Mechanism Of FK506 On Accelerating Functional Recovery After Sciatic Nerve Injury: A Study Of Rat Model

BackgroundGold first reported in 1994 that FK506 had the potentiality of acceleratingaxonal regeneration after sciatic nerve crush injury in rat model, consequentlypromoting nerve reconstruction and functional recovery. Results from spinal cordtransplanted with pre-degenerative tibial nerve suggested that FKS06 could not onlypromote axonal elongation, but also accelerate regeneration of extrapyramidal motorfasciculus. A series of following experimental researches in vivo/vitro indicatedcommon results that FK506 had powerful potentiality of promoting nerveregeneration, both in central and peripheral nerve systems.However, what is the mechanism of FK506 promoting nerve regeneration?Currently, there are two explanations. First, Complex FKSO6-FK506bp12(immunophilin) promotes nerve growth and regeneration by immunosuppression.Second, FKS06 takes neurotrophic action by one of its components. Other studiessuggested that both immunosuppression and neurotrophic action played roles. In aword, it is still a controversy requiring further work.Researches indicated that nuclear factor-κB (NF-κB) was closely related tonerve injury. Brain injury in rat activated NF-κB for long time. During the first 1-2hours after injury, activated NF-κB existed mainly in axon, and then appeared inastrocyte in the next 24 hours, lasted for 1 year in pallium. Meanwhile, activatedNF-κB also appeared in vascular endothelial cell in brain for more than 1 year. Long-term activation of NF-κB in neuroglia cells induced atrophy of involved brain.Spine injury could also induce activation of NF-κB, by triggering release of free fattyacid, especially arachidonic acid, which activated NF-κB and suppressed expressionof inhibiting protein, subsequently induced oxidative stress reaction, resulted indamage to spinal neuron.Activation of NF-κB irritates strong inflammatory reaction after injury. NF-κBup-regulates production of inflammatory cytokine, including TNF-α,IL-12,macrophage inflammatory protein(MIP-1a), nitrogen monoxidum (NO), howeverdegrade expression of IL-10. Increase of TNF-αcould reversely activate NF-κB,further enhance inflammatory reaction. In rat astrocyte, inflammatory factorsbradykinin induce secretion of IL-6 through activation of NF-κB, consequentlyresult in destroy of blood- brain barrier. This process is inhibited by Ca²⁺/calcineurin (KN-93). Over expression of NF-κB inhibiting protein (IκB) can inhibit secretionof IL-6 induced by bradykinin. In human astrocyte, IL-1 is an effective inducer ofinflammation related gene. Compared with TNF, IL-1 is more effective in activatingnuclear factor activating protein 1 (AP-1) and NF-κB.Results from current researches show that NF-κB cause neuron injury by path ofregulating kinds of apoptosis related genes. NF-κB enhances expression of Bcl-Xafter cerebral ischemia, so as to P53, C-myc. These genes play critical roles both incell apoptosis and pathway of nerve cell death. Continuous activation of NF-κB wasobserved in brain cells after injury, which suggested close relation between NF-κBinduced inflammatory reaction and progressive atrophy after brain injury. CytokineIL-1 and TNF-αboth are able to activate NF-κB, initiate NF-κB regulated geneproceeding, and maintain continuous inflammatory reaction.To summarize, NF-κB plays critical role after nerve injury by regulatinginflamatation and apoptosis.The immunosuppressive mechanism of FK506 is: after entering cell, FK506combine with FKBP-12 in cytoplasm. Complex FK506/FKBP competitively combinewith protein phosphatase calcineurin (CaN), subsequently inhibit dephosphorylationof transcription factor NF-AT and NF-κB, which result in inhibition of expression and production of cytokine including IL—2,γ-interferon and IL-3, in T-cell. Contrary tothe situation in T cells, FK506 activates transcription factor NF-kB in nonlymphoidcells such as fibroblasts and renal mesangial cells.In nerve system, what is the relationship between FKS06 and NF-kBactivation/IL-6 production? The understanding of this question might explain themechanism of FKS06 on promoting nerve regeneration.The idea of neural stem cell (NSC) evoked the hope of neuron regeneration,which was believed to be permenantly unregeneratalbe. Researches in the field ofNSC are trying to provide explanation for nervous system associated diseases, so asto find solution. One key point of NSC study is to control differentiation, so as to gettarget nerve cell. Retinoic acid (RA) and nerve growth factor (NGF) are able toinduce directional differentiation of embryonic stem cell (ESC). Besides, IL-1, IL-7,IL-9, IL-1, bNGF, EGF, GDNF are also involved in the event. Since FK506 showeddefinite potentiality of promoting nerve regeneration, it might take effect through thepathway of NSC.ObjectivePartâ… Sciatic nerve injury in rat model was established, in order to evaluate nerveregeneration and functional recovery after short-term FK506 (tacrolimus)administration. The goal was to identify the effect of FKS06 on peripheral nerveregenerationPartâ…¡In sciatic nerve injury rat model, immunosuppressant FK506 was administratedas intervention. Activity of NF-κB and expression of inflammatory factor IL-6 insciatic nerve and spine segment L4-6 were tested. The present study was conducted toinvestigate correlation between FK506 and NF-κB, IL-6 after nerve injury, in order toelucidate the role of FK506 in promoting peripheral nerve regeneration.Partâ…¢To observe generation and differentiation of NSC with FK506 intervention, and try to identify dose-dependent effect.MethodsPartâ… 1. Rat model of autogenous nerve regeneration under immunosuppression wasestablished. All the operated animals were divided into two groups: theexperimental group was immunosuppressed with FK506 systemically, while theother untreated-group as control group.2. Weight of soleus muscle and contractility of triceps leg muscle was measured incomparison with the unoperated contralateral leg. Sciatic function index (SFI)and Somato-Sensory Evoked Potentials (SSEP) evaluated recovery of function.Besides, 10 cases of clinical volunteer were treated with FK506, and regularexamination were done for functional evaluation.Partâ…¡1. 40 SD rats were randomly divided into 4 groups. Group A (N=10) wasnon-operated group. Group B (N=10) was operated control group withoutFK506 treatment. Group C (N=20) was operated group treated with two dose ofFK506 (C1:0.5mg/kg/day, C2:1mg/kg/day, SC).2. Animals were excised in 7 days after operation. Sciatic nerve and L_4-6 segmentsof spinal cord harvesting were done for test.3. Evaluation parametera) RT-PCR tested mRNA of NF-κB and IL-6 in nerve and spinal segment.b) Western-blot tested expression of NF-κB and IL-6 in nerve and spinalsegment.Partâ…¢Neural stem cell isolated from neonate SD rat were cultivated, passaged andidentificated. Differentiation of NSC was induce with 20ï¼…fetal bovine serum anddifferent dose FK506 individually. Morphological observation was done. ResultsPartâ… 1. Recovery rates of soleus muscle weight and contractility of triceps leg musclein animals given FK506 were significantly higher than that in control group.2. SFI demonstrated better functional recovery of the injuried leg inFK506-treated animals.3. No significant difference of functional recovery could be seen between animalstreated with FK506 temporarily and continuously.4. Mortality in two groups showed no significant difference.Partâ…¡In L_4-6 spine segment, expression of NF-κB and IL-6 both in gene and proteinlevel increase significantly after sciatic nerve injury. Results of inter-groupcomparison was: compared with Group-A (control rat), Group-B (operated withoutFK506 rat) increased significantly (P＜0.05); compared with Group-B, Group-C(operated with FK506-treat rat) decreased significantly (P＜0.05); Group-C wassignificantly higher than Group-A (P＜0.05). Result of intra-group comparison:compared with Group-C1(0.5mg/kg/day), expression in Group-C2 (1mg/kg/day)decreased significantly (P＜0.05).In transected sciatic nerve, expression of NF-κB and IL-6 both in gene andprotein level increase significantly after sciatic nerve injury. Results of inter-groupcomparison was: compared with Group-A (control rat), Group-B (operated withoutFK506 rat) increased significantly (P＜0.05); Group-C (operated with FK506-treatrat)was significantly higher than Group-A (P＜0.05). There was no significantdifference between Group-B and Group-C (P＞0.05).Partâ…¢FK506 facilitated proliferation and differentiation of neural stem cell,demonstrated by cell number increasing, premature of differentiation, maturity anddifferentiation rate increasing. Moreover, the effect showed dose-dependent. Conclusions:Partâ… 1. Immunosuppressant FK506 is benefit to nerve regeneration, muscle andfunction recovery.2. Temporary usage of FK506 has the same effect as continuous usage, with nosignificant influence on survival.3. FK506 could be used as a novel drug for peripheral nerve treatment byaccelerating nerve regeneration.Partâ…¡Expression of NF-κB and IL-6 increased significantly in nerve andcorresponding spinal segment after sciatic nerve transected injury.Immunosuppressant FK506 inhibited up-regulation of injury-induced activation andexpression of NF-κB and IL-6 in spine, while took no effect of inhabition inperipheral nerve. This indicated an alternative mechanism of FK506 in promotingperipheral nerve regeneration, may via blocking signal transduction pathway ofNF-κB in spine. The blocking effect was dose-dependent.Partâ…¢1. FK506 may play the role of accelerating nerve regeneration by pathway ofNSC.2. FK506 could be used as other specific factors for study on neural stem cell.