苯甲基磺酰氟预处理磷酸三邻甲苯酯染毒母鸡对其脊髓中神经丝的影响

摘要

目的 研究用苯甲基磺酰氟(PMSF)预处理对有机磷化合物诱导的迟发性神经病(OPIDN)母鸡脊髓中神经丝(NFs)动态变化的影响,探索OPIDN发生的可能机制,方法成年母鸡随机分为正常对照组、磷酸三邻甲苯酯(TOCP)组和PMSF+TOCP组,每组32只,PMSF+TOCP组的动物提前给予皮下注射60ms/ksPMSF,24 h后给予TOCP组和PMSF+TOCP组动物一次性经口灌胃750mykg TOCP,对照组给予等体积生理盐水.分别在TOCP染毒后1、5、10、21 d处死动物,取脊髓组织匀浆分离出上清和沉淀2种组分,利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS.PAGE)和蛋白免疫印迹法(Westem-blotting)检测上清和沉淀中高相对分子质量神经丝蛋白(NF-H)、中相对分子质量神经丝蛋白(NF-M)、低相对分子质量神经丝蛋白(NF-L)的相对含量.结果 TOCP染毒组母鸡在21 d内均出现瘫痪,PMSF+TOCP组动物未出现明显的OPIDN表现,与正常对照相比,脊髓沉淀中3种NF亚单位的含量均表现为先降低后恢复,均在10 d达到最低,差异有统计学意义(P<0.01).上清中3种NF亚单位的变化趋势与沉淀中基本一致,均在染毒后10d时明显降低,到21 d时恢复到正常水平.其中,10 d时NF-L、NF-M和NF-H的含量降低幅度分别达到51%、86%和38%,与对照组比较,差异有统计学意义(P<0.05).PMSF预处理再染毒TOCP组母鸡在21 d内未出现瘫痪,且脊髓中NFs紊乱也明显减轻.与正常对照相比,脊髓样品匀浆离心后的沉淀中只有NF-M在21 d时增加了21%,差异有统计学意义(P<0.05);上清中只有NF-H和NF-M在21 d时增加了19%和35%,差异有统计学意义(P<0.05).结论 TOCP染毒母鸡出现OPIDN,脊髓组织中NFs含量发生紊乱.用PMSF预处理后能阻断OPIDN的发生,并且可明显地抑制脊髓中NFs含量的紊乱.%Objective To investigate the dynamic changes of neurofilaments(NFs)proteins in spinal cords of hens with phenylmethylsulfonyl fluride(PMSF)pretreatment for exploring the mechanism of tri-o-cresyl phosphate (TOCP)-induced delayed neuropathy (OPIDN).Method Adult Roman hens were ran-domly divided into three groups,control,TOCP and PMSF+TOCP.Birds in PMSF+TOCP set were Dretrleated with PMSF,24 hours later,hens in both TOCP group and PMSF+TOCP group were administrated with TOCP at a single dosage of 750 mg/ks.Then all animals were sacrificed on the corresponding time-points of 1,5,10.and 21days respectively after dosing of 750 mg/kg TOCP.The spinal cords were dissected,homogenized and centrifuged at 100 000xg.The levels of high molecular neurofilament(NF-H),medium molecular neumilla.ment(NF-M)and low molecular neurofilament(NF-L)in both pellet and supernatant fractions of spinal cords were determined by SDS-PAGE and Western-blotting.Results The hens in TOCP group showed Daralysis gait at the end of 21-day experimental period.The levels of NFs proteins in spinal cords changed obviouslv.Compared with control,the NFs in pellet showed a dramatic decrease on day 10 and then followed bv arecov-cry.In the supernatant,the NFs proteins showed similar changes,which decreased significantly on dav 10 and almost recovered control on day 21.Such as,NF-L,NF-M and NF-H decreased by 51%,86%and 38%on dayl0.The OPIDN signs were not observed in PMSF+TOCP group,and imbalances of NFs were obyiouslv al-leviated.Compared with control,only NF-M in pellet increased by 21%(P<0.05)on day 21.others remained no changes:The levels of NF-H and NF-M in supernatant respectively increased by 19%and 35%on day 21,others were no significant statistical differences.Conclusion TOCP may induce imbalance of NFs levels in progress of OPIDN,and PMSF pretreatment may protect animals from OPIDN by reducing above changes,which may explain that TOCP-indueed imbalance of NFs may be connected with the occurrence and develop-ment of OPIDN.