Mechanism Of Calpeptin Antagonizing Acrylamide-induced Aberrant Phosphorylation Of Neurofilaments In NSCs

ObjectivesAcrylamide(ACR)is widely used in industrial production activities.Occupational groups who have been long time exposed to ACR mainly suffer from sensory-motor peripheral neuropathy.Previous researches showed that ACR induces aberrant phosphorylation of Neurofilaments(NFs).Calpain actived by Ca2+ regulates the activity of protein kinase.Those protein kinases phosphorylate NFs,thus affecting its assembly,transport and depolymerization,and eventually causing peripheral neuropathy.Therefore,we assumed that ACR induced abnormal phosphorylation of NFsassociated with the related kinases changes mediated by calpain.With Neural Stem Cells(NSCs)as experimental object,we established ACR poisoning and Calpeptin(CP)intervention model,determing the phosphorylation level of NFs,Protein Kinase A(PKA),Protein Kinase C(PKC),Cyclin-dependent Kinase 5(Cdk5)and calpain.In this report we try to explore the mechanism of NFs abnormal phosphorylation induced by ACR and how CP inhibits ACR toxicity,providing theoretical foundation for the prevention and treatment of ACR poisoning.Methods1.Primary cell culture and NSCs identity.In aseptic conditions,the Dorsal Root Ganglion(DRG)was isolated from the new born Wistar rats(24h).We used immunofluorescence chemical staining to detect the NSCs specific expressed Nestin,Neuron Specific expressed Neuron specificity enolization enzyme(NSE)and Glial cell Specific collagen fibre acid protein(GFAP)to identify the NSCs.2.Establishment of experimental model.On the 4th day of cell culturing,the cells,staying at logarithmic growth phase,were divided into 4 groups:blank control group(only complete medium),CP group(4 ?mol/L CP),the ACR infected group(760?mol/L ACR),the ACR infected by CP intervention group(760?mol/L ACR+4?mol/L CP).The processing time is 48h.3.Immunofluorescence methods were used to detect the phosphorylation level of NFs and observe its intracellular distribution.4.Western blot were used to analyze phosphorylated NFs(p-NF-H),?-calpain and m-calpain,PKA,PKC and Cdk5 in NSCs.5.SPSS 21.0 software was used for statistical analysis,and Dunnett-t test was used for comparison between groups,and P<0.05 was considered statisticallysignificant.Results1.Primary cell culture and identification of NSCs.The cells of NSCs were cultured in vitro,and adhered to the wall in 24h,covering the bottom of 100cm2 cell culture dish in 6?7d.Immunofluorescence staining showed that the NSCs were Nestin positive and got a purity of over 90%.The cells of the same source were induced by 1 mol/L of vitamin A to differentiate into NSE positive neurons and GFAP positive glial cells.2.Phosphorylation levels of NFs.2.1 Qualitative analysis of immunofluorescence.Compared with the blank control group,CP treatment didn't increase the expression of p-NF-H.The fluorescence intensity of ACR group was increased,and the p-NF-H were also distributed in the axons.The fluorescence intensity of ACR+CP group was similar with control group,and there are p-NF-H existing in the cell body and nucleus circumference,as well as the axons had a smaller distribution.Compared with the ACR model group,the fluorescence intensity of p-NF-H in ACR+CP group decreased slightly and the axonal distribution also decreased.2.2 Expression level of p-NF-H.Compared with the blank control group,CP treatment didn't increase the expression of p-NF-H.The level of p-NF-H in ACR group increased by 67.3%(P<0.05).The p-NF-H in ACR+CP group increased by 32.3%(P<0.05).Compared with the ACR model group,The level of p-NF-H in ACR+CP group decreased by 35%(P<0.05).3.The change of calpain3.1 Expression level of ?-calpainCompared with the blank control group,CP treatment didn't increase the expression of ?-calpain.In ACR group,the expression of ?-calpain increased by 56.1%(P<0.05).ACR+CP group was reduced by 3.3%(P>0.05).Compared with the ACR group,the ?-calpain level in the ACR+CP group decreased by 59.4%(P<0.05).3.2 Expression level of m-calpainCompared with the blank control group,CP treatment didn't increase the expression of ?-calpain.The expression of m-calpain in ACR group increased by 121.7%(P<0.05).The expression of m-calpain in ACR+CP group was increased by74.1%(P<0.05).Compared with ACR group,the m-calpain in ACR+CP group decreased by 47.6%(P<0.05).4.Changes of Protein kinases.4.1 Expression level of PKCCompared with the blank control group,CP treatment didn't increase the expression of PKC.The expression of PKC in ACR group decreased by 23.6%(P<0.05),and the PKC level in ACR+CP group was reduced by 0.9%(P>0.05).Compared with the ACR group,the expression of PKC in ACR+CP group increased by 22.7%(P<0.05).4.2 Expression level of PKACompared with the blank control group,CP treatment didn't increase the expression of PKA.The expression of PKA in ACR group decreased by 23.0%(P<0.05),and the PKA level in ACR+CP group increased(P>0.05).Compared with the ACR group,the expression of PKA in ACR+CP group increased 29.8%(P>0.05).4.3 Expression level of Cdk5Compared with the blank control group,t CP treatment didn't increase the expression of Cdk5.The expression of Cdk5 in ACR group increased 64.6%(P<0.05).The Cdk5 level in ACR+CP group increased by 49.4%(P<0.05).Compared with the ACR group,the expression of Cdk5 in ACR+CP group decreased by 15.2%(P>0.05).Conclusions1.Successfully established the NSCs model of ACR poisoning and CP intervention.2.ACR poisoning induces abnormal phosphorylation of NFs,CP produce protective effect by inhibiting activity of calpain.3.The abnormal phosphorylation of NFs induced by ACR may be related with calpain-mediated PKA and PKC changes,and CP antagonize ACR poisoning by inhibiting calpain,PKA and PKC.