| Objective:Explore the mechanism of nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4),epithelial splicing regulatory protein 1(ESRP1)and epithelial mesenchymal transdifferentiation(EMT)in chronic obstructive pulmonary disease(COPD).Methods:1.Approved by the Ethics Committee of the General Hospital of Ningxia Medical University,collected patients from January 2015 to December 2017 in the Department of Cardiothoracic Surgery of Ningxia Medical University General Hospital who required lobectomy due to lung tumors.The results of ventilatory function were divided into control group(20 cases)and COPD group(16 cases).Obtain normal lung tissue specimens at the distal end of the lesion during the operation,and observe the morphological changes of the small airways in the COPD group and the control group by HE staining;Detect the expression of fibronectin(fibronectin,FN)and E-cadherin(E-cadherin,E-ca)in the lung tissues of the COPD group and the control group by immunohistochemistry;ELISA was performed to detect the expression of 8-isoprostaglandin F2α(8-isoprostaglandin F2α,8-iso-PGF2α)and total antioxidant capacity(T-AOC)in the serum of peripheral blood of the COPD group and the control group;2.Culture the human airway epithelial cell line(16HBE)in vitro,and treat the cells with transforming growth factor β1(TGFβ1),Detection of epithelial splicing regulatory protein 1(Epithelial splicing regulatory protein 1,ESRP1),NOX4 full Chinese name(full English name,English abbreviation)and extracellular matrix(Extracellular matrix,ECM)markers(Alpha-smooth muscle actin(Alpha-smooth muscle actin,α-SMA),N-cadherin(N-ca)and FN)expression levels;Intervene 16 HBE cells with si RNA and lentivirus,silence and overexpress ESRP1 and NOX4,respectively,detect the expression of ESRP1,NOX4 and the expression of ECM markers(α-SMA,N-ca and FN);use flow cytometry to detect The expression of reactive oxygen species(ROS)in cells.Results:1.The changes of small airway morphology in the COPD group and the control group;The pathological changes of small airways in the COPD group were observed by conventional pathological HE staining: In the COPD group,airway epithelial cells proliferated significantly,the arrangement of epithelial cells was disordered,and the small airway smooth muscle cells proliferated.2.The expression of EMT-related proteins in small airway epithelial cells in the COPD group and the control group: The interstitial marker FN in the COPD group was significantly higher than that in the control group((0.072±0.058)and(0.156±0.043),P=0.035);The epithelial marker E-ca was significantly lower than the control group((0.256±0.067)vs(0.142±0.068),P=0.016).3.The expression of 8-iso-PGF2α and T-AOC in peripheral blood serum of COPD group and control group: The expression of 8-iso-PGF2α in the serum of the COPD group was higher than that of the control group((45.91±21.17pg/ml)vs(29.74±15.88),P=0.013);The expression of T-AOC in the serum of the COPD group was lower than that of the control group((8.49±3.43U/ml)vs(13.35±4.28),P=0.001).4.The expression of ECM markers and ESRP1 and NOX4 under the action of TGFβ1 in16HBE: TGFβ1 stimulated 16 HBE cells at different concentrations and at different times.The protein levels of ECM markers FN,α-SMA and N-ca detected by Western blot were higher than those of the control group(P<0.05),and the protein levels of ESRP1 and NOX4 were also higher Group(P<0.05).5.Use si RNA to knock down ESRP1 and observe the expression of ECM markers,NOX4 and ROS after 16 HBE acts on TGFβ1: ESRP1-si RNA silenced the expression of ESRP1 in 16 HBE cells,and the effect of TGFβ1 on the protein expression of FN,N-ca andα-SMA in the si ESRP1 group was detected by Western blotting.The results showed that the protein expression of FN,N-ca and α-SMA in the si ESRP1 group was lower than that of the Control group(P<0.05),and the protein expression of NOX4 was also lower than that of the Control group(P < 0.05);The protein expression level of FN and N-ca in the si ESRP1+TGFβ1 group was higher than that in the TGFβ1 group(P<0.05),and the protein expression level of NOX4 was lower than that in the TGFβ1 group(P<0.05);The expression level of ROS in the cells of the si ESRP1 group was lower than that of the Control group by flow cytometry(P<0.05);The expression level of intracellular ROS in the si ESRP1+TGFβ1group was lower than that in the TGFβ1 group(P<0.05).6.Using lentivirus to overexpress ESRP1,observe the expression of ECM markers,NOX4 and ROS after TGFβ1 stimulates 16 HBE cells: The ESRP1 lentiviral overexpression system was established.TGFβ1 stimulated lv-ESRP1 cells for 72 hours.Western blotting was used to detect the protein expression of FN,N-ca,α-SMA and NOX4 in the lv-ESRP1 group.The results showed that the protein expressions of FN,N-ca,α-SMA and NOX4 in the lv-ESRP1 group were higher than those in the Control group(P < 0.05);The protein expression of FN,N-ca,α-SMA and NOX4 in the lv-ESRP1+TGFβ1 group;the results showed that the protein expression of FN,N-ca,α-SMA and NOX4 in the lv-ESRP1+TGFβ1group was higher than that in the Control group(P<0.05);The expression level of ROS in the cells of the lv-ESRP1 group was higher than that of the Control group by flow cytometry(P<0.05);The expression level of intracellular ROS in the lv-ESRP1+TGFβ1 group was significantly higher than that in the TGFβ1 group(P<0.05).7.Use adenovirus to knock down NOX4 and observe the expression of ECM markers,ESRP1 and ROS after TGFβ1 stimulates 16HBE: TGFβ1 acted on 16 HBE cells of sh NOX4 for 72 hours,and Western blot was used to detect the protein expression of FN,N-ca andα-SMA in the sh NOX4 group and the control group;The results showed that the protein expression levels of FN,N-ca and α-SMA in the sh NOX4 group were lower than those in the Control group(P<0.05),and the protein expression levels of ESRP1 were also lower than those in the Control group(P<0.05);The protein expression levels of FN,N-ca and α-SMA in the sh NOX4+TGFβ1 group were lower than those in the TGFβ1 group(P<0.05),and the protein expression levels of ESRP1 were also lower than that in the TGFβ1 group(P<0.05);Flow cytometry showed that the expression of ROS in the cells of the sh NOX4 group was lower than that of the Control group(P<0.05);the expression of ROS in the cells of the sh NOX4+TGFβ1 group was also lower than that of the TGFβ1 group(P<0.05).8.Using adenovirus to overexpress NOX4,observe the expression of ECM markers,ESRP1 and ROS after TGFβ1 stimulates 16HBE: TGFβ1 acted on OENOX4 cells for 72 hours.The protein expression levels of FN,N-ca and α-SMA in the OENOX4 group were higher than those in the Control group(P<0.05),and the protein expression levels of ESRP1 were higher than those in the Control group(P<0.05);TGFβ1 acted on OENOX4 cells for 72 hours.The protein expression levels of FN,N-ca and α-SMA in the OENOX4 group were higher than those in the Control group(P<0.05),and the protein expression levels of ESRP1 were higher than those in the Control group(P<0.05);Flow cytometry detected that the intracellular ROS expression level of OENOX4 group was higher than that of Control group(P<0.05);the intracellular ROS expression level of OENOX4+TGFβ1 group was also higher than that of TGFβ1 group(P<0.05).Conclusion:1.The small airway of COPD patients has obvious EMT phenomenon.The low expression of ESRP1 and high expression of NOX4 in small airway epithelial cells of COPD patients indicate that NOX4 and ESRP1 are involved in the occurrence and development of COPD epithelial-mesenchymal transition.2.Peripheral blood oxidized substance 8-isoprostaglandin F2α in COPD patients was significantly higher than that in the control group;the antioxidant substance T-AOC was significantly lower than that in the control group;TGF obviously induces the production of ROS in 16 HBE cells.With the increase of ROS production,16 HBE cells have obvious EMT.This suggests that oxidative stress is involved in the epithelial-mesenchymal transition of COPD.3.TGFβ1 promotes the expression of NOX4 and ESRP1 in human airway epithelial cells,increases the production of endogenous ROS in cells,and induces EMT,suggesting that the TGFβ1-ESRP1/NOX4-ROS signaling pathway may participate in the study of the mechanism of epithelial-mesenchymal transition.4.Overexpression or interference with the expression of ESRP can cause corresponding changes in the expression of NOX4,suggesting that ESRP1 may participate in the regulation of NOX4 signaling pathways through a certain pathway,but the relationship between ESRP1 and NOX4 still needs further study. |
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