| FK506(tacrolimus) is a macrolide antibiotic isolated from streptomycete in1984and has extensive application in the area of organ transplantation. Recent years, it has been demonstrated that FK506has powerful effect of promoting nerve regeneration.ObjectivePart ⅠSciatic nerve injury in rat model was established, in order to investigate whether FK506intensively promotes peripheral nerve regeneration and functional recovery by directly inhibiting scar formation of nerval anastomotic stoma.Part IITo investigate whether FK506reduces scar formation of nerval anastomotic stoma through inducing fibroblast apoptosis.MethodsPart Ⅰ1. establishing the model of rat sciatic nerve injury, then the rats received intragastric administration of FK506according to the dose of4mg/kg/d. The rats were divided into5groups (n=15):model group:intragastric administration with physiologic saline for6weeks after operation;2weeks group:intragastric administration with FK506for2weeks and then with physiologic saline for4weeks after operation;4weeks group:intragastric administration with FK506for4weeks and then with physiologic saline for2weeks after operation;6weeks group:intragastric administration with FK506for6weeks after operation;control group:intragastric administration with physiologic saline for6weeks without operation.2. At6weeks after operation, MASSON staining of rat sciatic nerve was performed to observe collagen fibrils and scar formation. Scar areas was measured by Image-pro plus5.0analysis software.3. At6weeks after operation, HE staining of rat sciatic nerve was performed to observe the density of medullated nerve fibers and connective tissue formation. The density of medullated nerve fibers, average axonal diameter and thickness of medullary sheath were measured.4. At6weeks after operation, ultrathin section of rat sciatic nerve was performed to observe the ultramicrostructure of regenerated axon.5. At6weeks after operation, the recovery rate of gastrocnemius wet weight was calculated (operation side/normal side,%).6. At6weeks after operation, Sciatic Functional Index (SFI) was determined by de Medinaceli process.7. At6weeks after operation, the latency and wave amplitude of CMAP (compound muscle active potential) were detected.8. Dependablity between medullated nerve fibers or SFI and scar areas was analyzed.Part II1. Primary cultures of rat skin fibroblasts were obtained from male Sprague-Dawley newborn rats1-2days after birth and then subcultured. Supplied as a crystalline solid, FK506was dissolved in DMSO (Dimethyl sulfoxide) and stored at-20℃. The culture media containing different concentrations of FK506were freshly prepared for each experiment. Fibroblasts were treated with different stimulus:control group:fibroblasts were cultured with DMEM (Dulbecco’s modified Eagle’s medium) free of FBS (fetal bovine serum) for8hours;DMSO group:fibroblasts were cultured with DMEM containing DMSO for8hours;12.5μM FK506group:fibroblasts were cultured with DMEM containing12.5μM FK506for8hours;25μM FK506group:fibroblasts were cultured with DMEM containing25μM FK506for8hours;50μM FK506group:fibroblasts were cultured with DMEM containing50μM FK506for8hours;75μM FK506group:fibroblasts were cultured with DMEM containing75μM FK506for8hours;100μM FK506group:fibroblasts were cultured with DMEM containing100μM FK506for8hours;JNK (c-Jun N-terminal kinase) inhibitor pretreatment group: fibroblasts were pretreated with SP600125(40μM) for30min, then cultured with DMEM free of FBS for8hours;ERK (extracellular-signal regulated kinase) inhibitor pretreatment group:fibroblasts were pretreated with PD98059(60μM) for30min, then cultured with DMEM free of FBS for8hours;JNK inhibitor pretreatment and50μM FK506group:fibroblasts were pretreated with SP600125(40μM) for30min, then cultured with DMEM containing50μM FK506for8hours;ERK inhibitor pretreatment and50μM FK506group:fibroblasts were pretreated with PD98059(60μM) for30min, then cultured with DMEM containing50μM FK506for8hours.2. CCK-8(Cell Counting Kit-8) assay was used to evaluate the inhibitory activity of FK506on cultured fibroblasts.3. Hoechst33342staining was used to observe apoptotic fibroblasts.4. Flow cytometry was used to detect apoptosis percentage of cultured fibroblasts. 5. Western blotting was used to detect expression level of apoptosis associated proteins.ResultsPart Ⅰ1. FK506significantly reduced the formation of collagen fibrils and the scar areas of nerval anastomotic stoma.2. FK506significantly increased the density of medullated nerve fibers, average axonal diameter and thickness of medullary sheath.3. FK506significantly increased degree of maturity of medullated nerve fibers.4. FK506significantly increased the recovery rate of gastrocnemius wet weight.5. FK506significantly improved SFI.6. FK506significantly shortened the latency and elevated the wave amplitude of CMAP.7. Complete negative correlations existed between medullated nerve fibers or SFI and scar areas.Part II1. FK506reduced rat skin fibroblasts viability in a dose-dependent manner.2. FK506induced characteristic morphological changes of apoptosis in rat skin fibroblasts.3. FK506induced significant apoptosis of rat skin fibroblasts in a dose-dependent manner, and the apoptosis could be weakened by JNK inhibitor SP600125or ERK inhibitor PD98059.4. FK506increased the expressions of p-JNK (phosphorylation of c-Jun N-terminal kinase), p-ERK (phosphorylation of extracellular-signal regulated kinase), cytosolic cytochrome c and cleaved-caspase-3in a dose-dependent manner.5. Pretreatment with JNK inhibitor SP600125or ERK inhibitor PD98059significantly reduced the expressions of p-JNK or p-ERK and cleaved-caspase-3. ConclusionsPart Ⅰ1. FK506could significantly decrease the formation of collagen fibrils and the scar areas of nerval anastomotic stoma following repair of peripheral nerve injury.2. FK506could significantly increase the speed of nerve regeneration and the quality of regenerated nerve fibers following repair of peripheral nerve injury.3. FK506could significantly accelerate the neurofunctional recovery.4. FK506intensively promotes peripheral nerve regeneration and functional recovery by directly inhibiting scar formation of nerval anastomotic stoma.Part II1. FK506significantly decreased the viability of rat skin fibroblasts.2. FK506significantly increased apoptotic percentage of rat skin fibroblasts.3. FK506activated both JNK and ERK with or without mitochondrial cytochrome c release, followed by the cleavage of caspase-3, subsequently leading to the apoptosis of rat skin fibroblasts. |
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