Mechanism of cigarette smoke extract stimulate alveolar macrophages at different times influencing efferocytosis and phagocytosis function based on PPARγ signaling pathway
Objective:To observe the stimulation of cigarette smoke extract(CSE)at different times influencing the alveolar macrophage efferocytosis and phagocytosis function,and to explore the effect of CSE on inflammatory response in lung diseases and molecular mechanism.Methods:Rat alveolar macrophages NR8383 were intervened with 10%CSE for 6 h,12 h,24 h,48 h,divided into 6 h blank control group,6 h-10%CSE group,12 h blank control group,12 h-10%CSE group,24 h blank control group,24 h-10%CSE group,48 h blank control group,48 h-10%CSE group.10%CSE intervention 12 h in combination with PPAR inhibitor/ago-nist,divided into PPAR inhibitor group and PPAR agonist group.Alamar Blue colorimetry was used to detect the proliferation and toxi-city of PPAR agonist on NR8383 cells.Flow cytometry was used to detect the efferocytosis and phagocytosis of NR8383 cells,and M1 and M2 polarizations.The contents of TNF-α,TGF-β1 and MFG-E8 were detected by enzyme-linked immunosorbent assay.Western blot was used to detect the expressions of PPARγ and CD36 protein.mRNA expressions of PPARγ and CD36 were detected by qPCR.Results:After 6 hours of 10%CSE stimulation,the phagocytosis and efferocytosis of NR8383 cells were increased,the expression of PPARγ was down-regulated,the expression of CD36 mRNA was increased,and the expressions of TNF-α,TGF-β1 and MFG-E8 were increased,but there was no obvious polarization direction.After 12 hours CSE stimulation,the efferocytosis and phagocytosis functione of NR8383 cells were significantly decreased,the expressions of PPARγ and CD36 were significantly down-regulated,the expression of TNF-α was increased,the expressions of TGF-β1 and MFG-E8 were decreased,and the cells were polarized to M1-type macrophages.After 24 hours of intervention,the efferocytosis rate of NR8383 cells were decreased,but the phagocytosis function of E.coli was increased,the expression of PPARγ was down-regulated,the expressions of CD36 protein was decreased,the expression of TNF-α was decreased,while there was no statistical difference,the expressions of TGF-β1 and MFG-E8 were still decreased,and there was obvious tendency of M1 polarization.After 48 h,the efferocytosis rate of NR8383 cells were still decreased,but the phagocy-tosis ability was significantly increased,the expression of PPARγ was significantly decreased,the expression of CD36 was significantly increased,the expression of TNF-α was decreased,the expressions of TGF-β1 and MFG-E8 were increased,and the polarization of macrophages towards M1 and M2 were increased.After 12 h stimulation with 10%CSE combined with PPAR agonist and inhibitor,it was found that PPAR agonist enhanced the efferocytosis and phagocytosis of NR8383 cells,up-regulated the expression of PPARγ and CD36,inhibited the expression of inflammatory factor TNF-α,and promoted the expressions of anti-inflammatory factor TGF-β1 and cytokinesis cofactor MFG-E8.Conclusion:With the stimulation time of CSE,alveolar macrophages gradually change from the activated state of early inflammatory response to chronic inflammatory response,which leads to alveolar macrophage efferocytosis and phagocyto-sis dysfunction,and the mechanism is related to the inhibition of PPARγ pathway.
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