自噬在吗啡预处理减轻小鼠原代皮层神经元氧糖剥夺/复糖复氧损伤中的作用及其与JNK的关系

Role of autophagy in morphine preconditioning-induced reduction of OGD/R injury in primary cortical neurons of mice and the relationship with JNK

目的:评价自噬在吗啡预处理减轻小鼠原代皮层神经元氧糖剥夺/复糖复氧(OGD/R)损伤中的作用及其与c-Jun氨基末端激酶(JNK)的关系。方法:选择出生24 h内的C57BL/6乳鼠,培养原代皮层神经元,采用随机数字表法分为9组( n=24):对照组(C组)、OGD/R组、吗啡预处理组(M组)、自噬抑制剂3-甲基腺嘌呤(3-MA)组(3-MA组)、3-MA+吗啡预处理组(3-MA+M组)、自噬抑制剂氯喹组(Ch组)、氯喹+吗啡预处理组(Ch+M组)、JNK抑制剂SP600125组(SP组)和SP600125+吗啡预处理组(SP+M组)。吗啡预处理:OGD/R前加入吗啡3 μmol/L孵育2 h。3-MA组、Ch组和SP组分别加入3-MA 5 mmol/L、氯喹50 μmol/L、SP6001252 5 μmol/L,孵育150 min。3-MA+M组、Ch+M组和SP+M组于吗啡预处理前30 min时分别加入3-MA 5 mmol/L、氯喹50 μmol/L、SP600125 25 μmol/L。随后氧糖剥夺1 h,复糖复氧24 h。采用CCK-8法测定神经元活力;Western blot法测定JNK、磷酸化JNK(p-JNK)、微管相关蛋白1轻链3(LC3)、p62、Beclin1、caspase-3和cleaved-caspase-3的表达;LC3双荧光腺病毒转染法计数自噬小体和自噬溶酶体;TUNEL染色法检测神经元凋亡率。 结果:与C组比较,OGD/R组神经元活力降低,Beclin1表达上调,p62表达下调,LC3Ⅱ/LC3Ⅰ比值、p-JNK/JNK比值、自噬小体计数、自噬溶酶体计数、cleaved-caspase-3/caspase-3比值和神经元凋亡率升高( P<0.001)。与OGD/R组比较,M组神经元活力、p-JNK/JNK比值、LC3Ⅱ/LC3Ⅰ比值、自噬小体计数和自噬溶酶体计数升高,Beclin1表达上调,p62表达下调,3-MA组LC3Ⅱ/LC3Ⅰ比值降低,p62表达下调,Ch组LC3Ⅱ/LC3Ⅰ比值升高,p62表达上调( P<0.001),SP600125组上述指标差异无统计学意义( P>0.05)。与M组比较,M+3-MA组神经元活力和LC3Ⅱ/LC3Ⅰ比值降低,p62表达上调,M+Ch组神经元活力降低,LC3Ⅱ/LC3Ⅰ比值升高,p62表达上调,M+SP组神经元活力、p-JNK/JNK比值和LC3Ⅱ/LC3Ⅰ比值降低,p62表达上调,自噬小体计数和自噬溶酶体计数降低,Beclin1表达下调,cleaved-caspase-3/caspase-3比值和神经元凋亡率升高( P<0.001)。 结论:吗啡预处理可通过激活JNK,增强自噬,抑制凋亡,减轻小鼠原代皮层神经元OGD/R损伤。

Objective To evaluate the role of autophagy in morphine preconditioning-induced reduction of oxygen-glucose deprivation and restoration(OGD/R)injury in primary cortical neurons of mice and the relationship with c-Jun N-terminal kinase(JNK).Methods Primary cortical neurons extracted from C57BL/6 neonatal mice within 24 h after birth were divided into 9 groups(n=24 each)using a random number table method:control group(C group),OGD/R group,morphine preconditioning group(M group),autophagy inhibitor 3-methyladenine(3-MA)group(3-MA group),3-MA+morphine preconditioning group(3-MA+M group),autophagy inhibitor chloroquine group(Ch group),chloroquine+morphine preconditioning group(Ch+M group),JNK inhibitor SP600125 group(SP group)and SP600125+morphine preconditioning group(SP+M group).Morphine preconditioning:morphine was added at a final concentration of 3μmol/L before OGD/R,and the cells were incubated for 2 h in OGD/R group.In 3-MA,Ch and SP groups,3-MA 5 mmol/L,chloroquine 50μmol/L and SP60012525μmol/L were added,respectively,and the cells were incubated for 150 min.In 3-MA+M,Ch+M and SP+M groups,3-MA 5 mmol/L,chloroquine 50μmol/L and SP60012525μmol/L were added,respectively,at 30 min before morphine preconditioning.Then the cells were subjected to oxygen-glucose deprivation for 1 h followed by restoration of oxygen-glucose supply for 24 h.CCK-8 assay was used to detect the neuronal viability.The expression of JNK,phosphorylated JNK(p-JNK),microtubule-associated protein 1 light chain 3(LC3),p62,Beclin1,caspase-3,and cleaved-caspase-3 was determined by Western blot.The autophagosomes and autolysosomes were counted using LC3-double fluorescent adenovirus transfection,and the neuronal apoptosis rate was determined by TUNEL staining.Results Compared with C group,the neuronal viability was significantly decreased,the expression of Beclin1 was up-regulated,the expression of p62 was down-regulated,and the LC3Ⅱ/LC3Ⅰratio,p-JNK/JNK ratio,the number of autophagosomes and autolysosomes,cleaved-caspase-3/caspase-3 ratio and neuronal apoptosis rate were increased in OGD/R group(P<0.001).Compared with OGD/R group,the neuronal viability,p-JNK/JNK ratio,LC3Ⅱ/LC3Ⅰratio and the number of autophagosomes and autolysosomes were significantly increased,the expression of Beclin1 was up-regulated,and the expression of p62 was down-regulated in M group,the LC3Ⅱ/LC3Ⅰratio was significantly decreased,and the expression of p62 was down-regulated in 3-MA group,the LC3Ⅱ/LC3Ⅰratio was significantly increased,and the expression of p62 was up-regulated in Ch group(P<0.001),and no significant change was found in the parameters mentioned above in SP600125 group(P>0.05).Compared with M group,the neuronal viability was significantly decreased,the LC3Ⅱ/LC3Ⅰratio was decreased,and the expression of p62 was up-regulated in M+3-MA group,the neuronal viability was significantly decreased,the LC3Ⅱ/LC3Ⅰratio was increased,and the expression of p62 was up-regulated in M+Ch group,and the neuronal viability,LC3Ⅱ/LC3Ⅰratio and p-JNK/JNK ratio were significantly decreased,the expression of p62 was up-regulated,the number of autophagosomes and autolysosomes was decreased,the expression of Beclin1 was down-regulated,and the cleaved-caspase-3/caspase-3 ratio and neuronal apoptosis rate were increased in M+SP group(P<0.001).Conclusions Morphine preconditioning can attenuate OGD/R injury by activating JNK,enhancing autophagy and inhibiting apoptosis in primary cortical neurons of mice.