| ObjectivesAcrylamide (ACR), as a vinyl monomer, is the raw material of polyacrylamide, which is widely used in industrial field. The main toxic effects of ACR monomer are carcinogenicity, genotoxicity, neurotoxicity and reproductive toxicity. Recently, the neurotoxicity of ACR has attracted more and more attention. The clinical manifestations of occupational exposure to ACR are ataxia, skeletal muscle weakness, cognitive disorder and numbness of limbs. Histopathologically, the main neurotoxic effects involve the accumulation of neurofilaments (NFs) in the distal swellings of large axons. As one kind of neutral cysteine hydrolase, calpain has functions of degrading NFs which depend on Ca2+ to activate. Calpain plays an important role in the neuropathy induced by ACR. Therefore, the subchronic ACR poisoned model and calpain inhibitor calpeptin (CP) model were established, which were to assess the neurobehavioral changes, the concentration of Ca2+ and calpain activity changes, and the content of NFs changes in central nervous system and peripheral nervous system and to discuss the intervention mechanism of NFs changes induced by ACR.Methods1.36 specific pathogen free female adult Wistar rats were randomly divided into four groups, and each group had 9 rats. As blank control group, rats in group A received 0.9％ normal saline (NS) by intraperitoneal (i.p.) injection. Rats in group B (CP control group) received 200μg/kg CP (i.p.). Rats in group C (ACR poisoned model group) received 30mg/kg ACR (i.p.). Rats in group D (CP intervention model group) received200μg/kg CP (i.p.) after the administration of 30mg/kg ACR (i.p.). Rats in A, C, D groups were administered NS or ACR once a day,3 days a week for 4 weeks. Rats in B, D groups were administered CP twice a day,3 days a week for 4 weeks.2. During the 4 weeks, body weight, gait score and hind limb splay were assessed.(1) Gait analysis:To observe gait abnormalities, each rat was observed and scored 1-4. 1=a normal gait; 2=a slightly abnormal gait; 3=a moderately abnormal gait; 4=a severely abnormal gait. Every rat was assessed 3 times. The average was obtained.(2) Hind limb splay:Rats were dropped onto the ground at a height of 30 cm above ground. The distance between the center of the right and left heels was measured. Every rat was measured 3 times. The average was obtained.3. After the 4 weeks, rats were sacrificed by decapitation. Brain and sciatic nerve tissues were preserved. Cell suspension of brain was prepared, the survival ratio of cells was calculated by trypan blue exclusion, and to assess the fluorescence intensity by Flua-2/AM. The concentration of Ca2+ was calculated.4. Brain and sciatic nerve tissues in different groups were homogenized respectively. Protein concentration of supernatant fraction was administered after homogenate was centrifuged. Calpain substrate Ⅱ was added and fully reacted with calpain, and 7-amino-4-methylcoumarin was used for the standard control. The fluorescence intensity was assessed and calpain activity was calculated.5. To assess the content of NFs in brain and sciatic nerve tissue, supernatant fraction from the homogenate was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot by semi-quantitative analysis.Results1. Body weight changesIn the final, compared to blank control group, body weight has no significant difference (P>0.05) in CP control group; it decreased significantly by 11.3％(P﹤0.01) in ACR poisoned model group; it has no significant difference (P>0.05) in CP intervention model group. Compared to ACR poisoned model group, it increased significantly by 8.3％(P<0.01) in CP intervention model group.2. Gait score and hind limb splay changes(1) Gait score:In the final, compared to blank control group, CP control group has no significant difference (P>0.05); it increased by 167％ (P<0.01),100% (P<0.01) respectively in ACR poisoned model group and CP intervention model group. Compared to ACR poisoned model group, it decreased by 25.1％(P<0.01) in CP intervention model group.(2) Hind limb splay:In the final, compared to blank control group, CP control group has no significant difference (P>0.05); it increased by 76.7％(P<0.01),49.5% (P<0.01) respectively in ACR poisoned model group and CP intervention model group. Compared to ACR poisoned model group, it decreased by 15.4％(P<0.01) in CP intervention model group.3. Ca2+ changesCompared to blank control group, Ca2+ has no significant difference (P>0.05) in CP control group; it increased significantly by 44.6％(P<0.01) in ACR poisoned model group; however the increase in CP intervention model group has no significant difference (P>0.05). Compared to ACR poisoned model group, it decreased significantly by 22.6％(P﹤0.01) in CP intervention model group.4. Calpain activity changes(1) Brain calpain activity:Compared to blank control group, calpain activity in brain decreased by 14.3％(P﹤0.01) in CP control group; it increased by 21.9% (P﹤0.01),11.5％(P﹤0.01) respectively in ACR poisoned model group and CP intervention model group. Compared to ACR poisoned model group, it decreased by 8.5％(P﹤0.01) in CP intervention model group.(2) Sciatic nerve calpain activity:Compared to blank control group, it has no significant difference (P>0.05) in CP control group; it increased by 27.6% (P﹤0.01),11.7％(P﹤0.01) respectively in ACR poisoned model group and CP intervention model group. Compared to ACR poisoned model group, it decreased by 12.4％(P﹤0.01) in CP intervention model group.5. NFs changes(1) NFs changes in brain:Compared to blank control group, the levels of NF-Land NF-H subunits in brain increased by 2.3%(P>0.05),0.3%(P>0.05) respectively, while the level of NF-M subunit decreased by 0.8%(P>0.05) in CP control group; the levels of NF-L, NF-M and NF-H subunits increased by 85.3% (P﹤0.01),74.3%(P﹤0.01),82.9%(P﹤0.01) in ACR poisoned model group, and these subunits increased by 32.4%(P﹤0.01),30.6%(P﹤0.01),32.3%(P﹤0.01) in CP intervention model group. Compared with ACR poisoned model group, the all three decreased respectively by 28.5%(P﹤0.01),25.1%(P﹤0.01) and 27.7%(P﹤0.01) in CP intervention model group.(2) NFs changes in sciatic nerve:Compared to blank control group, the levels of NF-L,NF-M and NF-H subunits in sciatic nerve increased by 14.3%(P﹤0.05), 28.6% (P﹤0.01),10.1%(P>0.05) respectively in CP control group; they decreased by 56.0%(P﹤0.01),55.5%(P﹤0.01),39.3%(P﹤0.01) in ACR poisoned model group, and they decreased by 25.6%(P﹤0.01),33.6%(P﹤0.01), 16.9%(P﹤0.05) in CP intervention model group. Compared to ACR poisoned model group, the all three increased respectively by 69.1%(P﹤0.01),49.2% (P﹤0.01) and 36.9%(P﹤0.01) in CP intervention model group.Conclusion1. The subchronic poisoned model and CP intervention model of rats have been established successfully.2. CP could reverse the increasing tendency of Ca2+ concentration and calpain activity induced by ACR; CP could reverse variation tendency of NFs content induced by ACR both in brain and sciatic nerve.3. ACR might induce neuropathy by activating the channel of Ca2+ —calpain-NFs. |
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