乳铁蛋白在CCI大鼠的镇痛作用与NO-cGMP-PKG信号通路的激活
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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。
Objective
Lactoferrin(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 methods
1.The making of the CCI model
The 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 analysis
All 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.
Results
1.Antihyperalgesic effect of lactoferrin in neuropathic pain
Ligation 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 lactoferrin
Spinal 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 lactoferrin
Intrathecal 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 lactoferrin
Intrathecal 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.
Conclusions
1.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.
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。
Objective
Lactoferrin(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 methods
1.The making of the CCI model
The 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 analysis
All 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.
Results
1.Antihyperalgesic effect of lactoferrin in neuropathic pain
Ligation 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 lactoferrin
Spinal 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 lactoferrin
Intrathecal 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 lactoferrin
Intrathecal 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.
Conclusions
1.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.
引文
1 Abbadie C,Lombard MC,Besson JM,et al.Mu and delta opioid receptor-like immunoreactivity in the cervical spinal cord of the rat after dorsal rhizotomy or neonatal capsaicin:an analysis of preand postsynaptic receptor distributions.Brain Res.2002;930:150-162.
2 Aley KO,Levine JD.Different peripheral mechanisms mediate enhanced nociception in metabolic/toxic and traumatic painful peripheral neuropathies in the rat.Neuroscience.2002;111:389-397.
3 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.
4 Bolotina VM,Najibi,Palacino JJ,et al.Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle.Nature.1994;368:850-853.
5 Bouhassira D,et al.Prevalence of chronic pain with Neuropathic characteristics in the general population.Pain.2008;136:380-387.
6 Brock JH.The physiology of lactoferrin.Biochem Cell Biol.2002;80:1-6.
7 Busija DW,Lacza Z,Rajapakse N,et al.Targeting mitochondrial ATP-sensitive potassium channels-a novel approach to neuroprotection.Brain Res Rev.2004;46:282-294.
8 Cabot PJ,Immune-derived opioids and peripheral antinociception.Clin.Exp.Pharmacol.Physiol.2001;28:230-232.
9 Chen SR,Eisenach JC,Pan HL.Intrathecal S-nitroso-N-acetylpenicillamine and L-cysteine attenuate nerve injury-induced allodynia through noradrenergic activation in rats.Neuroscience.2000;101:759-765.
10 Chen SR,Khan GM,Pan HL.Antiallodynic effect of intrathecal neostigmine is mediated by spinal nitric oxide in a rat model of diabetic neuropathic pain.Anesthesiology.2001;95:1007-1012.
11 Chen SR,Khan M,Pan HL.Antiallodynic effect of intrathecal neostigmine is mediated by spinal nitric oxide in a rat model of diabetic neuropathic pain.Anesthesiology.2001;95:1007-1012.
12 Chen SR,Pan HL.Spinal endogenous acetylcholine contributes to the analgesic effect of systemic morphine in rats.Anesthesiology.2001;95:525-530.
13 Chen SR,Pan HL.Spinal nitric oxide contributes to the analgesic effect of intrathecal [d-pen2,d-pen5]-enkephalin in normal and diabetic rats.Anesthesiology.2003;98:217-222.
14 Deciga-Campos M,Lopez-Munoz FJ.Participation of the L-arginine-nitric oxide-cyclic GMP-ATP-sensitive K+ channel cascade in the antinociceptive effect of rofecoxib.Eur.J.Pharmacol.2004;484:193-199.
15 Eder C.Regulation of microglial behavior by ion channel activity.J Neurosci Res.2005;81:314-321.
16 Fillebeen C,Ruchoux MM,Mitchell V,et al.Lactofferin is synthesized by activated microglia in the human substantia nigra and its synthesis by the human microglial CHME cell line is upregulated by tumor necrosis factor alpha or 1-methyl-4phenylpyridinium treatment.Brain Res.Mol.2001;96:103-113.
17 Finnerup NB et al.An evidence-based algorithm for the treatment of neuropathic pain.Med Gen Med.2007;9:36.
18 Francis SH,Corbin JD.Cyclic nucleotide-dependent protein kinases:intracellular receptors for cAMP and cGMP action.Crit Rev Clin Lab Sci 1999;36:275-328.
19 Francis SH,Woodford TA,Wolfe L,et al.Types I alpha and I beta isozymes of cGMP-dependent protein kinase:Alternative mRNA splicing may produce different inhibitory domains.Second Messengers Phosphoproteins.1988;12:301-310.
20 Furchgott RF,Zawadzki JV.The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine.Nature.1980;288:373-376.
21 Galluzzi KE.Managing neuropathic pain.J Am Osteopath Assoc.2007;107:ES39-48.
22 Gao HM,Hong JS,Zhang W,et al.Distinct role for microglia in rotenoneinduced degeneration of dopaminergic neurons.J Neurosci.2002;22:782-790.
23 Garbers DL.Guanylyl cyclase receptors and their endocrine,paracrine,and autocrine ligands.Cell.1992;71:1-4.
24 Geiselhoringer A,Gaisa M,Hofmann F,et al.Distribution of IRAG and cGKI-isoforms in murine tissues.FEBS Lett.2004;575:19-22.
25 Glezer A,Simard RS,Rivest S.Neuroprotective role of the innate immune system by microglia.Neuroscience.2007;147:867-883
26 Han J,Kim N,Joo H,et al.ATP-sensitive K~+ channel activation by nitric oxide and protein kinase G in rabbit ventricular myocytes.Am.J.Physiol.Heart Circ.Physiol.2002;283:H1545-H1554.
27 Hargreaves K,Dubner R,Brown F,et al.A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia.Pain.1988;32:77-88.
28 Hayashida K,Takeuchi T,Harada E.Lactoferrin enhances peripheral opioid-mediated antinociception via nitric oxide in rats.Eur.J.Pharmacol.2004;484:175-181.
29 Hayashida K,Takeuchi T,Shimizu H,et al.Lactoferrin enhances opioid-mediated analgesia via nitric oxide in the rat spinal cord.Am.J.Physiol.2003b;285:R306-R312.
30 Hayashida K,Takeuchi T,Shimizu H,et al.Novel central function of milk-derived lactoferrin on anti-nociception mediated by the μ-opioid receptor.Brain Res.2003a;965:239-245.
31 Hu LF,Wang S,Shi XR,et al.ATP-sensitive potassium channel opener iptakalim protectedagainst the cytotoxicity of MPP+ on SH-SY5Y cells by decreasing extracellular glutamate level.J Neurochem.2005;94:1570-1579.
32 Hu SJ,Song XJ,Greenquist KW,et al.Protein kinase A modulates spontaneous activity in chronically compressed dorsal root ganglion neurons in the rat.Pain.2001;94:39-46.
33 Ignarro LJ,Buga GM,Wood KS,et al.Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.Proc Natl Acad Sci USA.1987;84:9265-9269.
34 Inoue T,Mashimo T,Shibata M,et al.Rapid development of nitric oxide-induced hyperalgesia depends on an alternate to the cGMP-mediated pathway in the rat neuropathic pain model.Brain Res.1998;792:263-270.
35 Jarchau T,Hausler C,Markert T,et al.Cloning,expression,and in situ localization of rat intestinal cGMP-dependent protein kinase Ⅱ.Proc Natl Acad Sci USA.1994;91:9426-9430.
36 Jiang H,Shabb JB,Corbin JD.Cross-activation:overriding cAMP/cGMP selectivities of protein kinases in tissues.Biochem Cell Biol.1992;70:1283-1289.
37 Kitagawa,H.,Yoshizawa,Y,Yokoyama,T.,et al.Persorption of bovine lactoferrin from the intestinal lumen into the systemic circulation via the portal vein and the mesenteric lymphatics in growing pigs.J.Vet.Med.Sci.2003;65:567-572.
38 Levay PF,Viljoen M.Lactoferrin:a general review.Haematologica.1995;80:252-267.
39 Levy D,Strassman AM.Modulation of dural nociceptor mechanosensitivity by the nitric oxide-cyclic GMP signaling cascade.J.Neurophysiol.2004;92:766-772.
40 Lincoln TM,Dey N,Sellak H.Invited review:cGMP-dependent protein kinase signaling mechanisms in smooth muscle:from the regulation of tone to gene expression.J Appl Physiol 2001;91:1421-1430.
41 Liu CN,Wall PD,Ben-Dor E,et al.Tactile allodynia in the absence of C-fiber activation:altered firing properties of DRG neurons following spinal nerve injury.Pain.2000;85:503-521.
42 Lohmann SM,Walter U.Tracking functions of cGMP-dependent protein kinases (cGK).Front Biosci.2005;10:1313-1328.
43 Lorenzetti BB,Ferreira SH.Activation of the arginine-nitric oxide pathway in primary sensory neurons contributes to dipyrone-induced spinal and peripheral analgesia.Inflamm.Res.1996;45:308-311.
44 Lucas KA,Pitari GM,Kazerounian S,et al.Guanylyl cyclases and signaling by cyclic GMP.Pharmacol.Rev.2000;52:375-414.
45 Ma C,Shu Y,Zheng Z,et al.Similar electrophysiological changes in axotomized and neighboring intact dorsal root ganglion neurons.J Neurophysiol.2003;89:1588-1602.
46 Maffei FA,Heine RP,Whalen MJ,et al.Levels of antimicrobial molecules defensin and lactoferrin are elevated in the cerebrospinal fluid of children with meningitis.Pediatrics.1999;103:987-992.
47 Manning G,Whyte DB,Martinez R,et al.The protein kinase complement of the human genome.Science.2002;298:1912-1934.
48 Merskey H,Bogduk N (Eds).Classification of Chronic Pain.IASP Press,1994.49 Miletic G,Pankratz MT,Miletic V.Increases in the phosphorylation of cyclic AMP response element binding protein (CREB) and decreases in the content of calcineurin accompany thermal hyperalgesia following chronic constriction injury in rats.Pain.2002;99:493-500.
50 Minghetti L,Ajmone-Cat MA,De Berardinis MA,et al.Microglial activation in chronic neurodegenerative diseases:roles of apoptotic neurons and chronic stimulation.Brain Res Rev.2005;48:251-256.
51 Mixcoatl-Zecuatl T,Flores-Murrieta FJ,Granados-Soto V.The nitric oxide-cyclic GMP-protein kinase G-K~+ channel pathway participates in the antiallodynic effect of spinal gabapentin.Eur J Pharmacol.2006;531:87-95.
52 Nguyen MD,Julie JP,Rivest,S.Innate immunity:the missing link in neuroprotection and neurodegeneration.Nature Rev.Neurosci.2002;3:216-227.
53 Orstavik S,Natarajan V,Tasken K,et al.Characterization of the human gene encoding the type I a and type 113 cGMP-dependent protein kinase (PRKG1).Genomics.1997;42:311-318.
54 Orstavik S,Solberg R,Tasken K,et al.Molecular cloning,cDNA structure,and chromosomal localization of the human type Ⅱ cGMP-dependent protein kinase.Biochem Biophys Res Commun.1996;220:759-765.
55 Pan HL,Chen SR,Eisenach JC.Role of spinal NO in antiallodynic effect of intrathecal clonidine in neuropathic rats.Anesthesiology.1998;89:1518-1523.
56 Rodrigues AR.,Durate ID.The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K+ channels.Br.J.Pharmacol.2000;129,110-114.
57 Ruth P.Cyclic GMP-dependent protein kinases:understanding in vivo functions by gene targeting.Pharmacol Ther.1999;82:355-372.
58 Sachs D,Cunha FQ,Ferreira SH.Peripheral analgesic blockade of hypernociception:activation or arginine/NO/cGMP/protein kinase G/ATP-sensitive K~+ channel pathway.Proc.Natl.Acad.Sci.U.S.A.2004;101:3680-3685.
59 Salter M,Strijbos PJ,Neale S,et al.The nitric oxide-cyclic GMP pathway is required for nociceptive signalling at specific loci within the somatosensory pathway.Neuroscience.1996;73:649-655.
60 Schmidtko A,Ruth P,Geisslmger G,et al.Inhibition of cyclic guanosine 5-monophosphate-dependent protein kinase Ⅰ (PKG-Ⅰ) in lumbar spinal cord reduces formalin-induced hyperalgesia and PKG upregulation.Nitric Oxide.2003;8:89-94.
61 Scholz J,Woolf CJ.The neuropathic pain triad:neurons,immune cells,and glia.Nature Neurosci.2007;10:1361-1368.
62 Segawa K,Minami K,Shiga Y,et al.Inhibitory effects of nicorandil on rat mesangial cell proliferation via the protein kinase G pathway.Nephron,2001;87:263-268.
63 Soares AC,Leite R.,Tatsuo MA.et al.Activation of ATP-sensitive K~+ channels mechanism of peripheral antinociceptive action of the nitric oxide donor,sodium nitroprusside.Eur.J.Pharmacol.2000;400:67-71.
64 Song HK,Pan HL,Eisenach JC.Spinal nitric oxide mediates antinociception from intravenous morphine.Anesthesiology.1998;89:215-221.
65 Song XJ,Wang ZB,Gan Q,et al.cAMP and cGMP contribute to sensory neuron hyperexcitability and hyperalgesia in rats with dorsal root ganglia compression.J Neurophysiol.2006;95:479-492.
66 Sorimachi K,Akimoto K,Hattori Y,et al.Activation of macrophages by lactoferrin:secretion of TNF-a,IL-8 and NO.Biochem Mol Biol Int.1997;43:79-87.
67 Takasu K,Honda M,Ono H,et al.Spinal a_2-adrenergic and muscarinic receptors and the NO release cascade mediate supraspinally produced effectiveness of gabapentin at decreasing mechanical hypersensitivity in mice after partial nerve injury.British Journal of Pharmacology.2006;148:233-244.
68 Takeuchi,T,Kitagawa,H.,Harada,E.Evidence of lactoferrin transportation into blood circulation from intestine via lymphatic pathway in adult rats.Exp.Physiol.2004;89:263-270.
69 Talukder MJR.,Takeuchi T,Harada E.Characteristics of lactoferrin receptor in bovine intestine:higher binding activity in Peyer's patches.J.Vet.Med.2003a;A 50:123-131.
70 Tegeder I,Del Turco D,Schmidtko A,et al.Reduced inflammatory hyperalgesia with preservation of acute thermal nociception in mice lacking cGMP dependent protein kinase I.Proc Natl Acad Sci USA.2004;101:3253-3257.
71 Truong W,Cheng C,Xu QG,et al.Opioid receptors and analgesia at the site of a peripheral nerve injury.Ann Neurol.2003;53:366-375.
72 Vivancos GG,Parada CA,Ferreira SH.Opposite nociceptive effects of the arginine/NO/cGMP pathway stimulation in dermal and subcutaneous tissues.Br.J.Pharmacol.2003;138:1351-1357.
73 Watkins,L.R.& Maier,S.F.Targeting glia to control clinical pain:an idea whose time has come.Drug Discov.Today Then Strat.2004;1:83-88.
74 Werhagen L,Hultling C,Molander C.The prevalence of neuropathic pain after non-traumatic spinal cord lesion.Spinal Cord.2007;45:609-615.
75 Wolfe L,Corbin JD,Francis SH.Characterization of a novel isozyme of cGMP-dependent protein kinase from bovine aorta.J Biol Chem.1989;264:7734-7741.
76 Xu Z.,Tong C,Pan HL,et al.Intravenous morphine increases release of nitric oxide from spinal cord by an α-adrenergic and cholinergic mechanism.Journal of Neurophysiology.1997;78:2072-2078.
77 Yaksh TL,Rudy TA.Chronic catheterization of the spinal subarachnoid space.Physiol.Behav.1976;17:1031-1036.
78 Yamada K,Inagaki N.Neuroprotection by K_(ATP) channels.J Mol Cell Cardiol.2005;38:945-949.
79 Yang Y,Liu X,Long Y,et al.Activation of mitochondrial ATP-sensitive potassium channels improves rotenone-related motor and neurochemical alterations in rats.Int J Neuropsychopharmacol.2006;9:51-61.
80 Yoon YW,Sung B,Chung JM.Nitric oxide mediates behavioral signs of neuropathic pain in an experimental rat model.NeuroReport.1998;9:367-372.
81 Zhuo M.,Meller ST.,Gebhart GF.Endogenous nitric oxide is required for tonic cholinergic inhibition of spinal mechanical transmission.Pain.1993;54:71-78.
82 Zochodne D,Max MB.An old acquaintance:opioids in neuropathic pain.Neurology.2003;60:894-895.
1 Andrews NC.Iron homeostasis:insights from genetics and animal models.Nat.Rev.Genet.2000;1:208-217.
2 Baker HM,Anderson BF,Baker EN.Dealing with iron,common structural principles in proteins that transport iron and heme.Proc.Natl.Acad.Sci.USA.2003;100:3579-3583.
3 Benaissa M,Peyrat JP,Hornez L,et al.Expression and prognostic value of lactoferrin mRNA isoforms in human breast cancer.Int.J.Cancer.2005;114:299-306.
4 Britigan BE,Lewis TS,Waldschmidt M,et al.Lactoferrin binds CpG-containing oligonucleotides and inhibits their immunostimulatory effects on human B cells.J.Immunol.2001;167:2921-2928.
5 Brock JH.The physiology of lactoferrin.Biochem Cell Biol.2002;80:1-6.
6 Chandra Mohan KV,Kumaraguruparan R,Prathiba,D,et al.Modulation of xenobiotic-metabolizing enzymes and redox status during chemoprevention of hamster buccal carcinogenesis by bovine lactoferrin.Nutrition.2006;22:940-946.
7 Chodaczek G.,Zimecki M,Lukasiewicz J,et al.A complex of lactoferrin with monophosphoryl lipid A is an efficient adjuvant of the humoral and cellular immune response in mice.Medical Microbiology and Immunology.2006;195:207-216.
8 Conte MP,Longhi C,Petrone G,et al.The anti-invasive effect of bovine lactoferrin requires an interaction with surface proteins of Listeria monocytogenes.Int.J.Immunopathol.Pharmacol.1999;12:149-155.
9 Cornish J,Callon KE,Naot D,et al.Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo.Endocrinology.2004;145:4366-4374.
10 Cumberbatch M,Bhushan M,Dearman RJ,et al.IL-1 beta-induced Langerhans' cell migration and TNF-alpha production in human skin,regulation by lactoferrin.Clin.Exp.Immunol.2003;132:352-359.
11 Cumberbatch M,Dearman RJ,Uribe-Luna S,et al.Regulation of epidermal Langerhans cell migration by lactoferrin.Immunology.2000;100:21-28.
12 Curran CS,Demick KP,Mansfield JM.Lactoferrin activates macrophages via TLR4-dependent and-independent signaling pathways.Cellular Immunology.2006;242:23-30.
13 Damiens E,El Yazidi I,Mazurier J,et al.Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells.J.Cell.Biochem.1999;74:486-498.
14 Damiens E,Mazurier J,el Yazidi I,et al.Effects of human lactoferrin on NK cell cytotoxicity against haematopoietic and epithelial tumour cells.Biochim.Biophys.Acta.1998;1402:277-287.
15 Dhennin-Duthille I,Masson M,Damiens E,et al.Lactoferrin upregulates the expression of CD4 antigen through the stimulation of the mitogen-activated protein kinase in the human lymphoblastic T Jurkat cell line.J.Cell.Biochem.2000;79:583-593.
16 Dial EJ,Dohrman AJ,Romero JJ,et al.Recombinant human lactoferrin prevents NSAID induced intestinal bleeding in rodents.J.Pharm.Pharmacol.2005;57:93-99.
17 Dial EJ,Lichtenberger LM.Effect of lactoferrin on Helicobacter felis induced gastritis.Biochem.Cell Biol.2002;80:113-117.
18 Dwarakanath AD,Finnie I A,Beesley CM,et al.Differential excretion of leucocyte granule components in inflammatory bowel disease,implications for pathogenesis.Clin.Sci.1997;92:307-313.
19 Dzitko K,Dziadek B,Dziadek J,et al.Toxoplasma gondii:inhibition of the intracellular growth by human lactoferrin.Pol J Microbiol.2007;56:25-32.
20 Elrod KC,Moore WR,Abraham WM.et al.Lactoferrin,a potent tryptase inhibitor,abolishes latephase airway responses in allergic sheep.Am.J.Respir.Crit.Care Med.1997;156:375-381.
21 Fillebeen C,Ruchoux MM,Mitchell V,et al.Lactoferrin is synthesized by activated microglia in the human substantia nigra and its synthesis by the human microglial CHME cell line is upregulated by tumor necrosis factor alpha or 1-methyl-4-phenylpyridinium treatment.Brain Res.Mol.Brain Res.2001;96:103-113.
22 Fischer R,Debbabi H,Dubarry M,et al.Regulation of physiological and pathological Thl and Th2 responses by lactoferrin.Biochemistry and Cell Biology.2006;84:303-311.
23 Fleming MD,Trenor CC 3rd,Su MA,et al.Microcytic anaemia mice have a mutation in Nramp2,a candidate iron transporter gene.Nat.Genet.1997;16:383-386.
24 Frazer DM,Anderson GJ.The orchestration of body iron intake:how and where do enterocytes receive their cues? Blood Cells Mol.Dis.2003;30:288-297.
25 Fujita K,Matsuda E,Sekine K,et al.Lactoferrin enhances Fas expression and apoptosis in the colon mucosa of azoxymethane-treated rats.Carcinogenesis.2004b;25:1961-1966.
26 Fujita K,Matsuda E,Sekine K,et al.Lactoferrin modifi es apoptosis-related gene expression in the colon of the azoxymethane-treated rat.Cancer Lett.2004a;213:21-29.
27 Fujita K,Ohnishi T,Sekine K,et al.Down-regulation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced CYP1A2 expression is associated with bovine lactoferrin inhibition of MelQx-induced liver and colon carcinogenesis in rats.Jpn.J.Cancer Res.2002;93:616-625.
28 Ghio AJ,Carter JD,Dailey LA,et al.Respiratory epithelial cells demonstrate lactoferrin receptors that increase after metal exposure.Am.J.Physiol.1999;276:L933-940.
29 Giuffre G,Barresi V,Skliros C,et al.Immunoexpression of lactoferrin in human sporadic renal cell carcinomas.Oncol Rep.2007;17:1021-1026.
30 Gomez HF,Ochoa TJ,Carlin LG,et al.Human lactoferrin impairs virulence of Shigella flexneri.J.Infect.Dis.2003;187:87-95.
31 Gomez-Trejo JC,Cores JA,Cuervo SI,et al.Amebiasis intestinal.Infection.2007;11:36-45.
32 Grey A,Banovic T,Zhu Q,et al.The low-density lipoprotein receptor-related protein 1 is a mitogenic receptor for lactoferrin in osteoblastic cells.Mol.Endocrinol.2004;18:2268-2278.
33 Griffi ths CE,Cumberbatch M,Tucker SC,et al.Exogenous topical lactoferrin inhibits allergen-induced Langerhans cell migration and cutaneous inflammation in humans.Br.J.Dermatol.2001;144:715-725.
34 Groot F,Geijtenbeek TB,Sanders RW,et al.Lactoferrin prevents dendritic cell-mediated human immunodefi ciency virus type 1 transmission by blocking the DC-SIGN-gp 120 interaction.J.Virol.2005;79:3009-3015.
35 Guillen C,Mclnnes IB,Vaughan D,et al.The effects of local administration of lactoferrin on inflammation in murine autoimmune and infectious arthritis.Arthritis Rheum.2000;43: 2073-2080.
36 Guillen C,McInnes IB,Vaughan DM,et al.Enhanced Th1 response to Staphylococcus aureus infection in human lactoferrin-transgenic mice.J.Immunol.2002;168:3950-3957.
37 Gunshin H,Mackenzie B,Berger UV,et al.Cloning and characterization of a mammalian proton-coupled metal-ion transporter.Nature.1997;388:482-488.
38 Harada E,Araki Y,Furumura E,et al.Characteristic transfer of colostrums derived biologically active substances into cerebrospinal fluid via blood in natural suckling neonatal pigs.J.Vet.Med.2002;A 49:358-364.
39 Harada E,Itoh Y,Sitizyo K,et al.Characteristic transport of lactoferrin from the intestinal lumen into the bile via the blood in piglets.Comp.Biochem.Physiol.1999b;124A:321-327.
40 Harada,E,Sugiyama A,Takeuchi T,et al.Characteristic transfer of colostral components into cerebrospinal fluid via serum in neonatal pigs.Biol.Neonate.1999a;76:33-43.
41 Haversen L,Ohlsson BG,Hahn-Zoric M,et al.Lactoferrin down-regulates the LPS induced cytokine production in monocytic cells via NF-kappa B.Cell.Immunol.2002;220:83-95.
42 Haversen LA,Baltzer L,Dolphin G,et al.Anti-inflammatory activities of human lactoferrin in acute dextransulphate-induced colitis in mice.Scand.J.Immunol.2003;57:2-10.
43 Hayashida K,Kaneko T,Takeuchi T,et al.Oral administration of lactoferrin inhibits inflammation and nociception in rat adjuvant-induced arthritis.J.Vet.Med.Sci.2004;66:149-154.
44 Hayashida K,Takeuchi T,Harada E.Lactoferrin enhances peripheral opioid-mediated antinociception via nitric oxide in rats.Eur.J.Pharmacol.2004;484:175-181.
45 Hayashida K,Takeuchi T,Shimizu H,et al.Lactoferrin enhances opioid-mediated analgesia via nitric oxide in the rat spinal cord.Am.J.Physiol.2003b;285:R306-R312.
46 Hayashida K,Takeuchi T,Shimizu H,et al.Novel central function of milk-derived lactoferrin on anti-nociception mediated by the u-opioid receptor.Brain Res.2003a;965:239-245.
47 Hendrixson DR,Qiu J,Shewry SC,et al.Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine-rich sites.Mol.Microbiol.2003;47:607-617.
48 Iigo M,Shimamura M,Matsuda E,et al.Orally administered bovine lactoferrin induces caspase-1 and interleukin-18 in the mouse intestinal mucosa:a possible explanation for inhibition of carcinogenesis and metastasis.Cytokine.2004;25:36-44.
49 Kaisho T,Akira S.Pleiotropic function of Toll-like receptors.Microbes Infect.2004;6:1388-1394.
50 Kim CW,Son KN,Choi SY,et al.Human lactoferrin upregulates expression of KDR/Flk-1 and stimulates VEGF-A-mediated endothelial cell proliferation and migration.FEBS Letters.2006;580:4332-4336.
51 Kimber I,Cumberbatch M,Dearman RJ,et al.Regulation by lactoferrin of epidermal Langerhans cell migration.Adv.Exp.Med.Biol.1998;443:251-255.
52 Kitagawa H,Yoshizawa Y,Yokoyama T,et al.Persorption of bovine lactoferrin from the intestinal lumen into the systemic circulation via the portal vein and the mesenteric lymphatics in growing pigs.J.Vet.Med.Sci.2003;65:567-572.
53 Konishi,M,Iwasa M,Yamauchi K,et al.Lactoferrin inhibits lipid peroxidation in patients with chronic hepatitis C.Hepatology Research.2006;36:27-32.
54 Kruzel ML,Harari Y,Chen CY,et al.Lactoferrin protects gut mucosal integrity during endotoxemia induced by lipopolysaccharide in mice.Inflammation.2000;24:33-44.
55 Kruzel,ML,Bacsi A,Choudhury B,et al.Lactoferrin decreases pollen antigen-induced allergic airway inflammation in a murine model of asthma.Immunology.2006;119:159-166.
56 Kuhara,T,Yamauchi K,Tamura Y,et al.Oral administration of lactoferrin increases NK cell activity in mice via increased production of IL-18 and type Ⅰ IFN in the small intestine.Journal of Interferon Cytokine Research.2006;26:489-499.
57 Lee WJ,Farmer JL,Hilty M,et al.The protective effects of lactoferrin feeding against endotoxin lethal shock in germfree piglets.Infect.Immun.1998;66:1421-1426.
58 Legrand D,Elass E,Carpentier M,et al.Interaction of lactoferrin with cells involved in immune function.Biochem Cell Biol.2006;84:282-290.
59 Legrand D,Elass E,Carpentier M,et al.Lactoferrin:A modulator of immune and inflammatory responses.Cellular and Molecular Life Sciences.2005;62:2549-2559.
60 Legrand D,Elass E,Pierce A,et al.Lactoferrin and host defence:an overview of its immuno-modulating and anti-infl ammatory properties.Biometals.2004b;17:225-229.
61 Legrand D,Pierce A,Elass E,et al.Lactoferrin structure and function.Adv Exp Med Biol.2008;606:163-194.
62 Legrand D,Vigie K,Said EA,et al.Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells.Eur.J.Biochem.2004a;271:303-317.
63 Leon-Sicairos N,Lopez-Soto F,Reyes-Lopez M,et al.Amoebicidal activity of milk,apo-lactoferrin,slgA and lysozyme.Clin Med Res.2006;4:106-113.
64 Ling JM,Schryvers AB.Perspectives on interactions between lactoferrin and bacteria.Biochemistry and Cell Biology.2006;84:275—281.
65 Lorget F,Clough J,Oliveira M,et al.Lactoferrin reduces in vitro osteoclast differentiation and resorbing activity.Biochem.Biophys.Res.Commun.2002;296:261-266.
66 Masuda C,Wanibuchi H,Sekine K,et al.Chemopreventive effects of bovine lactoferrin on N-butyl-N-(4-hydroxybutyl) nitrosamine-induced rat bladder carcinogenesis.Jpn.J.Cancer Res.2000;91:582-588.
67 Matsuda Y,Saoo K,Hosokawa K,et al.Post-initiation chemopreventive effects of dietary bovine lactoferrin on 4-(methylnitrosamino)-1-(3-pyridyl)-l-butanone-induced lung tumorigenesis in female A/J mice.Cancer Letters.2007;246:41-46.
68 Ochoa TJ,Noguera-Obenza M,Ebel F,et al.Lactoferrin impairs type Ⅲ secretory system function in enteropathogenic Escherichia coli.Infect.Immun.2003;71:5149-5155.
69 Oh SM,Hahm DH,Kim IH.et al.Human neutrophil lactoferrin trans-activates the matrix metalloproteinase 1 gene through stress-activated MAPK signaling modules.J.Biol.Chem.2001;276:42575^12579.
70 Oh SM,Pyo C W,Kim Y,et al.Neutrophil lactoferrin upregulates the human p53 gene through induction of NF-kappa B activation cascade.Oncogene.2004;23:8282-8291.
71 Orsi N.The antimicrobial activity of lactoferrin,current status and perspectives.Biometals.2004;17:189-196.
72 Penco S,Caligo MA,Cipollini G,et al.Lactoferrin expression in human breast cancer.Cancer Biochem.Biophys.1999;17:163-178.
73 Reddy P.Interleukin-18:recent advances.Curr.Opin.Hematol.2004;11:405-410.
74 Rogan MP,Taggart RB,Greene CM,et al.Loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity in patients with cystic fi brosis.J.Infect.Dis.2004;190:1245-1253.
75 Seganti L,Di Biase AM,Marchetti M,et al.Antiviral activity of lactoferrin towards naked viruses.Biometals.2004;17:295-299.
76 Sekine K,Watanabe E,Nakamura J,et al.Inhibition of azoxymethane-initiated colon tumor by bovine lactoferrin administration in F344 rats.Jpn.J.Cancer Res.1997;88:523-526.
77 Shaper M,Hollingshead SK,Benjamin WH,et al.PspA protects Streptococcus pneumoniae from killing by apolactoferrin,and antibody to PspA enhances killing of pneumococci by apolactoferrin [corrected].Infect.Immun.2004;72:5031-5040.
78 Shimamura M,Yamamoto Y,Ashino H,et al.Bovine lactoferrin inhibits tumorinduced angiogenesis.Int.J.Cancer.2004;111:111-116.
79 Siebert PD,Huang BC.Identifi cation of an alternative form of human lactoferrin mRNA that is expressed differentially in normal tissues and tumor-derived cell lines.Proc.Natl.Acad.Sci.USA.1997;94:2198-2203.
80 Singh PK,Parsek MR,Greenberg EP,et al.A component of innate immunity prevents bacterial biofilm development.Nature.2002;417:552-555.
81 Son KN,Park J,Chung CK,et al.Human lactoferrin activates transcription of IL-1beta gene in mammalian cells.Biochem.Biophys.Res.Commun.2002;290:236-241.
82 Suzuki YA,Lonnerdal B.Characterization of mammalian receptors for lactoferrin.Biochem.Cell Biol.2002;80:75-80.
83 Suzuki YA,Shin K,Lonnerdal B.Molecular cloning and functional expression of a human intestinal lactoferrin receptor.Biochemistry.2001;40:15771-15779.
84 Takeuchi T,Kitagawa H,Harada E.Evidence of lactoferrin transportation into blood circulation from intestine via lymphatic pathway in adult rats.Exp.Physiol.2004;89:263-270.
85 Talukder MJR,Takeuchi T,Harada E.Characteristics of lactoferrin receptor in bovine intestine:higher binding activity in Peyer's patches.J.Vet.Med.2003;A 50:123-131.
86 Tanaka T,Kawabata K,Kohno H,et al.Chemopreventive effect of bovine lactoferrin on 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in male F344 rats.Jpn.J.Cancer Res.2000;91:25-33.
87 Teng C,Gladwell W,Raphiou I,et al.Methylation and expression of the lactoferrin gene in human tissues and cancer cells.Biometals.2004;17:317-323.
88 Togawa J,Nagase H,Tanaka K,et al.Lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance.Am.J.Physiol.Gastrointest.Liver Physiol.2002a;283:G187-195.
89 Togawa J,Nagase H,Tanaka K,et al.Oral administration of lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance.J.Gastroenterol.Hepatol.2002b;17:1291-1298.
90 Tsuchiya T,Takeuchi T,Hayashida K,et al.Milk-derived lactoferrin may block tolerance to morphine analgesia.Brain Res.2006;1068:102-108.
91 Tsuda H,Sekine,Fujita K,et al.Cancer prevention by bovine lactoferrin and underlying mechanisms-A review of experimental and clinical studies.Biochemistry and Cell Biology.2002;80:131-136.
92 Tsuda H,Sekine K,Fujita K.et al.Cancer prevention by bovine lactoferrin and underlying mechanisms-a review of experimental and clinical studies.Biochem.Cell Biol.2002;80:131-136.
93 Ushida Y,Sekine K,Kuhara T,et al.Possible chemopreventive effects of bovine lactoferrin on esophagus and lung carcinogenesis in the rat.Jpn.J.Cancer Res.1999;90:262-267.
94 van der Strate BW,Beljaars L,Molerna G,et al.Antiviral activities of lactoferrin.Antiviral Res.2001;52:225-239.
95 Varadhachary A,Wolf JS,Petrak K,et al.Oral lactoferrin inhibits growth of established tumors and potentiates conventional chemotherapy.Int.J.Cancer.2004;111:398-403.
96 Wakabayashi H,Takakura N,Yamauchi K,et al.Modulation of immunerelated gene expression in small intestines of mice by oral administration of lactoferrin.Clin Vaccine Immunol.2007;13:239-245.
97 Wakabayashi H,Takase M,Tomita M.Lactoferricin derived from milk protein lactoferrin.Curr.Pharm.Des.2003;9:1277-1287.
98 Wang WP,Iigo M,Sato J,et al.Activation of intestinal mucosal immunity in tumor-bearing mice by lactoferrin.Jpn.J.Cancer Res.2000;91:1022-1027.
99 Ward PP,Mendoza-Meneses M,Cunningham GA.et al.Iron status in mice carrying a targeted disruption of lactoferrin.Mol.Cell.Biol.2003;23:178-185.
100 Ward PP,Uribe-Luna S,Conneely OM.Lactoferrin and host defense.Biochem.Cell Biol.2002;80:95-102.
101 Wessling-Resnick M.Iron transport.Annu.Rev.Nutr.2000;20:129-151.
102 Wolf JS,Li D,Taylor RJ,et al.Lactoferrin inhibits growth of malignant tumors of the head and neck.ORL J.Otorhinolaryngol Relat.Spec.2003;65:245-249.
103 Wolf JS,Li G,Varadhachary A,et al.Oral lactoferrin results in T cell-dependent tumor inhibition of head and neck squamous cell carcinoma in vivo.Clin Cancer Res.2007;13:1601-1610.
104 Xiao Y,Monitto CL,Minhas KM,et al.Lactoferrin down-regulates G1 cyclin-dependent kinases during growth arrest of head and neck cancer cells.Clin.Cancer Res.2004;10:8683-8686.
105 Yamauchi K,Wakabayashi H,Shin K,et al.Bovine lactoferrin:benefits and mechanism of action against infections.Biochem Cell Biol.2006;84:291-296.
106 Zarember KA,Sugui JA,Chang YC,et al.Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion.J Immunol.2007;178:6367-6373.
2 Aley KO,Levine JD.Different peripheral mechanisms mediate enhanced nociception in metabolic/toxic and traumatic painful peripheral neuropathies in the rat.Neuroscience.2002;111:389-397.
3 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.
4 Bolotina VM,Najibi,Palacino JJ,et al.Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle.Nature.1994;368:850-853.
5 Bouhassira D,et al.Prevalence of chronic pain with Neuropathic characteristics in the general population.Pain.2008;136:380-387.
6 Brock JH.The physiology of lactoferrin.Biochem Cell Biol.2002;80:1-6.
7 Busija DW,Lacza Z,Rajapakse N,et al.Targeting mitochondrial ATP-sensitive potassium channels-a novel approach to neuroprotection.Brain Res Rev.2004;46:282-294.
8 Cabot PJ,Immune-derived opioids and peripheral antinociception.Clin.Exp.Pharmacol.Physiol.2001;28:230-232.
9 Chen SR,Eisenach JC,Pan HL.Intrathecal S-nitroso-N-acetylpenicillamine and L-cysteine attenuate nerve injury-induced allodynia through noradrenergic activation in rats.Neuroscience.2000;101:759-765.
10 Chen SR,Khan GM,Pan HL.Antiallodynic effect of intrathecal neostigmine is mediated by spinal nitric oxide in a rat model of diabetic neuropathic pain.Anesthesiology.2001;95:1007-1012.
11 Chen SR,Khan M,Pan HL.Antiallodynic effect of intrathecal neostigmine is mediated by spinal nitric oxide in a rat model of diabetic neuropathic pain.Anesthesiology.2001;95:1007-1012.
12 Chen SR,Pan HL.Spinal endogenous acetylcholine contributes to the analgesic effect of systemic morphine in rats.Anesthesiology.2001;95:525-530.
13 Chen SR,Pan HL.Spinal nitric oxide contributes to the analgesic effect of intrathecal [d-pen2,d-pen5]-enkephalin in normal and diabetic rats.Anesthesiology.2003;98:217-222.
14 Deciga-Campos M,Lopez-Munoz FJ.Participation of the L-arginine-nitric oxide-cyclic GMP-ATP-sensitive K+ channel cascade in the antinociceptive effect of rofecoxib.Eur.J.Pharmacol.2004;484:193-199.
15 Eder C.Regulation of microglial behavior by ion channel activity.J Neurosci Res.2005;81:314-321.
16 Fillebeen C,Ruchoux MM,Mitchell V,et al.Lactofferin is synthesized by activated microglia in the human substantia nigra and its synthesis by the human microglial CHME cell line is upregulated by tumor necrosis factor alpha or 1-methyl-4phenylpyridinium treatment.Brain Res.Mol.2001;96:103-113.
17 Finnerup NB et al.An evidence-based algorithm for the treatment of neuropathic pain.Med Gen Med.2007;9:36.
18 Francis SH,Corbin JD.Cyclic nucleotide-dependent protein kinases:intracellular receptors for cAMP and cGMP action.Crit Rev Clin Lab Sci 1999;36:275-328.
19 Francis SH,Woodford TA,Wolfe L,et al.Types I alpha and I beta isozymes of cGMP-dependent protein kinase:Alternative mRNA splicing may produce different inhibitory domains.Second Messengers Phosphoproteins.1988;12:301-310.
20 Furchgott RF,Zawadzki JV.The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine.Nature.1980;288:373-376.
21 Galluzzi KE.Managing neuropathic pain.J Am Osteopath Assoc.2007;107:ES39-48.
22 Gao HM,Hong JS,Zhang W,et al.Distinct role for microglia in rotenoneinduced degeneration of dopaminergic neurons.J Neurosci.2002;22:782-790.
23 Garbers DL.Guanylyl cyclase receptors and their endocrine,paracrine,and autocrine ligands.Cell.1992;71:1-4.
24 Geiselhoringer A,Gaisa M,Hofmann F,et al.Distribution of IRAG and cGKI-isoforms in murine tissues.FEBS Lett.2004;575:19-22.
25 Glezer A,Simard RS,Rivest S.Neuroprotective role of the innate immune system by microglia.Neuroscience.2007;147:867-883
26 Han J,Kim N,Joo H,et al.ATP-sensitive K~+ channel activation by nitric oxide and protein kinase G in rabbit ventricular myocytes.Am.J.Physiol.Heart Circ.Physiol.2002;283:H1545-H1554.
27 Hargreaves K,Dubner R,Brown F,et al.A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia.Pain.1988;32:77-88.
28 Hayashida K,Takeuchi T,Harada E.Lactoferrin enhances peripheral opioid-mediated antinociception via nitric oxide in rats.Eur.J.Pharmacol.2004;484:175-181.
29 Hayashida K,Takeuchi T,Shimizu H,et al.Lactoferrin enhances opioid-mediated analgesia via nitric oxide in the rat spinal cord.Am.J.Physiol.2003b;285:R306-R312.
30 Hayashida K,Takeuchi T,Shimizu H,et al.Novel central function of milk-derived lactoferrin on anti-nociception mediated by the μ-opioid receptor.Brain Res.2003a;965:239-245.
31 Hu LF,Wang S,Shi XR,et al.ATP-sensitive potassium channel opener iptakalim protectedagainst the cytotoxicity of MPP+ on SH-SY5Y cells by decreasing extracellular glutamate level.J Neurochem.2005;94:1570-1579.
32 Hu SJ,Song XJ,Greenquist KW,et al.Protein kinase A modulates spontaneous activity in chronically compressed dorsal root ganglion neurons in the rat.Pain.2001;94:39-46.
33 Ignarro LJ,Buga GM,Wood KS,et al.Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.Proc Natl Acad Sci USA.1987;84:9265-9269.
34 Inoue T,Mashimo T,Shibata M,et al.Rapid development of nitric oxide-induced hyperalgesia depends on an alternate to the cGMP-mediated pathway in the rat neuropathic pain model.Brain Res.1998;792:263-270.
35 Jarchau T,Hausler C,Markert T,et al.Cloning,expression,and in situ localization of rat intestinal cGMP-dependent protein kinase Ⅱ.Proc Natl Acad Sci USA.1994;91:9426-9430.
36 Jiang H,Shabb JB,Corbin JD.Cross-activation:overriding cAMP/cGMP selectivities of protein kinases in tissues.Biochem Cell Biol.1992;70:1283-1289.
37 Kitagawa,H.,Yoshizawa,Y,Yokoyama,T.,et al.Persorption of bovine lactoferrin from the intestinal lumen into the systemic circulation via the portal vein and the mesenteric lymphatics in growing pigs.J.Vet.Med.Sci.2003;65:567-572.
38 Levay PF,Viljoen M.Lactoferrin:a general review.Haematologica.1995;80:252-267.
39 Levy D,Strassman AM.Modulation of dural nociceptor mechanosensitivity by the nitric oxide-cyclic GMP signaling cascade.J.Neurophysiol.2004;92:766-772.
40 Lincoln TM,Dey N,Sellak H.Invited review:cGMP-dependent protein kinase signaling mechanisms in smooth muscle:from the regulation of tone to gene expression.J Appl Physiol 2001;91:1421-1430.
41 Liu CN,Wall PD,Ben-Dor E,et al.Tactile allodynia in the absence of C-fiber activation:altered firing properties of DRG neurons following spinal nerve injury.Pain.2000;85:503-521.
42 Lohmann SM,Walter U.Tracking functions of cGMP-dependent protein kinases (cGK).Front Biosci.2005;10:1313-1328.
43 Lorenzetti BB,Ferreira SH.Activation of the arginine-nitric oxide pathway in primary sensory neurons contributes to dipyrone-induced spinal and peripheral analgesia.Inflamm.Res.1996;45:308-311.
44 Lucas KA,Pitari GM,Kazerounian S,et al.Guanylyl cyclases and signaling by cyclic GMP.Pharmacol.Rev.2000;52:375-414.
45 Ma C,Shu Y,Zheng Z,et al.Similar electrophysiological changes in axotomized and neighboring intact dorsal root ganglion neurons.J Neurophysiol.2003;89:1588-1602.
46 Maffei FA,Heine RP,Whalen MJ,et al.Levels of antimicrobial molecules defensin and lactoferrin are elevated in the cerebrospinal fluid of children with meningitis.Pediatrics.1999;103:987-992.
47 Manning G,Whyte DB,Martinez R,et al.The protein kinase complement of the human genome.Science.2002;298:1912-1934.
48 Merskey H,Bogduk N (Eds).Classification of Chronic Pain.IASP Press,1994.49 Miletic G,Pankratz MT,Miletic V.Increases in the phosphorylation of cyclic AMP response element binding protein (CREB) and decreases in the content of calcineurin accompany thermal hyperalgesia following chronic constriction injury in rats.Pain.2002;99:493-500.
50 Minghetti L,Ajmone-Cat MA,De Berardinis MA,et al.Microglial activation in chronic neurodegenerative diseases:roles of apoptotic neurons and chronic stimulation.Brain Res Rev.2005;48:251-256.
51 Mixcoatl-Zecuatl T,Flores-Murrieta FJ,Granados-Soto V.The nitric oxide-cyclic GMP-protein kinase G-K~+ channel pathway participates in the antiallodynic effect of spinal gabapentin.Eur J Pharmacol.2006;531:87-95.
52 Nguyen MD,Julie JP,Rivest,S.Innate immunity:the missing link in neuroprotection and neurodegeneration.Nature Rev.Neurosci.2002;3:216-227.
53 Orstavik S,Natarajan V,Tasken K,et al.Characterization of the human gene encoding the type I a and type 113 cGMP-dependent protein kinase (PRKG1).Genomics.1997;42:311-318.
54 Orstavik S,Solberg R,Tasken K,et al.Molecular cloning,cDNA structure,and chromosomal localization of the human type Ⅱ cGMP-dependent protein kinase.Biochem Biophys Res Commun.1996;220:759-765.
55 Pan HL,Chen SR,Eisenach JC.Role of spinal NO in antiallodynic effect of intrathecal clonidine in neuropathic rats.Anesthesiology.1998;89:1518-1523.
56 Rodrigues AR.,Durate ID.The peripheral antinociceptive effect induced by morphine is associated with ATP-sensitive K+ channels.Br.J.Pharmacol.2000;129,110-114.
57 Ruth P.Cyclic GMP-dependent protein kinases:understanding in vivo functions by gene targeting.Pharmacol Ther.1999;82:355-372.
58 Sachs D,Cunha FQ,Ferreira SH.Peripheral analgesic blockade of hypernociception:activation or arginine/NO/cGMP/protein kinase G/ATP-sensitive K~+ channel pathway.Proc.Natl.Acad.Sci.U.S.A.2004;101:3680-3685.
59 Salter M,Strijbos PJ,Neale S,et al.The nitric oxide-cyclic GMP pathway is required for nociceptive signalling at specific loci within the somatosensory pathway.Neuroscience.1996;73:649-655.
60 Schmidtko A,Ruth P,Geisslmger G,et al.Inhibition of cyclic guanosine 5-monophosphate-dependent protein kinase Ⅰ (PKG-Ⅰ) in lumbar spinal cord reduces formalin-induced hyperalgesia and PKG upregulation.Nitric Oxide.2003;8:89-94.
61 Scholz J,Woolf CJ.The neuropathic pain triad:neurons,immune cells,and glia.Nature Neurosci.2007;10:1361-1368.
62 Segawa K,Minami K,Shiga Y,et al.Inhibitory effects of nicorandil on rat mesangial cell proliferation via the protein kinase G pathway.Nephron,2001;87:263-268.
63 Soares AC,Leite R.,Tatsuo MA.et al.Activation of ATP-sensitive K~+ channels mechanism of peripheral antinociceptive action of the nitric oxide donor,sodium nitroprusside.Eur.J.Pharmacol.2000;400:67-71.
64 Song HK,Pan HL,Eisenach JC.Spinal nitric oxide mediates antinociception from intravenous morphine.Anesthesiology.1998;89:215-221.
65 Song XJ,Wang ZB,Gan Q,et al.cAMP and cGMP contribute to sensory neuron hyperexcitability and hyperalgesia in rats with dorsal root ganglia compression.J Neurophysiol.2006;95:479-492.
66 Sorimachi K,Akimoto K,Hattori Y,et al.Activation of macrophages by lactoferrin:secretion of TNF-a,IL-8 and NO.Biochem Mol Biol Int.1997;43:79-87.
67 Takasu K,Honda M,Ono H,et al.Spinal a_2-adrenergic and muscarinic receptors and the NO release cascade mediate supraspinally produced effectiveness of gabapentin at decreasing mechanical hypersensitivity in mice after partial nerve injury.British Journal of Pharmacology.2006;148:233-244.
68 Takeuchi,T,Kitagawa,H.,Harada,E.Evidence of lactoferrin transportation into blood circulation from intestine via lymphatic pathway in adult rats.Exp.Physiol.2004;89:263-270.
69 Talukder MJR.,Takeuchi T,Harada E.Characteristics of lactoferrin receptor in bovine intestine:higher binding activity in Peyer's patches.J.Vet.Med.2003a;A 50:123-131.
70 Tegeder I,Del Turco D,Schmidtko A,et al.Reduced inflammatory hyperalgesia with preservation of acute thermal nociception in mice lacking cGMP dependent protein kinase I.Proc Natl Acad Sci USA.2004;101:3253-3257.
71 Truong W,Cheng C,Xu QG,et al.Opioid receptors and analgesia at the site of a peripheral nerve injury.Ann Neurol.2003;53:366-375.
72 Vivancos GG,Parada CA,Ferreira SH.Opposite nociceptive effects of the arginine/NO/cGMP pathway stimulation in dermal and subcutaneous tissues.Br.J.Pharmacol.2003;138:1351-1357.
73 Watkins,L.R.& Maier,S.F.Targeting glia to control clinical pain:an idea whose time has come.Drug Discov.Today Then Strat.2004;1:83-88.
74 Werhagen L,Hultling C,Molander C.The prevalence of neuropathic pain after non-traumatic spinal cord lesion.Spinal Cord.2007;45:609-615.
75 Wolfe L,Corbin JD,Francis SH.Characterization of a novel isozyme of cGMP-dependent protein kinase from bovine aorta.J Biol Chem.1989;264:7734-7741.
76 Xu Z.,Tong C,Pan HL,et al.Intravenous morphine increases release of nitric oxide from spinal cord by an α-adrenergic and cholinergic mechanism.Journal of Neurophysiology.1997;78:2072-2078.
77 Yaksh TL,Rudy TA.Chronic catheterization of the spinal subarachnoid space.Physiol.Behav.1976;17:1031-1036.
78 Yamada K,Inagaki N.Neuroprotection by K_(ATP) channels.J Mol Cell Cardiol.2005;38:945-949.
79 Yang Y,Liu X,Long Y,et al.Activation of mitochondrial ATP-sensitive potassium channels improves rotenone-related motor and neurochemical alterations in rats.Int J Neuropsychopharmacol.2006;9:51-61.
80 Yoon YW,Sung B,Chung JM.Nitric oxide mediates behavioral signs of neuropathic pain in an experimental rat model.NeuroReport.1998;9:367-372.
81 Zhuo M.,Meller ST.,Gebhart GF.Endogenous nitric oxide is required for tonic cholinergic inhibition of spinal mechanical transmission.Pain.1993;54:71-78.
82 Zochodne D,Max MB.An old acquaintance:opioids in neuropathic pain.Neurology.2003;60:894-895.
1 Andrews NC.Iron homeostasis:insights from genetics and animal models.Nat.Rev.Genet.2000;1:208-217.
2 Baker HM,Anderson BF,Baker EN.Dealing with iron,common structural principles in proteins that transport iron and heme.Proc.Natl.Acad.Sci.USA.2003;100:3579-3583.
3 Benaissa M,Peyrat JP,Hornez L,et al.Expression and prognostic value of lactoferrin mRNA isoforms in human breast cancer.Int.J.Cancer.2005;114:299-306.
4 Britigan BE,Lewis TS,Waldschmidt M,et al.Lactoferrin binds CpG-containing oligonucleotides and inhibits their immunostimulatory effects on human B cells.J.Immunol.2001;167:2921-2928.
5 Brock JH.The physiology of lactoferrin.Biochem Cell Biol.2002;80:1-6.
6 Chandra Mohan KV,Kumaraguruparan R,Prathiba,D,et al.Modulation of xenobiotic-metabolizing enzymes and redox status during chemoprevention of hamster buccal carcinogenesis by bovine lactoferrin.Nutrition.2006;22:940-946.
7 Chodaczek G.,Zimecki M,Lukasiewicz J,et al.A complex of lactoferrin with monophosphoryl lipid A is an efficient adjuvant of the humoral and cellular immune response in mice.Medical Microbiology and Immunology.2006;195:207-216.
8 Conte MP,Longhi C,Petrone G,et al.The anti-invasive effect of bovine lactoferrin requires an interaction with surface proteins of Listeria monocytogenes.Int.J.Immunopathol.Pharmacol.1999;12:149-155.
9 Cornish J,Callon KE,Naot D,et al.Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo.Endocrinology.2004;145:4366-4374.
10 Cumberbatch M,Bhushan M,Dearman RJ,et al.IL-1 beta-induced Langerhans' cell migration and TNF-alpha production in human skin,regulation by lactoferrin.Clin.Exp.Immunol.2003;132:352-359.
11 Cumberbatch M,Dearman RJ,Uribe-Luna S,et al.Regulation of epidermal Langerhans cell migration by lactoferrin.Immunology.2000;100:21-28.
12 Curran CS,Demick KP,Mansfield JM.Lactoferrin activates macrophages via TLR4-dependent and-independent signaling pathways.Cellular Immunology.2006;242:23-30.
13 Damiens E,El Yazidi I,Mazurier J,et al.Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells.J.Cell.Biochem.1999;74:486-498.
14 Damiens E,Mazurier J,el Yazidi I,et al.Effects of human lactoferrin on NK cell cytotoxicity against haematopoietic and epithelial tumour cells.Biochim.Biophys.Acta.1998;1402:277-287.
15 Dhennin-Duthille I,Masson M,Damiens E,et al.Lactoferrin upregulates the expression of CD4 antigen through the stimulation of the mitogen-activated protein kinase in the human lymphoblastic T Jurkat cell line.J.Cell.Biochem.2000;79:583-593.
16 Dial EJ,Dohrman AJ,Romero JJ,et al.Recombinant human lactoferrin prevents NSAID induced intestinal bleeding in rodents.J.Pharm.Pharmacol.2005;57:93-99.
17 Dial EJ,Lichtenberger LM.Effect of lactoferrin on Helicobacter felis induced gastritis.Biochem.Cell Biol.2002;80:113-117.
18 Dwarakanath AD,Finnie I A,Beesley CM,et al.Differential excretion of leucocyte granule components in inflammatory bowel disease,implications for pathogenesis.Clin.Sci.1997;92:307-313.
19 Dzitko K,Dziadek B,Dziadek J,et al.Toxoplasma gondii:inhibition of the intracellular growth by human lactoferrin.Pol J Microbiol.2007;56:25-32.
20 Elrod KC,Moore WR,Abraham WM.et al.Lactoferrin,a potent tryptase inhibitor,abolishes latephase airway responses in allergic sheep.Am.J.Respir.Crit.Care Med.1997;156:375-381.
21 Fillebeen C,Ruchoux MM,Mitchell V,et al.Lactoferrin is synthesized by activated microglia in the human substantia nigra and its synthesis by the human microglial CHME cell line is upregulated by tumor necrosis factor alpha or 1-methyl-4-phenylpyridinium treatment.Brain Res.Mol.Brain Res.2001;96:103-113.
22 Fischer R,Debbabi H,Dubarry M,et al.Regulation of physiological and pathological Thl and Th2 responses by lactoferrin.Biochemistry and Cell Biology.2006;84:303-311.
23 Fleming MD,Trenor CC 3rd,Su MA,et al.Microcytic anaemia mice have a mutation in Nramp2,a candidate iron transporter gene.Nat.Genet.1997;16:383-386.
24 Frazer DM,Anderson GJ.The orchestration of body iron intake:how and where do enterocytes receive their cues? Blood Cells Mol.Dis.2003;30:288-297.
25 Fujita K,Matsuda E,Sekine K,et al.Lactoferrin enhances Fas expression and apoptosis in the colon mucosa of azoxymethane-treated rats.Carcinogenesis.2004b;25:1961-1966.
26 Fujita K,Matsuda E,Sekine K,et al.Lactoferrin modifi es apoptosis-related gene expression in the colon of the azoxymethane-treated rat.Cancer Lett.2004a;213:21-29.
27 Fujita K,Ohnishi T,Sekine K,et al.Down-regulation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced CYP1A2 expression is associated with bovine lactoferrin inhibition of MelQx-induced liver and colon carcinogenesis in rats.Jpn.J.Cancer Res.2002;93:616-625.
28 Ghio AJ,Carter JD,Dailey LA,et al.Respiratory epithelial cells demonstrate lactoferrin receptors that increase after metal exposure.Am.J.Physiol.1999;276:L933-940.
29 Giuffre G,Barresi V,Skliros C,et al.Immunoexpression of lactoferrin in human sporadic renal cell carcinomas.Oncol Rep.2007;17:1021-1026.
30 Gomez HF,Ochoa TJ,Carlin LG,et al.Human lactoferrin impairs virulence of Shigella flexneri.J.Infect.Dis.2003;187:87-95.
31 Gomez-Trejo JC,Cores JA,Cuervo SI,et al.Amebiasis intestinal.Infection.2007;11:36-45.
32 Grey A,Banovic T,Zhu Q,et al.The low-density lipoprotein receptor-related protein 1 is a mitogenic receptor for lactoferrin in osteoblastic cells.Mol.Endocrinol.2004;18:2268-2278.
33 Griffi ths CE,Cumberbatch M,Tucker SC,et al.Exogenous topical lactoferrin inhibits allergen-induced Langerhans cell migration and cutaneous inflammation in humans.Br.J.Dermatol.2001;144:715-725.
34 Groot F,Geijtenbeek TB,Sanders RW,et al.Lactoferrin prevents dendritic cell-mediated human immunodefi ciency virus type 1 transmission by blocking the DC-SIGN-gp 120 interaction.J.Virol.2005;79:3009-3015.
35 Guillen C,Mclnnes IB,Vaughan D,et al.The effects of local administration of lactoferrin on inflammation in murine autoimmune and infectious arthritis.Arthritis Rheum.2000;43: 2073-2080.
36 Guillen C,McInnes IB,Vaughan DM,et al.Enhanced Th1 response to Staphylococcus aureus infection in human lactoferrin-transgenic mice.J.Immunol.2002;168:3950-3957.
37 Gunshin H,Mackenzie B,Berger UV,et al.Cloning and characterization of a mammalian proton-coupled metal-ion transporter.Nature.1997;388:482-488.
38 Harada E,Araki Y,Furumura E,et al.Characteristic transfer of colostrums derived biologically active substances into cerebrospinal fluid via blood in natural suckling neonatal pigs.J.Vet.Med.2002;A 49:358-364.
39 Harada E,Itoh Y,Sitizyo K,et al.Characteristic transport of lactoferrin from the intestinal lumen into the bile via the blood in piglets.Comp.Biochem.Physiol.1999b;124A:321-327.
40 Harada,E,Sugiyama A,Takeuchi T,et al.Characteristic transfer of colostral components into cerebrospinal fluid via serum in neonatal pigs.Biol.Neonate.1999a;76:33-43.
41 Haversen L,Ohlsson BG,Hahn-Zoric M,et al.Lactoferrin down-regulates the LPS induced cytokine production in monocytic cells via NF-kappa B.Cell.Immunol.2002;220:83-95.
42 Haversen LA,Baltzer L,Dolphin G,et al.Anti-inflammatory activities of human lactoferrin in acute dextransulphate-induced colitis in mice.Scand.J.Immunol.2003;57:2-10.
43 Hayashida K,Kaneko T,Takeuchi T,et al.Oral administration of lactoferrin inhibits inflammation and nociception in rat adjuvant-induced arthritis.J.Vet.Med.Sci.2004;66:149-154.
44 Hayashida K,Takeuchi T,Harada E.Lactoferrin enhances peripheral opioid-mediated antinociception via nitric oxide in rats.Eur.J.Pharmacol.2004;484:175-181.
45 Hayashida K,Takeuchi T,Shimizu H,et al.Lactoferrin enhances opioid-mediated analgesia via nitric oxide in the rat spinal cord.Am.J.Physiol.2003b;285:R306-R312.
46 Hayashida K,Takeuchi T,Shimizu H,et al.Novel central function of milk-derived lactoferrin on anti-nociception mediated by the u-opioid receptor.Brain Res.2003a;965:239-245.
47 Hendrixson DR,Qiu J,Shewry SC,et al.Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine-rich sites.Mol.Microbiol.2003;47:607-617.
48 Iigo M,Shimamura M,Matsuda E,et al.Orally administered bovine lactoferrin induces caspase-1 and interleukin-18 in the mouse intestinal mucosa:a possible explanation for inhibition of carcinogenesis and metastasis.Cytokine.2004;25:36-44.
49 Kaisho T,Akira S.Pleiotropic function of Toll-like receptors.Microbes Infect.2004;6:1388-1394.
50 Kim CW,Son KN,Choi SY,et al.Human lactoferrin upregulates expression of KDR/Flk-1 and stimulates VEGF-A-mediated endothelial cell proliferation and migration.FEBS Letters.2006;580:4332-4336.
51 Kimber I,Cumberbatch M,Dearman RJ,et al.Regulation by lactoferrin of epidermal Langerhans cell migration.Adv.Exp.Med.Biol.1998;443:251-255.
52 Kitagawa H,Yoshizawa Y,Yokoyama T,et al.Persorption of bovine lactoferrin from the intestinal lumen into the systemic circulation via the portal vein and the mesenteric lymphatics in growing pigs.J.Vet.Med.Sci.2003;65:567-572.
53 Konishi,M,Iwasa M,Yamauchi K,et al.Lactoferrin inhibits lipid peroxidation in patients with chronic hepatitis C.Hepatology Research.2006;36:27-32.
54 Kruzel ML,Harari Y,Chen CY,et al.Lactoferrin protects gut mucosal integrity during endotoxemia induced by lipopolysaccharide in mice.Inflammation.2000;24:33-44.
55 Kruzel,ML,Bacsi A,Choudhury B,et al.Lactoferrin decreases pollen antigen-induced allergic airway inflammation in a murine model of asthma.Immunology.2006;119:159-166.
56 Kuhara,T,Yamauchi K,Tamura Y,et al.Oral administration of lactoferrin increases NK cell activity in mice via increased production of IL-18 and type Ⅰ IFN in the small intestine.Journal of Interferon Cytokine Research.2006;26:489-499.
57 Lee WJ,Farmer JL,Hilty M,et al.The protective effects of lactoferrin feeding against endotoxin lethal shock in germfree piglets.Infect.Immun.1998;66:1421-1426.
58 Legrand D,Elass E,Carpentier M,et al.Interaction of lactoferrin with cells involved in immune function.Biochem Cell Biol.2006;84:282-290.
59 Legrand D,Elass E,Carpentier M,et al.Lactoferrin:A modulator of immune and inflammatory responses.Cellular and Molecular Life Sciences.2005;62:2549-2559.
60 Legrand D,Elass E,Pierce A,et al.Lactoferrin and host defence:an overview of its immuno-modulating and anti-infl ammatory properties.Biometals.2004b;17:225-229.
61 Legrand D,Pierce A,Elass E,et al.Lactoferrin structure and function.Adv Exp Med Biol.2008;606:163-194.
62 Legrand D,Vigie K,Said EA,et al.Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells.Eur.J.Biochem.2004a;271:303-317.
63 Leon-Sicairos N,Lopez-Soto F,Reyes-Lopez M,et al.Amoebicidal activity of milk,apo-lactoferrin,slgA and lysozyme.Clin Med Res.2006;4:106-113.
64 Ling JM,Schryvers AB.Perspectives on interactions between lactoferrin and bacteria.Biochemistry and Cell Biology.2006;84:275—281.
65 Lorget F,Clough J,Oliveira M,et al.Lactoferrin reduces in vitro osteoclast differentiation and resorbing activity.Biochem.Biophys.Res.Commun.2002;296:261-266.
66 Masuda C,Wanibuchi H,Sekine K,et al.Chemopreventive effects of bovine lactoferrin on N-butyl-N-(4-hydroxybutyl) nitrosamine-induced rat bladder carcinogenesis.Jpn.J.Cancer Res.2000;91:582-588.
67 Matsuda Y,Saoo K,Hosokawa K,et al.Post-initiation chemopreventive effects of dietary bovine lactoferrin on 4-(methylnitrosamino)-1-(3-pyridyl)-l-butanone-induced lung tumorigenesis in female A/J mice.Cancer Letters.2007;246:41-46.
68 Ochoa TJ,Noguera-Obenza M,Ebel F,et al.Lactoferrin impairs type Ⅲ secretory system function in enteropathogenic Escherichia coli.Infect.Immun.2003;71:5149-5155.
69 Oh SM,Hahm DH,Kim IH.et al.Human neutrophil lactoferrin trans-activates the matrix metalloproteinase 1 gene through stress-activated MAPK signaling modules.J.Biol.Chem.2001;276:42575^12579.
70 Oh SM,Pyo C W,Kim Y,et al.Neutrophil lactoferrin upregulates the human p53 gene through induction of NF-kappa B activation cascade.Oncogene.2004;23:8282-8291.
71 Orsi N.The antimicrobial activity of lactoferrin,current status and perspectives.Biometals.2004;17:189-196.
72 Penco S,Caligo MA,Cipollini G,et al.Lactoferrin expression in human breast cancer.Cancer Biochem.Biophys.1999;17:163-178.
73 Reddy P.Interleukin-18:recent advances.Curr.Opin.Hematol.2004;11:405-410.
74 Rogan MP,Taggart RB,Greene CM,et al.Loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity in patients with cystic fi brosis.J.Infect.Dis.2004;190:1245-1253.
75 Seganti L,Di Biase AM,Marchetti M,et al.Antiviral activity of lactoferrin towards naked viruses.Biometals.2004;17:295-299.
76 Sekine K,Watanabe E,Nakamura J,et al.Inhibition of azoxymethane-initiated colon tumor by bovine lactoferrin administration in F344 rats.Jpn.J.Cancer Res.1997;88:523-526.
77 Shaper M,Hollingshead SK,Benjamin WH,et al.PspA protects Streptococcus pneumoniae from killing by apolactoferrin,and antibody to PspA enhances killing of pneumococci by apolactoferrin [corrected].Infect.Immun.2004;72:5031-5040.
78 Shimamura M,Yamamoto Y,Ashino H,et al.Bovine lactoferrin inhibits tumorinduced angiogenesis.Int.J.Cancer.2004;111:111-116.
79 Siebert PD,Huang BC.Identifi cation of an alternative form of human lactoferrin mRNA that is expressed differentially in normal tissues and tumor-derived cell lines.Proc.Natl.Acad.Sci.USA.1997;94:2198-2203.
80 Singh PK,Parsek MR,Greenberg EP,et al.A component of innate immunity prevents bacterial biofilm development.Nature.2002;417:552-555.
81 Son KN,Park J,Chung CK,et al.Human lactoferrin activates transcription of IL-1beta gene in mammalian cells.Biochem.Biophys.Res.Commun.2002;290:236-241.
82 Suzuki YA,Lonnerdal B.Characterization of mammalian receptors for lactoferrin.Biochem.Cell Biol.2002;80:75-80.
83 Suzuki YA,Shin K,Lonnerdal B.Molecular cloning and functional expression of a human intestinal lactoferrin receptor.Biochemistry.2001;40:15771-15779.
84 Takeuchi T,Kitagawa H,Harada E.Evidence of lactoferrin transportation into blood circulation from intestine via lymphatic pathway in adult rats.Exp.Physiol.2004;89:263-270.
85 Talukder MJR,Takeuchi T,Harada E.Characteristics of lactoferrin receptor in bovine intestine:higher binding activity in Peyer's patches.J.Vet.Med.2003;A 50:123-131.
86 Tanaka T,Kawabata K,Kohno H,et al.Chemopreventive effect of bovine lactoferrin on 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in male F344 rats.Jpn.J.Cancer Res.2000;91:25-33.
87 Teng C,Gladwell W,Raphiou I,et al.Methylation and expression of the lactoferrin gene in human tissues and cancer cells.Biometals.2004;17:317-323.
88 Togawa J,Nagase H,Tanaka K,et al.Lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance.Am.J.Physiol.Gastrointest.Liver Physiol.2002a;283:G187-195.
89 Togawa J,Nagase H,Tanaka K,et al.Oral administration of lactoferrin reduces colitis in rats via modulation of the immune system and correction of cytokine imbalance.J.Gastroenterol.Hepatol.2002b;17:1291-1298.
90 Tsuchiya T,Takeuchi T,Hayashida K,et al.Milk-derived lactoferrin may block tolerance to morphine analgesia.Brain Res.2006;1068:102-108.
91 Tsuda H,Sekine,Fujita K,et al.Cancer prevention by bovine lactoferrin and underlying mechanisms-A review of experimental and clinical studies.Biochemistry and Cell Biology.2002;80:131-136.
92 Tsuda H,Sekine K,Fujita K.et al.Cancer prevention by bovine lactoferrin and underlying mechanisms-a review of experimental and clinical studies.Biochem.Cell Biol.2002;80:131-136.
93 Ushida Y,Sekine K,Kuhara T,et al.Possible chemopreventive effects of bovine lactoferrin on esophagus and lung carcinogenesis in the rat.Jpn.J.Cancer Res.1999;90:262-267.
94 van der Strate BW,Beljaars L,Molerna G,et al.Antiviral activities of lactoferrin.Antiviral Res.2001;52:225-239.
95 Varadhachary A,Wolf JS,Petrak K,et al.Oral lactoferrin inhibits growth of established tumors and potentiates conventional chemotherapy.Int.J.Cancer.2004;111:398-403.
96 Wakabayashi H,Takakura N,Yamauchi K,et al.Modulation of immunerelated gene expression in small intestines of mice by oral administration of lactoferrin.Clin Vaccine Immunol.2007;13:239-245.
97 Wakabayashi H,Takase M,Tomita M.Lactoferricin derived from milk protein lactoferrin.Curr.Pharm.Des.2003;9:1277-1287.
98 Wang WP,Iigo M,Sato J,et al.Activation of intestinal mucosal immunity in tumor-bearing mice by lactoferrin.Jpn.J.Cancer Res.2000;91:1022-1027.
99 Ward PP,Mendoza-Meneses M,Cunningham GA.et al.Iron status in mice carrying a targeted disruption of lactoferrin.Mol.Cell.Biol.2003;23:178-185.
100 Ward PP,Uribe-Luna S,Conneely OM.Lactoferrin and host defense.Biochem.Cell Biol.2002;80:95-102.
101 Wessling-Resnick M.Iron transport.Annu.Rev.Nutr.2000;20:129-151.
102 Wolf JS,Li D,Taylor RJ,et al.Lactoferrin inhibits growth of malignant tumors of the head and neck.ORL J.Otorhinolaryngol Relat.Spec.2003;65:245-249.
103 Wolf JS,Li G,Varadhachary A,et al.Oral lactoferrin results in T cell-dependent tumor inhibition of head and neck squamous cell carcinoma in vivo.Clin Cancer Res.2007;13:1601-1610.
104 Xiao Y,Monitto CL,Minhas KM,et al.Lactoferrin down-regulates G1 cyclin-dependent kinases during growth arrest of head and neck cancer cells.Clin.Cancer Res.2004;10:8683-8686.
105 Yamauchi K,Wakabayashi H,Shin K,et al.Bovine lactoferrin:benefits and mechanism of action against infections.Biochem Cell Biol.2006;84:291-296.
106 Zarember KA,Sugui JA,Chang YC,et al.Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion.J Immunol.2007;178:6367-6373.