血管紧张素转换酶2激动剂三氮脒减轻小鼠肢体缺血再灌注引发的肺损伤

ACE2 agonist DIZE alleviates lung injury induced by limb ischemia-reperfusion in mice

本文旨在探讨血管紧张素转换酶2(angiotensin converting enzyme 2,ACE2)激动剂三氮脒(diminazene aceturate,DIZE)对小鼠肢体缺血再灌注(limb ischemia-reperfusion,LIR)引发的急性肺损伤(acute lung injury,ALI)的作用。雄性8周龄野生型和人ACE2(hACE2)转基因ICR小鼠随机分为:野生对照组(W组)、野生模型组(WL组)、野生模型DIZE干预组(WLD组)、hACE2转基因对照组(T组)、hACE2转基因模型组(TL组)和hACE2转基因模型DIZE干预组(TLD组),每组6只。采用常规止血带套扎双侧后肢的方法复制小鼠LIR模型。各DIZE干预组在LIR前预先腹腔注射DIZE(15 mg/kg),持续4周。LIR结束时,计算肺组织脏器系数和湿干质量比(wet/dry weight ratio,W/D);计数肺泡灌洗液细胞并检测蛋白浓度;HE染色后观察肺组织形态变化并进行病理损伤评分;用ELISA法测定肺组织中血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)和Ang(1-7)水平;用Western blot检测肺组织血管紧张素Ⅱ受体1(angiotensin Ⅱ type 1 receptor,AT1)和Mas受体蛋白表达变化。结果显示:(1)WL和TL组小鼠均有明显的肺损伤,TL组小鼠肺损伤轻于WL组,而DIZE可减轻WL和TL组小鼠肺损伤。(2)WL组小鼠肺组织Ang Ⅱ水平升高,Ang(1-7)水平降低,TL组小鼠这两种蛋白没有明显变化,而DIZE可降低WL和TL组Ang Ⅱ水平,升高WL组Ang(1-7)水平。(3)WL和TL组小鼠肺组织AT1和Mas受体蛋白表达升高,而DIZE可逆转WL和TL组AT1蛋白表达的变化,并进一步上调这两组Mas受体蛋白表达。以上结果提示,DIZE可能通过调控局部肺组织ACE2-Ang(1-7)-Mas轴改善肾素-血管紧张素系统稳态失衡,减轻LIR所致小鼠ALI,从而发挥保护作用。

This study was aimed to explore the effect of angiotensin converting enzyme 2（ACE2） agonist diminazene aceturate（DIZE） on acute lung injury（ALI） induced by limb ischemia-reperfusion（LIR） in mice. Male 8-week-old wild-type and h ACE2 transgenic ICR mice were randomly divided into 6 groups（6 in each group）, including wild-type control（W）, wild-type model（WL）, wild-type model with DIZE administration（WLD）, transgenic control（T）, transgenic model（TL）, and transgenic model with DIZE administration（TLD） groups. LIR model was established by 4 h reperfusion following 2 h ischemia of bilateral hindlimbs with rubber bands in mice. The WLD and TLD groups were pretreated with DIZE（15 mg/kg, i.p.） for 4 weeks before LIR. At the end of LIR, the mice were sacrificed and lung tissues were sampled. Indexes for evaluating lung injury include organ coefficient and wet/dry weight ratio（W/D）, cell count and protein concentration of bronchoalveolar lavage fluid（BALF）, as well as morphological change and pathological score were detected. Angiotensin Ⅱ（Ang Ⅱ） and Ang（1-7） levels in lung tissue were determined by using ELISA commercial kits. And the protein expressions of angiotensin Ⅱ type 1 receptor（AT1） and Mas receptor protein in lung tissue were detected by Western blot. The results were as follows：（1） There was obvious lung injury in both the WL and TL groups. The lung injury in the TL group was lighter than that in the WL group. DIZE could attenuate the lung injury in both the two groups.（2） The WL group showed increased Ang Ⅱ and decreased Ang（1-7） levels, whereas the TL group did not exhibit any changes of these two proteins. DIZE decreased the level of Ang Ⅱ in both the WL and TL groups, and increased the level of Ang（1-7） in the WL group.（3） In the WL and TL groups, AT1 and Mas receptor protein expressions were up-regulated. DIZE reversed the change of AT1 protein expression, whereas further increased Mas receptor expression in both the two groups. These results suggest that DIZE may improve the renin-angiotensin system homeostasis by regulating ACE2-Ang（1-7）-Mas axis in local lung tissue and play a protective role in LIR-induced ALI in mice.