论文标题:已酮可可碱对内毒素血症大鼠心肌损伤保护作用的实验研究
The Experimental Study of the Protective Effect of Pentoxifylline on Myocardial Injury in Rats with Endotoxemia
论文作者
论文导师 郑智,论文学位 博士,论文专业 急诊医学
论文单位 华中科技大学,点击次数 210,论文页数 85页File Size1913K
2006-04-01论文网 http://www.lw23.com/lunwen_1248872/
Lipopolysaccharide (LPS);; Endotoxemia (ETM);; InhibitoryκB (I-κB);; Nuclear factor-κB (NF-κB);; Tumor necrosis factor-α(TNF-α);; Interleukin-1β(IL-1β);; Intercellular adhesion molecule-1 (ICAM-1);; Heat shock protein70 (HSP70);; Pentoxifylline (PTX)
内毒素血症是现代临床危重病医学面临的棘手问题,也是影响患者预后的重要因素。在内毒素血症中,心脏是最常受累器官之一。一旦出现心脏并发症,患者病情急剧恶化,死亡率会大幅度上升。由于内毒素血症发病机制复杂,目前临床上尚缺乏足够有效的治疗方法。因此,寻找各种有效的治疗方法,加强内毒素血症患者心肌保护变得至关重要。已酮可可碱是甲基黄嘌呤衍生物,是一种非选择性磷酸二酯酶抑制剂,是一种非特异性的外周血管扩张剂,可以降低血管阻力,改善脑和周围血循环。同时,可以改善红细胞变形能力,降低纤维蛋白浓度,抑制血小板聚集,改善缺血组织微循环环境,多用于治疗周围血管性疾病。现在,越来越多研究发现PTX具有选择性抗炎作用,对内毒素血症机体组织和细胞可以产生有益的保护作用。本研究建立大鼠急性内毒素血症模型,以已酮可可碱作为研究对象,观察心肌组织内NF-κB、I-κB、ICAM-1、HSP70的表达及血清TNF-α、IL-1β水平的变化,旨在探讨内毒素血症大鼠心肌损伤的发生机制及PTX在内毒素血症大鼠心肌损伤中的保护作用及机制,从而为临床PTX的应用提供理论依据。 第一部分NF-κB、I-κB在内毒素血症大鼠心肌组织中的表达及已酮可可碱的影响 目的:探讨NF-κB、I-κB在内毒素血症大鼠心肌组织中的表达及已酮可可碱的影响。 方法:采用直接注射内毒素的方法建立大鼠急性内毒素血症模型。18只Wistar大鼠随机分成3组:①假手术组(S);②内毒素组(L);③PTX组(P)。内毒素组在10min内通过颈静脉注入内毒素5mg/Kg,其后给予生理盐水对照(总剂量5 ml/Kg),用法同PTX组。PTX组在10min内通过颈静脉注入内毒素5mg/Kg,其后在10min内静脉注入PTX(剂量10mg/Kg),注入完成后,通过微量输液泵泵注PTX(剂量15mg/Kg)维持50min。采用免疫组织化学法检测心肌组织NF-κB、I-κB蛋白,免疫印迹法检测心肌组织NF-κB蛋白,并观察各组大鼠心肌组织的病理形态。 结果:内毒素组免疫组织化学法检测心肌组织NF-κB、I-κB蛋白水平与假手术组比较明显增强(P<0.01),免疫印迹法检测心肌组织NF-κB蛋白水平与假手术组比较也明显增强(P<0.01);予PTX干预后,免疫组织化学法和免疫印迹法检测心肌组织NF-κB蛋白水平与内毒素组比较明显减弱(P<0.01),而免疫组织化学法检测心肌组织I-κB蛋白水平与内毒素组比较无明显差异(P>0.05),内毒素组心肌组织的病理损害明显加重,PTX干预后,损害减轻。 结论:NF-κB、I-κB均参与了内毒素血症心肌损伤的发生,PTX可以通过抑制NF-κB活化,减轻心肌损伤,保护心肌组织。 第二部分血清TNF-α、IL-1β和心肌组织ICAM-1在内毒素血症大鼠中的变化及已酮可可碱的影响 目的:探讨血清TNF-α、IL-1β和心肌组织ICAM-1在内毒素血症大鼠中的变化及已酮可可碱的影响。 方法:采用直接注射内毒素的方法建立大鼠急性内毒素血症模型。18只Wistar大鼠随机分成3组:①假手术组(S);②内毒素组(L);③PTX组(P)。内毒素组在10min内通过颈静脉注入内毒素5mg/Kg,其后给予生理盐水对照(总剂量5 ml/Kg),用法同PTX组。PTX组在10min内通过颈静脉注入内毒素5mg/Kg,其后在10min内静脉注入PTX(剂量10mg/Kg),注入完成后,通过微量输液泵泵注PTX(剂量15mg/Kg)维持50min。采用放射免疫分析法检测血清TNF-α、IL-1β水平,免疫组织化学法检测心肌组织ICAM-1蛋白水平。 结果:内毒素组放射免疫分析法检测血清TNF-α、IL-1β水平与假手术组比较明显增强(P<0.01),免疫组织化学法检测心肌组织ICAM-1蛋白水平与假手术组比较也明显增强(P<0.01);予PTX干预后,血清TNF-α、IL-1β水平与内毒素组比较明显减弱(P<0.01),免疫组织化学法检测心肌组织ICAM-1蛋白水平与内毒素组比较也明显减弱(P<0.01)。 结论:血清TNF-α、IL-1β及心肌组织内ICAM-1参与了内毒素所致心肌损伤,PTX可以作用于TNF-α、IL-1β及ICAM-1,下调其水平,从而减轻LPS对心肌组织造成的损伤,保护心肌组织。 第三部分热休克蛋白70在内毒素血症大鼠心肌组织中的表达及已酮可可碱的影响 目的:探讨热休克蛋白70在内毒素血症大鼠心肌组织中的表达及已酮可可碱的影响。 方法:采用直接注射内毒素的方法建立大鼠急性内毒素血症模型。18只Wistar大鼠随机分成3组:①假手术组(S);②内毒素组(L);③PTX组(P)。内毒素组在10min内通过颈静脉注入内毒素5mg/Kg,其后给予生理盐水对照(总剂量5 ml/Kg),用法同PTX组。PTX组在10min内通过颈静脉注入内毒素5mg/Kg,其后在10min内静脉注入PTX(剂量10mg/Kg),注入完成后,通过微量输液泵泵注PTX(剂量15mg/Kg)维持50min。分别采用免疫组织化学法和免疫印迹法检测心肌组织HSP70蛋白。 结果:内毒素组免疫组织化学法检测心肌组织HSP70蛋白水平与假手术组比较明显增强(P<0.01),免疫印迹法检测心肌组织HSP70蛋白水平与假手术组比较也明显增强(P<0.01);予PTX干预后,免疫组织化学法检测心肌组织HSP70蛋白水平与内毒素组比较明显增强(P<0.01),免疫印迹法检测心肌组织HSP70蛋白水平与内毒素组比较也明显增强(P<0.01)。 结论:内毒素血症大鼠心肌组织内的HSP70蛋白表达可明显增高,PTX干预可以进一步增高内毒素血症大鼠心肌组织内的HSP70蛋白表达,从而进一步加强对心肌的保护。
The endotoxemia is a delicate problem to modern critical care medicine, and is also an important factor affecting the patient’prognosis. In the endotoxemia, the heart is one of the organs, which are liable to be affected. Once the heart complication appearing, the patient’s condition will worsen suddenly and the mortality will increase. Because the endotoxemia’s pathogenesis is complex, there are not enough effective methods to treat it. Therefore, seeking a effective method of treatment and strengthening the protection of patient’s myocardium are more and more important. Pentoxifylline is a methylxanthine derivative, non-specific phosphodiesterase inhibitor and non-specific circumference blood vessel vasodilator. It can reduce the blood vessel resistance and improve the blood circulation of the brain and periphery. At the same time, it can improve the red blood cell distortion ability, reduce the fibrin density, suppress platelet gathering and improve the microcirculation. It is used in the diseases of periphery vessels. Now, more and more researches have discovered that PTX has the selective anti- inflammation function and may have the beneficial protective function to the tissue and cell in endotoxemia. In the research, we established the model of acute endotoxemia, took pentoxifylline as the research object and observed the changes of the expression of NF- kappa B, I- kappa B, ICAM-1 and HSP70 in myocardium and levels of TNF- alpha, IL-1 beta in serum. The aim is to discuss the mechanism of myocardial injury in rats with endotoxemia and PTX’s function and mechanism in the myocardial injury in rats with endotoxemia, which can provide the basic theory for PTX’s application. Part One Expression of NF-κB and I-κB in myocardial tissue in rats with endotoxemia and effects of pentoxifylline Objective:To study the expression of NF-κB and I-κB in myocardial tissue in rats with endotoxemia and effects of penyoxifylline. Methods:Set the models of rats with acute endotoxemia by the method of injecting LPS directly. Wistar rats were randomly divided into 3 groups①sham operated control (S);②LPS (L);③PTX (P). In the LPS group, rats were given LPS (5mg/Kg) through the neck vein in 10 minutes, then were given the physiological saline comparison (total dose 5 ml/Kg). Method is the same as the PTX group. In the PTX group, rats were given LPS (5mg/Kg) through the neck vein in 10 minutes, then were given the PTX(10mg/Kg) through the neck vein in 10 minutes. After injection, PTX(10mg/Kg) were pumped into the rats by micro-pump in 50 minutes. The immunohistochemistry was used to assess the expression of the NF-κB and I-κB in the myocardial tissues. The Western blotting was used to assess the expression of the NF-κB in the myocardial tissues. The pathologic lesions of myocardial tissues were observed. Results:The levels of NF-κB and I-κB in the myocardial tissues in L group are higher than those in sham operated control group (P<0.01) (immunohistochemistry). The levels of NF-κB in the myocardial tissues in L group are also higher than those in sham operated control group (P<0.01) (Western blotting). After PTX treatment ,the levels of NF-κB in the myocardial tissues in P group are lower than those in L group (P<0.01) (immunohistochemistry, Western blotting), but the levels of I-κB in the myocardial tissues in P group are no different with those in L group (P>0.05) (immunohistochemistry). The pathologic lesions of myocardial tissues in P group are less serious than those in L group. Conclusion: NF-κB and I-κB take part in the progress of myocardial injury in rats with endotoxemia. PTX can eliminate the myocardial injury and protect the myocardial tissues by inhibiting the activation of NF-κB. Part Two Expression of TNF-αand IL-1βin serum and ICAM-1 in myocardial tissue in rats with endotoxemia and effects of pentoxifylline Objective:To study the expression of TNF-αand IL-1βin serum and ICAM-1 in myocardial tissue in rats with endotoxemia and effects of penyoxifylline. Methods:Set the models of rats with acute endotoxemia by the method of injecting LPS directly. Wistar rats were randomly divided into 3 groups①sham operated control (S);②LPS (L);③PTX (P). In the LPS group, rats were given LPS (5mg/Kg) through the neck vein in 10 minutes, then were given the physiological saline comparison (total dose 5 ml/Kg). Method is the same as the PTX group. In the PTX group, rats were given LPS (5mg/Kg) through the neck vein in 10 minutes, then were given the PTX(10mg/Kg) through the neck vein in 10 minutes. After injection, PTX(10mg/Kg) were pumped into the rats by micro-pump in 50 minutes. The radioimmunoassay was used to assess the levels of TNF-αand IL-1βin serum. The immunohistochemistry was used to assess the expression of ICAM-1 in the myocardial tissues. Results:The levels of TNF-αand IL-1βin serum in L group are higher than those in sham operated control group (P<0.01) (radioimmunoassay). The levels of ICAM-1 in the myocardial tissues in L groups are higher than those in sham operated control group (P<0.01) (immunohistochemistry). After PTX treatment ,the levels of TNF-αand IL-1βin serum in P group are lower than those in L group (P<0.01) (radioimmunoassay), and the levels of ICAM-1 in the myocardial tissues in P group are also lower than those in L group (P<0.01) (immunohistochemistry). Conclusion: TNF-α, IL-1βand ICAM-1 take part in the progress of myocardial injury in rats with endotoxemia. PTX can eliminate the myocardial injury and protect the myocardial tissues by down regulating the levels of TNF-α, IL-1βand ICAM-1. Part Three Expression of heat shock protein 70 in myocardial tissue in rats with endotoxemia and effects of pentoxifylline Objective:To study the expression of HSP70 in myocardial tissue in rats with endotoxemia and effects of pentoxifylline. Methods:Set the models of rats with acute endotoxemia by the method of injecting LPS directly. Wistar rats were randomly divided into 3 groups①sham operated control (S);②LPS (L);③PTX (P). In the LPS group, rats were given LPS (5mg/Kg) through the neck vein in 10 minutes, then were given the physiological saline comparison (total dose 5 ml/Kg). Method is the same as the PTX group. In the PTX group, rats were given LPS (5mg/Kg) through the neck vein in 10 minutes, then were given the PTX(10mg/Kg) through the neck vein in 10 minutes. After injection, PTX(10mg/Kg) were pumped into the rats by micro-pump in 50 minutes. The immunohistochemistry and western blotting were all used to assess the expression of HSP70 in the myocardial tissues. Results:The levels of HSP70 in the myocardial tissues in L group are higher than those in sham operated control group (P<0.01) (immunohistochemistry). The levels of HSP70 in the myocardial tissues in L group are also higher than those in sham operated control group (P<0.01) (Western blotting). After PTX treatment, the levels of HSP70 in the myocardial tissues in P group are higher than those in L group (P<0.01) (immunohistochemistry), and the levels of HSP70 in the myocardial tissues in P group are also higher than those in L group (P<0.01) (western blotting). Conclusion: The expression of HSP70 in myocardial tissues elevated in rats with endotoxemia. PTX can eliminate the myocardial injury and protect the myocardial tissues by further elevating the expression of ICAM-1.
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