摘要
急性肺损伤(ALI)是指由心源性以外的各种肺内外致病因素导致的急性、进行性缺氧性呼吸衰竭,临床常见,死亡率高。内毒素是革兰阴性菌壁外膜成分,化学结构为脂多糖(LPS),是ALI的主要致病因素之一,目前尚无有效的抗内毒素治疗药物。
目的
戊乙奎醚是受体选择性抗胆碱药,具有细胞保护作用,能提高细胞对缺血、缺氧的耐受性,稳定溶酶体和线粒体等亚细胞结构,减少溶酶体酶的释放,抑制花生四稀酸代谢物的产生和休克因子的形成,减轻全身炎症反应。但戊乙奎醚减轻肺损伤的机制并不明确,本研究通过研究戊乙奎醚预处理对内毒素诱导新生大鼠急性肺损伤炎症反应的作用,探讨戊乙奎醚治疗急性肺损伤的机制。
方法
40只健康7 d Wistar大鼠随机分为4组(n=10)：生理盐水对照组(NS组,腹腔注射生理盐水)、模型组(LPS组,腹腔注射内毒素5mg/kg);低剂量戊乙奎醚干预急性肺损伤组(PL)和高剂量戊乙奎醚干预急性肺损伤组(PH),分别腹腔注射戊乙奎醚0.1mg/kg和0.5mg/kg,30min后腹腔注射内毒素5mg/kg。注射LPS4h,10%水合氯醛350mg/kg腹腔注射麻醉后活杀新生大鼠,留取组织标本。RT-PCR法测定肺组织TLR4及CD14 mRNA表达,ELISA法检测肺组织肿瘤坏死因子-α、白介素1β、白介素10的含量,光镜、电镜下观察肺组织病理形态学改变。
结果
光镜观察：NS组肺组织结构正常,LPS组肺组织大量中性粒细胞浸润,肺泡壁水肿,部分肺泡壁破坏严重,肺泡内可见大量红细胞和炎症细胞,PH组肺泡结构存在,中性粒细胞浸润明显减轻,肺泡内可见少量红细胞,与LPS组相比较,PL组肺损伤稍有减轻。
电镜观察：NS组肺组织超微结构正常,LPS组可见肺泡Ⅱ型上皮细胞游离面微绒毛断裂；板层小体体积增大,密度减低,排空明显增强；线粒体大部分嵴缺失、模糊不清；；PL组和PH组肺组织超微结构变化较LPS组减轻。
与NS组相比较,LPS组、PL组及PH组肺组织TLR4和CD14 mRNA表达水平均升高(P<0.05),与L组比较,PL组和PH组肺组织TLR4和CD14 mRNA表达水平均降低(P<0.05),与PL组比较,PH组TLR4和CD14 mRNA表达水平降低(P<0.05)。
与NS组相比较,TNF-α、IL-1β、IL-10含量皆升高(P<0.05),与L组比较,PL组和PH组肺组织TNF-α、IL-1β、IL-10含量均降低(P<0.05),与PL组比较,PH组TNF-α、IL-1β降低(P<0.05),IL-10含量的变化没有统计学意义(P>0.05)。
结论
盐酸戊乙奎醚肺保护作用机制可能与抑制LPS受体CD14和TLR4表达,阻断炎症信号传导通路有关,进而减轻炎症反应,阻止ALI的发展。
Acute lung injury(ALI) is an acute progressive respiratory failure induced by various reaseons except cardiogenic factors with high mortality. Lipopolysaccharide(LPS), a mojor component of Gram-negative bacterial endotoxin, is the leading cause of ALI. So far, there are still no effective antiendotoxin drugs.
Objective
Penehyclidine Hydrochoride injection is the new development anti-choline medicine, can improve the cells'toleration on ischemia and hypoxia, stabilize subcellular structure of cytolysosome and chondriosome,reduse the releasing of lysosomal enzyme,inhibite the information of metabolite of arachidonic acid and shock factors,release the systemic inflammatorome responded. However, the precise mechanism are not as yet fully understood. Our goal is to investigate the protective mechanism of penehychdine hydrochloride against acute lung injury induced by endotoxin in neonatal rats.
Methods
Forty 7 days old Wistar rats were randomly divided into 4 groups(n=10 each): group NS, which was treated identically to the group LPS except for the substitution of 0.9% saline for LPS; group LPS,which was given LPS injection intraperitoneally (5mg/kg) to reproduce the ALI rats models; group PL, which received intraperitoneal injection of penehychdine hydrochloride (0.1mg/kg) while they were induced by intraperitoneal infection of LPS; group PH,which received intraperitoneal injection of penehychdine hydrochloride (0.5 mg/kg) just after the infection of LPS. After 4 hours, they were killed. Then the lungs were removed.Levels of CD14 and TLR4 mRNA in lung were determined with RT-PCR.The expression of TNF-α, IL-1β, IL-10 in the lung tissues were detected by enzyme linked immune adsorbing analysis (ELISA). Furthermore, the lung pathologic structure were observed under the optical microscope and transmission electron microscope.
By microscopy,each group had classic change presenting acute lung injury which included, the infiltration of inflammatory cells, alveolar septum widen,the leakage of erythrocyte except group A,which had normal lung construction. Group LPS had most severe pathologic changes.In the group PH, the according pathological changes were slighter than group PL.
Under the transmission electron microscope, in the group LPS, the microvilli on the surface of ATⅡcells disappeared;lamellar bodyincreased enlarged in size and reduced in density; The number of mitochondria increased, and some of them became swollen and enlarged. The pathological changes were attenuated in group PL and PH with the latter being even more improved.
There was significant difference on expression of CD14 and TRL4 in lung of neonatal rats among four groups.Compared with NS group,the expression of CD14 and TRL4 of lung tissues in the group LPS increased obviously(P<0.05). Compared with LPS group, the level of CD14 and TRL4 of lung tissues in the group PL and PH decreased (P<0.05). Furthermore, group PH was lower than that of group PL.
Content of TNF-α, IL-1βand IL-10 protein were significantly higher in group L, PL and PH than in group C (P<0.05) and were significantly lower in group PH1 and PH2 than in group L (P<0.05).
Conclusions
Penehychdine hydrochloride has the protective effect on ALI.The mechanisms of the effect are due to its ability of inhibiting the expression of CD14 and TRL4, thereby inhibit the development of uncontrolled inflammation at the development of ALI.
引文
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