| ObjectivesAcrylamide (ACR) is widely used in various fields. As a raw material, ACR is applied to synthesize hundreds of industrial products. ACR induces central-peripheral neuropathy. Workers who are exposed to ACR begin to have clinical symptoms after several weeks. The typical clinical symptom is peripheral neuropathy. The manifestationsare were muscle weakness, ataxia and limb numbness and radiculalgia. There has not been final conclusion on the pathogenic mechanism. Now most researches focus on neuraxon. Recently, some researchers show that the subunit of neurofilament has changed induced by ACR, but no exact mechanism has been determined. So, further studies are needed to clarify. In this study, to determine the effect of subunit of neurofilament, we established the model of ACR poisoning and inhibit the activity of calpain by calpeptin. We decteced the changes of calcium(Ca2+),calpain activity and neurofilaments. Then we can clarify the effect of calpain during ACR poisoning and explore the mechanism of neuropathy.Methods1.30femal Wistar rats were housed lived in the animal room for1week and then were randomly divided into three groups (n=10rats per group). The rats in group1, served as control, and recived normal saline, the animals in group2and3were given ACR(30mg/kg, i.p., dissolved in0.9%saline) and then group3given calpeptin (250μg/kg, twice of day) for consecutive4weeks with3times a week. The cerebral cortex, spinal cord and sciatic nerves were dissected and then stored until analyzed. body weight were measured every week. 2. Neurobenhavioral indexes. The changes of general status, neurobenhavioral function were observed including spontaneous locomotion in an open field and foot splays in hind limb landing.3. Separated the spinal cord, that digested with trypsin into cells. The cells were loaded with fura-2/AM for determined with microplate reader.4. The activity of calpain were determined by using calpain substrate II with microplate reader to measure fluorescence value.5. Samples of spinal cord were collected and used for examination of NF-L and NF-M protein levels using Western blotting.Results1. The body weight of the rats in the control group increased readily within4weeks, beginning in the2st week show significant difference between the intoxicated and the control group(P<0.01, the followed is same). The body weight of the rats in the CP-treated groups was increased than that in the intoxicated group from2st week (P<0.01).2. The gait observations and landing foot splay (LFS), beginning in the2nd week, ACR-treated rats developed classic signs of ACR neuropathy, and with the time passing by, intoxication of ACR caused progressive abnormalities. Compared with the control, gait score and LFS increased remarkably by4th week (P<0.01). Compared with the ACR group, CP-treated rats reduced these motor deficits by26.92%and11.45%respcectively (P<0.01).3. Compared with control, cell calcium of spinal cord treated ACR was increased25.34%with CP-treated was increased17.03%(P<0.05). Compared with the ACR group, CP group was reduced6.63%(P<0.05).4. Results of calpain activity show that, compared with control, spinal cord increased33.33%from ACR-treated group and15.87%from CP-treated group(P<0.01), Compared with ACR-tread group, CP-treated decrease calpain activities of spinal cord13.10%(P<0.05).5. The protein levels of neurofilaments in the apinal cord. Compared to control, ACR-treated group were increased NF-L (30.0%, P<0.05) and NF-M(60.9%, P<0.05). CP-treated group were increased NF-L (14.1%, P<0.05) and NF-M(25.9%, P<0.05); Compared to ACR-treated group, CP-treated group were reduced NF-L (12.6%, P<0.05) and NF-M(21.7%,P<0.05).Conclusions1. CP could significantly improve mental state, gait score and hind limb splay of rats. It explained that CP had protective effects of motor nerves injury.2. CP could inhibit the activity of calpain and Ca2+in spinal cord. It was implied that calpain and Ca2+were related with the process of neuropathy induced by ACR.3. CP could induce the decrease of NF in nerve tissues, which was implied that the neuropretective effects of CP in ACR rats was related with high expression of calpain. |
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