The Study On Fasudil Improves ARDS Induced By Sepsis Through RhoA/ROCK1 Signaling Pathway And Its Mechanism

ObjectivesThe goal of our study is to observe the related effects of Fasudil(FAS)on the mice with acute respiratory distress syndrome(ARDS)caused by sepsis that damaged twice by lipopolysaccharide.By comparing lung pathology,apoptosis factor Caspase-3 and Endothelial Nitric Oxide Synthase(e NOS)related protein expression,we investigated the specific regulation of fasudil in regulating Rho A/ROCK1 pathway to protect lung tissue.We hope our study can provide new scientific ideas for and the application of clinical drugs and the exploration of methods to prevent and treat ARDS.MethodsThere are forty-five male healthy adult(4-6 weeks old)C57BL mice and they were randomly divided into control group,lipopolysaccharide(LPS)group,and Fasudil-intervention(FAS+LPS)group.LPS group: to establish the mice model of ARDS,the mice were given low-dose LPS solution(1mg/kg)by intraperitoneal injection.After 16 hours,the mice were anesthetized and LPS(5mg/kg)was given by intratracheal instillation;FAS+LPS group: Fasudil(10mg/kg)was injected into the abdominal cavity of mice,before30 minutes of intraperitoneal injection and after1 hour of LPS intratracheally injected;Control group: mice were given normal saline at the same time.After modeling,the mice in three groups were continued to be observed for4 hours.Finally,the mice were killed to get the lung tissues.(1)To observe the survival status and behavioral changes of the mice in each group;(2)To observe the appearance of fresh lung tissues.Pathological sections were stained with hematoxylin and eosin(HE)to observe the pathology of the lung tissues in each group;(3)To evaluate the edema and damage of inflammatory in mice by testing the wet /dry weight ratio(W/D),malondialdehyde(MDA)content and myeloperoxidase(MPO)activity of lung tissues;(4)To evaluate the apoptosis of lung cells by Immunohistochemistry(IHC)that was used to determine the expression level of caspase-3;(5)To determine the expression of Rho A,ROCK1,e NOS and p-e NOS protein in lung tissues in each group by Western-Blot.Results(1)The mice in the control group were in good mental state.And breath,the color of skin mucous membrane,eating and drinking were normal;The mice in LPS group were lethargic,short of breath,slow in movement,cyanosis,anorexia,and bleeding in the mouth and nose.The above symptoms were improved in the mice of FAS+LPS group.(2)Pathology of lung tissues showed: Compared with the control group,the lung volume of the LPS group was significantly increased with scattered bleeding points.Lungs are infiltrated by a large number of inflammatory cell(mainly neutrophils)were seen under the microscope.The lung tissue structures were severely damaged with pulmonary congestion,edema and hyaline;Compared with the LPS group,the inflammatory cell infiltration and red blood cell exudation in the lung tissues of the FAS+LPS group were significantly reduced,and the interstitial edema and alveolar structure damage were significantly reduced.(3)The W/D value,MDA content and MPO activity of the LPS group were significantly higher than those of the Control group(P<0.01);Compared with the LPS group,the W/D value,MDA content and MPO activity of the FAS+LPS group were significantly reduced(P<0.01).(4)Compared with the control group,the expression of Caspase-3 in the lungs of the LPS group was significantly up-regulated(P<0.01);Compared with the LPS group,the expression of Caspase-3 in the FAS+LPS group was significantly decreased(P<0.01).(5)Compared with the control group,the expression of Rho A and ROCK1 protein in the LPS group were increased,but the expression of p-e NOS protein was decreased(P<0.05);Compared with the LPS group,the expression of Rho A and ROCK1 protein in the fasudil group were down-regulated,but the expression of p-e NOS protein was up-regulated(P<0.05);There was no significantly difference in the expression of the total e NOS protein in the three groups(P>0.05).Conclusion(1)Fasudil may relieve inflammatory cell infiltration in lung tissue and resist oxidative stress by inhibiting the activity of Rho A/ROCK1 signaling pathway.Therefore,it can reduce inflammatory edema and improve the structural destruction of lung tissue.(2)Fasudil may prevent lung tissue cell apoptosis by inhibiting the activity of the Rho A/ROCK1 signaling pathway.Therefore,it can reduce lung tissue damage in ARDS mice induced by sepsis.(3)Fasudil may alleviate lung endothelial cell damage by promoting the phosphorylation of e NOS through the Rho A/ROCK1 pathway.