Imipramine对小鼠急性肺损伤肺泡上皮屏障功能保护作用的研究

发布时间：2018-03-08 14:26

  本文选题：imipramine　切入点：紧密连接蛋白　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的急性肺损伤(Acute Lung Injury,ALI)/急性呼吸窘迫综合症(Acute Respiratory Distress Syndrome,ARDS)均是呼吸内科常见的危重疾患,且具有较高的病死率。目前,临床上仍缺乏有效的治疗措施。在ALI发生发展中最显著的病理变化是弥漫性肺泡上皮细胞损伤,因此,增强肺泡上皮细胞屏障功能可成为治疗ALI一个靶点。Imipramine是常见的三环类抗抑郁药,也是动物实验中常用的酸性鞘磷脂酶(Acid Sphingomyelinase,ASMase)生物活性抑制剂,其具有抗炎、抗凋亡的作用。本文主要探讨imipramine对脂多糖(LPS)诱导的小鼠ALI肺泡上皮屏障功能是否有保护作用,并探讨其可能机制。方法将SPF级雄性Balb/c小鼠随机分为4组,即正常对照组、Imipramine组、LPS组、LPS+Imipramine组。采用LPS(20mg/kg)腹腔注射建立小鼠ALI模型,并在腹腔注射LPS前30分钟给予imipramine(25mg/kg)干预,处死前10 min,给予尾静脉注射FITC-FD4,12 h后麻醉下放血处死小鼠,随后取出支气管肺泡灌洗液(BALF)及肺组织。常规HE染色观察肺组织病理变化及病理学评分;肺组织湿/干比(W/D)和BALF/血清FD4比值的测定分别评估肺水肿和肺泡上皮细胞通透性;采用real-time PCR、Western blot和免疫组织化学方法检测不同组紧密连接蛋白Occludin、Claudin-4和ZO-1的表达情况;免疫荧光法检测每组ASMase的生物活性。数据分析采用SPSS 16.0软件,多组间的均数比较采用单因素方差分析,组间两两比较采用LSD-t检验。结果腹腔注射LPS诱导小鼠ALI,模型简单、稳定且均匀,肺组织病理学变化符合ALI病理改变。正常对照组和Imipramine组小鼠肺组织病理学无明显变化,LPS+Imipramine组肺组织病理学评分小于LPS组,差异具有统计学意义(P0.001);LPS+Imipramine组肺组织湿/干比小于LPS组,差异具有统计学意义(P=0.001);LPS+Imipramine组较LPS组相比,BALF/血清FD4比值下降差异具有统计学意义P=0.002);与LPS组相比,LPS+Imipramine组小鼠肺紧密连接蛋白Occludin、Claudin-4、ZO-1 m RNA及蛋白的表达水平均升高(P0.05);免疫组化结果显示,紧密连接蛋白Occludin、Claudin-4主要位于肺泡上皮细胞膜,ZO-1主要位于肺泡上皮细胞胞浆内,在正常对照组和Imipramine组蛋白表达明显,LPS组蛋白表达降低(P0.05),LPS+Imipramine组较LPS组蛋白表达有所恢复(P0.05);免疫荧光法检测ASMase活性结果显示,LPS+Imipramine组较LPS组相比,ASMase活性降低,差异具有统计学意义(P0.05),说明Imipramine能抑制应激状态下ASMase的活性,进而减少神经酰胺的生成。结论通过腹腔注射LPS建立小鼠ALI在ALI动物模型中值得推广;Imipramine可改善小鼠ALI病理变化,降低肺水肿程度;Imipramine可能通过抑制应激状态下ASMase的活性,减少神经酰胺的生成,进而改善肺泡上皮细胞紧密连接蛋白的表达以增强肺泡上皮细胞的屏障功能,最终保护LPS诱导的小鼠ALI。
[Abstract]:Objective Acute Lung injury / acute Respiratory Distress Syndrome ARDS are common serious diseases in respiratory medicine department, and have high mortality. The most significant pathological change in the development of ALI is diffuse alveolar epithelial cell injury. Enhancing the function of alveolar epithelial cell barrier is a target of ALI. Imipramine is a common tricyclic antidepressant and a bioactive inhibitor of acid sphingolipase acid sphingomyelinase (ASMase). To investigate the protective effect of imipramine on ALI alveolar epithelial barrier induced by lipopolysaccharide (LPS) and its possible mechanism. Methods male Balb/c mice of SPF grade were randomly divided into 4 groups. The ALI model of mice was established by intraperitoneal injection of LPS-20 mg / kg. The mice were treated with imipramine 25mg / kg 30 minutes before intraperitoneal injection of LPS. The mice were sacrificed 10 min before death and anesthetized with FITC-FD412 h after intravenous injection of FITC-FD412 h. Then the bronchoalveolar lavage fluid (BALF) and lung tissue were taken out. The pathological changes and pathological scores of lung tissue were observed by routine HE staining, the lung tissue wet / dry ratio (WR / D) and the ratio of serum FD4 of BALF/ were measured to evaluate pulmonary edema and alveolar epithelial cell permeability, respectively. Real-time PCR Western blot and immunohistochemistry were used to detect the expression of cladin-4 and ZO-1 in different groups, and immunofluorescence assay was used to detect the bioactivity of ASMase in each group. The data were analyzed by SPSS 16.0 software. Single factor analysis of variance (ANOVA) and LSD-t test were used to compare the mean of multiple groups. Results the model was simple, stable and uniform in mice induced by intraperitoneal injection of LPS. The lung histopathological changes were consistent with the pathological changes of ALI in normal control group and Imipramine group. The lung histopathological score in Imipramine group was lower than that in LPS group, and the difference was statistically significant (P 0.001). The wet / dry ratio of lung tissue in LPs Imipramine group was lower than that in LPS group. Compared with LPS group, the ratio of BALF-1 / serum FD4 in LPS-LPs group was significantly lower than that in LPS group, and the expression of clondin-4ZO-1 m RNA and protein in LPS-LPs Imipramine group was significantly higher than that in LPS-#en4# group (P 0.05). Claudin-4 is mainly located in the cytoplasm of alveolar epithelial cells, and ZO-1 is mainly located in the cytoplasm of alveolar epithelial cells. In the normal control group and the Imipramine histone expression group, the expression of LPS histone protein was significantly decreased in the Imipramine group compared with that in the LPS group, and the results of immunofluorescence assay showed that the ASMase activity in the LPS-#en4# group was lower than that in the LPS group. The difference was statistically significant (P 0.05), which indicated that Imipramine could inhibit the activity of ASMase and decrease the production of ceramide. Conclusion the establishment of mouse ALI by intraperitoneal injection of LPS is worth popularizing in ALI animal model, which can improve the pathological changes of ALI in mice. Reducing the degree of pulmonary edema and imipramine may enhance the barrier function of alveolar epithelial cells by inhibiting the activity of ASMase and reducing the production of ceramide, and then improving the expression of tight junction protein in alveolar epithelial cells. The final protection of LPS induced ALI in mice.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563.8
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本文编号：1584275