The Mechanism By Which Nur77 Regulates Airway Smooth Muscle Cell Activation And Its Influence On Airway Remodeling

Objective:To investigate the effect of regulation of Nur77 expression on proliferation and migration of primary airway smooth muscle cells in mice and its mechanism,as well as the effect on airway remodeling in asthmatic mice.Methods:?1?PDGF-BB stimulated mouse primary airway smooth muscle cells,and CCK8 method and scratch test to detect the proliferation and migration of airway smooth muscle cells.Western blot and qRT-PCR were used to detect the expression of proliferating cell nuclear antigen?PCNA?,cyclin D1 and NR4A family mRNA and protein.?2?Nur77 small interfering RNA was constructed to transfect airway smooth muscle cells,or primary airway smooth muscle cells were stimulated with Nur77natural agonist Cytosporone B?Csn-B?.CCK-8 method,scratch test,Western blot and qRT-PCR were used to detect the effect of Nur77 on the proliferation and migration of mouse primary airway smooth muscle cells.?3?The various small molecule inhibitors CP-673451,SP600125 and KG-501 inhibited PDGF receptor activity,JNK pathway and CREB transcription factor activity,respectively.The expression of Nur77 in airway smooth muscle cells induced by PDGF-BB was detected,and the molecular mechanism of Nur77 expression in airway smooth muscle cells was investigated.?4?Asthma mice were constructed using the classic OVA asthma model.The treatment group was intraperitoneally injected with Csn-B(10 mg·kg^-1).HE staining was used to detect pathological changes in lung tissue.The airway wall thickness and airway smooth muscle layer thickness of each group were calculated by IPP software.The expression of Nur77 in mouse lung tissue was detected by immunohistochemistry,Western blot and qRT-PCR.Results:?1??1?PDGF-BB induced the expression of Nur77 in airway smooth muscle cells in a concentration-and time-dependent manner.The expression of Nur77mRNA was highest at 1 hour after stimulation with 20 ng/mL PDGF-BB,and the expression of Nur77 protein was highest at 6 hours after stimulation,and then decreased rapidly.?2?PDGF-BB promotes the expression of PCNA and cyclin D1 in mouse primary airway smooth muscle cells,and enhances airway smooth muscle cell proliferation in a concentration-dependent manner and accelerates cell migration.?2??1?Csn-B can induce the expression of Nur77 in mouse primary airway smooth muscle cells.After Csn-B incubation,the expression of Nur77 in cells increased rapidly,and it was found that the level of Nur77 remained high at 24 hours.?2?After induction of Nur77 expression in mouse primary airway smooth muscle cells,the cell proliferation rate decreased significantly,and the PCNA protein expression level also decreased.The cell migration ability also decreased.?3?After knocking down the expression of Nur77in mouse primary airway smooth muscle cells,cell proliferation and migration ability were significantly enhanced.?3?PDGF-BB is capable of inducing phosphorylation of JNK protein and CREB transcription factors in mouse primary airway smooth muscle cells in a time-dependent manner.After inhibiting the phosphorylation of JNK protein,the phosphorylation degree of CREB was significantly decreased,and the expression of Nur77 was decreased in mouse primary airway smooth muscle cells.After inhibiting CREB phosphorylation in mouse primary airway smooth muscle cells,the expression of Nur77 was significantly decreased.?4??1?The expression of Nur77 in lung tissue of asthma model mice was slightly higher than that in the control group,but the level of Nur77 in the lung tissue of mice treated with Csn-B was higher than that of the control group and the asthma model group.?2?HE staining results showed that Csn-B significantly reduced the airway wall and airway smooth muscle layer thickness of model animals,and reduced the degree of airway remodeling in asthmatic mice.Conclusion:?1?In mouse primary airway smooth muscle cells,Nur77 can be rapidly induced and rapidly degraded by PDGF-BB,and cell proliferation and migration are significantly increased.Regulation of Nur77 expression in mouse primary airway smooth muscle cells significantly affects cell proliferation and migration.?2?In mouse primary airway smooth muscle cells,the regulation of Nur77may be through the JNK signaling pathway,and CREB transcription factors are also involved in the regulation of Nur77 expression.?3?Up-regulation the level of Nur77 in mouse lung tissue significantly reduced the airway wall and airway smooth muscle layer thickness in asthmatic mice and inhibited the development and progression of airway remodeling in asthmatic mice.In conclusion,this study demonstrates that orphan nuclear receptor Nur77 is an important transcription factor that regulates the proliferation and migration of mouse primary airway smooth muscle cells and airway remodeling in asthmatic mice,which provides a new target and research direction for the development of new drugs for the treatment of asthma.