钙蛋白酶抑制剂calpeptin对丙烯酰胺所致神经丝磷酸化及相关激酶异常表达的拮抗作用  

Antagonistic effect of calpain inhibitor calpeptin on abnormal expression of neurofilament phosphorylation and related kinases induced by acrylamide

作　　者：刘宁[1] 苏本玉 管强东 于素芳[1] LIU Ning;SU Ben-yu;GUAN Qiang-dong;YU Su-fang(Department of Occupational and Environmental Health,School of Pubfic Health,Shandong University,Jinan 250012,China)

机构地区：[1]山东大学公共卫生学院职业与环境健康学系,山东济南250012

出　　处：《中国药理学与毒理学杂志》2018年第8期607-613,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：国家自然科学基金(81372969)~~

摘　　要：目的通过制备神经干细胞(NSC)丙烯酰胺(ACR)中毒模型,探讨钙蛋白酶抑制剂calpeptin(CP)对ACR中毒的拮抗作用。方法将新生24 h内Wistar乳大鼠的脊髓背根神经节(DRG)培养48 h制备原代NSC,用抗巢蛋白、神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP)单抗,采用免疫荧光染色法鉴定。原代NSC培养至第4天,将其分为细胞对照组、CP组(CP 4μmol·L-1)、ACR染毒组(ACR760μmol·L-1)和ACR+CP组(ACR 760μmol·L-1+CP 4μmol·L-1),培养48 h。采用Western蛋白印迹法检测NSC中蛋白激酶A(PKA),PKC和磷酸化高相对分子质量神经丝(p-NF-H)蛋白表达水平,应用免疫荧光法在荧光显微镜下观察PKA,PKC和p-NF-H蛋白在NSC中的分布和表达。结果经免疫荧光染色法鉴定,制备的原代NSC巢蛋白免疫反应阳性,NSC纯度>90%;经维生素A诱导,该原代NSC可分化成NSE阳性的神经元和GFAP阳性的神经胶质细胞。Western蛋白印迹实验结果表明,与细胞对照组相比,CP组NSC中PKA,PKC和p-NF-H蛋白表达水平无明显变化;ACR组PKA和PKC蛋白表达分别下降41.6%和29.4%(P<0.05),p-NF-H蛋白表达升高66.4%(P<0.05)。与ACR组相比,ACR+CP组PKA和PKC蛋白表达升高68.9%和34.4%(P<0.05),p-NF-H蛋白表达降低18.6%(P<0.05)。免疫荧光法实验结果表明,与细胞对照组相比,CP组NSC中PKA,PKC和p-NF-H荧光强度相近,ACR组PKA和PKC荧光强度减少且分布不均匀,p-NF-H荧光强度增加;与ACR组相比,ACR+CP组NSC中PKA和PKC荧光强度升高且分布均匀,p-NF-H荧光强度减弱。结论 ACR可导致NSC中PKA和PKC蛋白表达降低,p-NF-H蛋白表达升高;CP对ACR导致的上述变化有拮抗作用。OBJECTIVE To explore the antagonistic effect of calpain inhibitor calpeptin (CP)on acrylamide (ACR)poisoning neural stem cells (NSCs)and provide data for the prevention and treatment of ACR poisoning.METHODS Primary NSCs were prepared by culturing spinal dorsal root ganglions (DRGs)of Wistar rats within 24h after birth for 48h,and the antibodies against nestin,neuron-specific enolase (NSE)and glial fibrillary acidic protein (GFAP)were used to identify the NSCs by immunofluorescence staining.The primary NSCs were cultured for 4d and divided into four groups:cell control group (complete medium only),CP group (CP 4μmol·L^-1),ACR group (ACR 760μmol·L^-1),and ACR+ CP group (ACR 760μmol·L^-1 and CP 4μmol·L^-1).Western blotting was used to analyze the protein expression of protein kinase A (PKA),PKC and phosphorylated high neurofilament (p-NF-H)in NSCs. Immunofluorescence assay was used to observe the distribution and expression of PKA,PKC,and p-NF-H in NSCs under a fluorescence microscope.RESULTS The primary NSCs were identified by immunofluorescence staining,and nestin on the prepared NSCs was immune response positive with a purity >90%.Upon induction by vitamin A,the primary NSCs differentiated into NSE-positive neurons and GFAP-positive glial cells.Western blotting results showed that there was no significant difference in the expressions of PKA,PKC and p-NF-H protein in CP group compared with the cell control group, and PKA and PKC protein expression in theACR group decreased by 41.6%and 29.4%(P<0.05), respectively,and p-NF-H protein expression increased by 66.4%(P<0.05).Compared with ACR group, PKA and PKC protein expressions in ACR+CP group increased by 68.9%and 34.4%(P<0.05),respectively,and p-NF-H protein expression decreased by 18.6%(P<0.05).The results of immunofluorescence assay showed that compared with the cell control group,the fluorescence intensity of PKA,PKC and p-NF-H in NSCs of CP group was not significantly different,that of PKA and PKC in ACR group decreased and the distribution was uneven,

关 键 词：丙烯酰胺 蛋白激酶 磷酸化高相对分子质量神经丝 calpeptin 神经干细胞 

分 类 号：R741[医药卫生—神经病学与精神病学]