摘要
目的研究碘化N-正丁基氟哌啶醇(F2)对大鼠心肌细胞无外钙(零钙液)缺氧/复氧(H/R)时环磷酸腺苷(c AMP)/蛋白激酶A(PKA)/细胞外信号调节激酶(ERK)1/2信号通路及细胞损伤的作用。方法建立新生大鼠心肌细胞零钙液H/R模型,于正常培养基、缺氧液和复氧液中加入F2(1×10-6 mol/L)。采用蛋白印迹法检测心肌细胞磷酸化ERK(p-ERK1/2)、总ERK1/2及PKA蛋白表达;酶联免疫吸附法(ELISA)测定心肌细胞c AMP的水平;比色法检测培养细胞上清液乳酸脱氢酶(LDH)的含量;Hoechst 33342染色法检测心肌细胞凋亡情况。结果零钙液H/R可激活心肌细胞ERK1/2,使心肌细胞培养液中LDH浓度升高,心肌细胞凋亡增加;F2和ERK1/2抑制剂U0126及PD98059可下调零钙液H/R所致p-ERK1/2蛋白高表达,抑制细胞LDH漏出,减少细胞凋亡;ERK1/2激动剂表皮生长因子(EGF)可拮抗F2对零钙液H/R心肌细胞p-ERK1/2高表达的抑制作用,进而拮抗F2的心肌保护作用。零钙液H/R对培养心肌细胞c AMP含量及PKA蛋白表达无影响,F2对零钙液H/R状态下心肌细胞c AMP含量及PKA蛋白表达无影响。结论 F2可通过非L-型钙通道依赖机制拮抗心肌细胞H/R损伤,这可能与其抑制非L型钙通道依赖的ERK1/2信号通路激活有关,但与c AMP/PKA信号通路无关。
Objective To investigate the effects of N-n-butyl haloperidol iodide（F2） on c AMP/PKA/ERK1/2signaling pathway and cell injury of cardiomyocytes during extracellular calcium-free（calcium-free）-hypoxia/reoxygenation（H/R） in rats. Methods The calcium-free-H/R models of neonatal rat cardiomyocytes were established. And F2（1×10^-6mol/L） was added to normal medium, hypoxia buffer and reoxygenation buffer. Western blot analysis was used to determine the changes of phosphorylated extracellular signal-regulated kinase（p-ERK1/2）, total ERK1/2 and PKA protein expressions in cardiomyocytes. Enzyme-linked immunosorbent assay（ELISA） was used to determine the level of c AMP in cardiomyocytes. Colorimetric assay was used to measure the content of LDH in the supernatant. Hoechst33342 staining method was used to detect apoptosis in cardiomyocytes. Results Calcium-free-H/R could activate ERK1/2 in the cardiomyocytes, and therefore elevate LDH concentration in the cardiomyocytes supernatant and in-crease the apoptosis of cardiomyocytes. F2 and the ERK1/2 inhibitors U0126 and PD98059 were found to down-regulate the high expression of calcium-free-H/R-induced ERK1/2 protein, suppress LDH leakage and reduce apoptosis.The ERK1/2 agonist EGF was found to antagonize the inhibition of F2 on the high expression of calcium-free-H/R cardiomyocytes p-ERK1/2 and to further antagonize F2 protection. Calcium-free-H/R had no effect on the c AMP content and PKA protein expression in the cardiomycytes. Also, F2 had no effect on the c AMP content and PKA protein expression in the cultured cardiomycytes under H/R condition. Conclusion F2 can antagonize cardiomyocytes H/R injury through non-L-type calcium channel-independent mechanism, which may be related to its inhibition on the non-Ltype calcium channel-independent ERK1/2 signaling pathway instead of the c AMP/PKA signaling pathway.
出处
《中国药物与临床》
CAS
2015年第2期149-153,共5页
Chinese Remedies & Clinics
基金
国家自然科学基金-广东省自然科学基金联合资助基金(U0932005)
国家自然科学基金(81072633
81473215)
广东省汕头市科技计划项目(汕市财教【2013】244号/73号)
关键词
碘化合物
心肌
缺氧
细胞外信号调节MAP激酶类
Iodide compounds
Myocardium
Anoxia
Extracellular signal-regulated MAP kinase
