Gu Wei, Li Chunsheng, Yin Wenpeng, Hou Xiaomin
Department of Emergency Medicine, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China. Corresponding author: Li Chunsheng, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2015 Mar;27(3):190-6. doi: 10.3760/cma.j.issn.2095-4352.2015.03.007.
To examine whether Shenfu injection (SFI) reduces post-resuscitation myocardial dysfunction in a pig model by modulating expression imbalance of transcription factors of regulatory T cell, namely GATA-3 and T-bet.
Thirty pigs were randomly divided into sham group (n = 6) and cardiopulmonary resuscitation (CPR) group (n = 24) according to the random number table method, and the pigs in the CPR group were randomly subdivided into normal saline (NS) group, epinephrine (EP) group, and SFI group (n = 8 per group). After 8 minutes of untreated ventricular fibrillation (VF) followed by 2 minutes of CPR, animals in three groups respectively received central venous injection of either 20 mL SFI (1.0 mL/kg, SFI group), EP (0.02 mg/kg, EP group) or NS (NS group). Blood samples were obtained before VF and 0.5, 2, 6 hours after restoration of spontaneous circulation (ROSC), and the parameters of hemodynamics and oxygen metabolism were determined. Surviving pigs were sacrificed at 24 hours after ROSC, the pathological changes in myocardium were observed, the levels of interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α) and γ-interferon (IFN-γ) were measured by enzyme linked immunosorbent assay (ELISA), and expressions of protein and mRNA of GATA-3 and T-bet were determined by Western Blot and quantitative real-time polymerase chain reaction (RT-qPCR), respectively.
Six pigs of three resuscitation groups were successfully resuscitated. The CPR time, number of defibrillation, defibrillation energy, and ROSC time were significantly decreased in the EP and SFI groups compared with those in the NS group. Compared with the sham group, the parameters of left ventricular systolic function and oxygen metabolism were significantly decreased, myofibril organelles were extensively damaged, and progressive and severe deterioration of the myocardium was found, and mitochondrial structure was not recognizable in the NS group; the level of IL-4 in myocardium were markedly decreased, while that of TNF-α, IFN-γ and IFN-γ/ IL-4 [reflecting helper T cell 1/2 (Th1/Th2)] were significantly increased. Protein and mRNA expressions of GATA-3 were markedly reduced in the myocardium of pigs in the NS group compared with that of the sham group at 24 hours after ROSC, while T-bet was significantly increased. Compared with the NS group, animals treated with SFI had minimal myocardial intracellular damage, with decreased heart rate (HR, bpm: 90.33 ± 3.79 vs. 106.83 ± 5.36) and increased mean arterial pressure (MAP), cardiac output (CO), oxygen delivery (DO₂), and oxygen consumption (VO₂) at 6 hours after ROSC [MAP (mmHg, 1 mmHg = 0.133 kPa): 107.67 ± 1.96 vs. 86.83 ± 1.85, CO (L/min): 2.47 ± 0.08 vs. 2.09 ± 0.04, DO₂ (mL/min): 364.31 ± 4.21 vs. 272.33 ± 3.29, VO₂(mL/min): 95.00±2.22 vs. 82.50 ±2 .28, all P < 0.05]. Compared with the NS groups at 24 hours after ROSC, level of IL-4 was markedly increased in myocardial cells (ng/L: 33.80 ± 3.06 vs. 16.15 ± 1.34, P < 0.05), while the levels of TNF-α, IFN-γ and IFN-γ/IL-4 were lowered significantly [TNF-α (ng/L): 18.16 ± 0.71 vs. 29.64 ± 1.89, IFN-γ (ng/L): 373.75 ± 18.36 vs. 512.86 ± 27.86, IFN-γ/IL-4: 16.15 ± 1.34 vs. 33.80 ± 3.06, all P < 0.05], and myocardial T-bet protein and mRNA expressions were reduced [T-bet protein (gray value): 0.41 ± 0.07 vs. 0.59 ± 0.11, T-bet mRNA (2(-ΔΔCt)): 4.37 ± 0.21 vs. 7.57 ± 0.55, both P < 0.05], furthermore, myocardial GATA-3 protein and mRNA expressions were significantly up-regulated in SFI group [GATA-3 protein (gray value): 0.25 ± 0.07 vs. 0.16 ± 0.07, GATA-3 mRNA (2(-ΔΔCt)): 0.63 ± 0.07 vs. 0.34 ± 0.05, both P < 0.05]. The parameters in SFI group were significantly improved compared with those of the EP group.
Myocardial immune dysfunction is induced by Th1/Th2 imbalance following myocardial injury subsequent to CPR in pigs. SFI can attenuate myocardial injury and regulate myocardial immune disorders, protect post-resuscitation myocardial injury by modulating expression imbalance of transcription factors GATA-3 and T-bet.
通过调节调节性T细胞转录因子GATA-3和T-bet的表达失衡，探讨参附注射液（SFI）对猪模型复苏后心肌功能障碍的影响。
采用随机数字表法将30头猪随机分为假手术组（n = 6）和心肺复苏（CPR）组（n = 24），CPR组猪再随机分为生理盐水（NS）组、肾上腺素（EP）组和SFI组（每组8头）。经8分钟非处理性室颤（VF）后进行2分钟CPR，三组动物分别经中心静脉注射20 mL SFI（1.0 mL/kg，SFI组）、EP（0.02 mg/kg，EP组）或NS（NS组）。于VF前及自主循环恢复（ROSC）后0.5、2、6小时采集血样，测定血流动力学和氧代谢参数。存活猪于ROSC后24小时处死，观察心肌病理变化，采用酶联免疫吸附测定（ELISA）法检测白细胞介素-4（IL-4）、肿瘤坏死因子-α（TNF-α）和γ-干扰素（IFN-γ）水平，分别采用蛋白质印迹法和定量实时聚合酶链反应（RT-qPCR）法测定GATA-3和T-bet的蛋白及mRNA表达。
三个复苏组各有6头猪成功复苏。与NS组相比，EP组和SFI组的CPR时间、除颤次数、除颤能量及ROSC时间均显著缩短。与假手术组相比，NS组左心室收缩功能和氧代谢参数显著降低，肌原纤维细胞器广泛受损，心肌呈进行性、重度恶化，线粒体结构无法辨认；心肌中IL-4水平显著降低，而TNF-α、IFN-γ及IFN-γ/IL-4[反映辅助性T细胞1/2（Th1/Th2）]显著升高。ROSC后24小时，NS组猪心肌中GATA-3的蛋白和mRNA表达较假手术组显著降低，而T-bet显著升高。与NS组相比，SFI处理的动物心肌细胞内损伤最小，ROSC后第6小时心率（HR，bpm：90.33±3.79比106.83±5.36）降低，平均动脉压（MAP）、心输出量（CO）、氧输送（DO₂）及氧消耗（VO₂）升高[MAP（mmHg，1 mmHg = 0.133 kPa）：107.67±1.96比86.83±1.85，CO（L/min）：2.47±0.08比2.09±0.04，DO₂（mL/min）：364.31±4.21比272.33±3.29，VO₂（mL/min）：95.00±2.22比82.50±2.28，均P < 0.05]。与ROSC后24小时的NS组相比，SFI组心肌细胞中IL-4水平显著升高（ng/L：33.80±3.06比16.15±1.34，P < 0.05），而TNF-α、IFN-γ及IFN-γ/IL-4水平显著降低[TNF-α（ng/L）：18.16±0.71比29.64±1.89，IFN-γ（ng/L）：373.75±18.36比512.86±27.86，IFN-γ/IL-4：16.15±1.34比33.80±3.06，均P < 0.05]，且心肌T-bet蛋白和mRNA表达降低[T-bet蛋白（灰度值）：0.41±0.07比0.59±0.11，T-bet mRNA（2(-ΔΔCt)）：4.37±0.21比7.57±0.55，均P < 0.05]，此外，SFI组心肌GATA-3蛋白和mRNA表达显著上调[GATA-3蛋白（灰度值）：0.25±0.07比0.16±0.07，GATA-3 mRNA（2(-ΔΔCt)）：0.63±0.07比0.34±0.05，均P < 0.05]。SFI组的参数较EP组显著改善。
猪CPR后心肌损伤可导致Th1/Th2失衡，进而引起心肌免疫功能障碍。SFI可减轻心肌损伤，调节心肌免疫紊乱，通过调节转录因子GATA-3和T-bet的表达失衡保护复苏后心肌损伤。
