| 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. |
