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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 investigate the protective mechanism of Shenfu injection on lung injury induced by pigs with cardiac arrest and asphyxia model. Methods Twenty-four healthy inbred Wuzhishan miniature pigs were treated with tracheal intubation and occlusion to establish the model of cardiac arrest in cardiac arrest and standard cardiopulmonary resuscitation (CPR). Twenty-eight of them successfully returned to spontaneous circulation (ROSC) The random number table was divided into two groups: Shenfu group (n = 9): continuous intravenous injection of Shenfu injection at 0.24mg / min after ROSC until 6h after resuscitation; saline group (n = 9) Immediately after ROSC, continuous intravenous infusion of the same dose and speed of saline until 6h after resuscitation. Oxygenation index (OI), respiration index (RI) and oxygen delivery (Oxygen delivery) were measured by gas analyzer at baseline, immediately after ROSC, and after 15min, 30min, 1h, 2h, 4h and 6h after ROSC. DO2, VO2, O2ER, PaCO2 and LAC were measured and the animals were monitored for lung compliance (Cdyn), airway resistance (Raw), extravascular Pulmonary Water Index (EVLWI) and Pulmonary Vascular Permeability Index (PVPI)]. The activities of Na + -K + -ATPase, Ca2 + -ATPase, SOD and MDA, TNF- α and IFN- γ were measured by enzyme-linked immunosorbent assay The levels of IL-4 and IL-4 were measured and the IFN-γ / IL-4 levels were calculated. Apoptosis was measured by TUNEL method and the apoptotic index (AI) was calculated. The Bcl-2 and Bax protein concentrations were detected by immunohistochemical staining. 2 value; Western blot detection of Caspase-3 protein. Results The survival rate in SHP group was 88.9% (8/9) and the survival rate in saline group was 66.7% (6/9) 6 h after ROSC. The mean survival time in SHP group (5.77 ± 0.71) h was longer than that in saline group (4.77 ± 0.59) h, no significant difference between the two groups (P> 0.05). OI and Cdyn decreased significantly at ROSC and RI, DO_2, VO_2, O_2ER, Raw, EVLWI, PVPI, PaCO_2 and LAC immediately after ROSC in both groups compared with basal state (P < 0.05), and all the indexes recovered with the prolongation of time. Compared with the saline group, the levels of OI, Cdyn, DO_2, VO_2 and O2ER in the Shenfu group were significantly higher than those in the saline group at various time points after ROSC (P < 0.05, P <0.01), while RI, Raw, EVLWI, PVPI, PaCO_2 and LAC were lower than saline group (P <0.05, P <0.01). Compared with saline group, the levels of Na ~ + -K ~ + -ATPase, Ca ~ (2 +) - ATPase, SOD, IFN-γ, IFN- γ / IL-4 and Bcl- While the levels of MDA, TNF-α, IL-4, AI, Bax / Bcl-2 and Caspase-3 were decreased (P <0.05, P <0.01). Conclusion Shenfu injection can improve cellular energy metabolism, increase cellular antioxidant capacity and reduce oxidative stress, reduce the release of inflammatory mediators and regulate Thl / Th2 balance, while reducing lung cell apoptosis in order to achieve cardiopulmonary resuscitation After lung injury protection.