脑泰方含药血清对缺氧缺糖/复氧PC12细胞损伤的保护作用

Protective effects of Naotai Formula medicated serum on oxygen-glucose deprivation/reoxygenation-induced injury of PC12 cells

目的探讨脑泰方含药血清通过内质网应激相关通路调控缺氧缺糖/复氧PC12细胞凋亡,从而阐明脑泰方对脑缺血再灌注损伤的保护作用机制。方法将PC12细胞分为正常组(常规培养)、模型组(OGD/R)、抑制剂(4-PBA)组及脑泰方低浓度(10%)、中浓度(15%)、高浓度(20%)组。CCK8法检测细胞活力;采用乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒检测LDH活性;Hoechst 33258染色观察细胞核形态变化;Tunel染色检测细胞凋亡率;免疫荧光法和Western blot检测细胞Beclin-1、LC3、GRP78、CHOP、PERK蛋白表达情况。结果模型组LDH活性增加,细胞凋亡率增加,细胞存活率下降(P<0.01),GRP78、PERK、CHOP、LC3Ⅱ、Beclin-1蛋白表达明显增加,LC3Ⅰ蛋白表达减少(P<0.05,P<0.01);与模型组比较,抑制剂(4-PBA)组、脑泰方中浓度组、脑泰方高浓度组LDH活性降低,细胞凋亡率减少,细胞存活率明显增加(P<0.05,P<0.01),GRP78、PERK、CHOP、LC3Ⅱ、Beclin-1蛋白表达明显降低,LC3Ⅰ蛋白表达明显增加(P<0.05,P<0.01)。结论脑泰方含药血清可有效抑制缺氧缺糖/复氧PC12细胞的凋亡,调控GRP78/PERK/CHOP信号通路,表明脑泰方可能缓解受损神经细胞内质网应激导致的过度自噬状态,进而抑制细胞凋亡,发挥一定的神经保护作用。

Objective To explore the regulation of Naotai Formula medicated serum on the apoptosis of PC12 cells induced by oxygen-glucose deprivation/reoxygenation(OGD/R)through endoplasmic reticulum stress-related pathways,so as to demonstrate the mechanism of Naotai Formula's protective effects on cerebral ischemia-reperfusion injury(CIRI).Methods PC12 cells were divided into normal group(conventional culture),model group(OGD/R),inhibitor(4-PBA)group and low-(10%),medium-(15%)and high-(20%)concentration Naotai Formula groups.CCK8 method was used to examine cell viability;LDH activity was detected by LDH quantification kit.Hoechst 33258 staining was used to observe the nuclear morphological changes and the apoptosis rate was checked by Tunel staining.The expressions of Beclin-1,LC3,GRP78,CHOP and PERK proteins was examined by immunofluorescence method and the Western blot.Results In the model group,the LDH activity of PC12 cells increased,the apoptosis rate increased and the cell survival rate decreased(P<0.01),the expressions of GRP78,PERK,CHOP,LC3Ⅱ,and Beclin1 were notably elevated,while LC3Ⅰexpression was lower(P<0.05,P<0.01).Compared with the model group,the LDH activity of PC12 cells in the inhibitor(4-PBA)group,the medium-and high-concentration groups of Naotai Formula decreased,the apoptosis rate was lower and the cell survival rate increased significantly(P<0.05,P<0.01).The expressions of GRP78,PERK,CHOP,LC3Ⅱand Beclin-1 proteins were reduced significantly,while the expression of LC3Ⅰprotein increased significantly(P<0.05,P<0.01).Conclusion Naotai Formula medicated serum can effectively inhibit the apoptosis of PC12 cells induced by oxygen-glucose deprivation/reoxygenation and regulate the GRP78/PERK/CHOP signaling pathway.It indicates that Naotai Formula may alleviate the excessive autophagy caused by endoplasmic reticulum stress of the injured nerve cells,and then inhibit the apoptosis of cells,so as to play a certain neuroprotective role.