Protective Mechanism Of M2 Macrophages On Apoptosis Of Renal Tubular Epithelial Cells Induced By Calcium Oxalate Crystals

Objective To investigate the sequential induction of human monocytic leukemia cells(THP-1)into M1/M2 macrophages,and the protective mechanism of M2 macrophages on oxidative stress injury and apoptosis induced by calcium oxalate crystals(CaOx)in renal tubular epithelial cells(HK-2)under co-culture conditions.Methods 10ng/ml Phorbol-12-myristate-13-acetate(PMA)were used to induce THP-1 cells to polarize M0 macrophages at 24 h,100ng/ml lipopolysaccharide(LPS)and 10ng/ml Interferon-?(IFN-?)were used to induce M0 macrophages to polarize M1 macrophages at 48 h,20ng/ml IL-4 and 20ng/ml IL-13 were used to induce M0 macrophages to polarize M2 macrophages at 48 h,To identify macrophage polarization,using morphological observation,Real-time quantitative PCR was used to detect CD68,TNF-?,IL-1?,IL-1ra and CD206 mRNA,ELISA was used to detect IL-6,IL-10,Western Blot was used to detect TGF-?,MCP-1 protein;HK-2 cells were stimulated with 0.5 mg/ml CaOx crystals,M2 macrophages and Apocynin were co-cultured with HK-2 cells induced by CaOx crystals The experime-ntal groups:HK-2 group,HK-2+CaOx group,HK-2+CaOx+M2 group,HK-2+CaOx+Apo group,HK-2+M2 group and HK-2+Apo group.The viability of HK-2 cells in each group was detected by CCK-8 assay,The lactate dehydrogenase(LDH)activity of HK-2 cells in each group was detected by microplate reader,The apoptosis of HK-2 cells was observed by flow cytometry and Hoechst33258 staining,The damage and apoptosis of HK-2 cells in each group was observed.To observe the effects of M2 macrophages on the apoptosis of HK-2 cells induced by calcium oxalate crystals in NADPH oxidase-ROS-P38 MAPK signaling pathway,The expression of ROS and mitochondrial membrane potential in HK-2 cells of each group were detected by fluorescence microplate reader,Western Blot was used to detect the expression of NADPH oxidase p47 phox,apoptosis-related protein Bcl-2,Cleave caspase-3,Cytochrome c and mitogen-activated protein kinases p38 MAPK and Phospho-p38 MAPK,To observe the effect of M2 macrophages on the proliferation of HK-2 cells.Western Blot was used to detect protein kinase Akt,Phospho-Akt protein expression,scratch test Detect the proliferation and migration of HK-2.Results The results of RT-PCR,Western Blot and ELISA showed that the expression of CD68 mRNA in M0?M1 and M2 macrophages was significantly higher than that in THP-1 cells(P<0.05),The expression of TNF-? mRNA,IL-1? mRNA,IL-6 and MCP-1 protein in M1 macrophages was significantly higher than M0 and M2 macrophages(P<0.05),M2 macrophages showed significantly higher expression of IL-1ra mRNA,CD206 mRNA,IL-10,and TGF-? protein than M0 and M1 macrophages(P<0.05),Morphology showed that THP-1 cells were polarized to M0 type.M1 and M2 macrophages.The results of co-culture of M2 macrophages and Apocynin after HK-2 cells were stimulated by CaOx crystals suggest that M2 macrophages and Apocynin significantly increased the viability of HK-2 cells,the release of mitochondrial membrane potential and the expression of Bcl-2 protein(P<0.05),LDH activity,ROS release,apoptosis rate,p47-phox,Phospho-p38 MAPK,Cleave caspase-3 and Cytochrome c protein expression in HK-2 cells after CaOx crystals were significantly reduced(P<0.05),Scratches suggested that M2 macrophages significantly promoted the migration of HK-2 cells(P<0.05).M2 macrophages promoted the Akt phosphorylation in HK-2 cells(P <0.05).Conclusions THP-1 cells were polarized to M0 macrophages after intervention with 10 ng/ml PMA for 24 h,M0 macrophages were able to be polarized to M1 macrophages by interfering with 100ng/ml LPS and 10 ng/ml IFN-? for 48 h,M0 macrophages were able to be polarized to M2 macrophages by interfering with 20ng/ml IL-4 and 20ng/ml IL-13 for 48 h.M2 macrophages may reduce the oxidative stress injury and apoptosis of renal tubular epithelial cells by inhibiting the activation of NADPH oxidase,reduce the production of ROS and inhibiting the phosphorylation of p38 MARK.M2 macrophages may promote the proliferation and repair of renal tubular epithelial cells after CaOx crystal injury by enhancing Akt phosphorylation.