Function Regulation Of Histone Deacetylase 2 In Lung Fibroblasts

Research backgroundIdiopathic pulmonary interstitial fibrosis(IPF)has a high morbidity and mortality in recent years,with an average survival of 3-5 years after onset,poor clinical treatment effect,and hormone and immunosuppressive drugs have been proved to be ineffective by evidence-based medicine1-5.Pulmonary fibrosis pathogenesis is still not clear,may be related to the excessive lung fibroblasts hyperplasia and alveolar epithelial cell apoptosis imbalance,pathological pulmonary fibrosis patients exist serious fibroblast proliferation and extracellular matrix proliferation,lung tissue stiffness due to these reasons,compliance,patients increased work of breathing.To treatment of pulmonary fibrosis,we need to reduce the lung fibroblast proliferation and extracellular matrix degradation,restore lung compliance,there are only acquaintances who pirfenidone and nintedanib can slow disease progression,pirfenidone' s main function is inhibition of TGF-betas to fibroblasts muscular transformation,main effect,nintedanib inhibit fibroblasts hyperplasia,but clinical practice found that effect is not exact,be badly in need of new treatment method solve the problem of the treatment of idiopathic pulmonary fibrosis1-5,16-18.Fibroblasts have the ability to differentiate into myofibroblasts,a special type of contractile cell that can synthesize large amounts of extracellular matrix(ECM),and myofibroblasts play an important role in normal wound healing.However,in fibrosis,apoptosis is not part of the normal metabolic process,which leads to excessive accumulation of ECM,tissue dysfunction,and replacement of normal tissue with scar tissue.The accumulation of myofibroblasts in the lung is positively correlated with the reduction of lung volume and the development of the severity of symptoms.TGF-beta 1 is considered to promote normal wound healing and tissue fibrosis as the primary mediator.Studies have shown that Smad3 is the key to enhanced contraction of human lung fibroblasts with TGF-beta 1,and that TGF-beta 1 induced alpha SMA expression via Smad3 signal transduction is also the key to contraction.Muscle fibroblasts in smooth muscle actin(alpha SMA)expressed as features,is widely considered to be involved in the process,illustrates the alpha SMA expression on the importance of the collagen gel contraction,alpha SMA has been confirmed and fibroblast contraction and tissue remodeling,and fibroblast contraction and participated in the normal wound healing and tissue remodeling fiber necrosis.TGF-beta 1 activates fibroblasts that express mesenchymal marker alpha-sma and collagen secreted into the extracellular matrix.TGF-beta 1 expression and myofibroblast activation were highly correlated with disease progression.Antagonizing TGF-beta 1 significantly inhibited collagen deposition and gingival fibrosis.In addition,studies have shown that TGF-beta 1 continuously promotes the formation of fibrosis by stimulating the expression of alpha-sma in fibroblasts to induce the transformation of fibroblasts.Therefore,the reduction of alpha-sma and collagen production is an important factor in the treatment of idiopathic pulmonary interstitial fibrosis7,9,13,15.Bleomycin-induced decreased Fas expression in fibroblasts in mice with pulmonary fibrosis and IPF was associated with increased HDAC2 and HDAC4 expression.Fibroblasts in IPF patients exhibit antiapoptotic behavior,especially fas-mediated apoptosis.Histone modification plays a crucial role in anti-apoptosis of fibroblasts.TGF-beta 1 induced myofibroblast differentiation is dependent on the hdac4-activated AKT signaling pathway.Trichostatin A(TSA),suberoy-lanilide hydroxamic acid(SAHA),and spA(Spiruchostatin A)can inhibit TGF-beta 1 induced myofibroblast differentiation.Eventually make a-SMA,the expression of collagen and collagen ? and the release of soluble collagen type is reduced,at the same time the TSA can make AKT phosphorylation.TGF-beta 1 induced myofibroblast differentiation produces large amounts of collagen and fibronectin,leading to the accumulation of ECM,which induces airway remodeling by stimulating myofibroblast differentiation.In this study,the effects of TGF-beta 1 on RNA and protein expression levels were reversed.The balance between histone acetyltransferases(HAT)and histone deacetylases(HDAC)regulates gene expression and cellular function by modifying core histone or non-histone precursors.Regions of DNA associated with highly acetylated histones are usually actively transcribed.HDACs deacetylize histone proteins to produce dense chromatin,which is not conducive to transcription.Non-histone acetylation can affect stability;protein-protein interactions,localization,or DNA binding.HDACs have been shown to be involved in ECM production of various organs.In mice,hdac2-related Hop overexpression leads to cardiac interstitial fibrosis,which is reversed by HDAC inhibitors.It was found that the expression of cyclooxygenase 2(cox-2)in the lungs of IPF patients was decreased,and HDAC may reduce cox-2 expression by causing histone deacetylation.It was also found that HDAC might decrease the expression of ip-10 in histone deacetylation f-ipf.Previous studies have shown increased expression of HDAC2 in TGF-beta 1 induced nasal polyp fibroblasts,and inhibition of TGF-beta 1 induced myofibroblast differentiation and ECM production by inhibiting expression of HDAC2 and histone hyperacetylation in nasal polyp organ culture7-11,13-14.Epigenetic studies have shown that histone deacetylase(HDACs)activation is necessary to regulate gene expression and cell proliferation.When histone is acetylated,chromosomes are opened to allow transcription factors and mRNA synthases to bind to the transcription site to increase transcription and expression,whereas when histone is deacetylated,chromosomes return to a tight state,reducing gene transcription.HDACs is a group of enzymes that catalyze the deacetylation of lysine residues in histones and some non-histone proteins associated with cell proliferation,such as tyrosine kinases and transcription factor3(STAT3).Among mammals,there are 18 hdacs,which are divided into four categories.Class ? HDACs includes HDAC1,2,3,8;The first ? HDACs including HDAC4,5,6,7,9,10;The first class ? HDACs called sirtuins;The fourth type of HDAC is HDAC11.In HDACs,HDAC1 and HDAC2 are reported to require the organ development and proliferation of embryonic stem cells and embryonic fibroblasts.HDAC inhibitors,such as trichostatin A(TSA),inhibit the activity of class ?and class ? HDACs and effectively inhibit cell cycle progression and cell proliferation of many cell types.Recent studies have shown that sk-7041 is a selective inhibitor of class I HDACs,and its antiproliferative effect is similar to that of class ?/? HDAC inhibitors,suggesting that class ? HDACs plays a leading role in the progression of fibrosis.At present,HDAC is widely used in basic medical research,especially in tumor and fibrosis,including pulmonary interstitial fibrosis,myocardial fibrosis,liver fibrosis and glomerular fibrosis.Moreover,studies have shown that almost all HDAC enzymes of class ? and ? are overexpressed and up-regulated in IPF lung tissues19,20.The silencing of HDAC2 mRNA in normal lung fibroblasts by siRNA gene silencing was performed in the laboratory.The behavior of silencing HDAC2 mRNA in normal lung fibroblasts showed that the metal matrix enzyme increased and the proliferation decreased6.However,there is still a lack of research on the role of HDAC2 in functional regulation of human lung fibroblasts.How does HDAC2 cell expression change during human lung fibroblast proliferation and muscular transformation?Does silencing HDAC2 by gene silencing potentially alter lung fibroblast function?The above is the purpose of this research.Function regulation of histone deacetylase 2 in lung fibroblastsObjective1.The success of human lung fibroblast culture was confirmed.2.After TGF-beta 1 stimulation of human lung fibroblasts,HDAC2 and alpha-smooth muscle actin(alpha-sma)expression levels were determined by Western blotting.3.Human lung fibroblasts were transfected with small interfering RNA using Endofectin-max to determine whether HDAC2 could be silenced by siRNA specific transfection of human lung fibroblasts.4.To explore the effect of histone deacetylase 2(HDAC2)on the activation and proliferation of human lung fibroblasts.MethodsSpecimen source:thoracic surgery operation in the process of normal lung tissue surrounding the volume about 20mm*20mm,male,60 lung nodules surgery patients,in history,no chronic smoking history of 20 years,400 smoking index,no exposure to toxic industrial history,serum-fee DMEM(biopsy specimens preserved in ice compress or 4 C),the specimens were collected for the generation of human lung fibroblasts.Experimental steps:2?4generations of lung fibroblasts experiment,TGF-beta 1 was used to stimulate 48 hours,using Western blot technique for testing HDAC2 and a-SMA expression;After transfection of HDAC2 into human lung fibroblasts by endofectin-max for 24 hours,the expression of HDAC2 was determined by Western blot.Application of HDAC2 siRNA specificity after transfection man lung fibroblasts,2 groups of cells respectively subculture for 24 hours,again using different concentrations of TGF-beta 1 to stimulate cell 48 hours,using Western blot technique for testinga-SMA expression quantity.To observe the effect of HDAC2 on proliferation of human lung fibroblasts.Results1.Human lung fibroblasts were self-cultured and confirmed by vimentin immunocytochemical stainingAlmost all cells in the visual field showed positive expression after Vimentin staining.In this study,adult peripheral normal lung tissue was used to culture fibroblasts in lung tissue by adherent method,and the success of primary cell culture was identified(see figure 1).2.a-SMA is a well known marker of myofibroblast differentiation.After TGF-beta 1 stimulation of human lung fibroblasts,the expression levels of HDAC2 and alpha-smooth muscle actin(alpha-sma)were significantly increased by Westernblotting.As can be seen from figure 2,the expression levels of HDAC2 cells in the control group were lower(Mean=1),while the expression levels of HDAC2 cells in the TGF-beta 1 group were higher(Mean=1.74519),The difference was statistically significant(P<0.05).As can be seen from figure 3,the expression level of alpha-sma cells in the control group was lower(Mean=1),while the expression level of alpha-sma cells in the TGF-beta 1 group was higher(Mean=3.17678),The difference was statistically significant(P<0.05).3.The transfection human lung fibroblasts siRNA-HDAC2 HDAC2 group and ?-SMA protein expression decreased obviously as shown in figure 4,HDAC2 cells in the control group had higher expression levels(Mean=1)and HDAC2 siRNA the expression level of HDAC2 cells in the control group was lower(Mean=0.438732),and the difference was statistically significant(P<0.05).Can be seen from the figure 5 controla-SMA cell expression level is higher,the control group a-SMA expression level of TGF-beta 1 concentration respectively 0 mol/L group(Mean=1),concentration of TGF-beta 1 mol/L 50 group(Mean=1.95305),concentration of TGF-beta 1 100 mol/L group(Mean=2.47244);HDAC2 siRNA group ?-SMA cell expression level is low,the control ?-SMA expression level of TGF-beta 1 concentration respectively 0 mol/L group(Mean=0.788312),concentration of TGF-beta 1 mol/L 50 group(Mean=1.46341),concentration of TGF-beta 1 100 mol/L group(Mean=2.14431),The difference was statistically significant(P<0.05).4.Through the above studies,it was confirmed that sirna-hdac2 transfection significantly inhibited the proliferation activity of human lung fibroblasts(see the attached figure for the specific figure).ConclusionHuman lung fibroblasts HDAC2 expression increases,the application of targeted gene silencing technique HDAC2 silence,?-SMA expression,can obviously reduce both into positive correlation,HDAC2,?-SMA expression and lung fibroblast proliferation and extracellular matrix proliferation have close relations,prompt HDAC2 expression increase involved in lung fibroblast proliferation and extracellular matrix proliferation of pathogenesis,suggests that targeted silence HDAC2 inhibits human lung fibroblasts proliferation and activation of prompt its may play a role of regulation in idiopathic pulmonary fibrosis,further in-depth study,It can provide a new therapeutic target for idiopathic pulmonary interstitial fibrosis.