Intervention Effect Of Calpain Inhabitor On NSCs Injury By Acrylamide And It’s Mechanism

ObjectivesAcrylamide (ACR) as chemical raw materials were widely used in industrial production.People were exposured in production and use of ACR. Starchy foods under high temperature (> 120 ℃) cooking also can produce microscale ACR.ACR poisoning is given priority damage to the nervous system including central nervous system and peripheral nervous system,that has been proved by animal experiments and epidemiological data.Some research and pathological results showed that ACR can cause axon swelling and Neurofilaments(NFs) accumulation.NFs are the major ingredient of cytoskeleton NFs triplet including NF-L,NF-M and NF-H.NFs subunits expression can lead to NFs structure or function disorder,cause axoplasmic transport change and the axon disease.It was showed that ACR poisoning increased intracellular Ca2+ concentration,the increased Ca2+ could activate the calpain activity. But the relation between them have not clear.We assume that the ACR cause NFs changed were associated with Calpain activity changes.We choose Neural Stem Cells (NSCs) as our experimental object establishing model with ACR and Calpeptin(CP).We will determinate the intracellular Ca2+ concentration.the activity of Calpain,as well as the change of the NFs content to explore the mechanism of ACR nerve poisoning and provide theoretical basis for the prevention and treatment of ACR poisoning.Methods1. The primary culture and identification of NSCsTake the DRG of Wistar rats (birth within 24 h) under aseptic circumstance for in vitro cell culture.By immunofluorescence chemical staining method to detect the NSCs specificity expressed nest Protein (Nestin), Neuron Specific expressive objects Neuron Specific Enolase(NSE) and Glial cell Specific Protein expression content Glial Fibrillary Acidic Protein(GFAP) to identify the NSCs.2. The ACR poisoning and CP intervention modelDetermine OD value, by MTT method,then calculatecell survival rate to determine the ACR and CP dose in this model.3. The determination of intracellular [Ca2+] and Calpain activityThe cells were loaded with Fura-2/AM to determine the intracellular Ca2+ concentration.The activity of Calpain were determined by using Calpain substate Ⅱ.4. The amount of NFsSodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE) and Western Blotting were used to determine the amount of NFs.5. Statistical AnalysisComparision among many groups was analyzed with one-way ANOVA, between two groups to compare with Dunnett-t test and Games-Howell test,which wrer all provided by SPSS 19.0 statistical software.For all comparisons described above, the differences were considered statistically significant at P<0.05.Results1. The primary culture and identification of NSCsNSCs could group strongly in vitro culture and cover the bottom of 25cm2cell culture bottle in 7～10d.According to the results ofImmunofluorescence chemical dyeing method,Nestin staining was positive, and the purity can reach more than 90%.Cells from same source could differentiate NSE positive neurons and GFAP positive neural glial cells by vitamin A(1μ mol/L).2. The ACR poisoning and CP intervention modelCompared with control group:ACR group(screening 760 μ mol/L),CP group (screening concentration 4 u mol/L), ACR+CP group (ACR 760 μ mol/L+CP 4μ mol/L),OD values were all lower than control group.ACR group compared with the control group,difference was statistically significant (P< 0.05).Compared with the ACR group:OD values were higher than in ACR group, which compared with ACR+CP group, the difference was statistically significant (P< 0.05).3. The intracellular [Ca2+]Compared with control group:ACR group, ACR+CP group intracellular Ca2+ concentration increased by 47.5％,12.4％respectively (P< 0.05).CP group of intracellular Ca2+ concentration was reduced by 2.7％(P> 0.05). Compared with the ACR group:ACR+CP group of intracellular Ca2+ concentration was reduced by 23.8% (P<0.05).4. The Calpain activityCompared with control group:ACR group Calpain activity increased 23.6％(P< 0.05), CP group, ACR+CP group Calpain activity was reduced by 2.8％and 4.1％ respectively (P>0.05). Compared with the ACR group:ACR+CP group of Calpain activity was reduced by 22.4％(P< 0.05). 5. The amount of NFsCompared with control group:CP group, ACR group,ACR+CP group of NF-L, NF-M, NF-H content are all increased. ACR group of NF-L, NF-M, NF-H respectively increased by 60.92％,48.31％,35.84％ (P<0.05).ACR+CP group of NF-L increased by 19.87％ (P<0.05),NF-M, NF-H were increased 6.01％,10.21％ (P>0.05). Compared with the ACR group:ACR+CP group of NF-L, NF-M. NF-H was reduced by 25.51％and 25.51％,18.87％(P<0.05).Conclusion1. Successfully established the model of ACR poisoning and CP intervention with the subjects of NSCs.2. CP has effective intervention to the changes of NSCs survival rate, concentration of Ca2+, Calpain activity and NFs amount by ACR.3. NFs amount changed by ACR was associated with Calpain activity changing, CP produce protective effect by inhibiting activity of Calpain.