| Acrylamide(ACR)is a water-soluble olefin that is used as a chemical raw material to produce polyacrylamide.The neurotoxicity of ACR has been fully verified in population epidemiological studies.The main clinical manifestations of patients with chronic ACR poisoning are:sweaty palms,ecdysis,numbness of limbs,weakening or disappearance of the Achilles tendon reflex,and so on.The number of microtubules(MT)at the end of the swollen axon is abnormal under electron microscope,but the mechanism by which ACR causes MT damage is unclear.The most abundant of the microtubule-associated proteins(MAPs)is MAP2.The most important function of MAP2 is to participate in the process of regulating the MT assembly.Calpain,as a Ca2+ dependent neutral cysteine hydrolytic enzyme,has the function of hydrolyzing signal enzymes,cytoskeleton proteins(MAP2,NFs,and Tau)and membrane proteins,and is related to the development of neuropathy.Calpeptin(CP),as a synthetic peptide calpain inhibitor,has the advantages of high efficiency,specificity and good cell permeability.It can reversibly inhibit the activity of calpain by binding to the activity site of calpain.It is hypothesized that MT injury induced by ACR is associated with increased calpain activity and MAP2 degradationObjectivesIn order to verify the hypothesis that ACR-induced MT injury is related to the increase of calpain activity and the degradation of MAP2,the intervention effect and mechanism of CP on MT injury in ACR poisoned rats were explored,which provided a basis for the prevention and treatment of ACR poisoning.Methods1.Animal treatment:After adaptive feeding,the Wistar rats were randomly divided into 4 groups(10 in each group),which were recorded as control group,CP group,ACR group and ACR+CP group.Rats in the control group were given normal saline by intraperitoneal injection(i.p.);rats in CP group were given CP 200 ?g/kg(i.p.);rats in ACR and ACRR+CP group were given ACR 30 mg/kg(ip).One hour after ACR,the ACR+ CP group was given CP 200 ?g/kg(ip).ACR was administered on alternate days,3 times/week for 4 weeks.CP intervention was given the next day,once in the morning and evening,6 times/week for 4 weeks.ACR was dissolved in 0.9%normal saline(concentration:30 mg/ml)and administered to rats at 1 ml/kg.2.Body weight and neurobehavioral function observation:Body weight,hind limb splay,accelerated rod test,and gait score were measured weekly during the exposure period.3.Morphological observation:HE staining.Morphological changes of spinal cord sections were observed under a light microscope.4.Immunohistochemical analysis of protein expression:The expression of m-calpain,?-calpain,MAP2,?-tubulin and ?-tubulin protein in the spinal cord was observed under light microscope by immunohistochemistry.5.Immunoblotting protein semi-quantitative:The protein content of m-calpain,?-calpain,MAP2,a-tubulin and ?-tubulin in spinal cord was determined by gel electrophoresis and semi-quantitative immunoblotting.6.Statistical methods:All experimental data are expressed as mean ± standard deviation(X±s).Gait scores were analyzed using the Mann-Whitney U test.Other data were analyzed by one-way analysis of variance(ANOVA).The pairwise comparison between groups was performed using the LSD methodResults1.WeightThe control group and the CP group were basically similar for 4 weeks.Compared with the control group,since the second week,the weight of the rats in ACR group decreased by 5.6%(P<0.05).This trend became more obvious as the exposure continued.By the fourth week,the weight of rats in the ACR group decreased by 8.8%(P<0.01);Compared with the ACR group,since the third week,the weight of rats in the ACR+CP group increased by 4.2%(P<0.05);By the fourth week,the weight of the rats in the ACR+CP group increased by 6.2%(P<0.01).There was no significant difference compared with the control group(P>0.05).2.Behavioral indicators(1)Hind limb splay:the control group and the CP group were basically similar for 4 weeks.Compared with the control group,since the second week,the hind limb splay of rats in the ACR group increased by 26.9%(P<0.01).This trend became more obvious as the exposure continued.By the fourth week,the hind limb splay of rats in the ACR group increased by 34.4%(P<0.01).Compared with the ACR group,since the third week,the hind limb splay of rats in the ACR+CP group was reduced by 15.9%(P<0.01).By the fourth week,the hind limb splay of rats in the ACR+CP group was decreased by 16.3%(P<0.01).There was still a significant difference compared with the control group(P<0.05).(2)Accelerated rotating rod test:the control group and the CP group were basically similar for 4 weeks.Compared with the control group,since the third week,the latency of rats in the ACR group decreased by 27.0%(P<0.01).This trend became more pronounced as the exposure continued.By week 4,the latency of rats in the ACR group decreased by 61.7%(P<0.01).Compared with the ACR group,since the third week,the latency of rats in the ACR+CP group increased by 20.2%(P<0.01).By the fourth week,the latency of rats in the ACR+CP group increased by 70.7%(P<0.01).There was still a significant difference compared with the control group(P<0.05).(3)Gait score:the control group and the CP group were basically similar for 4 weeks.Compared with the control group,since the second week,the gait score of rats in the ACR group increased by 40%(P<0.05).This trend became more pronounced as the exposure continued,and by the fourth week,the gait score of rats in the ACR group increased by 150%(P<0.01).Compared with the ACR group,since the third week,the gait score of the ACR+CP group was reduced by 35%(P<0.05).By the fourth week,the gait score of the ACR+CP group was reduced 32%(P<0.05).There was still a significant difference compared with the control group(P<0.05).3.HistopathologyAt the end of the exposure,the spinal cord tissue of the control group and the CP group was normal and the tissue structure was intact;the nucleus of motor neurons were mainly located in the center of the cell,and the nucleolus was clear.However,the spinal cord tissue of the ACR group was loosely arranged and vesicularly changed;the number of motor neurons decreased,some of the cells condensed,and the nucleolus dissolved or even disappeared.Compared with the ACR group,the motoneuron morphology of the ACR+CP group tend to be nonnal and the number was slightly increased,but it still did not return to the normal level of the control group.4.ImmunochemistryCompared with the control group,there was no significant difference in the expression of ?-calpain between ACR group and ACR+CP group.Compared with the control group,m-calpain over-expressed in the ACR group;and the expression of m-calpain decreased in the ACR+CP group compared with the ACR group.Compared with the control group,MAP2 expression decreased in the ACR group;and the expression of MAP2 increased in the ACR+CP group compared with the ACR group.Compared with the control group,there was no significant difference in the expression of a-tubulin between ACR group and ACR+CP group.Compared with the control group,?-tubulin over-expressed in the ACR group;and the expression of?-tubulin decreased in the ACR+CP group compared with the ACR group.5.Western blotting(1)CalpainThere was no statistically significant change in ?-calpain content among the CP group,the ACR group,and the ACR+CP group compared with the control group(P>0.05).Compared with the control group,the change of m-calpain protein content in CP group was not statistically significant(P>0.05),the m-calpain content in ACR group increased by 41.7%(P<0.01),and the m-calpain protein content in ACR+CP group increased by 14.6%(P<0.05).However,compared with the ACR group,the m-calpain protein content of the ACR+CP group decreased by 19.1%(P<0.01).(2)MAP2Compared with the control group,the change of MAP2 content in CP group was not statistically significant(P>0.05),the content of MAP2 in ACR group decreased by 43.4%(P<0.01).However,compared with the ACR group,the MAP2 content of the ACR+CP group increased by 67.5%(P<0.01).(3)MTCompared with the control group,the changes of a-tubulin content in CP group,ACR group and ACR+CP group were not statistically significant(P>0.05).Compared with the control group,the change of ?-tubulin content in the CP group was not statistically significant(P>0.05),and the content of ?-tubulin in ACR group increased by 23%(P<0.01).However,compared with the ACR group,the content of?-tubulin in ACR+CP group decreased by 13.1%(P<0.05).Conclusions1.ACR-induced MT injury is associated with increased calpain activity and MAP2 degradation,which may be one of the mechanisms of ACR-induced MT injury.2.CP interferes with MT injury caused by ACR by altering calpain and MAP2. |
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