高频振荡通气联合呼气末正压递增法对烟雾吸入性损伤犬心肌缺血缺氧和心肌细胞凋亡的影响

Effects of high frequency oscillatory ventilation combined with incremental positive end-expiratory pressure on myocardial ischemia and hypoxia and apoptosis of cardiomyocytes in dogs with smoke inhalation injury

目的比较高频振荡通气（HFOV）联合呼气末正压递增法（IP）和单纯HFOV对烟雾吸入性损伤犬心肌缺血缺氧及心肌细胞凋亡的影响。方法将12只健康雄性犬按随机数字表法分为HFOV组和HFOV＋IP组，每组6只。常规机械通气后将犬造成重度烟雾吸入性损伤，随后分别行相应通气治疗8h。治疗结束后立即心脏采血收集血浆标本，检测心肌型肌酸激酶同工酶（CK－MB）、乳酸脱氢酶同工酶1（LDH1）活性。之后处死犬，取部分心肌组织，采用ELISA法检测每克心肌组织中TNF－α含量；取部分心肌组织，采用流式细胞仪检测细胞凋亡率；取部分心肌组织行HE染色，观察心肌组织的缺氧情况；取部分心肌组织行生物素－链霉亲和素-过氧化物酶染色，检测肌动蛋白的定性及定量表达（数据以累积吸光度值表示）。对数据行t检验，对大鼠每克心肌组织中TNF．仪含量及心肌细胞凋亡率行Spearman直线相关分析。结果（1）HFOV＋IP组犬治疗8h时血浆CK－MB和LDH1活性分别为（734±70）、（182±15）U／L，均明显低于HFOV组的（831±79）、（203±16）U／L，t值分别为2．25、2．35，P值均小于0．05。（2）与HFOV组的（0．060±0．018）仙g比较，HFOV＋IP组犬治疗8h时每克心肌组织中TNF—d含量明显降低[（0．040±0．011）μg，t=2．32，P〈0．05]。（3）与HFOV组的（33．4±2．2）％比较，HFOV＋IP组犬治疗8h时心肌细胞凋亡率显著降低[（28．2±3．4）％，t=3．15，P〈0．05]；每克心肌组织中TNF—α的含量与心肌细胞凋亡率呈显著正相关（r=0．677，P〈0．05）。（4）HE染色显示，2组犬心肌纤维均呈现不同程度的波浪状排列，即有不同程度的缺氧表现，其中HFOV＋IP组的缺氧程度较轻。（5）免疫组织化学染色显示，与HFOV组比较，HFOV＋IP组犬心肌纤维中肌动蛋白脱失程度较轻。HFOV＋IP组犬治疗8h时心肌组织肌动蛋白表达量为194．7±3．1，明显高于HFOV组的172．9±2．6，t=13．20，P〈0．01。结论与单纯HFOV模式比较，HFOV联合IP可减轻犬心肌组织炎症反应，减少心肌细胞凋亡，缓解心肌缺血缺氧。

Objective To compare the effects of high frequency oscillatory ventilation （HFOV） combined with incremental positive end-expiratory pressure （IP） and those of pure HFOV on myocardial ischemia and hypoxia and apoptosis of cardiomyocytes in dogs with smoke inhalation injury. Methods Twelve healthy male dogs were divided into group HFOV and group HFOV ＋ IP according to the random number table, with 6 dogs in each group. After being treated with conventional mechanical ventilation, dogs in both groups were inflicted with severe smoke inhalation injury, and then they received corresponding ventilation for 8 hours respectively. After treatment, the blood samples were collected from heart to determine the activity of creatine kinase-MB （CK-MB） and lactate dehydrogenase 1 （LDH1） in plasma. The dogs were sacrificed later. Myocardium was obtained for determination of content of TNF-α per gram myocardium by ELISA, apoptotic rate of cardiomyocytes by flow cytometer, degree of hypoxia with HE staining, and qualita-tive and quantitative expression of actin （denoted as integral absorbance value） with streptavidin-biotin-peroxidase staining. Data were processed with t test. The relationship between the content of TNF-α per gram myoeardium and the apoptotic rate of cardiomyocytes was assessed by Spearman linear correlation analysis. Results （ 1 ） After treatment for 8 h, the values of activity of CK-MB and LDH1 in plasma of dogs in group HFOV ＋ IP were respectively （734 ＋70） and （ 182 ＋ 15） U/L, which were both lower than those in group HFOV [ （ 831 ± 79） and （203 ±16） U/L, with t values respectively 2.25 and 2.35, P values below 0.05 ]. （2） Compared with that in group HFOV [ （0. 060 ± 0.018 ） μg ], the content of TNF-α per gram myocardium of dogs in group HFOV ＋ IP after treatment for 8 h was decreased significantly [ （0. 040 ± 0. 011 ） txg, t = 2.32, P 〈 0.051. （3） Compared with that in group HFOV [ （33.4± 2.2） % , the apptotic rate of cardiomyocytes of dogs in group HFOV ＋ IP after treatment for 8 h was significantly decreased [ （28.2 ± 3.4） % , t = 3.15, P 〈 0.05 ]. There was a positive correlation between the content of TNF-α per gram myocardium and the apoptotic rate of cardiomyocytes （ r = 0. 677, P 〈 0.05 ）. （4） HE staining showed that myocardial fibers of dogs in both groups were arranged in wave shape in different degrees, indicating there was myocardial hypoxia in different degrees. Compared with that of group HFOV, the degree of hypoxia in group HFOV ＋ IP was slighter. （5） The results of immunohistochemical staining showed that there was less loss of actin in myocardial fibers of dogs in group HFOV ＋ IP than in group HFOV. The expression level of actin in myocardium of dogs in group HFOV ＋ IP after treatment for 8 h （ 194.7 ± 3.1 ） was obviously higher than that in group HFOV （ 172.9 ± 2.6, t = 13.20, P 〈 0.01 ）. Conclusions Compared with pure HFOV, HFOV combined with IP can alleviate the inflammatory reaction in myocardium of dogs, reduce the apoptosis of cardiomyocytes, and ameliorate the myocardial damage due to ischemia and hypoxia.