参附注射液对窒息法心脏骤停动物模型复苏后肺损伤的影响

Protective Effect of Shenfu Injection on Postresuscitation Lung Injury in a Porcine Model of Asphysia-induced Cardiac Arrest

目的通过猪心脏骤停窒息模型探讨参附注射液对此类肺损伤保护作用的可能机制。方法34只健康近交系五指山小型猪使用气管插管夹闭方法制作窒息型心脏骤停动物模型及行标准的心肺复苏术,其中18只成功恢复自主循环(return of spontaneous circulation,ROSC),使用随机数字表法分为两组:参附组(n=9):于ROSC后即刻以参附注射液0.24mg/min的剂量持续静脉泵入直至复苏后6h;盐水组(n=9):于ROSC后即刻持续静脉泵入相同剂量及速度的生理盐水直至复苏后6h。通过血气分析仪测量基础状态、ROSC即刻、ROSC后15min、30min、1h、2h、4h及6h的氧代谢及呼吸力学指标[包括:氧合指数(OI)、呼吸指数(RI)、氧输送(DO2)、氧消耗(VO2)、氧摄取(O2ER)、二氧化碳分压(PaCO2)及血乳酸(LAC),并监测同一时刻动物的肺顺应性(Cdyn)、气道阻力(Raw)、血管外肺水指数(EVLWI)和肺血管通透性指数(PVPI)];酶联免疫吸附法测定Na+-K+-ATP酶活性、Ca2+-ATP酶活性、SOD及MDA含量、TNF-α、IFN-γ和IL-4浓度,计算IFN-γ/IL-4值;利用TUNEL法检测细胞凋亡并计算凋亡指数(AI);免疫组织化学法检测Bcl-2、Bax蛋白浓度,计算BAX/BCL-2值;Western blot法检测Caspase-3蛋白定量。结果至ROSC后6h,参附组存活率为88.9%(8/9),盐水组存活率为66.7%(6/9);参附组平均生存时间(5.77±0.71)h长于盐水组(4.77±0.59)h,两组比较差异无统计学意义(P>0.05)。与基础状态比较,两组的OI和Cdyn在ROSC即刻明显降低(P<0.05),而RI、DO_2、VO_2、O_2ER、Raw、EVLWI、PVPI、PaCO_2和LAC在ROSC即刻则显著升高(P<0.05),各指标不论升高或降低均随时间延长而恢复;与盐水组比较,参附组在ROSC后的各个时间点,OI、Cdyn、DO_2、VO_2和O2ER显著高于盐水组(P<0.05,P<0.01),而RI、Raw、EVLWI、PVPI、PaCO_2和LAC则低于盐水组(P<0.05,P<0.01)。与盐水组比较,参附组Na^+-K^+-ATP酶、Ca^(2+)-ATP酶、SOD水平、IFN-γ水平、IFN-γ/IL-4值及Bcl-2浓度升高,而MDA、TNF-α、IL-4水平、AI、Bax/Bcl-2及Caspase-3蛋白水平降低(P<0.05,P<0.01)。结论参附注射液能改善细胞能量代谢,提高细胞的抗氧化能力并减少氧化应激,减少炎症介质的释放和并调节Thl/Th2的平衡,同时减轻肺组织细胞凋亡,从而实现对心肺复苏后肺损伤的保护。

Objective To observe the protective effect of Shenfu Injection （SFI） on post-resusci- tation lung injury in a porcine model of asphyxia-induced cardiac arrest. Methods Thirty-four anaesthe- tized Wuzhi Mountain inbred miniature piglets of both sexes were subjected to asphyxia by intubation clipping, followed by standard cardiopulmonary resuscitation. Eighteen successfully resuscitated pigs [ with recovery of return of spontaneous circulation （ROSC） ] were divided into the SFI group and the normal saline （NS） group according to random digit table, 9 in each group. SFI at 0.24 mg/min was intravenously pumped to piglets in the SFI group immediately from ROSC to 6 h after resuscitation, while NS at 0.24 mg/min was intravenously pumped to piglets in the NS group immediately from ROSC to 6 h after resuscitation. Oxygen metabolism, respiratory mechanics indices including oxygenation index （OI）, respiration index （RI）, oxygen delivery （DO2）, oxygen consumption （VO2）, oxygen extraction ratio （O2ER）, PaCO2, lactic acid （LAC） were detected using blood gas analyzer at basic state, immediately after ROSC, 15 and 30 min, 1,2,4, and 6 h after ROSC. Dynamic lung compliance （Cdyn）, airway resistance （Raw）, external vascular lung water index （EVLWI）, pulmonary vascular permeability index （PVPI） were monitored at each aforesaid time point. Activities of Na ＋-K ＋-ATPase and Ca2＋-ATPase, contents of SOD and MDA, concentrations of TNF-α, IFN-γ, and IL-4 were determined using ELISA.IFN-,y/IL-4 ratio was calculated. Cell apoptosis was detected using TUNEL and apoptotic index （AI） calculated. Protein concentrations of Bcl-2 and Bax were detected using immunohistochemical assay, and Bax/Bcl-2 ratio calculated. Caspase-3 protein was quantitatively detected using Western blot. Results The survival rate was 88.9% （8/9） in the SFI group and 66.7% （6/9） in the NS group at 6 h after ROSC. The mean survival time was （5.77±0.71） h in the SFI group, longer than that in the NS group [ （4.77 ±0.59）h, P 〉0.05]. Compared with the basic state, OI and Cdyn obviously decreased immediately after ROSC （P 〈0.05）; RI, DO2, VO2, O2ER, Raw, EVLWI, PVPI, PaCO2, and LAC obviously increased immediately after ROSC （P 〈0.05）. All indices were recovered as time went by. Compared with the NS group, OI, Cdyn, DO2, VO2, and 02 ER at each time points after ROSC were significantly higher in the SFI group than in the NS group （P 〈0.05, P 〈0.01 ） ; RI, Raw, EVLWI, PVPI, PaCO2, and LAC were significantly lower in the SFI group than in the NS group （P 〈0.05, P 〈0.01 ）. Compared with the NS group, activities of Na＋-K ＋-AT- Pase and Ca2＋-ATPase, contents of SOD, level of IFN-γ, IFN-γ/IL-4 ratio, concentrations of Bcl-2 in- creased more; MDA, TNF-（x, IL-4 level, AI, Bax/Bcl-2 ratio, Caspase-3 protein level decreased more （P 〈0.05, P 〈0.01 ）. Conclusion SFI could improve cell energy metabolism, enhance antioxidant capacity of cells, reduce the release of inflammatory mediators, regulate the Thl/Th2 balance, and attenuate cell apoptosis of lung tissue, thereby protecting post-resuscitation lung injury.