Luteolin Regulates Macrophage Polarization Via The PI3K/Akt Pathway To Inhibit The Apoptosis Stimulated By Angiotensin ?

Backgrounds and Aims:Atherosclerosis(AS)is a chronic inflammatory vascular disease.Macrophages are involved in the occurrence and development of the overall process of AS.Therefore,macrophages have become a vital target for the prevention and treatment of AS.In particular,macrophage polarization has become an important research topic.Studies have shown that macrophage polarization can impact the occurrence and development of AS.Previous study demonstrated that Lut suppresses Ang ?-induced oxidative stress and apoptosis in murine peritoneal macrophages.However,the specific mechanism for the anti-apoptotic effect of Lut on murine peritoneal macrophages is not yet clear.Akt is a member of the protein kinase B family and is a downstream target of phosphoinositide 3-kinase(PI3K).The PI3K/serine/threonine kinase(PI3K/Akt)pathway is an important regulator of apoptosis/proliferation signaling pathways,which play critical roles in normal cellular functions,including proliferation,adhesion,migration,invasion,energy metabolism and protein synthesis.Accordingly,we speculated that Lut may protect macrophages from Ang II-induced apoptosis by inducing macrophage polarization via the PI3K/Akt pathway.The aim of the present study was to explore the underlying mechanisms for the anti-apoptotic effects of Lut on macrophages as well as to determine the role of the PI3K/Akt pathway in this process.Methods:C57BL/6 mice(female,8-weeks old)were used in the study.Murine peritoneal macrophages were cultured.Isolation and culture of peritoneal macrophages,and identified the anti-CD68 monoclonal antibodies for macrophages by Immunofluorescence.To select the appropriate concentration of Lut,the cell viability was evaluated using CCK-8,and the levels of LDH in the cell supernatants were determined using a commercially available kit.Experimental group:the experiment groups were assigned:control group,Ang ? group,Ang ? + LY294002 group,Ang ? +Luteolin group,Ang ? +Luteolin+LY294002 group.The fluorescence intensities of the annexin V/Pl-stained cells were analyzed using flow cytometry within 1h.The apoptotic cells,including Annexin V +/PI-were counted.Next,the cells(100 ?l)were incubated with 5?1 of a fluorescein isothiocyanate-labeled CD 16/32 antibody and 5 ?l of a p-phycoerythrin-labeled CD206 antibody for 15 min in the dark at room temperature.The fluorescence intensities of the stained cells were analyzed using flow cytometry within 1 h.The supernatants from the M1 and M2 macrophages were tested for the presence of cytokines and growth factors using commercially available ELISAs for IL-6,TNF-?,iNOS,Dectin-1,IL-10 and Arg-1.To investigate the mechanisms underlying the protective effects of Lut on the peritoneal macrophages that had been stimulated with Ang ?,Akt,phospho-Akt308,phospho-Akt473,Bax,Bcl-2,cleaved caspase-3 and caspase-3 were measured using western blotting with ?-actin as an internal standard.Results:1.The macrophage viability in the presence of Lut(6.25,12.5,25 ?M)was similar to that of the control group whereas Lut treatment at 50 ?M caused a decrease.LDH was also measured at the various concentrations of Lut,Lut(50 ?M)treatment significantly increased the content of LDH compared to the control group.Based on these results,the 25?M concentration of Lut was selected as the optimal condition for the following experiments.2.Compared to the Ang II-treated group,the Bcl-2/Bax and caspase-3/cleaved caspase-3 ratios were slightly increased in the LY294002-treated group(all P<0.05),and these indices were further iincreased in the Lut group compared to the LY294002 group(all P<0.05).The values for the Lut group and the Lut+LY294002 group had nowere not statistically different.3.Annexin V/PI staining and flow cytometric analysis showed that the apoptosis rate for LY294002 pretreatment,the apoptosis rate decreased compared with the Ang ? group(P<0.001),and Lut pretreatment tfurther inhibited the macrophage apoptosis compared with the LY294002-pretreated group(P<0.01).The difference between the Lut group and the Lut+LY294002 group was not statistically significant.4.The expression levels ofMI(IL-6,TNF-?,iNOS)and M2(Dectin-1,IL-10,Arg-1)macrophage phenotypic markers showed a slight decrease in the LY294002-treated group compared with the Ang ?-treated group(P<0.001).In addition,the Lut-treated group showed a further decrease in the expression of' these markers compared with the LY294002-treated group(P<0.05).No significant difference was observed between the Lut-treated group and the Lut+LY294002-treated group.5.The percentage of CD16/32 cells was slightly lower in the LY294002-treated group compared with the Ang ?-treated group(P<0.001),and there was a further reduction in the Lut-treated group compared with the LY294002-treated group(P<0.01).The difference between the Lut-treated group and the Lut+LY294002-treated group was not significant.In contrast,the percentage of CD206 cells in the LY294002-treated group was increased compared with the Ang II-treated group(P<0.001).and the percentage of CD206 cells in the Lut-treated group were further increased compared with the LY294002-treated group(P<0.05).The difference between the Lut-treated group and the Lut+LY294002-treated group was not significant.6.Lut caused a further decline in the protein level of p-Akt(473)in a time-and concentration-dependent manner.Lut pretreatment(25 ?M for 12 h)also significantly decreased the protein level of p-Akt(308).The effects of Lut were similar to those of LY294002,a specific inhibitor of the PI3K/Akt pathway.Conclusions:These results indicated that Lut has anti-apoptotic effects on macrophages that were stimulated with Ang ? through the promotion of macrophage polarization,which might be associated with the PI3K/Akt signaling pathway.