The Effects Of Autophagy On Classical Activation Of Macrophages Induced By High Glucose

Objectives:Macrophage infiltration is an important histopathological feature of diabetic nephropathy,and the activation state of macrophages in the local microenvironment is the main factor that determines the prognosis of the disease.Previous studies confirmed that infiltration of macrophages in the renal tissue of DN patients was dominated by classically activated macrophages(M1),and the proportion of alternatively activated macrophages(M2)was significantly decreased.Autophagy is a lysosome-mediated process of excessive or abnormal protein degradation.It is a highly dynamic,multi-phase process,and also known as autophagic flow that mainly includes two phases of autophagosomes and autolysosome.Recent studies have found that autophagy plays an important role in the regulation of many cell functions.However,its role in the macrophage M1/M2 phenotype transition and the specific mechanisms still need to be further explored.In this study,macrophages were cultured in vitro to investigate the effect of autophagy on macrophage activation in high glucose conditions.Then we further explored the effect of each phase of autophagy on the phenotypic transformation of macrophages.These studies will provide basis for the use of autophagy to regulate macrophage activation status to prevent the occurrence and development of diseases.Methods:(1)Mouse macrophage RAW264.7 was incubated with 30.0 mmol/L glucose and treated with autophagy activator(RAPA)and inhibitor(3-MA).Western blot and immunofluorescence were used to detect the expression of M1 type marker iNOS,TNF-? and M2 markers MR,Arg-1,and autophagy markers LC3,Beclin-1,and p62.(2)RAW264.7 cells were stimulated with 100 U/mL IFN-? + 5 ng/mL LPS to induce M1 activation and stimulated with 10 ng/mL IL-4 stimulation induced M2 activation.Using RAPA,3-MA,Bafi,and CQ to interfere with the autophagic flow in the classic M1/M2 model,the levels of M1,M2,and autophagy markers were again detected by the above method.Then using an electron microscope to observe autophagic flow.Results:(1)(1)High glucose promoted the expression of iNOS and TNF-? in RAW264.7 cells,but inhibited the expression of MR and Arg-1,then induced M1 activation.(2)The high glucose environment inhibited the expression of LC3 and Beclin-1,but promoted the expression of p62,so decreased the autophagy level in RAW264.7 cells.(3)Using RAPA to increase the level of autophagy resulted in a decrease in the expression of iNOS in RAW264.7 cells induced by high glucose and an increase in MR expression,suggesting that increased autophagy tends to M2 activation.After the autophagy was reduced by 3-MA,the expression of iNOS was further increased,and the expression of MR decreased significantly.That is,when the level of autophagy was low,M1 activation was tended to occur.(2)(1)The M1/M2 model of RAW264.7 cells was constructed.M1 cells inhibited the expression of LC3 and Beclin-1 and promoted the expression of p62.The autophagy level of M2 cells was higher than that of M1 cells.(2)Under the electron microscope,autophagic flow process of macrophages was observed,such as autophagosome formation,fusion of autophagosomes and lysosomes,autolysosome formation,and substrate degradation.(3)In M1 cells,RAPA promoted the production of autophagosomes,and the expression of LC3,Beclin-1,and p62 increased;3-MA inhibited the production of autophagosomes,and the expression of LC3,Beclin-1,and p62 decreased;Bafi inhibited the fusion of autophagosomes and lysosomes,the expression of LC3 and Beclin-1 decreased,while the expression of p62 increased significantly;CQ inhibited autolysosome degradation,and the expression of autophagy-related proteins LC3-II,Beclin-1,and p62 showed a tendency to increase.(4)In the M1/M2 model of RAW264.7 cells,it was observed that the intervention of autophagic flow could affect the M1/M2 phenotype transition.That is,activation of autophagy induces M2-type transformation of the cells,while inhibition of autophagy induces M1-type activation.Conclusions: 1.High glucose induces classical activation of macrophages(M1),and that is associated with decreased autophagy.2.Autophagic flow can regulate the mutual transformation of macrophage M1/M2 phenotype.3.Changes in a single marker don't determine the level of autophagy in the cell,and it need to assesse the dynamic changes in autophagy flow.