Role And Mechanism Research Of NOX4 And ESRP1 In Airway Epithelial-mesenchymal Transition

Objectives: To investigate the Mechanism research between the expression of Nicotinamide-adenine dinucleotide phosphate oxidase 4(NOX4)and Epithelial splicing regulatory protein 1(ESRP1)in Epithelial-mesenchymal transition(EMT)of Chronic Obstructive Pulmonary Disease(COPD).Methods:1.Collect lung cancer patients undergoing pulmonary surgical operation,Preoperative lung function tests were divided into lung cancer with COPD groups(referred to as COPD groups)and lung tumor normal lung function groups(referred to as the Control groups),each of the groups have 23 examples.The normal lung tissues distal to the tumor lesions were taken during operation.Detect the expression of ESRP1,NOX4,and ECM markers in the COPD groups and Control groups.Detect the levels of SOD3 and MDA in the COPD groups and Control groups.2.Cultivate people airway epithelial cell line in vitro,with TGF?1 and NAC to detect the expression of ESRP1,NOX4 and EMT markers in BEAS-2B;Detect the levels of ROS in BEAS-2B.Results:1.Expression of EMT Related Markers in Small Airway Epithelial Cells in COPD Group: Immunohistochemistry and Western blotting showed that the expression of EMT-associated marker proteins such as FN,LN,?-SMA,COI-I and TGF?1 in COPD group were higher than those in control group.In the group,the expression of E-Ca was lower thanthat of the control group.2.The expression of ESRP1 and NOX4 in small airway epithelial cells in COPD group:Immunohistochemistry,Western blotting and real-time fluorescence quantitative detection showed that the protein and m RNA levels of ESRP1 in COPD group were lower than those in the control group(P<0.05).The protein and m RNA levels of NOX4 were higher than those of the control group(P<0.05).3.COPD patients serum test results: ELISA showed that COPD patients serum SOD3 levels were lower than the control group(P <0.05),MDA levels were higher than the control group(P <0.05).4.TGF?1 induces the expression of EMT-related markers in BEAS-2B cells: BEAS-2B cells were stimulated by different concentrations and time points of TGF?1.Western blot analysis showed that the protein levels of FN,LN,?-SMA,COI-I,N-Ca,Vimentin were higher than those of the control group(P<0.05).5.TGF?1 induces the expression of ESRP1 and NOX4 in BEAS-2B cells: BEAS-2B cells were stimulated with different concentrations and time points of TGF?1,Western blot analysis showed that the protein and mRNA levels of ESRP1 and NOX4 were higher than those of the control group(P<0.05).6.TGF?1 induces the ROS expression of BEAS-2B cells: Flow cytometry showed that TGF?1 stimulation of BEAS-2B cells,ROS expression was higher than the control group(P<0.05).7.The effect of NAC on TGF?1-induced EMT antagonism: After NAC pretreatment of cells,BEAS-2B cells(NAC+ TGF?1 group)were treated with TGF?1,and EMT-related markers FN,LN,?-SMA,N-Ca,Vimentin and COI-I were detected by Western blotting.The protein levels expression of FN,LN,?-SMA,COI-I,N-Ca and Vimentin in NAC+ TGF?1group were lower than those in TGF?1 alone group(P<0.05).8.The effects of NAC on TGF?1-mediated expression of ESRP1 and NOX4: After NACpretreatment of cells,BEAS-2B cells(NAC+ TGF?1 group)were treated with TGF?1,and the expression of ESRP1 and NOX4 were quantitatively detected by Western blot and real-time fluorescence.The results showed that: The mRNA and protein levels of ESRP1 and NOX4 in NAC+TGF?1 group were lower than those in TGF?1 alone group(P<0.05).Conclusions:1.There is a significant EMT phenomenon in the small airway of COPD patients,and low expression of ESRP1 and high expression of NOX4 in small airway epithelial cells suggest that NOX4 and ESRP1 may participate in the occurrence and development of COPD epithelial-mesenchymal transition through certain signaling mechanisms.2.TGF?1 up-regulates the expression of ESRP1,an EMT-related regulator,and induces EMT in human airway epithelial cells.It is suggested that TGF?1-ESRP1-EMT signaling pathway plays an important role in the development of EMT in human small airway epithelial cells.3.The level of MDA in circulating oxygenated products was significantly higher in patients with COPD and the SOD of antioxidative enzymes was significantly decreased.At the cellular level,TGF?1 upregulates the expression of NOX4 in airway epithelial cells and induces release of intracellular ROS,suggesting that the oxidation/antioxidation imbalance is slow-resistance.Lungs play an important role in the occurrence/development of the lungs4.Antioxidant NAC can significantly antagonize the TGF?1-induced EMT;antagonize TGF?1-mediated NOX4 overexpression and intracellular ROS release,suggesting that NOX4-mediated ROS signaling pathways and ESRP-mediated EMT signaling pathways have some cross-linking.NAC can be used as a new drug target for COPD.