Regulation Of 1,25（OH）₂D₃ On Polarization And Autophagy Of Head Kidney Macrophages In Yellow Catfish Pelteobagrus Fulvidraco

As a key class of immune cells,macrophages are not only the first line of defense against pathogen invasion,but also play different functions through polarization.Macrophages can secrete pro-inflammatory factors and cause excessive inflammatory response,resulting in a variety of inflammatory diseases when polarizing into M1 type.Macrophages can secrete anti-inflammatory factors and play an anti-inflammatory role when polarizing into M2 type.Its dynamic plasticity regulates cell-mediated tissue damage and repair functions,and plays an important role in the stages of inflammation,proliferation,and healing.Autophagy repairs damaged organelles,regulates protein recycling.It plays an important role in cell survival,development,immune regulation,intracellular homeostasis and the occurrence and development of diseases.Therefore,maintaining cell homeostasis by regulating macrophage phenotype balance and autophagy regulation is a topic worthy of study.At present,studies have found that 1,25（OH）₂D₃plays a very important role in immune regulation.The role of 1,25（OH）₂D₃in macrophage autophagy is reflected through different pathways,which can regulate the balance between autophagy and apoptosis to reduce excessive inflammation.At the same time,1,25（OH）₂D₃can reduce macrophage infiltration and improve tissue damage caused by inflammation by balancing polarization phenotype.However,the regulatory mechanism of1,25（OH）₂D₃on macrophage polarization and autophagy is not clear.Therefore,the purpose of this study is to explore how 1,25（OH）₂D₃affects macrophage polarization and autophagy.The details are as follows:（1）In this study,the primary macrophage cells of Pelteobagrus fulvidraco head kidney were isolated and cultured,and studied the effect of 1,25（OH）₂D₃on the polarization of the head kidney macrophages of Pelteobagrus fulvidraco by morphological analysis,biological function analysis and gene expression analysis.After being challenged by LPS and c AMP to induce macrophage polarization,the cell morphological changes were observed by inverted light microscopy,and the functional changes were studied by measuring survival rate,phagocytosis,reactive oxygen species and nitric oxide production,superoxide anion radical and arginase activity,as well as the related gene expression charactering in different macrophages polarization states.The results are as follows:The results showed that 1,25（OH）₂D₃reduced the mortality of M1 and M2macrophages and enhanced the phagocytic activity of macrophages.In M1macrophages,1,25（OH）₂D₃inhibited the production of reactive oxygen species（ROS）and inflammatory mediator nitric oxide（NO）,reduced the activity of superoxide anion free radical,and decreased the expression of interleukin-1β（IL-1β）and tumor necrosis factor-α（TNF-α）（P<0.05）.The activity of arginase as well as the expression of interleukin-10（IL-10）and transforming growth factor（TGF-β）in M2 cells was up-regulated by 1,25（OH）₂D₃（P<0.05）.In conclusion,1,25（OH）₂D₃inhibited the polarization of macrophages to M1 phenotype,promoted the polarization of macrophages to M2 phenotype,and played an anti-inflammatory role.In the current study,Nos2 and arg2 were found to be biomarker genes of M1 and M2 macrophages,respectively.These results preliminarily reveal the mechanism of 1,25（OH）₂D₃on the polarization of head kidney macrophages of Pelteobagrus fulvidraco,and favor the regulation of macrophage phenotypes in the treatment of inflammation,which provides useful information for further study on the polarization of fish macrophages and the effect of 1,25（OH）₂D₃on polarization regulation.（2）In this experiment,after macrophages were incubated with chloroquine,an autophagy inhibitor,and 1,25（OH）₂D₃,after being challenged by LPS and c AMP to induce macrophage polarization,MDC staining was used to detect autophagy level,oil red O staining was used to detect lipid droplet accumulation,and the changes of intracellular total cholesterol and free cholesterol were detected.At the same time,polarization markers such as reactive oxygen species,nitric oxide,superoxide anion free radical and arginase were also measured.Finally,the key autophagy genes,cholesterol efflux related genes,NLRP3 inflammatory corpuscle genes and polarization marker genes were detected by real-time quantitative PCR,so as to further reveal the effect of 1,25（OH）₂D₃on autophagy and polarization of head kidney macrophages of Pelteobagrus fulvidraco.The results are as follows:After inhibiting autophagy,the differentiation of cells into M1 type increased significantly,while the differentiation into M2 type cells decreased significantly.Inhibition of autophagy could change the direction of polarization,but polarization could not change the expression of autophagy.Autophagy behavior existed in M1 and M2 cells.Autophagy inhibitor treatment could inhibit the level of autophagy,and1,25（OH）₂D₃incubation could increase the level of autophagy in M2 cells.The results showed that there was a negative correlation between the activation of NLRP3inflammatory body and autophagy.Increasing autophagy could inhibit the expression of NLRP3 inflammatory body.1,25（OH）₂D₃may inhibit the expression of NLRP3inflammatory body and promote the expression of anti-inflammatory factors by increasing autophagy activity and promoting cholesterol outflow,and finally promote the cell polarization to M2 type through autophagy.