化痰开窍方通过调控自噬改善体外神经元低糖低氧损伤的机制初探

Preliminary exploration on mechanism of Huatan Kaiqiao formula in regulating autophagy and improving neuronal low glucose and low oxygen injury in vitro

中文关键词: 低糖低氧    化痰开窍方    自噬    线粒体稳态    血管性认知障碍

英文关键词: low glucose and low oxygenHuatan Kaiqiao formulaautophagymitochondrial homeostasisvascular cognitive impairment

中文摘要:

      目的 研究化痰开窍方对体外神经元低糖低氧(LGLO)损伤细胞的保护作用及其机制。方法 采用小鼠神经母细胞瘤(N2a)细胞系模拟体外神经元，构建LGLO损伤模型。将N2a细胞分为正常组、模型组、空白血清组、化痰开窍方低剂量组、化痰开窍方中剂量组、化痰开窍方高剂量组。采用细胞计数试剂盒-8(CCK-8)法检测各组细胞存活率;蛋白质印迹法检测各组细胞中微管相关蛋白1轻链3B(LC3B)、P62、Beclin 1及线粒体外膜转位酶20(TOMM20)的蛋白表达水平。结果 与造模前相比，不同LGLO诱导时间的N2a细胞存活率差异有统计学意义(P <0.001)。LGLO诱导2、4、6、8 h细胞存活率分别为(92.81±3.58)%、(76.92±7.15)%、(63.67±5.27)%和(48.15±5.02)%，细胞存活率随诱导时间延长呈下降趋势。与模型组相比，化痰开窍方中剂量组(P=0.010)和高剂量组(P=0.005)均提高LGLO诱导6 h N2a细胞存活率。与正常组相比，模型组LC3B-Ⅱ/LC3B-Ⅰ比值(P <0.001)、LC3B-Ⅱ(P <0.001)、P62 (P=0.015)、Beclin 1(P=0.010)及TOMM20(P=0.017)蛋白表达均升高。与模型组相比，化痰开窍方中剂量组LC3B-Ⅱ/LC3B-Ⅰ比值(P <0.001)、LC3B-Ⅱ(P <0.001)、P62(P=0.003)及TOMM20(P=0.003)蛋白表达下调。结论 LGLO随时间延长可逐渐加重体外神经元损伤。化痰开窍方对LGLO损伤细胞具有一定保护作用，其机制可能与调节自噬异常、改善线粒体稳态并促进受损线粒体清除有关。

英文摘要:

      Objective To investigate the protective effects of Huatan Kaiqiao formula(HKF)on neuronal low glucose and low oxygen(LGLO)injury in vitro and explore its underlying mechanism.Methods Mouse neuroblastoma(N2a)cells were used to simulate the neurons in vitro,and a LGLO injury model was constructed.N2a cells were randomly divided into six groups:normal group,model group,blank serum group,low-dose HKF group,medium-dose HKF group,and high-dose HKF group.The cell counting kit-8 assay was used to measure cell survival rate.The protein expression levels of microtubule-associated protein 1 light chain 3 beta(LC3B),P62,Beclin 1,and translocase of the outer mitochondrial membrane 20(TOMM20)were detected by western blotting.Results Compared with before modeling,there was a statistically significant difference in the survival rates of N2a cells induced by different LGLO times(P＜0.001).The survival rates of N2a cells at 2,4,6,and 8 h after LGLO induction were(92.81±3.58)％,(76.92±7.15)％,(63.67±5.27)％,and(48.15±5.02)％,respectively.The cell survival rate showed a downward trend as the induction time prolonged.Compared with the model group,the medium-dose HKF group(P=0.010)and high-dose HKF group(P=0.005)significantly increased the survival rate of N2a cells after 6 h of LGLO induction.Compared with the normal group,the model group showed a significantly increased LC3B-Ⅱ/LC3BⅠ-ratio(P＜0.001)and significantly increased protein expression levels of LC3B-Ⅱ(P＜0.001),P62(P=0.015),Beclin1(P=0.010),and TOMM20(P=0.017).Compared with the model group,the medium-dose HKF group showed a significantly decreased LC3B-Ⅱ/LC3B-Ⅰratio(P＜0.001)and significantly decreased protein expression levels of LC3B-Ⅱ(P＜0.001),P62(P=0.003),and TOMM20(P=0.003).Conclusion LGLO can progressively exacerbate neuronal injury in vitro over time.HKF exerts a protective effect on LGLO-induced injury,and its mechanism may be related to regulating abnormal autophagy,improving mitochondrial homeostasis,and promoting the clearance of damaged mitochondria.