射组进一步分为1μg(IT1组)、10μg(IT10组)、100μg(IT100组)3个剂量组,对照组注射等容积生理盐水,每组动物为8只,用热刺激仪,观测大鼠后爪回缩潜伏期(paw withdrawal latency,PWL),每30min一次,直至180min。腹腔内注射组,将PWL换算为最大可能效应百分数(maximum possible effect,MPE%)来评价乳铁蛋白的镇痛效应。MPE%=(给药后PWL-给药前PWL)×100/(20-给药前PWL)。鞘内注射组,采用曲线下面积法来评价乳铁蛋白的镇痛效应。3.乳铁蛋白的镇痛效应与NO-cGMP-PKG信号通路的激活将CCI大鼠分为5大组,鞘内给予100μg乳铁蛋白10min后,各组分别鞘内注射不同剂量的工具药及其等容积溶媒:L-NAME(非特异性一氧化氮合酶抑制剂)、7-NI(神经性一氧化氮合酶抑制剂)、ODQ(鸟苷环化酶抑制剂)、KT-5823(特异性PKG抑制剂)、格列本脲(ATP敏感K~+通道阻断剂)。每个剂量组8只大鼠,通过热刺激仪记录大鼠PWL,采用曲线下面积法,分析各工具药对鞘内注射乳铁蛋白对神经病理性痛大鼠的镇痛作用的影响。4.乳铁蛋白的镇痛作用与阿片受体的关系将CCI大鼠随机分为4组,每组8只,鞘内给予100μg乳铁蛋白10min后,各组分别鞘内注射纳洛酮(非选择性阿片受体拮抗剂)10μg,CTOP(选择性μ阿片受体拮抗剂)0.1μg、1.0μg及等容积生理盐水对照,通过热刺激仪记录大鼠PWL,采用曲线下面积法,分析纳洛酮和CTOP对鞘内注射乳铁蛋白在神经病理性痛大鼠的镇痛作用的影响。5.鞘内乳铁蛋白镇痛过程中PKG的表达将CCI大鼠分为6组,每组24只,分别为空白对照组(不给药)、生理盐水对照组(等容积生理盐水)、乳铁蛋白组(乳铁蛋白100μg)、DMSO组(乳铁蛋白100μg+DMSO10μl)、ODQ组(乳铁蛋白100μg+ODQ10μg)及KT-5823组(乳铁蛋白100μg+KT5823 10μg),给药后30min、60min、90min等时间点各取4只大鼠脊髓标本,免疫组化和western blot检测PKG的表达的改变。6.统计学分析所有计量资料数据以均数±标准差(mean±SD)表示,SPSS13.0统计学软件分析数据,组间比较采用单因素方差分析,Tukey’s检验。P＜0.05认为有统计学意义。实验结果1.乳铁蛋白在神经病理性痛的镇痛作用CCI大鼠腹腔内注射乳铁蛋白后30min,即产生镇痛效应,最大效应发生在注射乳铁蛋白后60min,各剂量组MPE分别为6.98±2.07(IP10)、14.76±2.44(IP100)、42.57±5.48(IP300),显著高于对照组MPE值3.14±1.82(P＜0.05)。给药后180min,镇痛效应消失。CCI大鼠鞘内注射乳铁蛋白后30min,即开始产生镇痛效应,持续约180min,最大效应产生于注射乳铁蛋白后60min,各剂量组曲线下面积(AUC)分别为1103.41±104.82(IT1)、1347.98±111.94(IT10)、1426.85±70.46(IT100),显著高于对照组AUC值904.83±50.17(P＜0.05)。2.乳铁蛋白镇痛的镇痛效应与NO-cGMP-PKG信号通路的激活各工具药组的曲线下面积分别为:L-NAME(1374.52±77.61,1287.96±50.74,950.99±68.73),7-NI(1325.7±70.81,1173.75±67.49,976.58±5.89),ODQ(1394.68±99.43,1254.54±65.17,964.52±100.24),KT-5823(1274.25±103.21,971.87±87.63,940.65±64.28),格列本脲(1375.95±95.83,1065.62±58.39,987.91±60.85),其中部分中小剂量组和所有大剂量组的AUC显著低于各自的对照组(P＜0.05)。单独应用各工具药最大剂量本身不产生镇痛效应,其AUC与对照组的差异无显著性。3.乳铁蛋白的镇痛效应与阿片受体的关系各工具药组曲线下面积分别为:纳洛酮(1298.47±89.42),CTOP0.1(1347.98±97.55),CTOP1.0(1287.45±68.41),与对照组(1421.85±70.56)相比,差异无显著性(P＞0.05)。4.鞘内乳铁蛋白镇痛过程中PKG表达免疫荧光结果显示,大鼠脊髓PKG阳性反应为绿色荧光,弥漫性分布,与空白对照组相比,生理盐水组的荧光强度无明显改变,而乳铁蛋白组的荧光强度则明显增强(P＜0.05);与乳铁蛋白组相比,DMSO组荧光强度无明显改变,而ODQ组及KT-5823组的荧光强度明显降低(P＜0.05)。与生理盐水组相比,大鼠鞘内注射乳铁蛋白后30min、60min及90min,荧光强度增强(P＜0.05),在60min时荧光强度最大(P＜0.05);免疫印迹结果显示,与空白对照组相比,生理盐水组PKG表达无明显改变,而乳铁蛋白组PKG表达则明显增多(P＜0.05);与乳铁蛋白组相比,DMSO组PKG表达无明显改变,而ODQ组及KT-5823组PKG表达显著减少(P＜0.05)。与生理盐水组相比,大鼠鞘内注射乳铁蛋白后30min、60min及90min,脊髓PKG表达均增多(P＜0.05),在60min时PKG表达最多(P＜0.05)。而乳铁蛋白产生的镇痛作用的最大效应发生于注射乳铁蛋白后60min,这也正是脊髓PKG表达量增加最显著的时间。免疫荧光与免疫印迹结果均提示,鞘内注射乳铁蛋白引起脊髓PKG表达增加,鞘内注射鸟苷环化酶(guanylyl-cyclase,GC)和PKG抑制剂则明显抑制了脊髓PKG的表达,乳铁蛋白在CCI大鼠产生的镇痛作用与脊髓PKG表达增多有关。结论1.腹腔内及鞘内注射乳铁蛋白均在神经病理性痛产生镇痛效应,其镇痛效应呈剂量依赖性,最大镇痛效应产生于注射乳铁蛋白后60min;2.鞘内注射乳铁蛋白在神经病理性痛产生镇痛效应与NO-cGMP-PKG信号通路的激活有关;3.鞘内注射乳铁蛋白对CCI大鼠的镇痛作用在脊髓水平与阿片受体的激活无关;4.鞘内注射乳铁蛋白诱导脊髓PKG表达增加,PKG在脊髓的表达随着鞘内注射乳铁蛋白产生的镇痛作用的增强而增加。更多还原

【Abstract】 ObjectiveLactoferrin(LF) is a single-chain glycoprotein with a molecular weight of about 80,000 that belongs to the family of transferrins.Lactoferrin is found in many mammalian species and their various biological fluids such as milk,blood,saliva,nasal secretions,tears,bronchial mucus,hepatic bile,pancreatic juice,and seminal fluid. Lactoferrin has been noticed as a multifunctional protein,and it is well known that lactoferrin induces the primary defense against bacterial and viral infection,antitumor activity,immunomodulation,and cell growth regulation.Recently,it has been reported that lactoferrin elicited an analgesic effect in a formalin pain model.Despite hypothesis to explain the actions of lactoferrin,the exact site and mechanisms responsible for its analgesic effect remain unclear,especially in a model of neuropathic pain.In an attempt to widen our basic knowledge concerning this issue,we aimed to explore(1)whether lactoferrin elicite analgesic effect in a model of neuropathic pain;(2)whether NO-cGMP-PKG pathway could be underlying the analgesic effect induced by lactoferrin in the chronic constriction nerve injury(CCI) model of neuropathic pain in rats;(3)whether theμ-opioid receptor is involved in the analgesic effect of lactoferrin; (4) protein kinase G expression during the analgesic process induced by lactoferrin.Material and methods1.The making of the CCI modelThe surgical procedure was performed aseptically under sodium pentobarbital anesthesia(50mg/kg,i.p.).CCI rats were produced by loosely ligating the common sciatic nerve according to the method of Bennett and Xie(1988).Briefly,on one side the rat’s sciatic nerve was exposed in the mid-thigh and four loose ligatures of 4.0 chromic gut were made around the dissected nerve with a 1.0-1.5mm interval between each of them.The skin wound was closed using 6.0 nylon sutures.Sham rats were made following the same surgical procedure except for nerve ligation.2.To explore whether lactoferrin elicite analgesic effect in a model of neuropathic pain,animals were divided into different groups according to given doses: 30mg/kg(i.p.),100mg/kg(i.p.),300mg/kg(i.p.) and 1μg(i.t.),10μg(i.t.),100μg(i.t.).The method of Hargreaves et al.(1988) was used to assess paw withdrawal latency(PWL) to a thermal nociceptive stimulus.A significant reduction in PWL compared with normal baseline was interpreted as thermal hyperalgesia.3.Rats received a spinal injection of vehicle or increasing doses of lactoferrin (1μg,10μg,100μg) 15min before evaluation of withdrawal threshold.To determine whether lactoferrin-induced antihyperalgesia was mediated by NO-cGMP-PKG pathway activation,effect of pretreatment(-5min) with the appropriate vehicle or L-NAME(1μg,10μg,100μg),D-NAME(100μg),7-NI(1μg,10μg,100μg),ODQ(1μg, 3μg,10μg) or KT-5823(1μg,3μg,10μg) on the antihyperalgesic effect induced by lactoferrin(-15min,100μg) was assessed.4.To determine whether K~+ channel blockers affect lactoferrin-induced antihyperalgesia,effect of pretreatment(-5min) with the appropriate vehicle or glybenclamide(12.5μg,25μg,50μg) on the antihyperalgesic effect induced by lactoferrin(-15min,100μg) was assessed.5.To test the possible participation of theμ-receptor in the lactoferrin’s antihyperalgesic activity,the effect of pretreatment(-5min) with the appropriate vehicle, CTOP(0.1μg,1.0μg) or naloxone(10μg) on the antihyperalgesic effect induced by lactoferrin(-15min,100μg) was assessed.All drugs were injected intrathecally in a 10μl volume followed by a 10μl saline flush.6.Statistical analysisAll data are expressed as the mean±SD for seven to eight animals per group. Curves were constructed plotting the PWL as a function of time.An increase of PWL was considered as antihyperalgesic effect.Area under the PWL against time curve (AUC) for a period of 180min was calculated by the trapezoidal method.Differences between treatment groups were assessed by analysis of variance(ANOVA),followed by Tukey’s test.The difference between sham group and CCI group was assessed by student’s t-test.In all cases,P＜0.05 was considered to indicate statistical significance.Results1.Antihyperalgesic effect of lactoferrin in neuropathic painLigation of the common sciatic nerve produced a clear-cut hyperalgesia in rats submitted to the surgery compared to the sham operated rats(P＜0.05).Intrathecal administration of lactoferrin(1μg,10μg,100μg),but not vehicle,reduced thermal hyperalgesia induced by CCI.2.Effect of L-NAME,D-NAME and 7-nitroindazole on the antihyperalgesic activity of lactoferrinSpinal pretreatment with the non-specific inhibitor of NO synthase L-NAME(1μg, 10μg,100μg) and the neuronal NO synthase inhibitor 7-nitroindazole(1μg,10μg, 100μg),but not the inactive isomer of L-NAME,D-NAME(100μg) or vehicle, significantly(P＜0.05) reversed the antihyperalgesic effect induced by the spinal administration of lactoferrin 100μg.At the greatest tested doses,the NO synthesis inhibitors did not modify thermal hyperalgesia in the CCI rats.3.Effect of ODQ and KT-5823 on the antihyperalgesic activity of lactoferrinIntrathecal pretreatment with the guanylyl cyclase inhibitor ODQ(1μg,3μg,10μg) and KT-5823(1μg,3μg,10μg) significantly blocked the antihyperalgesic activity of intrathecal lactoferrin 100μg(P＜0.05).In contrast,intrathecal pretreatment with the greatest tested doses of ODQ(10μg) and KT-5823(10μg) did not produce any effect on CCI-induced thermal hyperalgesia.4.Effect of glybenclamide on the antihyperalgesic activity of lactoferrin Intrathecal pretreatment with glybenclamide(12.5μg,25μg,50μg) blocked the antihyperalgesic activity of intrathecal lactoferrin 100μg(P＜0.05) and intrathecal pretreatment with glybenclamide(50μg) alone did not produce any effect on CCI-induced thermal hyperalgesia.5.Effect of naloxone and CTOP on the antihyperalgesic activity of lactoferrinIntrathecal pretreatment with naloxone(10μg) and CTOP(0.1μg,1.0μg) did not blocked the antihyperalgesic activity of intrathecal lactoferrin 100μg and intrathecal pretreatment with naloxone(10μg) and CTOP(1.0μg) did not produce any effect on CCI-induced thermal hyperalgesia.Conclusions1.Lactoferrin elicite analgesic effect in a model of neuropathic pain;2.NO-cGMP-PKG pathway could be underlying the analgesic effect induced by lactoferrin in the chronic constriction nerve injury(CCI) model of neuropathic pain in rats;3.Theμ-opioid receptor is not involved in the analgesic effect of lactoferrin at the spinal level;4.The expression of protein kinase G in the spinal cord during the analgesic process induced by lactoferrin is in accordance with the analgesic effect of lactoferrin.更多还原