地佐辛对β淀粉样多肽处理的原代大鼠皮质星形胶质细胞的保护作用

摘要

Objective To investigate hyperpolarization-activated cyclic nucleotide-gated (HCN) channels inhibited by dezocine when primary rat cortical astrocytes were exposed to β amyloid peptide.Methods Cultured primary cortical astrocytes from new-born SD rats (within 24 h) were divided into 7 groups (n=5) according to random number table.The astrocytes in groups ZA and CDA were pretreated with 7-CH-cAMP (30 mmol/L),an agonist of HCN channels and ZD7288 (30 mmol/L),an inhibitor of HCN channels for 24 h,respectively.And then the cells in groups DA1,DA2,DA3 and CDA were treated with dezocine 10-7,10-6,10-5and 10-16 mmol/L for 24 h,respectively.Following with the treatments above,the cells in groups A,DA1,DA2,DA3,ZA and CDA were exposed to Aβ1-42 (15μmol/L) for 24 h,but the cells in group C were cultured normally.The effects of cell apoptosis,viability and injury were assessed by Annexin V-FITC/PI assay,MTT assay and lactate de hydrogenase (LDH) release assay.IL-1β was assessed by ELISA assay.The expressions of Cu/ZnSOD and Mn-SOD protein were assessed by Western blot assay.Results Compared with group C,there were significant increases of cell apoptosis,injury and IL-1β level in group A (P＜0.05).Compared with group A,there were significant decreases of cell apoptosis,injury,and IL-1β level but increase of Cu/Zn-SOD and Mn-SOD expressions in groups ZA,DA2 and DA3 (P＜0.05),while the neuroprotection of dezocine was partially restored by 7-CH-cAMP in group CDA (P ＜ 0.05).Conclusion The neuroprotection of dezocine against apoptosis induced by β amyloid peptide could be associated with up-regulation of anti-oxidative stress related Cu/Zn-SOD and Mn-SOD mediated by inhibition of HCN channels.%目的 探讨地佐辛对接受β淀粉样多肽处理的原代大鼠皮质星形胶质细胞的影响以及与超极化激活的环核苷酸门控(hyperpolarization-activated cyclic nucleotide-gated,HCN)通道的关系.方法 出生24 h内的SD大鼠,体外培养皮质星形胶质细胞并接种于培养瓶或35 mm培养皿,采用随机数字表法分为七组(n=5).ZA组和CDA组预先24 h分别向培养基中加入HCN通道激动剂7-CH-cAMP(30μmol/L)和阻断剂ZD7288(30μmol/L),随后DA1、DA2、DA3和CDA组分别向培养基中加入地佐辛10-7、10-6、10 5和10-6 mmol/L孵育24 h,最后A、DA1、DA2、DA3、ZA和CDA组分别向培养基中加入Aβ1-42(15 μmol/L)孵育24 h作为β淀粉样多肽处理,而对照组(C组)细胞正常培养.按照各组处理收集星形胶质细胞,采用Annexin V-FITC/PI双染色流式细胞法测定细胞凋亡率,乳酸脱氢酶(LDH)法测定细胞损伤率,ELISA法测定细胞前炎性因子IL-1β浓度,Westem blot法测定细胞内Cu/Zn-SOD和Mn-SOD蛋白含量.结果 与C组比较,A组星形胶质细胞凋亡率、损伤率和IL-1β浓度明显升高(P＜0.05);与A组比较,DA2、DA3和ZA组星形胶质细胞凋亡率、损伤率和IL-1β浓度明显下降,Cu/Zn-SOD和Mn-SOD蛋白含量明显升高(P＜0.05);与CDA组比较,DA2、DA3和ZA组星形胶质细胞凋亡率、损伤率和IL-1β浓度明显下降,Cu/Zn-SOD和Mn-SOD蛋白含量明显升高(P＜0.05).结论 地佐辛抑制β淀粉样多肽诱发的原代大鼠皮质星形胶质细胞凋亡的机制可能与HCN通道开放被抑制后,抗氧化应激相关蛋白Cu/Zn-SOD和Mn-SOD含量增加相关.