Intervention And Mechanism Of Calpeptin On Microtubules Injury Of CNS Induced By Acrylamide

Acrylamide(ACR)is widely used in industrial production.It is mainly used as raw material for the production of polyacrylamide.Polyacrylamide is widely used in water treatment,textile industry and paper industry.The toxicity of ACR to rodents is mainly manifested in neurotoxicity,reproductive toxicity,carcinogenicity and mutagenicity.Occupational ACR poisoning mainly leads to nervous system damage,sensory-motor peripheral and central nervous system(CNS)neuropathy.Pathological features are neuronal damage,and the axons at the end of the swelling have cytoskeletal damage such as reduction of microtubules(MT).When the intracellular Ca22 concentration is increased caused by exogenous ACR exposure,the Ca2+-dependent proteolytic enzyme Calpain can be activated,which leads to the change of the stability of MT and cytoskeleton(CSK),then causes the hydrolysis of MT,and finally leads to the destruction of CSK morphology.The p35-CDK5/p25 pathway can be involved in neural cell migration,synaptic growth,axonal pattern formation,and neurodegenerative disease processes.We hypothesized that MT injury during ACR poisoning was associated with Calpain activation and abnormalities in the p35-CDK5/p25 pathway,and the Calpain inhibitor Calpeptin(CP)interacts.ObjectiveThe ACR neurotoxicity model and CP intervention model of female Wistar rats were established to determine the calpain activity of brain and cerebellum tissue using of immunofluorescence,as well as the contents of ?-tubulin,?-tubulin,CAM,CAMKII,CDK5,p25 and p35 protein using of Western Blotting,and to explore the effect and its mechanism of CP on ACR-induced MT injury of CNS.This study was to provide a theoretical basis for prevention and treatment of ACR-induced nerve damage.Method1.Animal model establishment and grouping There were 40 adult female Wistar rats with specific pathogens(SPF).The initial body weight of the rats was 180?210 g.After 7 days of adaptive breeding,they were randomly divided into control group,CP group,ACR group and ACR+CP group,with 10 rats in each group,and the record was the 0 week of the experiment.The control group was given intraperitoneal injection(i.p.);CP group was given 200 ?g/kg b.w.CP(i.p.);ACR group was given 30 mg/kg b.w.ACR(i.p.);ACR+CP group was given 200 ?g/kg b.w.CP(i.p.)1 h after 30 mg/kg b.w.ACR(i.p.)intervention.ACR and CP were administered in a volume of 1 ml/kg b.w.3 times a week for 4 weeks.The rats were weighed once a week.After the terminal exposure and intervention treatment,the rats were decapitated and the brain and cerebellum tissues were quickly stripped,frozen in liquid nitrogen,and stored in a refrigerator at-80?.2.Gait score The rats were placed in a transparent plexiglass box(90cm × 90 cm)for 3 minutes.According to the gait scoring standard,the score was divided into 1 to 4 points,of which 1=unaffected normal gait;2=slightly abnormal gait;3=moderate abnormal gait;4=severe abnormal gait.3.Rotarod test The rats were placed on a rotating rod for training and gradually accelerated from 0 r/min to 40 r/min in 200 s.The residence time of the rats on the rotating rod was recorded,and each rat was continuously performed three times at intervals of 30 min each time to calculate the average residence time.4.Tissue homogenate The brain and cerebellum tissue frozen in a refrigerator at-80?was weighed and weighed 0.5 g into a homogenizer.Solution was well homogenized in an ice bath;Centrifuged and removed the supernatant and transfered to Eppendorf tube for labeling,and froze in liquid nitrogen.5.Protein quantification and Calpain activity assay BCA protein quantification kit was used to determine the protein contents and the AMC calpain activity.6.Gel electrophoresis and Western blotting The brain and cerebellar tissue were homogenized and the contents of ?-tubulin,?-tubulin,CAM,CAMKII,CDK5,p25 and p35 were determined using by Western blotting.7.Statistical analysis Data of body gain weight,rotarod test,calpain activity and Western blotting were expressed as(?),and one-way comparison analysis was performed using one-way analysis of variance,LSD or Dunnett's test.Data of gait score was analyzed using Mam-Whitney U test.All of the data were analyzed using SPSS 24.0 software and the inspection level ?=0.05.Result1.Body gain weight changesDuring the whole experiment,the control and CP rats did not show abnormal gain weight,and there was no significant difference in gain weight between the initial and first week treatment(P>0.05),which were basically at the same level.The gain weight of the ACR group was shortened from the second week,and was significantly lower than that of the control group.By the 4 week the experiment,Contrasted to the the initial body weight,the average body weight of the four groups increased by 58.69 g,54.57 g,36.20 g and 50.13 g,respectively.The gain weight of the ACR group was significantly lower than the control group;Contrasted to the the ACR group,the gain weight of the ACR+CP group increased,and there was a statistically significant difference(P<0.05).2.Gait scoreDuring the whole experiment,the control and CP rats did not show abnormal gait,and there was no significant difference in gait score between the initial and first week treatment(P>0.05),which were basically at the same level.At the second week,rats in the ACR group showed mild gait abnormalities.After 4 weeks of treatment,the mean gait score of the ACR group was increased by 1.6 points and the mean gait score of the CP group was increased by 0.8 points Contrasted to the the control group;the mean gait score of the ACR+CP group was reduced by 0.8 points Contrasted to the the ACR group,and the difference was statistically significant(P<0.05).3.Rotarod testDuring the whole experiment,the control and CP rats did not show abnormal residence time of rotarod test,and there was no significant difference between the initial rotation rod test residence time and the first week treatment time of the four groups of rats(P>0.05),which were basically at the same level.The residence time of the ACR group was shortened from the third week,and was significantly lower than that of the control group.The residence time of the control and CP groups did not change much at the end of the experiment,but the ACR group was significantly lower than the control group;Contrasted to the the ACR+CP group,the residence time increased,and there was a statistically significant difference(P<0.05).4.Calpain activityContrasted to the the control group,Calpain activity in the brain and cerebellar tissue homogenate of ACR group showed a significant increase,increasing by 59.86%and 31.17%(P<0.05),respectively.Contrasted to the the ACR group,the Calpain activity of the ACR+CP group showed a significant decrease trend,which was reduced by 32.80%and 21.25(P?0.05),respectively.5.MT proteinContrasted to the the control group,the contents of ?-tubulin and ?-tubulin in the ACR group showed a significant increase,which increased by 12.63%and 21.14%in the brain tissue homogenate,respectively(P<0.05),then 15.54%and 33.35%in the cerebellar tissue homogenate,respectively(P<0.05).Contrasted to the the ACR group,the contents of a-tubulin and ?-tubulin in the ACR+CP group showed a significant decrease,which was decreased by 13.10%and 10.96%in the brain tissue homogenate,respectively(P<0.05),then 24.03%and 19.41%in the cerebellar tissue homogenate,respectively(P<0.05).6.CAM and CAMKIIContrasted to the the control group,the contents of CAM and C AMKII in the ACR group showed a significant increase in the Calpain activation pathway,which increased by 57.33%and 46.00%in the brain tissue homogenate,respectively(P<0.05),then 42.18%and 54.18%in the cerebellar tissue homogenate,respectively(P<0.05).Contrasted to the the ACR group,the contents of CAM and CAMKII in the ACR+CP group showed a significant decrease,which was 18.88%and 32.58%in the brain tissue homogenate,respectively(P<0.05),then16.51%and 18.51%in the cerebellar tissue homogenate,respectively(P<0.05).7.CDK5,p35,p25Brain homogenate:Contrasted to the the control group,the contents of CDK5 and p35 protein in the CP group showed a significant increase in the p35-CDK5/p25 pathway,which increased by 31.01%and 19.04%,respectively;the contents of CDK5,p35 and p25 protein in the ACR group showed a significant increase in the p35-CDK5/p25 pathway,which increased by 52.81%,40.58%and 25.05%,respectively;the contents of CDK5 and p35 protein in the ACR+CP group showed a significant increase in the p35-CDK5/p25 pathway,which increased by 42.80%,and 22.60%,respectively(P?0.05).Contrasted to the the ACR group,the contents of CDK5,p35 and p25 in the ACR+CP group showed a significant decrease trend,which decreased by 6.55%,12.79%and 25.65%,respectively(P<0.05).Cerebellum homogenate:Contrasted to the the control group,the contents of CDK5 and p25 protein in the CP group showed a significant increase in the p35-CDK5/p25 pathway,which increased by 25.45%and 44.44%,respectively;the contents of CDK5,p35 and p25 protein in the ACR group showed a significant increase,which increased by 47.11%,15.60%and 76.84%,respectively;the contents of CDK5 and p25 protein in the ACR+CP group showed a significant increase in the p35-CDK5/p25 pathway,which increased by 37.25%and 31.37%,respectively(P<0.05).Contrasted to the the ACR group,the contents of CDK5,p35 and p25 in the ACR+CP group showed a significant decrease trend,which decreased by 6.70%,23.17%and 24.43%,respectively(P<0.05).Conclusions1.CP has an intervention effect on ACR-induced increase in Calpain activity and MT injury.2.The intervention mechanism of CP on ACR-induced MT injury may be that CP maintains the stability of MT by regulating Calpain activation and p35-CDK5/p25-tubulin pathway.