基于PARP9-DTX3L/Notch轴探讨平喘颗粒抑制树突状细胞成熟的机制研究OA

Objective:To investigate the effects of Pingchuan Granules on dendritic cell maturation mediated by the PARP9-DTX3L/Notch axis,thereby elucidating its molecular mechanism in inhibiting airway inflammation in asthma.Methods:Bone marrow-derived mononuclear cells from C57BL/6J mice were differentiated into dendritic cells using granulocyte-macrophage colony-stimulating factor and interleukin-4,and an in vitro maturation model was established by stimulation with tumor necrosis factor-α.The experiment included the control group,the model group,the Pingchuan Granule group,the inhibitor group,and the inhibitor+Pingchuan Granule group.Cell morphology was observed under an inverted microscope.Flow cytometry was employed to detect dendritic cell purity and the expression of surface molecules CD80,CD86,MHC-Ⅱ,and FcεRI.Western blot and RT-qPCR were used to measure the expression of PARP9,DTX3L,and N1ICD at both protein and mRNA levels,and the nu-cleocytoplasmic translocation of N1ICD was analyzed.Co-immunoprecipitation was performed to detect the PARP9-DTX3L inter-action,N1ICD ubiquitination,and the ADP-ribosylation level of Ub.Results:Compared to the model group,Pingchuan Granule-containing serum significantly inhibited dendritic cell maturation and reduced the expression levels of CD80,CD86,MHC-Ⅱ,and FcεRI(P<0.01).At the molecular level,Pingchuan Granules downregulated the expression of PARP9 and N1ICD proteins,as well as the mRNA levels of PARP9,Hes1,Hes5,and Hey1(P<0.01),and inhibited the nuclear import of N1ICD(P<0.05),whereas they had no significant effect on the protein and mRNA expression of DTX3L as well as the mRNA expression of Notch1(P>0.05).Furthermore,Pingchuan Granules significantly inhibited the interaction between PARP9 and DTX3L,reduced the levels of ADP-ribosylation of Ub,and promoted the ubiquitination of N1ICD,with all differences being statistically significant(P<0.01).Conclusion:Pingchuan Granules can effectively inhibit the maturation of dendritic cells,its mechanism may be related to interfering with the formation of the PARP9-DTX3L complex,thereby suppressing the activation and nuclear translocation of N1ICD in the Notch signaling pathway,and the transcription of its downstream target genes.