Mechanism Of Calpeptin Antagonizing Acrylamide-induced Neurofilament-associated Phosphorylation Kinases In Rat

ObjectivesAcrylamide?ACR?is a water-soluble,vinyl white crystal,which is widely used in various industries.Previous studies have shown that ACR could mainly lead to neurotoxicity,ataxia,skeletal muscle weakness,tremor and other peripheral nerve degeneration.Protein Kinase A?PKA?,Protein Kinase C?PKC?and Cyclin-dependent kinase 5?Cdk5?could phosphorylate neurofilament?NF?protein which is rich in Serine/threonine.These phosphorylated NFs could change its function,and finally could affect the cytoskeleton.Calpain is a class of calcium-dependent enzyme,which could change the content of PKA,PKC,Cdk5,etc.We speculate that ACR may activate calpain by increasing intracellular Ca²⁺,and then activate PKA,PKC,Cdk5 in order to phosphorylate NFs which play an important role in ACR-induced neuropathy.Therefore,we established a rat model of ACR subchronic poisoning and given calpeptin?CP?for intervention in this study.The neurobehavioral and pathological changes were observed after giving CP.The concentration of Ca²⁺,PKA,PKC and Cdk5 were detected at the end.This study try to explore the neurological changes caused by ACR poisoning and its mechanism.Methods1.60 female adult Wistar rats were randomly divided into four groups:blank control group,CP control group,ACR model group,ACR + CP intervention group?n?15?.Rats in ACR and ACR + CP group were given 3 times per week by intraperitoneal injection of 30mg/kg · bw;Rats in the blank control group was given equal volume of saline.ACR + CP intervention group was given 200ug/kg-bw CP,6 times per week;CP control group given equal volume of CP.Four weeks later,part of rats were sacrificed by decapitation.Others were perfused intracardially with saline at first and then a solution of 4%paraformaldehyde for pathological detection.Then spinal cord and sciatic nerve were reserved from each group.During the exposure period,the body weight,gait score and hind limbs were assessed.2.Calcium ion:Cells suspension of spinal cord was prepared,and then were assess the fluorescence intensity by Flua-2/AM.The concentration of Ca²⁺ was calculated.3.Determination of protein kinase:Supernatant fraction from the homogenate was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis?SDS-PAGE?and western blot?WB?by semi-quantitative analysis.4.NFs phosphorylation:Changes of NFs phosphorylation of spinal cord and sciatic nerves were observed by immunohistochemistry method.5.Histomorphological observation:Pathological changes of spinal cord and sciatic nerves were observed through hematoxylin-eosin?HE?staining and toluidine blue staining;changes of myelinated nerve fiber of sciatic nerves were observed by electron microscopy.Results1.body weightCompared with blank control group at the same period,body weight in CP control group has no significant difference?P>0.05?,body weight in ACR group were significantly lower than that in blank control group?P<0.05?;Compared with ACR group at the same period,body weight in ACR+CP group were significantly higher than that in ACR group?P<0.05?.2.Neurobehaviors?1?Gait score:Compared with blank control group at the same period,scores in CP control group has no significant difference?P>0.05?,scores in ACR group were significantly higher than that in blank control group?P<0.05?;Compared with ACR group at the same period,scores in ACR+CP group were significantly lower than that in ACR group?P<0.05?.?2?Hind limb grip strength:Compared with blank control group at the same period,grip strengths in CP control group has no significant difference?P>0.05?,grip strengths in ACR group were significantly lower than that in blank control group?P<0.05?;Compared with ACR group at the same period,grip strengths in ACR+CP group were significantly higher than that in ACR group?P<0.05?.3.Ca²⁺ in Spinal nervesCompare with blank control group,Ca²⁺ has no significant difference?P>0.05?in CP control group,Ca²⁺ was significantly increased in ACR group?P<0.05?.Compare with ACR group,Ca²⁺ was significantly decreased in ACR+CP group?P<0.05?.4.Protein kinase:Compare with blank control group,PKA?in sciatic nerves?,PKC?in spinal cord and sciatic nerves?and Cdk5?in sciatic nerves?were significantly increased in ACR group?P<0.05?;PKA in spinal cord of ACR group has no significant difference?P>0.05?.Compare with ACR group,PKA?in sciatic nerves?,PKC?in spinal cord and sciatic nerves?and Cdk5?in sciatic nerves?were significantly decreased in ACR+CP group?P<0.05?;PKA in spinal cord of ACR+CP group was significantly increased?P>0.05?.5.Phosphorylation of NFsIn spinal cord and sciatic nerves,phosphorylation of NFs in the ACR model group was higher than that in other three groups,and the ACR + CP group was more than that in control group.But non-phosphorylation of NFs was the opposite.6.Histomorphology?1?Spinal cord:Under the HE and toluidine blue staining,the number of motor neurons was decreased and nuclear pyknosis/membrane disoperation were found in ACR group.Compared with ACR group,the number and morphology of motor neurons in ACR + CP group was more normal,and the distribution of Nissl bodies around the nucleus was increased.?2?Sciatic nerves:Under the HE and electron microscopy,axonal cytoplasmic vacuolization and degeneration in the axons of the sciatic nerve were seen in the ACR group;compared with the ACR group,less axonal cytoplasmic vacuolization and degeneration were obseved in the ACR+CP group.Under the electron microscopy,compare with blank control group,the g-Ratio was were significantly decrease in ACR group?P<0.05?.Compare with ACR group,and the g-Ratio was significantly increased in ACR+CP group?P<0.05?.Conclusions1.CP could antagonize neurobehavioral and pathological changes of spinal cord and sciatic nerves induced by ACR.2.CP could antagonize the changes of calcium concentration,neurofilament-associated phosphorylation kinases and phosphorylate level induced by ACR.3.CP could antagonize ACR-induced neuropathy by inhibiting calcium and neurofilament-associated phosphorylation kinases through Ca²⁺-Calpain-Kinase-NFs pathway.