Protective Effect Of Calpeptin On Acrylamide-induced Microtubule Injury In Sciatic Nerve And Its Mechanism

Acrylamide(ACR)is a chemical poison widely used in industrial production.People may be exposed to ACR when they are in the workplace or when ingesting starchy foods that have been heated at high temperatures.Studies have shown that ACR has many toxicities such as neurotoxicity,reproductive toxicity,carcinogenicity and genotoxicity,but it is mainly neurotoxic in the human population.Numerous studies have found that the major neurotoxic effects of ACR involved cytoskeletal proteins which play an important role in axonal transport.As one of the main components of the cytoskeleton,Microtubule(MT)consists of ?-tubulin and?-tubulin.MT is involved in the maintenance of cell morphology,intracellular maternal transport and signal transduction.The injury of MT is likely to cause neuronal death further leading to neurodegenerative lesions.Calpain plays an important role in ACR-induced MT injury and neurodegenerative diseases,but the specific mechanism remains unclear,and it may be related to the effect of calpain on Tan,Microtubule-associated Protein 2(MAP2),Protein Kinase C(PKC)and Cyclin-dependent Kinase 5(CDK5).Caleptin(CP),as a Calpain inhibitor,has received increasing attention.ObjectiveBy establishing ACR poisoning and CP intervention models,neuropathology and histopathological changes were observed,and the protein level of tubulin,Calpain,MAPs,and protein kinase in the sciatic nerve were determined to explore the intervention effect and mechanism of CP on ACR-induced MT injury.Methods1.Establishment of animal models40 female Wistar rats were randomly divided into 4 groups(Control group,CP group,ACR group and ACR+CP group).And 1 ml/kg·bw of normal saline,30 mg/kg bw of ACR and 200 ?g/kg·bw of CP were administered to the corresponding rats by intraperitoneal injection,3 days a week(normal saline and ACR once a day,CP twice a day),for 4 weeks.After the end of the administration,the rats were sacrificed and the sciatic nerve was taken for use.2.Assessment of body weight and neurobehavioral indicatorsBody weight and neurobehavioral indicators(gait score,hind-limb splay,and rotarod test)were measured weekly during the administration pernod.3.Determination of protein levelsThe protein content of ?-tubulin,a-tubulin,Calpain ?,Calpain ?,Tau,MAP2,PKC and CDK5 in each group of sciatic nerve were detected by Western blot.The expression of the above proteins in the sciatic nerve were detected by immunohistochemistry.4.Histomorphological observationThe histomorphological changes of the sciatic nerve was observed by hematoxylin-eosin(HE)staining.Results1.Body weightAfter 4 weeks of administration,the body weight of Control group,CP group,ACR group and ACR+CP group increased by 30.3%,28.2%,18.7%and 25.9%respectively.Compared with the Control group,the ACR group had significantly lower body weight(P<0.05);while the ACR+CP group had significantly higher body weight than the ACR group(P<0.05).2.Neurobehavioral indicatorsAfter 4 weeks of administration,compared with the Control group,the ACR group showed a phenomenon of hind-limb paralysis and dragging of the feet,and the gait score was significantly increased(P<0.05),the hind-limb splay index was significantly increased(P<0.05),and the rotarod test time was significantly shortened(P<0.05).The gait score and hind-limb splay index of ACR+CP group was significantly lower than the ACR group(P<0.05).3.Changes in protein levels(1)Changes in ?-tubulin and a-tubulinThe results of western blot showed that the p-tubulin content in the sciatic nerve of the ACR group was increased by 22.0%compared with the Control group(P<0.05),while the ACR+CP group was reduced by 16.0%compared with the ACR group(P<0.05).The results of immunohistochemistry showed that ?-tubulin wasover-expressed in the ACR group compared with the Control group,whereas?-tubulin over-expression was significantly reduced in the ACR+CP group compared with the ACR group.The results of western blot and immunohistochemistry showed no significant difference in a-tubulin protein levels between the 4 groups.(2)Changes in Calpain ? and Calpain ?The results of western blot showed that the contents of Calpain ? and Calpain ?in the ACR group were increased respectively by 53.1%(P<0.05)and 28.1%(P<0.05)compared with the Control group,while the contents of Calpain ? and Calpain ? in the ACR+CP group was decreased respectively by 26.3%(P<0.05)and 18.1%(P<0.05)compared with the ACR group.The results of immunohistochemistry showed that Calpain I and Calpain II were over-expressed in the ACR group compared with the Control group,whereas the overexpression of Calpain ? and Calpain ? in the ACR+CP group was significantly reduced compared with the ACR group.(3)Changes in Tau and MAP2The results of western blot showed that the contents of Tau and MAP2 in theACR group were increased respectively by 37.3%(P<0.05)and 38.8%(P<0.05)compared with the control group,while the contents of Tau and MAP2 in the ACR+CP group were decreased respectively by 15.4%(P<0.05)and 17.2%(P<0.05)compared with the ACR group.The results of immunohistochemistry showed that Tau and MAP2 were over-expressed in the ACR group compared with the Control group,whereas the overexpression of Tau and MAP2in the ACR+CP group was significantly reduced compared with the ACR group.(4)Changes in PKC and CDK5The results of western blot showed that the contents of PKC and CDK5 in the ACR group were increased respectively by 149.9%(P<0.05)and 44.4%(P<0.05)compared with the control group,while the contents of PKC and CDK5 in the ACR+CP group were decreased respectively by 56.5%(P<0.05)and 15.2%(P<0.05)compared with the ACR group.The results of immunohistochemistry showed that PKC and CDK5were over-expressed in the ACR group compared with the Control group,whereas the overexpression of PKC and CDK5in the ACR+CP group was significantly reduced compared with the ACR group.4.HistomorphologyStained by HE and observed under a high power microscope(400x),there was no significant difference between the control and CP groups,and the cross sections of the sciatic nerve in both groups were complete with close arrangement,ordered structure,and even dyeing.Loose arrangement,disorganised structure,uneven density,and exfoliated perineurium were observed in the ACR group;CP intervention significantly improved these changes.5.Statistical analysisThe data of body weight,hindlimb splay,and rotarod were analyzed with repeated measures analysis of variance,and the data of gait score was analysed with generalized estimated equation.The data of western blotting were analysed with one-way analysis of variance,followed by LSD's or Dunnett's post hoc tests,by using SPSS 23.0.Data are expressed as the mean ± standard deviation.The differences were deemed statistically significant at P<0.05.Conclusions1.ACR poisoning model and CP intervention model of rats were successfully established.2.CP has an intervention effect on abnormal changes of Calpain,protein kinase,MAPs and tubulin in sciatic nerve caused by ACR.3.CP could antagonize ACR-induced MT damage in sciatic nerve by inhibiting the Calpain-PKC/CDK5-MAPs-tubulin pathway.