Astaxanthin Inhibit Pulmonary Epithelial-Mesenchymal Transition By Ameliorating Mitochondrial Respiratory Chain Complex Ⅰ And Ⅲ

BackgroundIdiopathic pulmonary fibrosis(IPF),the most common form of the idiopathic interstitial pneumonias,is a chronic,progressive,irreversible,and usually lethal lung disease of unknown cause.In recent years,the incidence of IPF is increasing year by year,and the pathogenic mechanisms remain are unclear,resulting in lack of effective pharmacotherapy.There is an urgent need to address the mechanisms of pulmonary fibrosis and find novel target of pharmacotherapy.In the currently accepted paradigm,a major factor in IPF pathogenesis is thought to be recurrent alveolar epithelial cell(AEC)injury repeatedly that provoke the migration,proliferation,and activation of mesenchymal cells,with the formation of fibroblast and myofibroblast foci.Epithelial to mesenchymal transition(EMT)has long been considered an important source of fibroblasts and myofibroblasts.EMT is defined as a process in which epithelial cells lose their phenotypic characteristics and acquire features of mesenchymal cells.Previous studies have suggested that TGF-β1 is a major inducement factor for EMT,and recent studies have shown that reactive oxygen species(ROS)also plays an important role in the process of pulmonary fibrosis by regulating the EMT.The mitochondrial respiratory chain complex I and III is a major source of ROS,which could be a contributing factor to pulmonary fibrosis.There is few literature focusing on this issue.Astaxanthin,one kind of lipid-soluble non-vitamin A carotenoid,has a variety of pharmacological effects,such as anti-inflammatory,antioxidant,anti-tumor and enhance immunity.Recent studies have shown that it could inhibit the formation of hepatic fibrosis,renal fibrosis and peritoneal fibrosis,and could improve the pulmonary fibrosis by improving the redox state of cells and promoting the apoptosis of myofibroblasts.However,much uncertainty still exists about the relationship between astaxanthin and pulmonary fibrosis.The aim of our study is to explore the relationship between astaxanthin,mitochondrial respiratory chain and pulmonary fibrosis and study specific therapeutic targets.Methods1.Astaxanthin improve the pulmonary fibrosis induced by bleomycin in rats1.1 Adult male Sprague-Dawley rats,each weighing 200–220 g,were obtained from Third Military Medical University,Chongqing,China.All the animals were acclimatized in a room(12/12 h light/dark cycle;25 ± 2 °C)and allowed free access to diet and water ad libitum.128 SD rats were randomly divided into four groups(32 rats each): control group(Sham Operation Group),AX Group,BLM Group and AX+BLM Group.Pulmonary fibrosis was induced by a single intratracheal instillation of 5 mg/kg bleomycin dissolved in normal saline,and the control rats received an equal volume of saline only.Beginning on the first day,the AX Group and the AX+BLM Group were orally administered daily with astaxanthin(2 mg/kg)oil solution,and the BLM stimulated rats were orally an equal volume of vegetable oil daily,respectively.All rats were killed on day 3,7,14 and 21,and lung tissue sections were removed and immediately frozen in liquid nitrogen for further analysis.Blood were collected from abdominal aorta.All of lung tissue was used to examine the lung coefficient and observe pulmonary lesions by HE staining and Ashcroft score.The lung tissues of 14 th day only were stained with Masson and the protein expression of E-cadherin,Fibronectin and α-SMA were detected by immunohistochemistry.The lung tissues were used to measure the concentration of Hyp and MDA and enzyme activity of GSH-PX by biochemical detection assay.The level of TGF-β1 in serum was detected by ELISA.The protein level of Smad2/3,p-Smad2 and p-Smad3 were detected by western bolt.1.2 Three rats were taken randomly in each group to carry on micro-CT scanning on day 7th,14 th and 21 th after the initial instillation.2.Astaxanthin inhibit EMT by ameliorating mitochondrial respiratory chain complex I and III2.1 Human lung adenocarcinoma 549(A549)cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences(Shanghai,China).The cells were divided into five groups: Control group,AX group,TGF-β1 group,AX+TGF-β1 group,and MitoQ+TGF-β1 group.The cells were pre-treated with 0.1% DMSO,0.1 ?M AX or 200 n M Mito Q for 30 minutes then co-treated with or without TGF-β1(5 ng/mL)for another 48 hrs.The activity of cell is measured by WST-1.E-cadherin,Fibronectin,Vimentin,α-SMA and NOX4 expression were detected by RT-PCR and immunofluorescence.2.2 Atfer adding the TGF-β1 or rotenone(0.5 ?M)or antimycin A(3 ?M)to the A549 cells 30 min,the level of ROS in the cell or mitochondria was measured by flow cytometry.The production of ATP was detected by ATP Reagent Kit.The enzymatic activity of mitochondrial respiratory chain complex I and III were detected by Spectrophotometric method.The expression of mitochondrial complex I and III expression was detected by immunofluorescence and RT-PCR.And then siRNA is transfected into the A549 cells for interfering with QPC expression.Accordingly,the level of ROS was measured by flow cytometry.Furthermore,the m RNA expression of E-cadherin,Fibronectin,Vimentin,α-SMA and NOX4,and the protein expression of Smad2/3,p-Smad2 and p-Smad3 were detected by RT-PCR and Western Blot.The expression of QPC in lung tissue was detected by immunohistochemistry.Results:1.Astaxanthin ameliorate the BLM-induced pulmonary fibrosis in ratsThere is severe inflammation in the lung of BLM group rats,supporting by observing lung HE stain,Ashcroft score and micro-CT,and when it comes to 14 th and 21 th day,the lung got apparent pulmonary fibrosis.Comparing with control and AX group,BLM group got substantial deposition of collagen fibers,highly Hyp and MDA,and the acivity of GSH-PX is down.The level of E-cadherin was down.Correspondingly,the level of Fibronectin and α-SMA get up as well as the level of TGF-β1 in the serum.The protein expression of p-Smad2 and p-Smad3 was significantly higher.However,after oral administration of astaxanthin,these change as above described induced by BLM could significantly ameliorate.2.Astaxanthin inhibit TGF-β1-induced EMT by ameliorating mitochondrial respiratory chain complex I and III2.1 WST-1 showed that AX at a concentration greater than 0.1 ?M inhibits cell growth,while AX at a concentration below 0.1 ?M promotes cell growth when 0.01~1 ?M AX worked on the TGF-β1-induced EMT in A549.Comparing with control group,TGF-β1 could significantly decrease the mRNA and protein of E-cadherin and increased the mRNA and protein of α-SMA as well as the mRNA of Vimentin,Fibronectin and NOX4.Both AX(0.1 ?M)and MitoQ(200 n M)could inhibit these change of the TGF-β1-induced EMT in A549.2.2 After adding the TGF-β1 into A459 30 minutes,the level of ROS intracellular and mitochondria was increased.And both AX and MitoQ could remarkably decrease the level of ROS intracellular and mitochondria.2.3 When A549 cells were exposed to TGF-β1 48 hrs,the yield of ATP and the enzymatic activity of mitochondrial respiratory chain complex I and III declined significantly and increased after AX and MitoQ pretreatment.2.4 Comparing with control group,there were three subunits of mitochondrial respiratory chain complex I(NDUFA1、NDUFB11、NDUFS5)getting down in TGF-β1 group and increased by AX and MitoQ.Specially,there were no differences in NDUFA2,NDUFA9,NDUFB1,NDUFB7,NDUFB10,NDUFC1,NDUFC2 and NDUFV1.QPC,one subunit of mitochondrial respiratory chain complex III,had outstanding declined and increased by AX and MitoQ.But there were no differences in UQCRFS1.And immunofluorescence show that the mitochondrial respiratory chain complex I and QPC down-regulation in TGF-β1 group and the up-regulation in AX treatment group and MitoQ treatment group.2.5 After inhibiting mitochondrial respiratory chain complex I and III successfully by rotenone(0.5 ?M)and antimycin A(3 ?M)in A549 cells,the level of ROS in the cell were remarkably increased.The mRNA expression of α-SMA increased while the mRNA expression of E-cadherin decreased.2.6 After transfecting siRNA of QPC into the A549 cells successfully,the protein of QPC are get down remarkably.Compared with the control group,the ROS in the QPCkd group and the QPCkd+TGF-β1 group increased markedly.Compared with the TGF-β 1 group,the mRNA expression level of Fibronectin was significantly elevated in QPCkd+TGF-β1 group.Compared with AX+TGF-β1 group,the mRNA expression level of Fibronectin was significantly elevated in QPCkd+AX+TGF-β1 group.2.7 The expression of QPC was down-regulated in the BLM group and increased by AX which could inhibit the expression of TGF-β1-induced p-Smad2 and p-Smad3.Conclusion:1.AX could ameliorate the BLM-induced lung injury and pulmonary fibrosis by inhibiting oxidative Stress,EMT and regulating TGF-β1/ Smads pathway2.Mitochondrial respiratory chain complex I and III may paly vital role in EMT.The down-regulation of NDUFA1,NDUFB11,NDUFS5 and QPC were involved in the progress of EMT.AX could inhibit TGF-β1-induced EMT by ameliorating mitochondrial respiratory chain complex I and III and decreasing ROS derived from mitochondria.3.Knowing QPC down may deteriorate the EMT,and AX may inhibit EMT by promoting QPC expression.AX could increase the expression of QPC in pulmonary fibrosis.These results indicated that QPC may serve as a potential therapeutic target.4.AX could inhibit the activation of Smads pathway caused by TGF-β1.