辛伐他汀对COPD大鼠肺组织细胞凋亡的影响
发布时间：2019-06-02 00:34
【摘要】：[目的]通过观察辛伐他汀对慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)模型大鼠肺组织凋亡细胞及凋亡相关因子内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)及半胱氨酸蛋白酶-3(Caspase-3)蛋白和mRNA表达的影响,探讨辛伐他汀在COPD中的作用机制,为其在COPD临床治疗中的应用提供理论依据。 [方法] 1、将30只健康雄性Wistar大鼠随机分为：正常组(n=10)、对照组(n=10)和治疗组(n=10)。 2、采用熏香烟和气管内滴入脂多糖(lipopolysaccharides,LPS)的方法建立COPD大鼠模型,造模开始两周后,治疗组同时给予辛伐他汀(2.5mg/kg)灌胃治疗,治疗6周。 3、HE染色观察各组大鼠肺组织病理改变；采用脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记技术(TUNEL)观察各组大鼠肺组织中凋亡的细胞,并计算凋亡指数(AI)；应用免疫组织化学技术及RT-PCR检测各组大鼠肺组织中eNOS、iNOS和Caspase-3蛋白及mRNA的表达。 [结果] 1、HE染色肺组织病理学 正常组大鼠肺组织中气管黏膜假复层纤毛柱状上皮完整,气道周围无炎症细胞浸润,毛细血管无扩张充血,肺泡大小及结构正常。对照组大鼠气管和支气管黏膜纤毛柱状上皮层增厚,纤毛倒伏、粘连、坏死、脱落,粘膜下腺体肥大,粘膜下层和肌层大量炎症细胞浸润,血管周围大量炎性细胞浸润,毛细血管扩张充血,肺泡结构破坏,肺泡壁变薄断裂融合。治疗组大鼠气道纤毛柱状上皮细胞脱落、杯状细胞增生、血管周围炎性细胞浸润较对照组呈不同程度减轻,肺泡破坏断裂融合较对照组少。 2、病理形态学定量 对照组和治疗组大鼠肺组织中肺平均内衬间隔(Mean linear intercept,MLI)高于正常组(P0.05),治疗组MLI与对照组比较降低(P0.05)；对照组和治疗组大鼠肺组织中平均肺泡数(Mean alveolar numbers,MAN)少于正常组(P0.05),治疗组MAN与对照组比较增多(P0.05)。 3、TUNEL法检测肺组织细胞凋亡 大鼠肺组织中凋亡细胞的细胞核可被染为淡棕色至深棕色不等,主要为肺泡上皮细胞、气道上皮细胞,亦可见血管内皮细胞、血管、支气管平滑肌细胞及少量炎症细胞。对照组及治疗组与正常组比较,肺泡上皮细胞和气道上皮细胞的凋亡增多(P0.05)；与对照组比较,治疗组肺泡上皮细胞和气道上皮细胞凋亡减少(P0.05)。 4、SP法检测肺组织蛋白表达 eNOS表达于气道上皮细胞、肺泡上皮细胞和血管内皮细胞,对照组及治疗组与正常组比较,大鼠肺组织中eNOS表达减少(P0.05),与对照组比较,治疗组eNOS表达增多(P0.05)。iNOS主要表达于肺泡上皮细胞、血管内皮细胞和炎症细胞,对照组及治疗组与正常组比较,大鼠肺组织中iNOS表达增多P0.05),与对照组比较,治疗组iNOS表达减少(P0.05)。Caspase-3的表达分布于肺泡上皮细胞、气道上皮细胞、血管内皮细胞、气道和血管平滑肌细胞,对照组和治疗组与正常组比较,大鼠肺组织中Caspase-3表达增加(P0.05),与对照组比较,治疗组Caspase-3表达减少(P0.05)。 5、RT-PCR检测基因的表达 对照组及治疗组与正常组比较,大鼠肺组织中eNOS mRNA表达减少(P0.05),与对照组比较,治疗组eNOS mRNA表达增多(P0.05)。对照组和治疗组与正常组比较,iNOS和Caspase-3mRNA表达增多(P0.05),与对照组比较,治疗组iNOS和Caspase-3mRNA表达减少(P0.05)。 6、大鼠肺组织Caspase-3与eNOS及iNOS蛋白表达之间的相关性分析 正常组大鼠肺组织中Caspase-3与eNOS蛋白表达之间呈负相关(r=0.86,P0.05),与iNOS呈正相关(r=0.80,P0.05)；对照组Caspase-3与eNOS蛋白表达之间呈负相关(r=-0.67,P0.05),与iNOS呈正相关(r=0.77,P0.05)；治疗组Caspase-3与eNOS蛋白表达之间呈负相关(r=-0.85,P0.05),与iNOS呈正相关(r=0.88,P0.05)。 7、各组大鼠肺组织中Caspase-3与eNOS及iNOS mRNA表达之间的相关性分析 正常组大鼠肺组织Caspase-3与eNOS mRNA表达之间呈负相关(r=-0.86,P0.05),与iNOS mRNA表达呈正相关(r=0.94,P0.05)；对照组Caspase-3与eNOS mRNA表达之间呈负相关(r=-0.65,P0.05),与iNOS mRNA表达呈正相关(r=0.89,P0.05)；治疗组Caspase-3与eNOS mRNA表达之间呈负相关(r=-0.92,P0.05),与iNOS mRNA的表达呈正相关(r=O.90,PO.05)。 [结论] 1、辛伐他汀可以减轻COPD模型大鼠支气管及肺血管周围炎症。 2、辛伐他汀可以减轻COPD模型大鼠支气管及肺组织细胞凋亡。 3、辛伐他汀通过增加肺组织eNOS的表达,降低iNOS的表达,进而减少Caspase-3的表达,从而减轻COPD肺组织细胞凋亡,在COPD中发挥保护性作用,为其在COPD I临床治疗中的应用提供理论依据。
[Abstract]:[Objective] To observe the effect of simvastatin on the apoptosis and apoptosis-related factor endothelial nitric oxide synthase (eNOS) in the lung of rats with chronic obstructive pulmonary disease (COPD). The effect of simvastatin on the expression of inducible nitric oxide synthase (iNOS) and caspase-3 (Caspase-3) protein and mRNA was discussed. [Party Method] 1. Thirty healthy male Wistar rats were randomly divided into normal group (n = 10), control group (n = 10) and treatment group (n = 10). 10).2. The model of chronic obstructive pulmonary disease (COPD) was established by the method of dropping the lipopolysaccharides (LPS) into the cigarette and the trachea, and the treatment group was given simvastatin (2.5 mg/ kg) in the treatment group at the same time after the start of the model for two weeks. 3, HE staining was used to observe the pathological changes of lung tissue in each group, and the apoptotic cells in the lung tissues of each group were observed by the end-end labeling (TUNEL) of dUTP nick end-transferase mediated dUTP nick end labeling (TUNEL). The expression of eNOS, iNOS and Caspase-3 in lung tissues of each group was detected by immunohistochemistry and RT-PCR. mRNA Expression.[Results] 1. The tracheal mucosa pseudostratified ciliated columnar epithelium in the lung tissue of the normal group of HE stained lung tissue was intact, there was no inflammatory cell infiltration around the airway, and the capillary was not dilated. In the control group, the cilia of the trachea and the bronchial mucosa of the control group were thickened, the cilia lodging, the adhesion, the necrosis, the shedding, the hypertrophy of the submucosal glands, the infiltration of the submucosal and muscular layers of the inflammatory cells, the infiltration of a large number of inflammatory cells around the blood vessels, the capillary, vasodilation, congestion, alveolar junction in that treatment group, the ciliated columnar epithelial cells of the group of the treatment group fall off, the goblet cell hyperplasia and the infiltration of the peripheral inflammatory cells of the blood vessel are reduced to a different extent than the control group, Alveolar destruction and fracture fusion In the control group, the mean pulmonary lining interval (MLI) in the lung tissue of the control group and the treatment group was higher than that in the normal group (P0.05), and the mean number of alveoli in the lung tissues of the control group and the treatment group (Me An alveolar numbers (MAN) was less than that of the normal group (P0.05), and the treatment group M The number of AN and the control group increased (P0.05). .3. TUNEL method in the detection of apoptosis in the lung tissue of the lung tissue of rats can be stained with light brown to dark brown, mainly for the alveolar epithelial cells, airway epithelial cells, and also to the vascular endothelial cells. In the control group and the treatment group, the apoptosis of the alveolar epithelial cells and the airway epithelial cells increased (P0.05); and compared with the control group, the treatment group's alveoli Reduction of apoptosis in skin cells and airway epithelial cells (P0.05). The expression of eNOS in the lung tissue was detected by SP method in airway epithelial cells, alveolar epithelial cells and vascular endothelial cells. The expression of eNOS in the tissue was decreased (P0.05). The expression of eNOS in the treatment group was increased in comparison with the control group (P0.05). iNOS was mainly expressed in the alveolar epithelial cells, the vascular endothelial cells and the inflammatory cells, and the control group and the treatment group were compared with the normal group. The expression of iNOS in the lung tissue was increased (P0.05). The expression of Caspase-3 was decreased in the treatment group compared with the control group (P0.05). The expression of Caspase-3 was distributed in the alveolar epithelial cells, the airway epithelial cells, the vascular endothelial cells, the airway and the vascular smooth muscle cells, the control group and the treatment group were compared with the normal group, and the calcium in the lung tissues of the rats The expression of spase-3 was increased (P0.05) and compared with the control group. Table of Caspase-3 in the Treatment Group The expression of eNOS mRNA in the lung of rats was decreased (P0.05). Compared with the control group, the expression of eNOS mRNA in the treatment group increased (P0.05). Compared with the control group, the expression of iNOS and Caspase-3 mRNA was increased (P0.05). The expression of iNOS and Caspase-3 mRNA was decreased (P0.05). The correlation between the expression of Caspase-3 and eNOS and iNOS protein was negatively correlated with the expression of the expression of Caspase-3 and eNOS in the normal group (r = 0.86, P0.05). There was a negative correlation between the expression of e-3 and eNOS (r =-0.67, P <0.05) and positive correlation with iNOS (r = 0.77, P0.05); and the expression of Caspase-3 and eNOS in the treatment group was negatively correlated (r =-0. (85, P0.05), positive correlation with iNOS (r = 0.88, P0.05). There was a negative correlation between the expression of Caspase-3 and eNOS and iNOS mRNA in the normal group (r =-0.86, P0.05) and positive correlation with the expression of iNOS mRNA (r = 0.94, P0.05). There was a negative correlation between the expression of ase-3 and eNOS mRNA in the treatment group (r =-0.89, P0.05), and the expression of Caspase-3 and eNOS mRNA in the treatment group was negatively correlated (r =-0.92, P <0.05). P0.0 5) The expression of iNOS mRNA was positively correlated with the expression of iNOS mRNA (r = O.90, PO. 05).[Conclusion] 1, Simvastatin can reduce the COPD model rats 2. Simvastatin can reduce the expression of iNOS and decrease the expression of Caspase-3 by increasing the expression of eNOS in the lung, so as to reduce the COP.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.9
本文编号：2490693
[Abstract]:[Objective] To observe the effect of simvastatin on the apoptosis and apoptosis-related factor endothelial nitric oxide synthase (eNOS) in the lung of rats with chronic obstructive pulmonary disease (COPD). The effect of simvastatin on the expression of inducible nitric oxide synthase (iNOS) and caspase-3 (Caspase-3) protein and mRNA was discussed. [Party Method] 1. Thirty healthy male Wistar rats were randomly divided into normal group (n = 10), control group (n = 10) and treatment group (n = 10). 10).2. The model of chronic obstructive pulmonary disease (COPD) was established by the method of dropping the lipopolysaccharides (LPS) into the cigarette and the trachea, and the treatment group was given simvastatin (2.5 mg/ kg) in the treatment group at the same time after the start of the model for two weeks. 3, HE staining was used to observe the pathological changes of lung tissue in each group, and the apoptotic cells in the lung tissues of each group were observed by the end-end labeling (TUNEL) of dUTP nick end-transferase mediated dUTP nick end labeling (TUNEL). The expression of eNOS, iNOS and Caspase-3 in lung tissues of each group was detected by immunohistochemistry and RT-PCR. mRNA Expression.[Results] 1. The tracheal mucosa pseudostratified ciliated columnar epithelium in the lung tissue of the normal group of HE stained lung tissue was intact, there was no inflammatory cell infiltration around the airway, and the capillary was not dilated. In the control group, the cilia of the trachea and the bronchial mucosa of the control group were thickened, the cilia lodging, the adhesion, the necrosis, the shedding, the hypertrophy of the submucosal glands, the infiltration of the submucosal and muscular layers of the inflammatory cells, the infiltration of a large number of inflammatory cells around the blood vessels, the capillary, vasodilation, congestion, alveolar junction in that treatment group, the ciliated columnar epithelial cells of the group of the treatment group fall off, the goblet cell hyperplasia and the infiltration of the peripheral inflammatory cells of the blood vessel are reduced to a different extent than the control group, Alveolar destruction and fracture fusion In the control group, the mean pulmonary lining interval (MLI) in the lung tissue of the control group and the treatment group was higher than that in the normal group (P0.05), and the mean number of alveoli in the lung tissues of the control group and the treatment group (Me An alveolar numbers (MAN) was less than that of the normal group (P0.05), and the treatment group M The number of AN and the control group increased (P0.05). .3. TUNEL method in the detection of apoptosis in the lung tissue of the lung tissue of rats can be stained with light brown to dark brown, mainly for the alveolar epithelial cells, airway epithelial cells, and also to the vascular endothelial cells. In the control group and the treatment group, the apoptosis of the alveolar epithelial cells and the airway epithelial cells increased (P0.05); and compared with the control group, the treatment group's alveoli Reduction of apoptosis in skin cells and airway epithelial cells (P0.05). The expression of eNOS in the lung tissue was detected by SP method in airway epithelial cells, alveolar epithelial cells and vascular endothelial cells. The expression of eNOS in the tissue was decreased (P0.05). The expression of eNOS in the treatment group was increased in comparison with the control group (P0.05). iNOS was mainly expressed in the alveolar epithelial cells, the vascular endothelial cells and the inflammatory cells, and the control group and the treatment group were compared with the normal group. The expression of iNOS in the lung tissue was increased (P0.05). The expression of Caspase-3 was decreased in the treatment group compared with the control group (P0.05). The expression of Caspase-3 was distributed in the alveolar epithelial cells, the airway epithelial cells, the vascular endothelial cells, the airway and the vascular smooth muscle cells, the control group and the treatment group were compared with the normal group, and the calcium in the lung tissues of the rats The expression of spase-3 was increased (P0.05) and compared with the control group. Table of Caspase-3 in the Treatment Group The expression of eNOS mRNA in the lung of rats was decreased (P0.05). Compared with the control group, the expression of eNOS mRNA in the treatment group increased (P0.05). Compared with the control group, the expression of iNOS and Caspase-3 mRNA was increased (P0.05). The expression of iNOS and Caspase-3 mRNA was decreased (P0.05). The correlation between the expression of Caspase-3 and eNOS and iNOS protein was negatively correlated with the expression of the expression of Caspase-3 and eNOS in the normal group (r = 0.86, P0.05). There was a negative correlation between the expression of e-3 and eNOS (r =-0.67, P <0.05) and positive correlation with iNOS (r = 0.77, P0.05); and the expression of Caspase-3 and eNOS in the treatment group was negatively correlated (r =-0. (85, P0.05), positive correlation with iNOS (r = 0.88, P0.05). There was a negative correlation between the expression of Caspase-3 and eNOS and iNOS mRNA in the normal group (r =-0.86, P0.05) and positive correlation with the expression of iNOS mRNA (r = 0.94, P0.05). There was a negative correlation between the expression of ase-3 and eNOS mRNA in the treatment group (r =-0.89, P0.05), and the expression of Caspase-3 and eNOS mRNA in the treatment group was negatively correlated (r =-0.92, P <0.05). P0.0 5) The expression of iNOS mRNA was positively correlated with the expression of iNOS mRNA (r = O.90, PO. 05).[Conclusion] 1, Simvastatin can reduce the COPD model rats 2. Simvastatin can reduce the expression of iNOS and decrease the expression of Caspase-3 by increasing the expression of eNOS in the lung, so as to reduce the COP.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.9
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