摘要
第一部分慢性间歇性低氧对大鼠左心功能的影响及作用机制研究
目的
本研究旨在建立慢性间歇性低氧大鼠模型模拟睡眠呼吸暂停,通过特异性的NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)在转录水平进行干预,了解慢性间歇性低氧对大鼠左心室功能的影响,及NF-κB和心肌细胞凋亡在其中的作用,初步探讨睡眠呼吸暂停对左心室功能的影响及发生机制。
材料和方法
选取体重200g左右的雄性大鼠24只,随机分为慢性间歇性低氧组(CIH组)、干预组(NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(PDTC组))、正常对照组(NC组),每组8只。CIH组大鼠每天9:00am～5:00pm置于慢性间歇性低氧舱内8小时,PDTC组大鼠根据体重腹腔注射PDTC 10mg/kg·d,与CIH组大鼠同时置于相同的慢性间歇性低氧舱内8小时,NC组大鼠在与CIH组相同的饲养舱但为正常氧浓度环境饲养。每周测量体重,饲养35天结束时,鼠尾测压仪测定血压和心率,超声心动图检查测定左心室舒张末期内径、左心室收缩末期内径、左心室短轴缩短率和左心室射血分数。对大鼠麻醉进行心导管检查,测定平均动脉压、左心室舒张末期压力、左心室收缩期压力上升最大速率、左心室舒张期压力下降最大速率等参数,检查结束后将大鼠处死取材,称量整体心脏重量,并分离心室后称量左心室重量,然后取靠近心尖部心肌组织置于10%福尔马林中固定24h,常规石蜡包埋切片,TUNEL方法检测心肌细胞凋亡。选取约100mg环状左心室心肌组织冷冻,用于提取蛋白进行Western blot检测心肌细胞caspase 3、caspase 8、Fas、Bax、Bcl-2及NF-κB蛋白表达水平。采用SPSS 13.0进行数据资料分析。P＜0.05认为具有统计学显著差异。
结果
1.慢性间歇性低氧对大鼠心功能的影响及NF-κB的作用
第5周实验结束时NC组体重明显大于CIH组和PDTC组。左心室占体重的比值CIH组明显高于NC组(分别为3.04±0.24‰和2.53±0.16‰,P＜0.001),PDTC组左心室占体重比值较CIH组明显减小(分别为2.82±0.21‰和3.04±0.24‰,P=0.035)。CIH组和PDTC组血压明显高于NC组(三组分别为136.3±6.8mmHg、134.3±6.7mmHg和122.3±4.1mmHg,P＜0.001)。超声心动图检查显示左室射血分数CIH组明显低于NC组(分别为73.0±5.6%和86.0±4.3%,P＜0.001),PDTC组较CIH组明显升高(分别为84.0±4.1%和73.0±5.6%,P＜0.001),与NC组没有明显差异(P=0.462)。
5周时的导管数据显示CIH组和PDTC组平均动脉压明显高于NC组,分别为86.7±4.7mmHg、85.2±4.1mmHg和80.7±3.2mmHg,P=0.013。左心室舒张末压力CIH组明显高于NC组(分别为13.6±1.0和5.7±0.5,P＜0.001),CIH组±dp/dt_(max)明显低于NC组;PDTC组左心室舒张末压力较CIH组明显降低(分别为6.2±3.0mmHg和13.6±1.0mmHg,P＜0.001),±dp/dt_(max)较CIH组明显升高。PDTC组与NC组比较没有明显差异(P＞0.05)。各组间心率无明显差异。
2.慢性间歇性低氧对大鼠心肌细胞凋亡的影响及NF-κB的作用
TUNEL方法检测凋亡细胞,CIH组凋亡指数明显高于NC组(分别为17.5%和1.9%,P＜0.001),PDTC组调亡指数为10.1%,较CIH组明显降低(P＜0.001),但仍高于NC组(P＜0.001)。此外我们采用Western blot测定凋亡实施蛋白caspase3表达水平,半定量分析显示caspase 3与β-actin灰度比值CIH明显高于NC组(分别为0.035和0.010,P＜0.001),进一步证实慢性间歇性低氧大鼠心肌细胞凋亡增多。相关性分析显示超声心动图检查左心室射血分数与心肌细胞凋亡指数呈负相关(r=-0.719,P＜0.001),与caspase3呈负相关(r=-0.784,P＜0.001)。心导管测量左心室舒张末压力与心肌细胞凋亡指数呈正相关(r=0.807,P＜0.001),与caspase3呈正相关(r=0.847,P＜0.001)。
3.慢性间歇性低氧对大鼠心肌细胞凋亡影响的通路及NF-κB的作用
Western blot检测左室心肌细胞凋亡途径相关的蛋白水平,发现与NC组相比,CIH组Fas、caspase 8表达均明显增加(P＜0.001),而抑制凋亡调节蛋白Bcl-2表达降低(P＜0.001),促凋亡蛋白Bax两组间没有明显差异(P=0.213);与PDTC相比也有类似发现,但PDTC组与NC组间没有统计学差异(P＞0.05)。
结论
1.慢性间歇性低氧可引起大鼠平均动脉压增高,左心室收缩和舒张功能降低,经PDTC干预后左心室功能改善;
2.慢性间歇性低氧可引起大鼠心肌细胞凋亡增加,经PDTC干预后心肌细胞凋亡减少,大鼠左心室功能降低与心肌细胞凋亡相关;
3.慢性间歇性低氧可能通过NF-κB活化,转录调节使促凋亡基因Fas表达增加而抑凋亡基因Bcl-2表达降低,心肌细胞凋亡增加,影响左心室收缩和舒张功能。
第二部分睡眠呼吸暂停对心力衰竭患者预后的影响
目的
睡眠呼吸暂停的特点是睡眠中反复的呼吸暂停或低通气,导致间歇性低氧和复氧、睡眠结构异常、交感激活等病理生理改变,从而对循环系统产生不利影响。但是,目前文献有关睡眠呼吸暂停对心力衰竭预后的影响报道尚不一致,本研究目的在于评价睡眠呼吸暂停对心力衰竭预后的影响,包括死亡、心脏移植和因心力衰竭加重再次住院等。
方法
前瞻性病例对照研究。2004年11月～2005年5月,阜外心血管病医院心衰病房和五病房中,符合以下条件的患者:①年龄大于18岁;②超声心动图检查左室射血分数小于45%;③NYHA心功能分级Ⅱ-Ⅳ级,进行多导睡眠图监测,根据监测结果分为心力衰竭不合并睡眠呼吸暂停组(CHF-N)、心力衰竭合并阻塞性睡眠呼吸暂停组(CHF-OSA)和心力衰竭合并中枢性睡眠呼吸暂停组(CHF-CSA),定期进行随访,随访过程中进行最优化药物治疗,随访至2008年5月9日结束。主要终点事件为全因死亡,次要终点事件为死亡、心脏移植和因心力衰竭加重再次住院。随访结束时比较各组间死亡率以及联合事件的发生,观察睡眠呼吸暂停对心力衰竭患者预后的影响。
结果
148例入选患者进行睡眠监测,其中20例患者由于接受了大于7天的无创呼吸机正压通气治疗被排除。128例患者(105例男性,23例女性),平均年龄55.6岁(18～80岁),左室射血分数平均为35.4%。睡眠呼吸监测发现,23例患者(18%)不合并睡眠呼吸暂停(CHF-N),55例(43%)患者合并阻塞性睡眠呼吸暂停(CHF-OSA),50例患者(39%)合并中枢性睡眠呼吸暂停(CHF-CSA)。平均随访35个月(1～50月)。CHF-N组死亡率明显低于CSA组(分别为6.7每一百人年和18.7每一百人年,P=0.017),死亡组中SA发生率高于存活者(分别为89.5%和78.9%,P=0.008)。但是多元分析显示,矫正年龄和NYHA心功能分级等混杂因素后,CSA、OSA或SA的严重程度(呼吸暂停低通气指数和最低血氧饱和度)均不是心力衰竭患者死亡的独立预测因子。此外,三组患者中联合事件(死亡、心脏移植和因心力衰竭住院)的发生没有明显差别。以呼吸暂停低通气指数分别为10/h、15/h为睡眠呼吸暂停的诊断阈值,评价睡眠呼吸暂停对心力衰竭死亡率和预后的影响,生存分析显示尽管睡眠呼吸暂停组心力衰竭患者死亡率有增高的趋势,但是没有达到统计学意义。
结论
本组资料中单因素分析心力衰竭合并CSA患者存活率低于单纯心力衰竭患者,但矫正其它因素后SA(包括CSA或OSA)对心力衰竭患者的死亡率或预后未达到独立相关的统计学意义。
Objective:The aim of this study was to investigate the impact of chronic intermittent hypoxia(CIH) on cardiac function,and explore the role of NF-κB in the mechanisms of cardiac dysfunction due to CIH.
Methods:Twenty four adult male Sprague-Dawley rats(body weight about 200g) were randomly divided into three groups.The CIH group exposed to CIH(nadir O_2 6～7%),the PDTC group exposed to CIH and intraperitoneal injected proline dithiocarbamate(PDTC,special inhibitor of NF-κB)(10mg/kg·d),and the NC group was handled normoxic controls.Exposure last 8h/d for 5 weeks.At the end of the exposure,blood pressure was measured by tail cuff plethysmograph;left global function was accessed by echocardiographic and invasive cardiac catheterization. Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL). Expressions of apoptosis related proteins caspase 3,Fas,caspase 8,Bax,Bcl-2,and nucleoprotein NF-κB were detected by Western blot.
Results:1.The impact of CIH on cardiac function in rats.
At the end of the 5-weeks exposure,there were no significant differences among the groups in the weight gain.Compared with NC,CIH and PDTC animals demonstrated elevations in arterial pressure(122.3±4.1 mmHg vs.136.3±6.8mmHg, P＜0.001;122.3±4.1mmHg vs.134.3±6.7 mmHg,P＜0.001).Left ventriaclar ejection fraction measured by echocardiogram was lower in CIH than in NC(73.0±5.6%vs. 86.0±4.3%,P＜0.001),and was not significantly different between NC and PDTC (84.0±4.1%vs.86.0±4.3%,P=0.117).Compared with NC,left ventricular end-diastolic pressure was higher(6.2±3.0mmHg vs 13.6±1.0mmHg,P＜0.001),and maximal rates of left ventricular pressure in systole and diastole were lower in CIH (3764.3±784.8 vs 4894.9±651.7,P＜0.001).However,there were no significant differences between NC and PDTC.There were no significant differences in heart rate among the groups.
2.Apoptosis assays.
The incidences of TUNEL-positive cells from the CIH group was approximately seven folds greater compared with the NC group(17.5%vs.1.9%,P＜0.001). Expression of caspase 3 increased in CIH than in NC(3.5%vs.0.1%,P＜0.001), which showed myocyte apoptosis increases too.Pearson anylasis showed that LVEF was negatively related with apoptosis index(r=-0.792,P＜0.001) and with caspase 3 activity(r=-0.784,P＜0.001).Left ventricular end-diastolic pressure was positively related with apoptosis index(r=0.807,P＜0.001) and with caspase 3 activity(r= 0.847,P＜0.001).
3.Expression of apoptosis related proteins and the role of NF-κB.
Compared with NC,the expression of Fas,caspase 8 increased,Bcl-2 decreased in CIH(P＜0.001),but the expression of Bax was not different(P=0.213).Compared with PDTC,the findings in CIH are similar.However,there were no significant differences in Fas,caspase 8,Bcl-2 and Bax between the PDTC group and the NC group.
Conclusion:CIH may activate NF-κB,which upregulates Fas and downregulates Bcl-2.CIH may lead to increased apoptosis through activated NF-κB.NF-κB is the important factor in the impact of CIH on cardiac dysfunction.
Objective:The aim of this study was to assess the impact of untreated sleep apnea (SA) on mortality,heart transplantation,and hospitalization in patients with congestive heart failure(CHF) in China.
Methods:This was a prospective case controlled study.Patients who met criterias followed:age less than 18 years,left ventricular ejection fraction(LVEF) less than 35.4%and NYHA classⅡ～Ⅳ,were enrolled.The patients were performed ploysomonlography and were divided into three groups.They were followed up regularly.At the end of follow-up,we compared mortality and combined events (including death,transplantation,and hospitalization due to heart failure) among the three groups.
Results:128 CHF patients were enrolled in this study.Eighteen percent had no SA (CHF-N),43%had obstructive sleep apnea(CHF-OSA),and 39%had central sleep apnea(CHF-CSA).Mortality was significantly greater in the CHF-CSA group than in the CHF-N group(18.2 vs 6.7 per 100 person-years,P=0.017),and the survivors had more prevalence of SA(78.9%) than the non-survivors(89.5%).However, multivariate analysis identified age and NYHA class function but not CSA,OSA,or the severity of SA as predictors for survival in heart failure.In addition,the percentages of combined events were not significantly different among three groups. When we defined presence of SA as apnea-hyponea index above 10/h and 15/h respectively,mortality and combined etents rate were not significantly different among the groups.
Conclusion:Untreated SA(OSA or CSA) has no significant impact on the prognosis of patients with congestive heart failure in China.
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