Effects Of Trichostatin A And Cd4 + T Cell Differentiation On Bleomycin Induced Pulmonary Fibrosis In Mice And Its Mechanism

Background: Idiopathic pulmonary fibrosis(IPF)is one of the most common interstitial lung diseases.Its etiology and pathogenesis are not very clear.It is a chronic,progressive and irreversible lung disease.The main pathological features are the proliferation and aggregation of fibroblasts,the deposition of collagen fibers in lung interstitium,and the activation and infiltration of lymphocytes,macrophages and monocytes,resulting in the destruction of alveolar structure.At present,the clinical treatment drugs for IPF mainly include antioxidants,glucocorticoids,immunosuppressants,new anti pulmonary fibrosis drugs(such as: pirfenidone,nidanib),etc.whether the above drugs are used alone or in combination,the effect is not good,they can not stop the progress of pulmonary fibrosis,and eventually lead to death.Therefore,it is of great practical significance to further study the molecular mechanism of the occurrence and development of pulmonary fibrosis,find new therapeutic targets and delay the progress of pulmonary fibrosis.Recent studies have shown that HDACs are not only involved in the development of tumor,but also related to the process of organ fibrosis A.TSA is the most widely used HDACi.What is its mechanism of action in idiopathic pulmonary fibrosis,whether it can alleviate pulmonary fibrosis by inhibiting TGF-β1/Smads pathway,and whether it has different effects compared with traditional anti fibrosis drugs? It is necessary to conduct in-depth research on this,and further clarify the role of histone deacetylase inhibitors in pulmonary fibrosis Objective to find a new therapeutic target for pulmonary fibrosis.Objective:1.Pulmonary fibrosis model was established by intranasal instillation of bleomycin in mice.2.To observe the expression of pulmonary fibrosis in different time after modeling.3.Observe the expression changes of HDAC1,TGF-β1 and Smads in the model of pulmonary fibrosis,and clarify the relationship between HDAC1 and TGF-β1/Smads in the progress of pulmonary fibrosis.4.To compare the effects of different antifibrotic drugs on HDAC1 and TGF-β1/Smads pathway protein expression.5.Clarify the antifibrotic effect of TSA.6.Observe the expression level of related cytokines in different stages of pulmonary fibrosis model.7.To clarify the relationship between the differentiation of CD4 + T cell subsets and pulmonary fibrosis.8.The effect of IL-17 A on HDAC1 activity was determined in vitro.9.The effect of IL-17 A on TGF-β1/Smads pathway was confirmed in vitro.Method:1.Pulmonary fibrosis model was established by intranasal instillation of bleomycin in mice.Sixty healthy C57 BL / 6 mice were randomly divided into five groups: control group(CON),model group(MOD),dexamethasone group(DEX),pirfenidone group(PIR)and TSA group(TSA).12 rats in the control group were randomly selected,and the remaining 48 rats were given bleomycin intranasal drip for modeling.After anesthesia with chloral hydrate,200 UL of bleomycin(5mg/kg)was dripped into the nostrils of mice with micropipette.On the next day,the treatment group was given dexamethasone(3mg/kg.d,intraperitoneal injection),pirfenidone(300mg/kg.d,intragastric administration)and TSA(40mg/kg.d,intraperitoneal injection).2.On the 7th,14 th and 28 th day after modeling,the mice were killed in batches,and the peripheral blood and lung tissue were collected.The morphological changes of lung tissue were observed by HE staining and Masson staining.3.The expression of vimentin,Collagen-I and a-SMA was detected by IHC.4.Hydroxyproline kit was used to detect the content of hydroxyproline in lung tissue of mice in each group.5.The expression levels of TGF-β1,vimentin,a-SMA,Collagen-I,HDAC1,Smad2,Smad3,Smad7,p-Smad2 and p-smad3 were detected by Western blot.6.Western blot was used to detect the expression of NF-k B,p38-MAPK,PI3 K and other proteins to explore the role of related signaling pathways in pulmonary fibrosis.7.The expression levels of IL-17 A,IL-6,TGF-β1,IL-10 and other cytokines in peripheral blood were detected by enzyme-linked immunosorbent assay(ELISA).8.Real time fluorescent quantitative polymerase chain reaction(QRT-PCR)was used to detect the changes of Collagen-I m RNA,a-SMA m RNA and vimentin m RNA.9.The peripheral blood CD4+T cell subsets of mice in each group were sorted by flow cytometry,and the changes of Th17,Treg,Th1,Th2 ratio were observed.10.In vitro,human lung fibroblasts(MRC5)were identified by cell immunofluorescence(IF),and the effects of IL-17 A and TSA on the proliferation of MRC5 were detected by MTT assay.11.HDAC1 kit was used to detect the changes of HDAC1 activity in MRC5 cells under different treatment conditions.12.MRC5 was divided into control group,IL-17 A stimulation group,IL-17 A and TSA co-action group.The expression of vimentin,Collagen-I and a-SMA was detected by cell immunofluorescence(IF).13.Western blot was used to detect the expression of vimentin,a-SMA,HDAC1,Smad2,Smad3,Smad7,p-Smad2 and p-smad3.Result:The first part is in vivo experiment:1.48 mice were successfully established by intranasal instillation of bleomycin.2.HE staining results showed that the lung tissue structure of the control group was complete,the alveolar cavity was clear,and there was no exudation and filling in the alveolar cavity.On the 7th day after modeling,a large number of lymphocytes and a small number of macrophages were seen in the lung tissues of the model group,dexamethasone group and TSA group,and more thrombosis was seen in some vascular cavities;on the 14 th day after modeling,alveolar structural disorder,hemorrhage in the alveolar cavity,slightly less thrombosis in the vascular cavity,and alveolar edema were seen in the model group,dexamethasone group,pirfenidone group and TSA group Septal thickening,deposition of collagen fibers,proliferation of fibroblasts and type II alveolar epithelial cells could be seen in the lung interstitium;a large number of macrophages could be seen in dexamethasone group and pirfenidone group.On the 28 th day after modeling,alveolar fusion,deposition of collagen fibers and destruction of alveolar structure were observed in the model group,dexamethasone group,pirfenidone group and TSA group.The alveolar cavity could not be identified.A large number of basal cells proliferated in clusters,alveolar hemorrhage,decreased intravascular microthrombosis and obvious proliferation of alveolar type II epithelial cells were observed in dexamethasone group and pirfenidone group A large number of macrophages proliferated and aggregated in nisolone group and TSA group.3.Masson staining results showed that the alveolar structure of the control group was complete,the alveolar septum was not widened,there was no collagen deposition,and a little blue purple collagen fibers were arranged around the bronchus.Seven days after modeling,collagen deposition was found in the alveolar septum of the model group and dexamethasone group,but the amount was less.14 days after modeling,alveolar structure was destroyed,alveolar septum widened,normal lung tissue structure disappeared and collagen fiber deposition increased in the model group.28 days after modeling,the alveolar structure disappeared in the model group,dexamethasone group and pirfenidone group,a large number of collagen fibers were deposited in the lung interstitium,collagen fibers were filled in the alveolar,the damage degree of lung tissue was further aggravated,and the lesion site completely lost the lung tissue structure.4.IHC staining results showed that: the control group only showed a small amount of a-SMA expression around the blood vessels;the expression of vimentin,Collagen-I,a-SMA in model group,dexamethasone group and pirfenidone group were significantly increased compared with the control group,and the expression increased with time after modeling;the expression of vimentin,Collagen-I,a-SMA in TSA group was significantly less than that in model group,dexamethasone group and pirfenidone group Fenidone group.5.Hydroxyproline content test results showed that: after modeling,the hydroxyproline content of model group,dexamethasone group and pirfenidone group increased significantly,dexamethasone group was significantly higher than pirfenidone group and TSA group,and the hydroxyproline content of TSA group was the least.6.Western The results of blot showed that: the expression of vimentin,Collagen-I,a-SMA,HDAC1,p-Smad2,p-smad3 in the model group was significantly higher than that in the control group;the expression of the above proteins in dexamethasone,pirfenidone and TSA group,was significantly lower than that in the model group,especially in TSA group.The expression of Smad2 and Smad3 protein had no significant difference between each group and each time period after modeling;the expression of NF-k B and PI3 K protein in model group was significantly higher than that in control group,and the expression of above proteins in dexamethasone group,pirfenidone group and TSA group decreased in varying degrees compared with that in model group,and TSA group was lower than that in control group.There was no significant difference in the expression of p38-MAPK protein between each group and each time period after modeling.7.RT-PCR results showed that the expression of vimentin m RNA,Collagen-I m RNA,a-SMA m RNA increased gradually with time in the model group,28 days > 14 days >7 days > 0 days.In dexamethasone group,the expression of vimentin m RNA,Collagen-I m RNA and a-SMA m RNA remained at a high level,while in TSA group,the expression of vimentin m RNA,Collagen-I m RNA and a-SMA m RNA gradually decreased with time.The second part is the sorting of CD4 + T lymphocyte subsets in peripheral blood of mice and the detection of cytokine levels:1.The proportion of Th1 cells and Th2 cells decreased in the model group,and the proportion of Th1 cells and Th2 cells increased with time in dexamethasone group,pirfenidone group and TSA group;the proportion of Th17 cells increased in the model group,and the proportion of Th17 cells in dexamethasone group was significantly higher than that in pirfenidone group and TSA group.The proportion of Treg cells in the model group was not significantly different from that in the control group.The proportion of Treg cells in the dexamethasone group was significantly higher than that in other groups.2.ELISA results showed that: IL-17 A,IL-6,TGF-β1 increased in the early stage after modeling,and the expression level of IL-17 A in the model group was significantly higher than that in the control group,and the expression level of IL-17 A in the TSA group was the lowest;the level of IL-6 in the model group decreased gradually with time;IL-17 A showed a continuous increase,the expression level of IL-10 did not increase significantly after modeling,but the levels of pirfenidone group and TSA group were significantly higher Significantly higher than other groups.The third part is in vitro experiment:1.IL-17 A can activate MRC5 cells.With the increase of IL-17 A concentration,the proliferation activity of MRC5 cells increases.TSA inhibited the proliferation of MRC5 cells.2.IL-17 A can increase the activity of HDAC1 in MRC5 cells.With the increase of IL-17 A concentration on MRC5 cells,the activity of HDAC1 gradually increased.3.After grouping,the activity of HDAC1 increased,the expression of vimentin,a-SMA,HDAC1,p-Smad2,p-smad3 increased and the expression of Smad7 decreased in IL-17 A and TSA treated MRC5 cells;after IL-17 A and TSA treatment,the activity of HDAC1 decreased,the expression of vimentin,a-SMA,HDAC1,p-Smad2,p-smad3 decreased and the expression of Smad7 increased.Conclusion:1.Intranasal instillation of bleomycin can successfully simulate the lung fiber model and minimize the impact of exogenous stimulation or injury on the internal environment.2.In the process of pulmonary fibrosis,there are obvious destruction of lung tissue structure,alveolar hemorrhage,microvascular thrombosis,a large number of collagen fiber deposition in the lung interstitium and alveolar cavity,forming a large number of basal cell proliferation clusters.After drug intervention,there are a large number of macrophages infiltration in the lung tissue.3.With the progress of fibrosis,the expression of interstitial related proteins in lung tissue increased progressively,leading to the destruction of lung structure in a time-dependent manner.On the 28 th day,the lung injury was the most serious,and the expression of interstitial related proteins was the most serious in dexamethasone group.4.TSA can improve pulmonary fibrosis by inhibiting the activity of HDAC1 and TGF-β 1 / Smads pathway.5.Through the exploration and analysis of other signaling pathways,TSA may also inhibit the process of pulmonary fibrosis by inhibiting NF-k B,PI3 K pathways;the results did not show that p38-MAPK pathway is involved in the process of pulmonary fibrosis.6.Th1 cells and Th2 cells play a key role in the chronic stage of pulmonary fibrosis,Th17 cells play a key role in the early stage of pulmonary fibrosis,and Treg cells participate in the inhibition of the progress of pulmonary fibrosis.7.IL-17 A,TGF-β 1 and IL-6 can promote pulmonary fibrosis.IL-10 plays an anti fibrosis role in the progression of pulmonary fibrosis.It can be seen that TSA can reduce the expression of IL-17 A to achieve anti fibrosis effect.8.IL-17 A can activate the transformation of MRC5 cells into myofibroblasts and the expression of interstitial proteins,and TSA can significantly inhibit this process.