Effects And Mechanisms Of Edaravone On Acute Respiratory Distress Syndrome Associated Early Pulmonary Fibro-proliferation

BackgroundAcute respiratory distress syndrome(ARDS)is a clinical syndrome secondary to infections,shocks and other pathological injuries.Progressive hypoxemia and dyspnea are the main clinical manifestations.In the Berlin definition of 2012,the mortality of patients with mild,moderate,and severe ARDS was 27%,32%,and 45%,respectively.Progressive fibrosis in the lungs was an important factor affecting the survival rate and prognosis of ARDS patients.However,the progress and mechanism of pulmonary fibrosis in ARDS have not been fully elucidated so far,and there is still no effective pharmacological treatment.Therefore,it is of great scientific value to study the mechanism and effective drugs of pulmonary fibrosis in ARDS.The studies have shown that transforming growth factor-β1(TGF-β1)is a key factor leading to fibrosis,and Smads signaling pathway is equally important in the development of fibrosis.At the same time,more and more studies have shown that oxidative stress is one of the important mechanisms of pulmonary fibrosis.In the course of ARDS,free radicals,especially oxygen free radicals and other oxidative stress reactions to the development of disease has important implications,such as early promotion of oxygen radical scavenging,or permitting the inhibition of pulmonary fibrosis and improving the prognosis of ARDS.Edaravone(EDA)is a potent free radical scavenger and has been widely used in patients with acute cerebral infarction.In recent years,it has also been found to have protective effects on lung injury.Therefore,we induced rat ARDS model by constructing lipopolysaccharide(LPS),and explored whether EDA could inhibit the fibro-proliferation of lung in the early stage of ARDS from the perspective of pathology and molecular biology and elucidate its possible mechanism.ObjectiveTo investigate the mechanism of EDA regulating TGF-β1/Smads signaling pathway,we can further elucidate the role of oxidative stress in early lung fibrosis of ARDS,and provide evidences for basic research and clinical treatment in this field.MethodNinety-six male SD rats were randomly divided into three groups(n=32): sham-injured group(Sham group),lipopolysaccharide group(LPS group),and lipopolysaccharide + edaravone group(EDA group).The groups were divided into four time points of 1,7,14 and 28 days(n=8).The LPS group and EDA group were injected with LPS 5 mg/kg via the tail vein.The Sham group was given the same volume of physiological saline.After the injection of LPS,the EDA group was intraperitoneally injected with EDA 6 mg/kg,and the same dose was continuously administered for 7 days.The Sham group and LPS group were given intraperitoneal saline at the appropriate time points.Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital(40mg/kg)at each time point.The rats were sacrificed after cardiac blood was drawn.The lung wet mass was measured and calculated the coefficient value of lung according to each body weight and the lung tissue was left to be examined.Paraffin-embedded lung tissues were stained with hematoxylin-eosine(HE)to observe pathological inflammatory changes;Masson staining was performed to assess the distribution of fibrous hyperplasia;and the levels of α-smooth muscle contractile proteins(α-SMA)and collagen I(Col I)were observed by immunohistochemistry.The lung tissues for detection of the hydroxyproline(HYP)content with alkaline hydrolysis.The expression of TGF-β1 mRNA,Smad3 and Smad4 protein in lung tissue were detected by RT-qPCR and WB assay,respectively.ELISA determined the levels of Col I,TGF-β1,TNF-α and IL6 in lung tissue.The expressions of MDA,SOD and GSH-PX in lung tissue were detected by thiobarbituric acid(TBA)method,xanthine oxidase method(hydroxylamine method)and colorimetry.Result1.General status: The rats of Sham group’s behavioral activity,breathing was smooth,diet were unchanged,glossiness of hair and body weight gradually increased;LPS group had the phenomena response was gradually dull,activity decreased,huddled in the corner of the cage,breathing faster,deeper,feeding and drinking water decreased.The above abnormal performance,LPS group lasted about 7 days,EDA group lasted about 5 days,then the response to stimulation gradually became better,activities increased,weight gradually increased.2.Lung index: Compared with Sham group,the LPS group had significant increase in lung index at each time point.Compared with LPS group,from the 7th day onwards,the EDA group had a different degree of decline in lung index,and the difference was statistically significant.3.Pathomorphology: The structure of the lung in the Sham group was clear,the alveolar septum was not thickened,there was no congestion,edema,and inflammatory cell infiltration.Compared with Sham group,LPS group had alveolar septum edema and thickening of alveolar wall.There was obvious inflammatory cell infiltration in the bronchial wall,some alveolar compensatory expansion,collagen fiber deposition at each level of the bronchial wall,and focal fibrosis at alveolar wall.There is no diffuse pulmonary fibrosis.Compared with LPS group,the level of inflammation and fibrous hyperplasia in the lungs of the EDA group ameliorate to some extent.4.TGF-β1/Smads signaling pathway: Compared with Sham group,the expression of TGF-β1 mRNA,Smad3 and Smad4 protein in lung tissue,and the expression of TGF-β1 protein in serum of LPS group were significantly increased.Compared with the LPS group,the TGF-β1 mRNA,TGF-β1,Smad3 and Smad4 proteins were decreased to varying degrees in the EDA group at the same time point.5.Inflammatory factor expression: The serum levels of TNF-α and IL-6 in LPS group and EDA group rats increased significantly on the first day,and then gradually decreased.Compared with Sham group at the same time points,the levels of TNF-α and IL-6 in LPS group were significantly increased.Compared with LPS group,the extents of TNF-α and IL-6 in EDA group were significantly reduced.6.Markers of oxidative stress: LPS and EDA group rats showed a significant increase in MDA content in lung tissue at 1 day,with the highest level on the 7th day and then decreased gradually.Compared with the Sham group at the same time point,the MDA content in the lung tissue of the LPS group increased significantly;compared with the LPS group at the same time point,the MDA content in the lung tissue of the EDA group was significantly reduced.Compared with Sham group,the two antioxidant substances(SOD,GSH-PX)decreased significantly on the first day,SOD of LPS group decreased gradually,GSH-PX decreased to the lowest level on the 7th day,and then gradually recovered;Compared with LPS group,the content of SOD and GSH-PX in EDA group decreased significantly at the same time points.ConclusionIn the rat model of ARDS,edaravone can effectively ameliorate lung inflammation and early fibrous hyperplasia of ARDS.Its possible mechanism is to improve inflammatory response through anti-oxygen free radicals,thereby inhibiting TGF-β1/Smads.In turn,it reduces fibrotic hyperplasia in lung tissue.