Lu Enming, Zhuo Qi, Chen Falu, Lin Qingming, Wang Xiaoping
Shengli Clinical Medical College of Fujian Medical University, Fuzhou 350001, Fujian, China.
Fujian Provincial Key Laboratory of Emergency Medicine, Fuzhou 350001, Fujian, China.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2022 Dec;34(12):1280-1284. doi: 10.3760/cma.j.cn121430-20220411-00355.
To explore the effects and the possible mechanism of bone marrow mesenchymal stem cell (BMMSC) transplantation on apoptosis in rats cerebral cortex after cardiac arrest/cardiopulmonary resuscitation (CA/CPR).
The BMMSC of 2 Sprague-Dawley (SD) rats aged 4-5weeks was extracted, and the 3rd passage was used in experimental study. Eighteen Sprague-Dawley (SD) rats were divided into sham group, model group (CA/CPR group) and intervention group (BMMSC group) according to random number table method, with 6 rats in each group. CPR was performed 6 minutes after asphyxia induced CA. In sham group, CA was not induced except performing general surgical procedure. At 1 hour after return of spontaneous circulation (ROSC), 0.5 mL phosphate buffered saline (PBS) was injected through tail vein in CA/CPR group. 2×10/L green fluorescence protein (GFP)-labeled BMMSC was injected through tail vein 1 hour after ROSC in BMMSC group. Neurological deficit score (NDS) were assessed in every group at 72 hours after CPR. Serum S100 calcium binding protein B (S100B) levels were assayed by enzyme linked immunosorbent assay (ELISA). Distribution of BMMSC in brain was observed under a fluorescent microscope. Apoptosis rate in cerebral cortex was assayed by TdT-mediated dUTP nick-end labeling (TUNEL). Western blotting was performed to measure the expression levels of active aspartic acid specific cysteine proteinase (caspase-8 and caspase-9) in cerebral cortex.
At 3 days after CPR, compared with sham group, the apoptosis of cerebral cortex cells was increased and brain damage was obvious, NDS score was decreased significantly (56.6±5.5 vs. 80.0±0.0, P < 0.05), and serum S100B was increased markedly (ng/L: 45.1±4.7 vs. 19.1±1.4, P < 0.05), apoptosis rate of cerebral cortex cells increased significantly [(52.9±11.8)% vs. (10.1±1.5)%, P < 0.05], the level of active caspase-8 expression in cerebral cortex was significantly higher (caspase-8/GAPDH: 0.689±0.047 vs. 0.330±0.108, P < 0.05), and there was no significant difference in active caspase-9 protein expression (caspase-9/GAPDH: 0.428±0.014 vs. 0.426±0.021, P > 0.05) in CA/CPR group. After BMMSC transplantation, GFP-labeled BMMSC were primarily detected in cerebral cortex, compared with CA/CPR group, the apoptosis of cerebral cortex cells and brain injury were significantly improved in BMMSC group, NDS score increased significantly (70.6±2.1 vs. 56.6±5.5, P < 0.05), serum S100B levels in BMMSC group were lower (ng/L: 32.0±3.2 vs. 45.1±4.7, P < 0.05), apoptosis rate of cerebral cortex cells decreased significantly [(31.1±3.4)% vs. (52.9±11.8)%, P < 0.05], and the active caspase-8 expression in cerebral cortex in BMMSC group was significantly decreased (caspase-8/GAPDH: 0.427±0.067 vs. 0.689±0.047, P < 0.05). The active caspase-9 expression in cerebral cortex in BMMSC group and CA/CPR group were not significantly different (caspase-9/GAPDH: 0.431±0.022 vs. 0.428±0.014, P > 0.05).
BMMSC transplantation can alleviate rat brain damage after CA/CPR possibly by inhibiting the death receptor mediated apoptotic pathway to inhibit the apoptosis of brain cells.
探讨骨髓间充质干细胞（BMMSC）移植对心脏骤停/心肺复苏（CA/CPR）后大鼠大脑皮质细胞凋亡的影响及其可能机制。
取2只4-5周龄Sprague-Dawley（SD）大鼠的BMMSC，取第3代用于实验研究。将18只Sprague-Dawley（SD）大鼠按随机数字表法分为假手术组、模型组（CA/CPR组）和干预组（BMMSC组），每组6只。窒息诱导CA后6分钟进行心肺复苏。假手术组除进行常规手术操作外，不诱导CA。自主循环恢复（ROSC）后1小时，CA/CPR组经尾静脉注射0.5 mL磷酸盐缓冲液（PBS）。BMMSC组在ROSC后1小时经尾静脉注射2×10/L绿色荧光蛋白（GFP）标记的BMMSC。心肺复苏后72小时对每组进行神经功能缺损评分（NDS）。采用酶联免疫吸附测定（ELISA）法检测血清S100钙结合蛋白B（S100B）水平。在荧光显微镜下观察BMMSC在脑内的分布。采用TdT介导的dUTP缺口末端标记法（TUNEL）检测大脑皮质细胞凋亡率。采用蛋白质免疫印迹法检测大脑皮质中活性天冬氨酸特异性半胱氨酸蛋白酶（caspase-8和caspase-9）的表达水平。
心肺复苏后3天，与假手术组相比，CA/CPR组大脑皮质细胞凋亡增加，脑损伤明显，NDS评分显著降低（56.6±5.5 vs. 80.0±0.0，P<0.05），血清S100B显著升高（ng/L：45.1±4.7 vs. 19.1±1.4，P<0.05），大脑皮质细胞凋亡率显著升高[（52.9±11.8）% vs.（10.1±1.5）%，P<0.05]，大脑皮质中活性caspase-8表达水平显著升高（caspase-8/GAPDH：0.689±0.047 vs. 0.330±0.108，P<0.05），活性caspase-9蛋白表达无显著差异（caspase-9/GAPDH：0.428±0.014 vs. 0.426±0.021，P>0.05）。BMMSC移植后，在大脑皮质中主要检测到GFP标记的BMMSC，与CA/CPR组相比，BMMSC组大脑皮质细胞凋亡和脑损伤明显改善，NDS评分显著升高（70.6±2.1 vs. 56.6±5.5，P<0.05），BMMSC组血清S100B水平较低（ng/L：32.0±3.2 vs. 45.1±4.7，P<0.05），大脑皮质细胞凋亡率显著降低[（31.1±3.4）% vs.（52.9±11.8）%，P<0.05]，BMMSC组大脑皮质中活性caspase-8表达显著降低（caspase-8/GAPDH：0.
