法舒地尔调控RhoA/ROCK1通路改善脓毒症小鼠肺损伤

Fasudil alleviates lung injury via targeting Rhoa/ROCK signal pathway in septic mice

目的探讨法舒地尔对脓毒症小鼠急性肺损伤的保护作用。方法4~6周龄雄性C57BL小鼠45只随机(随机数字法)分为对照组(Control组)、脂多糖组(LPS组)、法舒地尔干预组(FAS+LPS组),每组15只。LPS溶液腹腔注射及气道内滴注建立脓毒症小鼠急性肺损伤模型。法舒地尔干预组分别于腹腔注射LPS前30 min和气道滴注LPS后1 h,腹腔内注射盐酸法舒地尔溶液(10 mg/kg)。造模后4 h处死小鼠取肺组织。HE染色观察病理形态学变化;测定肺组织湿重/干重比(W/D);TBA比色法测定MDA含量及MPO活性;IHC测定caspase-3表达水平;Western Blot法检测肺组织中RhoA、ROCK1、eNOS及p-eNOS的蛋白表达水平。结果FAS预处理后肺组织中炎性细胞浸润和红细胞渗出显著减少,间质水肿及肺泡结构破坏显著减轻。与LPS组相比,FAS+LPS组的W/D值、MDA含量及MPO活性较LPS组均显著降低(P<0.01)。FAS+LPS组的Caspase-3表达较LPS组显著下降(P<0.01)。与Control组比,LPS组的RhoA、ROCK1的蛋白表达水平增高(P<0.05),p-eNOS的蛋白表达水平下降(P<0.05);与LPS组比,FAS+LPS组的RhoA和ROCK1的蛋白表达下调(P<0.05),但p-eNOS的蛋白表达上调(P<0.05);三组的eNOS总蛋白表达水平差异无统计学意义(P>0.05)。结论法舒地尔可减轻脓毒症小鼠肺组织炎性细胞浸润程度,下调肺组织细胞凋亡,抑制RhoA/ROCK1信号通路活性并促进eNOS的磷酸化表达。

Objective To determine the protective effect of fasudil on acute lung injury in septic mice.Methods Forty-five 4-6-week-old male C57BL mice were randomly(random number)assigned to three groups(n=15 each group):control group,lipopolysaccharide(LPS)group and Fasudil intervention group(FAS+LPS).Acute lung injury model of septic mice was established with an intraperitoneal injection and intratracheal infusion of LPS.The mice in the FAS+LPS group were injected with fasudil hydrochloride intraperitoneally 30 min before intraperitoneal LPS injection and 1 h after intratracheal LPS infusion,respectively.All mice were sacrificed at 4 h after modeling,and lung tissues were collected.Hematoxylin-eosin staining was preformed to observe the morphological changes in the lung tissue.The wet/dry weight(W/D)ratio,malondialdehyde(MDA)content and the activity of myeloperoxidase(MPO)in the lung tissues were detected.Caspase-3 expression was examined by immunohistochemical(IHC)staining.Western blot was employed to detect the expression of RhoA,ROCK1,endothelial nitric oxide synthase(eNOS),and p-eNOS.Results Inflammatory cell infiltration and erythrocyte exudation were significantly reduced,and the degree of interstitial oedema and derangement of alveolar structure appeared in a decreasing degree after FAS intervention.Compared with the LPS group,the W/D ratio,MDA content,MPO activity and the expression of Caspase-3 in the FAS+LPS group were significantly reduced(all P<0.01).Meanwhile,the expression of RhoA and ROCK1 of the LPS group were obviously higher than those in the control group(P<0.05),and p-eNOS was obviously lower than that in the control group(P<0.05).Furthermore,the expression of RhoA and ROCK1 of the FAS+LPS group were obviously lower than those in the LPS group,and p-eNOS was obviously higher than that in the LPS group.There was no significant difference on the expression of eNOS among the three groups.Conclusions Fasudil can alleviate the degree of inflammatory cell infiltration,reduce apoptosis in lung tissue,inhibit the RhoA/ROCK1 signaling activity,and promote the phosphorylation expression of eNOS in septic mice.