盐酸法舒地尔对体外培养星形胶质细胞氧糖剥夺损伤的保护作用

发布时间：2018-12-26 08:20

【摘要】：目的研究盐酸法舒地尔(FH)对大鼠皮质星形胶质细胞氧糖剥夺损伤(OGD)的保护作用。方法传代培养的大鼠皮质星形胶质细胞与无糖Na2S2O42 mmol·L-1孵育4 h,制备OGD模型。FH 5和10μmol·L-1在OGD前2 h加入。采用噻唑蓝(MTT)法检测细胞存活;比色法检测乳酸脱氢酶(LDH);乙炔基脱氧尿苷(Ed U)掺入法检测星形胶质细胞DNA合成;Hoechst染色和丫啶橙(AO)/溴化乙啶(EB)荧光双染观察细胞凋亡;2,7-二氯氢化荧光素(DHCF-DA)荧光染色和硝基四氮唑蓝(NBT)还原实验检测细胞内活性氧(ROS)水平。结果与正常对照组比较,OGD模型组星形胶质细胞存活率为(24±6)%,下降了4倍(P0.01);OGD模型组LDH释放量显著升高至(366±7)U·L-1(P0.01)。加入FH 5和10μmol·L-1的预处理组,细胞存活率分别上升至(42±5)%和(43±5)%,LDH释放分别下降至290±18和(268±20)U·L-1(P0.01),细胞Ed U阳性率由OGD模型组的(47±6)%,分别下降到(35±6)%和(29±5)%(P0.01)。Hoechst染色及AO/EB双染实验结显示,FH能显著减少OGD损伤后细胞的凋亡及坏死,使晚期凋亡细胞比例减少(P0.05);DCFH-DA荧光和NBT还原实验显示,FH降低OGD损伤后ROS水平,且FH10μmol·L-1的预处理组降低的幅度比5μmol·L-1的预处理组更明显,但两组差异无统计学意义。结论FH对传代培养的大鼠皮质星形胶质细胞OGD具有保护作用,其作用机制可能与降低细胞内ROS水平有关。
[Abstract]:Objective to study the protective effect of fasudil hydrochloride (FH) on (OGD) injury induced by oxygen glucose deprivation in rat cortical astrocytes. Methods cultured rat cortical astrocytes were incubated with glycos-free Na2S2O42 mmol L-1 for 4 h to prepare OGD model. FH 5 and 10 渭 mol L-1 were added at 2 h before OGD. The cell survival was detected by thiazolyl (MTT) assay, and the DNA synthesis of astrocytes was detected by colorimetric assay for lactate dehydrogenase (LDH); acetyldeoxyuridine (Ed U) incorporation. Hoechst staining and acridine orange (AO) / ethidium bromide (EB) fluorescence double staining were used to observe the apoptosis of the cells, and the levels of reactive oxygen (ROS) in the cells were detected by 2C7-dichlorofluorescein (DHCF-DA) fluorescence staining and (NBT) reduction assay of nitro tetrazolium blue. Results compared with the control group, the survival rate of astrocytes in the OGD model group was (24 卤6)%, which decreased by 4 times (P0.01); OGD model group) significantly increased the LDH release to (366 卤7) U L-1 (P0.01). The cell survival rate increased to (42 卤5)% and (43 卤5)%, and the LDH release decreased to (290 卤18) and (268 卤20) U L-1 (P0.01) in the pretreatment group with FH 5 and 10 渭 mol L-1, respectively. The positive rate of Ed U decreased from (47 卤6)% in the OGD model group to (35 卤6)% and (29 卤5)% in the OGD model group (P0.01). Hoechst staining and AO/EB double staining respectively). The results showed that FH could significantly reduce the apoptosis and necrosis of the cells after OGD injury. The proportion of late apoptotic cells was decreased (P0.05). DCFH-DA fluorescence and NBT reduction experiments showed that FH decreased the level of ROS after OGD injury, and the reduction of ROS level in FH10 渭 mol L-1 preconditioning group was more significant than that in 5 渭 mol L-1 preconditioning group, but there was no significant difference between the two groups. Conclusion FH has protective effect on OGD of cultured rat cortical astrocytes, and its mechanism may be related to the reduction of intracellular ROS level.
【作者单位】：长江航运总医院武汉脑科医院神经内科;华中科技大学同济医学院药理学系;
【基金】：交通运输部长江航务管理局课题(201210011) 武汉市卫计委临床医学科研项目(WX14D12)~~
【分类号】：R965

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