【作者】 沈伟锋；

【导师】 江观玉；

【作者基本信息】 浙江大学 ， 外科学， 2006， 博士

【摘要】 急性肺损伤(acute lung injury,ALI)是一种多种原因引起的肺部失控性炎症反应导致的肺泡毛细血管损伤为基本病理生理特点的综合征,常继发于休克、严重创伤、感染等,病情凶险、预后差,可进一步发展为多器官功能衰竭,因此,防治本类疾病在外科急危重领域具有非常重要的意义。 药物干预在ALI防治中具有重要作用,目前,较为确定的ALI治疗药物有一氧化氮吸入、外源性肺泡表面活性物质、糖皮质激素等,但存在疗效不满意和/或不良反应严重的问题,至今仍无安全有效的治疗药物。莨菪类药物已应用于ALI治疗多年,但因传统莨菪类药物如山莨菪碱等的作用机制不甚明了和副作用大,限制了莨菪类药物的临床应用。盐酸戊乙奎醚(penehyclidine hydrochloride,PHC)是中国原创的新型选择性的莨菪类药物,因对M胆碱能受体亚型具有选择性,临床应用副作用少,扩大了莨菪类药物的应用范围和适应证,近来研究表明,盐酸戊乙奎醚能改善微循环、降低毛细血管壁的通透性、细胞保护和减少溶酶体释放等作用,提示盐酸戊乙奎醚可能对急性肺损伤具有治疗作用,因此,本项目将对新型莨菪类药物盐酸戊乙奎醚对急性肺损伤的保护作用进行深入的研究。综合大量文献,中性粒细胞大量肺内扣押、活化和进一步的脂质过氧化等造成肺微血管内皮细胞和肺泡上皮细胞等损伤,最终导致更多还原

【Abstract】 Acute lung injury (ALI) is a cause of acute respiratory failure that develops in patients of all ages from a variety of clinical disorders, including sepsis (pulmonary and nonpulmonary), pneumonia (bacterial, viral, and fungal), aspiration of gastric and oropharyngeal contents, major trauma, and several other clinical disorders including severe acute pancreatitis, drug overdose, and blood products. ALI is common and remains a significant cause of morbidity and mortality in critically ill patients.Penehyclidine hydrochloride (PHC), a new anticholinerigic drug invented by the Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences in the People’s Republic of China. It is noteworthy that PHC can decrease significantly the nasal mucosa capillary permeability. Thus, PHC is potentially beneficial for treatment of ALI. Recently, studies have confirmed PHC can improve the function of microcirculation during open heart surgery under cardiopulmonary bypass (CPB) with heart beating. In this study we aimed to investigate whether PHC has in vivo protective effects on ALI induced by LPS.Neutrophils mediate tissue injury and have been implicated in the pathogenesis of several important diseases, including those that may trigger ALI. Neutrophilssequestered in the lung play a central role in the pathogenesis of ALI induced by LPS. The acute phase of ALI is characterized by the influx of protein-rich edema fluid into the air spaces as a consequence of increased permeability of the alveolar-capillary barrier. The importance of endothelial injury and increased vascular permeability to the formation of pulmonary edema in this disorder has been well established. Therefore, the protective effect of PHC in vivo models of ALI in this study has been examined by these indices as we mentioned above.Elevated levels of proinfiammatory mediators combined with a decreased expression of anti-inflammatory molecules are critical components of lung inflammation and play a main role in ALI. The signals that lead to increased gene expression and biosynthesis of proinflammatory mediators by inflammatory and epithelial cells are thus of considerable interest. Recent experimental studies appeared to have shed some light on the intracellular signaling pathway in the inflammatory cascade in ALI. For example, recent evidence strongly suggested the crucial role of NF-kB in the initiation of ALI. Additionally, recent evidence has also shown that MAP kinases are involved in modulating the expression of many cytokines and other proinflammatory mediators implicated in the development and progression of ALI. An in vitro experiment showed that cytokine expression of PMNs was mediated by two separate signal pathways, i.e, NF-kB and MAP kinase, of which MAP kinase is now suggested to play a major role. Four subfamilies of MAP kinases, i.e. ERK, JNK, p38 and ERK5 participate in intracellular signaling cascades resulting in inflammatory responses. Inhibition of these pathways has been advocated as a novel therapeutic strategy for inflammatory diseases. Inhibition of the MAP kinases pathways may form the basis of a new strategy for treatment of ALI. The coordination and interaction of MAP kinase pathways and NF-kB cascade may play a key role in LPS induced gene expression. In this study, we also aimed to investigate whether the therapeutic effect of PHC in the treatment of ALI could—atleast in part-be brought about by its direct interference with the LPS-induced activation of NF-kappaB, p38, ERK and JNK.This study was divided into two chapters and four parts.Chapter onePartiProtective effect of penehyclidine hydrochloride pretreatment onlipopolysaccharide-induced acute lung injury in ratsObjective The aim of this study was to examine the effects of PHC pretreatmenton LPS-induced ALI in rats.Methods Forty eight adult male Sprague-Dawley rats weighing between 190 and230g were used. ALI was induced by intravenous injection of lipopolysaccharide(LPS). Five groups of rats were treated with LPS (5 mg/kg), LPS plus PHC (3.0, 1.0,0.3 mg/kg) or LPS plus methylprednisolone (MP) (30 mg/kg) for 6 hours. PHC andMP were administered at 30 minutes before LPS challenge. One group of ratsdesigned as control was treated with saline. Arterial blood gas analysis, lungpermeability index (LPI^ wet to dry weight ratio (W/D) and protein content inbronchoalveolar lavage fluid (BALF) were measured. Pathological changes of lungtissue were measured by light and electron microscopy.Results LPS-challenged rats had significantly increased arterial partial pressure ofcarbon dioxiden W/D ratio ^ LPL protein content in BALF and decreased partialpressure of oxygen of arterial blood, compared to controls. Compared with LPStreatment, PHC pretreatment significantly decreased arterial partial pressure ofcarbon dioxide n W/D ratio > LPL protein content in BALF and enhanced partialpressure of oxygen of arterial blood. Lung interstitial edema, diffuse hemorrhage, alarge amount of red blood cells and neutrophils infiltration in the alveoli, increase ofplasma protein exudate, formation of transparent membrane, and thickening of thealveolar wall were seen under light microscopy and the defects of endothelial cells and type II pneumocytes were shown under transmission electron microscope in LPS-challenged rats, PHC pretreatment significantly attenuated these pathological changes.Conclusions These results demonstrate that PHC can attenuate acute lung injury and exert protective effect on ALI induced by LPS. Further studies are required to evaluate the safety and clinical benefits of penehyclidine hydrochloride administration in ALI.Part IIPretreatment of PHC is beneficial to LPS-induced ALI in rats through inhibiting the lung neutrophil sequestration and oxygen free radicals injury Objective The aim of this study was to examine whether pretreatment of PHC is beneficial to LPS-induced ALI in rats through inhibiting the lung neutrophil sequestration and oxygen free radicals injury.Methods Forty eight adult male Sprague-Dawley rats weighing between 190 and 230g were used. ALI was induced by intravenous injection of LPS. Five groups of rats were treated with LPS (5 mg/kg), LPS plus PHC (3.0, 1.0, 0.3 mg/kg) or LPS plus methylprednisolone (MP) (30 mg/kg) for 6 hours. PHC and MP were administered at 30 minutes before LPS challenge. One group of rats designed as control was treated with saline. The ratio of neutrophiles in bronchoalveolar lavage fluid (BALF^ the activities of superoxide dismutase (SOD^ myeloperoxidase (MPO), lactate dehydrogenase (LDH) and malondialdehyde (MDA) content in blood serum and lung were measured.Results LPS-challenged rats had significantly increased MDA leveK MPO and LDH activity % the ratio of neutrophiles in BALF, and decreased SOD activity compared to controls. Compared with LPS treatment, PHC pretreatment significantlydecreased MDA level > MPO and LDH activity, the ratio of neutrophiles in BALF, and dramatically enhanced SOD activity.Conclusions It is suggested that pretreatment of penehyclidine hydrochloride is beneficial to LPS-induced ALI in rats through inhibiting the lung neutrophil sequestration and oxygen free radicals injury.Chapter two PartiProtective effect of penehyclidine hydrochloride on ALI rats induced by lipopolysaccharide via inhibition of NF-kappaB activation Objective We aimed to investigate whether the therapeutic effect of PHC in the treatment of ALI could~at least in part-be brought about by its direct interference with the LPS-induced activation of NF-kappaB.Methods ALI was induced by intravenous injection of lipopolysaccharide (LPS). It is projected that PHC at dose of 3.0 mg/kg will be administered at 0.5h before injection of LPS or saline and lung tissues at Oh, 2h, 4h, 6h, and 12h were taken for measurement of the levels of phosphorylated NF-kappaB by western blot analysis to examine the effects of PHC on activation of NF-kappaB in time-dependent manners. PHC at doses of 3.0 mg/kg, 1.0 mg/kg, and 0.3 mg/kg were administered at 0.5h before injection of LPS or saline and lung tissues at 6 hours were taken for measurement of the levels of phosphorylated NF-kappaB by western blot analysis to examine the effects of PHC on activation of NF-kappaB in dose-dependent manners. Results Compared to controls, LPS -challenged rats had significantly increased the levels of phosphorylated NF-kB in lung tissues. A high and middle level dose of PHC pretreatment resulted in a drastic down-regulation of phospho-NF-kappaB. PHC significantly attenuated LPS-induced phospho-NF-kappaB expression in dose-dependent and time-dependent manners.Conclusions These results suggest that NF-kappaB participates in the signal transduction in LPS induced acute lung injury. PHC relieves the inflammation in acute lung injury rats possibly through the inhibition of NF-kappaB activation.Part IIProtective effect of penehyclidine hydrochloride on ALI rats induced by lipopolysaccharide via inhibition of p38 mitogen-activated protein kinase-* c-Jun N-Terminal kinase and extracellular signal-regulated kinase activation Objective We aimed to investigate whether the therapeutic effect of PHC in the treatment of ALI could~at least in part—be brought about by its direct interference with the LPS-induced activation of p38 mitogen-activated protein kinase > c-Jun N-Terminal kinase and extracellular signal-regulated kinase activation. Methods ALI was induced by intravenous injection of lipopolysaccharide (LPS). It is projected that PHC at dose of 3.0 mg/kg will be administered at 0.5h before injection of LPS or saline and lung tissues at Oh, 2h, 4h, 6h, and 12h were taken for measurement of the levels of phosphorylated p38 mitogen-activated protein kinase % c-Jun N-Terminal kinase and extracellular signal-regulated kinase by western blot analysis to examine the effects of PHC on activation of p38 mitogen-activated protein kinase > c-Jun N-Terminal kinase and extracellular signal-regulated kinase in time-dependent manners. PHC at doses of 3.0 mg/kg, 1.0 mg/kg, and 0.3 mg/kg were administered at 0.5h before injection of LPS or saline and lung tissues at 6 hours were taken for measurement of the levels of phosphorylated p38 mitogen-activated protein kinase, c-Jun N-Terminal kinase and extracellular signal-regulated kinase by western blot analysis to examine the effects of PHC on activation of p38 mitogen-activated protein kinase ^ c-Jun N-Terminal kinase and extracellular signal-regulated kinase in dose-dependent manners. Results Compared to controls, LPS-challenged rats had significantly increasedthe levels of phosphorylated p38 mitogen-activated protein kinase ^ c-Jun N-Terminal kinase and extracellular signal-regulated kinase activation in lung tissues. A high level dose of PHC pretreatment resulted in a drastic down-regulation of phospho-p38 mitogen-activated protein kinase and phosphor -extracellular signal-regulated kinase. PHC attenuated LPS-induced phospho-p38 mitogen-activated protein kinase > phospho-extracellular signal-regulated kinase expression in dose-dependent and time-dependent manners. The expression of phospho-c-Jun N-Terminal kinase was not significantly down-regulated in PHC pretreatment group. Conclusions These results suggest that p38 mitogen-activated protein kinase and extracellular signal-regulated kinase participate in the signal transduction in LPS induced acute lung injury. PHC relieves the inflammation in acute lung injury rats possibly through the inhibition of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase activation.In conclusion, our data suggest that PHC pretreatment attenuated LPS-induced lung injury in rats. This beneficial effect of PHC may involve, in part, inhibition of lung neutrophil sequestration and oxygen free radicals injury, possibly through inhibition of lung NF-kappaB,p38 mitogen-activated protein kinase and extracellular signal-regulated kinase activation. If these findings are confirmed in additional models, PHC may become a candidate for clinical testing in critical care.更多还原