Carboxy-terminal-binding Protein 1 (CTBP1) Aggravates Proliferation And Activation Idiopathic Pulmonary Fibrosis By Promoting Fibroblast

Extensive deposition of extracellular matrix(ECM)in idiopathic pulmonary fibrosis(IPF)is due to excessive activation and proliferation of lung fibroblasts.Pulmonary fibrosis(PF)is a serious interstitial lung disease.The main pathological characteristics are diffuse alveolar inflammation in the early stage,pathological transformation of fibroblasts in the late stage and abnormal accumulation of extracellular matrix(ECM)in place of normal lung tissue.But its pathogenesis remains unclear.pulmonary fibroblast(FB)plays an important role in the pathogenesis of IPF.Under pathological conditions,FB secretes a large amount of extracellular matrix through abnormal proliferation and transformation,which directly causes IPF.In addition,FB can also act on epithelial cells,and some inflammatory cells are indirectly involved in the PF process.However,the exact mechanism remains unclear.Therefore,more mechanisms of the role of lung fibroblasts in IPF remain to be explored.C-terminal binding protein(CTBP)is an evolutionarily conserved transcriptional corepressor that regulates the expression of many genes.In recent years,a large number of studies have shown that CTBP is highly expressed in lung cancer,prostate cancer,breast cancer and other tumor tissues or cells.CTBP is closely related to the occurrence,development and prognosis of tumors.E-cadherin has been down-regulated by overexpression of CTBP in a variety of tumor cells.In cholangiocarcinoma,the expression of CTBP1 protein is significantly upregulated and negatively correlated with the expression of E-cadherin protein.CTBP1 may be involved in regulating and inhibiting the expression of E-cadherin protein in cholangiocarcinoma,thereby promoting tumor EMT.However,the relationship between CTBP1 and pulmonary fibrosis is rarely reported.Zinc finger E-box binding homeobox 1(ZEB1)is the key transcription factor of EMT.ZEB1 can promote the cell proliferation,migration and collagen formation and induce the fibrosis through EMT.Toosendanin is a tetracyclic triterpenoid compound derived from the fruit of Melia toosendan.Toosendanin has a certain degree of toxicity and was mainly used in agriculture in the early days.In recent years,in vitro studies have found that Toosendanin has a certain inhibitory effect on breast cancer cells,liver cancer cells,human leukemia cells,and other cells.Toosendanin can inhibit the proliferation and activation of various cells to a certain extent.This study focused on the role of CTBP1 in lung fibroblast function,elaborated its regulatory mechanism,and analyzed the relationship between CTBP1 and ZEB1.Meanwhile,the anti-pulmonary fibrosis effect and molecular mechanism of Toosendanin were studied.The main contents of this paper as follows:Part Ⅰ Study on the expression of carboxy-terminal binding protein 1(CTBP1)in lung fibrosis patients’ tissuesObjective: To explore the changes in the expression of CTBP1 in lung fibrosis tissues.Methods: Immunohistochemistry was used to detect the expression of CTBP1 in control and IPF patient lung tissues: This study collected lung tissue specimens from control and IPF patients,fixed them in 10% neutral buffered formalin,and then embedded them in paraffin.The paraffin blocks were cut into 4μm sections using a microtome,and the sections were placed on glass slides.After processing the slides with immunohistochemistry,they were incubated with CTBP1 antibody.Immunohistochemical staining was then performed.Image analysis software was used to analyze the results of immunohistochemical staining.The expression of CTBP1 in control and IPF patient lung tissues was calculated and statistically analyzed.Results: The results showed that the expression of CTBP1 was increased in fibrotic tissues in clinical samples with statistical difference.Conclusion: The high expression of CTBP1 may be closely related to the occurrence and development of pulmonary fibrosis.Part Ⅱ The interaction between CTBP1 and ZEB1 promotes the proliferation and activation of fibroblasts and aggravates idiopathic pulmonary fibrosisObjective: The second part of this study explores whether CTBP1 can promote the progression of pulmonary fibrosis and whether CTBP1 can interact with ZEB1。Methods:(1)Human IPF fibroblast cell lines(LL-97 A and LL-29)and normal fibroblast cell line(LL-24)were cultured in vitro.Cell proliferation was detected by BrdU method.Cells were divided into control and experimental groups.Cells in the control group were treated with control siRNA,and cells in the experimental group were treated with CTBP1 siRNA.After the cells were cultured for 48 hours,the corresponding reagents were added according to the operation requirements of the BrdU kit to detect the cell proliferation.(2)Western blot was used to detect the expression levels of lung fibrosis-related indicators in cells.After the cells with different treatments were cultured for 48 hours,the cells were lysed with RIPA cell lysate,and the proteins were collected for Western blot detection.(3)Use C57BL/6 mice to establish a model of pulmonary fibrosis,and analyze the effect of CTBP1 silencing on pulmonary fibrosis and lung function in mice.The mice were divided into control group,model group and experimental group.According to the classic bleomycin-induced pulmonary fibrosis mouse model modeling method,the mice were modeled with pulmonary fibrosis.Before modeling,on the 10 th day and the 21 st day,indicators such as tidal volume and minute ventilation of experimental animals were detected.After the experiment,the lung tissues of the animals were taken for subsequent experiments.(4)Detection of collagen 1a1(COL1A1),collagen 3a1(COL3A1),laminin(LN),fibronectin(FN)and α-smooth muscle actin(α-SMA)by cell proliferation assay and qRT-PCR expression.Results: Cell and animal studies showed that CTBP1 was upregulated in IPF lung fibroblasts,suggesting that CTBP1 may play a role in promoting lung fibrosis.Silencing of CTBP1 inhibits growth factor-driven proliferation and activation of lung fibroblasts.Overexpression of CTBP1 promotes growth factor-driven proliferation and activation of lung fibroblasts.After overexpression of CBTP1,1a1 collagen(COL1A1),3a1 collagen(COL3A1),laminin(LN),fibronectin(FN),and α-smooth muscle actin(α-SMA)protein expressions were all up-regulated.Silencing of CTBP1 reduces the degree of lung fibrosis in mice with pulmonary fibrosis.After BLM treatment,the total lung capacity of the mice was significantly reduced.However,lung capacity in mice increased with CTBP1 silencing.Lung compliance decreased 2 weeks after BLM stimulation.However,lung compliance in mice was enhanced in response to silencing of CTBP1.Tissue electrical resistance was measured 2weeks after BLM stimulation.The results showed that mice treated with adenoviral silencing of CTBP1 exhibited improved lung tissue resistance.Hydroxyproline in lung homogenate was determined with a hydroxyproline kit.Bleomycin-treated mice treated with adenovirus-silenced CTBP1 had reduced hydroxyproline content in lung tissue compared with bleomycin-treated mice.Western blot,CO-IP and BrdU assays confirmed that CTBP1 interacts with ZEB1 and promotes the activation of lung fibroblasts.Conclusion: CTBP1 and ZEB-1 are interacting proteins.CTBP1 promotes the activation of lung fibroblasts through ZEB1,thereby increasing the excessive deposition of ECM and aggravating IPF.Part Ⅲ Toosendanin inhibits the proliferation and activation of lung fibroblasts by inhibiting CTBP1Objective: To investigate the effect and mechanism of Toosendanin on proliferation and activation of lung fibroblasts.Methods: In this part of the study,a cell model of pulmonary fibrosis was established: PDGF-BB inducer was used to stimulate LL-29 cells to establish a cell model of pulmonary fibrosis.Toosendanin treatment:Toosendanin was added to the medium to make the final concentration of toosendanin to 1 μM and 10 μM respectively,and the pulmonary fibrosis cells were treated for 24 hours.Cell proliferation was detected by cell proliferation assay.The expression of collagen 1a1(COL1A1),collagen 3a1(COL3A1),laminin(LN),fibronectin(FN)and α-smooth muscle actin(α-SMA)was detected by qRT-PCR to verify the expression of Toosendan Inhibits the proliferation and activation of lung fibroblasts.Results: Through the third part of the study,we found that the expression of CTBP1 and ZEB1 in cells was significantly reduced when treated with toosendanin.Further qRT-PCR results showed that toosendanin could decrease the expression of COL1A1,COL3A1,LN,FN and α-SMA.Conclusion: This study found that toosendanin,a monomer molecule of traditional Chinese medicine,can inhibit the proliferation and activation of lung fibroblasts by inhibiting CTBP1.