内皮素-1受体拮抗剂对肺气肿大鼠肺组织的保护作用及机制

目的 探讨内皮素-1受体拮抗剂对肺气肿大鼠肺组织的保护作用及机制.方法 将24只SD大鼠随机分为健康对照组、肺气肿模型组、BQ123干预组、Bosentan干预组,每组6只.测4组大鼠平均内衬间隔(MLI)和肺泡破坏指数(DI).用缺口末端标记法(TUNEL)测肺泡间隔细胞凋亡;用免疫组化法、Western blot测caspase-3表达;用明胶酶谱法测基质金属蛋白酶(MMP)-2、MMP-9活性;用ELISA测TNFα、IL-1β浓度.结果 (1)肺气肿模型组大鼠出现典型肺气肿变化,MLI[(108.7±6.8)μm]和DI[(62.2±7.0)%]较健康对照组显著增高[(69.8±6.6)μm;(13.9±2.7)%;P<0.01];BQ123干预组[MLI(89.0±7.4)μm,DI(41.5±4.5)%]、Bosentan干预组[MLI(81.9±6.1)μm,DI(44.0±8.5)%]均较肺气肿模型组显著降低,但2组间差异无统计学意义.(2)4组大鼠肺内均可见凋亡细胞,肺气肿模型组凋亡指数(AI)较健康对照组明显增高,BQ123干预组、Bnsentan干预组AJ较肺气肿模型组明显减低,但仍高于健康对照组(P<0.01).(3)肺气肿模型组大鼠肺组织caspnse-3表达明显增高,BQ123干预组、Bosentan干预组caspase-3表达较肺气肿模型组明显降低.(4)肺气肿模型组大鼠肺内MMP-2、MMP-9活性较健康对照组明显增高,BQ123干预组、Bosentan干预组MMP-2、MMP-9活性降低,但差异无统计学意义.(5)肺气肿模型组大鼠肺组织匀浆上清中TNFα、IL-1β水平较健康对照组明显增高,BQ123干预组、Bosentan干预组TNFα、IL-1β水平较肺气肿模型组明显减低.结论 内皮素受体拮抗剂可通过抑制肺气肿大鼠凋亡基因表达,降低MMPs活性和减少炎性因子释放而起到部分保护作用.

The mechanism and pulmonary-protective effects of endothelin-1 receptor antagonist in chronic obstructive pulmonary diseases rat model

Objective To investigate whether the endothelin (ET) receptor antagonists have protective role in the development of emphysema. Methods Spragne-Dawley rats (n = 24) were divided into four groups: control group, cigarette smoke extract (CSE) group, BQ123 group and Bosentan group. CSE was injected intraperitoneally once a week for three weeks and BQ123 and Bosentan were administered daily for the same duration. TdT-mediated dUTP nick end labeling(TUNEL) was performed to observe the deoxyribonucleic acid (DNA) damaged cells and the expression of caspase-3 was determined by immunohistochemistry and Western blot. Matrix metalloproteinase-2 (MMP-2) and MMP-9 activities were investigated by gelatin zymography and tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) concentrations were measured by enzyme linked immunosorbent assay (ELISA). Results We confirmed the emphysematous destruction in the lungs of experimental rats induced by the intraperitoneal injection of CSE within 3 weeks. The mean lining inteval (MLI) and damage index (DI) were significantly increased in the CSE group compared with control group. However, the MLI and DI were significantly decreased in the BQI23 and bosentan groups compared with CSE rats (P < 0. 01, respectively). The TUNEL-positive cells were markedly distributed in the peribronchioles, intra-alveoli, and septal areas of the emphysematous lungs in CSE rats comparing with the lungs of control rats. The apoptosis index (AI) was significantly higher in CSE group than control group. And the AI was significantly reduced in BQ123 group and bosentan group compared with that in CSE group. The caspase-3 positive ceils were markedly distributed in the emphysematous lungs of CSE group comparing with the stained cells in the lungs of control rats. These positive cells were apparently reduced in the BQ123 and bosentan groups compared with the stained cells in CSE group. Comparing with the control group, expression of caspase-3 was prominently enhanced in CSE groups, but both BQ123 and bosentan treatments markedly inhabited the increases of the cleaved caspase-3 protein levels in rats injected with CSE. Rats injected with CSE showed increased MMP-2 and MMP-9 activities in their lung tissue homogenates and MMP-2 and MMP-9 activities were reduced significantly in both BQ123 and bosentan groups. The levels of TNFα and IL-1β were significantly increased in the CSE group in comparison to those in controls. BQ123 and bosentan significantly prevented the increases of the levels of TNFα and IL-1β in lungs of rats with injection of CSE. Condusions ET-1 may have an important role in the pathological process of emphysema and ET receptor antagonists protect against the development of emphysema probably by decelerating apoptosis, inhibiting proteolytic enzyme activity and reducing inflammatory cytokine levels.