HAMI 3379抑制自噬减轻BV2细胞缺氧缺糖损伤的机制
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摘要
目的探讨HAMI 3379抑制自噬减轻BV2细胞缺氧缺糖损伤的机制。方法随机将对数生长期BV2细胞分为正常对照组、氧糖剥夺(OGD)组和1、10、100 nM HAMI 3379组,然后采用OGD处理对细胞进行造模,培养48 h,采用MTT检测、LDH释放检测及蛋白印记检测观察HAMI 3379对OGD处理后BV2细胞活性及自噬的影响。结果与正常对照组比较,1、10、100 nMHAMI 3379组细胞均有明显损伤,且LDH释放增加(P<0.05);而与OGD组比较,给予1 nM HAMI 3379有改善细胞活性趋势,但差异无统计学意义(P>0.05);10、100 nM HAMI 3379可明显增加细胞存活率,减少LDH的释放(P<0.05)。与正常对照组比较,OGD组和10、100 n M HAMI 3379组Beclin-1表达量显著升高(P<0.05);而与OGD组比较,10、100 nM HAMI 3379组的BV2细胞Beclin-1表达量明显降低(P<0.05)。与正常对照组比较,OGD组和10、100 nM HAMI 3379组LC3Ⅱ/Ⅰ表达量显著升高,差异具有统计学意义(P<0.05);与OGD组比较,10、100 nM HAMI 3379组BV2细胞LC3Ⅱ/Ⅰ表达量明显降低(P<0.05)。结论HAMI 3379可改善OGD处理后BV2细胞活性,减少LDH释放,也可降低OGD处理激活的自噬信号分子Beclin-1和LC3的表达,说明HAMI 3379可通过调节自噬信号通路分子部分逆转OGD处理后BV2细胞的损伤。
Objective To explore the mechanism of HAMI 3379 inhibiting autophagy to alleviate the damage of BV2 cells caused by hypoxia and glucose deficiency. Methods BV2 cells in logarithmic phase were randomly divided into normal control group, oxygen and glucose deprivation(OGD) group and 1, 10, 100 nM HAMI 3379 group. Then the cells were modeled by OGD treatment. After 48 hours of incubation, the effects of HAMI 3379 on the activity and autophagy of BV2 cells treated with OGD were observed by MTT assay, LDH release assay and protein imprinting assay. Results Compared with the normal control group, the 1, 10, 100 nM HAMI 3379 group had significant cell damage and the release of LDH increased(P <0.05); compared with the OGD group, 1 nM HAMI 3379 had a tendency to improve cell viability, but there was no significant difference(P>0.05); 10, 100 nM HAMI 3379 could significantly increase cell viability and reduce the release of LDH(P<0.05). Compared with the normal control group, the expression of Beclin-1 in the OGD group and 10, 100 nM HAMI3379 group increased significantly(P<0.05); compared with the OGD group, the expression of Beclin-1 in BV2 cells in the 10,100 nM HAMI 3379 group decreased significantly(P<0.05). Compared with the normal control group, the expression of LC3Ⅱ/Ⅰ in the OGD group and 10, 100 nM HAMI 3379 group increased significantly(P<0.05); compared with the OGD group,the expression of LC3 Ⅱ/Ⅰ of BV2 cells in the 10, 100 nM HAMI 3379 group decreased significantly(P<0.05). Conclusion HAMI 3379 can improve the activity of BV2 cells after OGD treatment, reduce the release of LDH, and also reduce the expression of autophagic signal molecules Beclin-1 and LC3 activated by OGD treatment. This suggests that HAMI 3379 can partially reverse the damage of BV2 cells after OGD treatment by regulating autophagic signal pathway molecules.
Objective To explore the mechanism of HAMI 3379 inhibiting autophagy to alleviate the damage of BV2 cells caused by hypoxia and glucose deficiency. Methods BV2 cells in logarithmic phase were randomly divided into normal control group, oxygen and glucose deprivation(OGD) group and 1, 10, 100 nM HAMI 3379 group. Then the cells were modeled by OGD treatment. After 48 hours of incubation, the effects of HAMI 3379 on the activity and autophagy of BV2 cells treated with OGD were observed by MTT assay, LDH release assay and protein imprinting assay. Results Compared with the normal control group, the 1, 10, 100 nM HAMI 3379 group had significant cell damage and the release of LDH increased(P <0.05); compared with the OGD group, 1 nM HAMI 3379 had a tendency to improve cell viability, but there was no significant difference(P>0.05); 10, 100 nM HAMI 3379 could significantly increase cell viability and reduce the release of LDH(P<0.05). Compared with the normal control group, the expression of Beclin-1 in the OGD group and 10, 100 nM HAMI3379 group increased significantly(P<0.05); compared with the OGD group, the expression of Beclin-1 in BV2 cells in the 10,100 nM HAMI 3379 group decreased significantly(P<0.05). Compared with the normal control group, the expression of LC3Ⅱ/Ⅰ in the OGD group and 10, 100 nM HAMI 3379 group increased significantly(P<0.05); compared with the OGD group,the expression of LC3 Ⅱ/Ⅰ of BV2 cells in the 10, 100 nM HAMI 3379 group decreased significantly(P<0.05). Conclusion HAMI 3379 can improve the activity of BV2 cells after OGD treatment, reduce the release of LDH, and also reduce the expression of autophagic signal molecules Beclin-1 and LC3 activated by OGD treatment. This suggests that HAMI 3379 can partially reverse the damage of BV2 cells after OGD treatment by regulating autophagic signal pathway molecules.
引文
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[13]SHI QJ,WANG H,LIU ZX,et al.HAMI 3379,a CysLT2R antagonist,dose-and time-dependently attenuates brain injury and inhibits microglial inflammation after focal cerebral ischemia in rats[J].Neuroscience,2015,291:53-69.
[14]ZHANG XY,WANG XR,XU DM,et al.HAMI 3379,a CysLT2 receptor antagonist,attenuates ischemia-like neuronal injury by inhibiting microglial activation[J].J Pharmacol Exp Ther,2013,346(2):328-341.
[2]YU GL,WEI EQ,WANG ML,et al.Pranlukast,a cysteinyl leukotriene receptor-1 antagonist,protects against chronic ischemic brain injury and inhibits the glial scar formation in mice[J].Brain Res,2005,1053(1-2):116-125.
[3]YU GL,WEI EQ,ZHANG SH,et al.Montelukast,a cysteinyl leukotriene receptor-1 antagonist,dose-and time-dependently protects against focal cerebral ischemia in mice[J].Pharmacology,2005,73(1):31-40.
[4]CHEN LP,XU HM,ZHAO W,et al.Photomacrography of brain surface for evaluating blood-brain barrier disruption within 24 h after focal cerebral ischemia in mice[J].Zhejiang Da Xue Xue Bao Yi Xue Ban,2005,34(6):523-528.
[5]CHU LS,WEI EQ,YU GL,et al.Pranlukast reduces neutrophil but not macrophage/microglial accumu-lation in brain after focal cerebral ischemia in mice[J].Acta Pharmacol Sin,2006,27(3):282-288.
[6]NI NC,YAN D,BALLANTYNE LL,et al.A selective cysteinyl leukotriene recepto r 2 antagonist blocks myocardial ischemia/reperfusion injury and vascular permeability in mice[J].J Pharmacol Exp Ther,2011,339(3):768-778.
[7]WUNDER F,TINEL H,KAST R,et al.Pharmacological characterization of the first potent and selective antagonist at the cysteinyl leukotriene 2[CysLT(2)]receptor[J].Br J Pharmacol,2010,160(2):399-409.
[8]SHI QJ,XIAO L,ZHAO B,et al.Intracerebroventricular injection of HAMI 3379,a selective cysteinyl leukotriene receptor 2 antagonist,protects against acute brain injury after focal cerebral ischemia in rats[J].Brain Res,2012,1484(16):57-67.
[9]PARZYCH KR,KLIONSKY DJ.An overview of autophagy:morphology,mechanism,and regulation[J].Antioxid Redox Signal,2014,20(3):460-473.
[10]WANG P,SHAO BZ,DENG Z,et al.Autophagy in ischemic stroke[J].Prog Neurobiol,2018,163-164:98-117.
[11]齐倩倩,常明则,张格娟,等.雷帕霉素对PC12细胞氧糖剥夺损伤的保护作用[J].中风与神经疾病杂志,2017,34(6):494-496.
[12]FITZWALTER BE,THORBURN A.Recent insights into cell death and autophagy[J].FEBS J,2015,282(22):4279-4288.
[13]SHI QJ,WANG H,LIU ZX,et al.HAMI 3379,a CysLT2R antagonist,dose-and time-dependently attenuates brain injury and inhibits microglial inflammation after focal cerebral ischemia in rats[J].Neuroscience,2015,291:53-69.
[14]ZHANG XY,WANG XR,XU DM,et al.HAMI 3379,a CysLT2 receptor antagonist,attenuates ischemia-like neuronal injury by inhibiting microglial activation[J].J Pharmacol Exp Ther,2013,346(2):328-341.