Regulation And Mechanism Of NaCl On M1 Macrophage Polarization

Objective:To study on the regulation of high salt on M1 macrophage polarization,and verify on the inflammation disease that endotoxic shock model,which provides a theoretical basis for clinical intervention and treatment measures of some diseases.At the same time,these lay a solid foundation for our next step to explore its mechanism.Method : 1.GM-CSF was used to induce mouse bone marrow-derived primary macrophages(BMDMs),before adding LPS(200 ng/ml)and IFN-?(10 ng/ml),BMDMs were pretreated with different concentrations of NaCl for 2 hours.(1)M1 macrophages activity were detected by CCK8 in the presence of different concentrations of NaCl,aiming to determine a suitable high salt concentration used in the following experiments.(2)With the appropriate high salt concentration pretreating BMDMs,We acquired cell culture supernatant in 6,12 and 24 hours respectively,ELISA was used to detect the expression of M1 macrophage factor IL-6 and IL-12p40.(3)With the different concentrations of NaCl(5,10 and 20 mM)pretreating BMDMs,after 24 hours,we got the cells,and the expression of IL-12p40 and IL-6 in mRNA level were detected by RT-PCR.(4)The average fluorescence intensity of IL-12p40 was detected by flow cytometry.(5)Flow cytometry was used to detect the expression of CD86 and MHCII on the surface of M1 macrophages.(6)The expression of IRF5 protein was detected by Western Blot.2.BMDMs cells were divided into two groups,one group were pretreated with NaCl for 6 hours,then,cells wre collected and made the concentration of 2×106/200 ?l.20 mice were divided into two groups,each group had 10 mice,twogroups of cells were injected into mice respectively.(1)After 24 hours,each mouse was injected intraperitoneally with 800 ?g LPS,and the mortality rate was observed.(2)After 24 hours,each mouse was injected intraperitoneally with 200 ?g of LPS.And after 12 hours,the mice were taken out of eye blood and killed by cutting off the neck,and then we obtained peritoneal cells,lung,liver and spleen.(1)Pathological section H&E staining showed the lung and liver injury.(2)ELISA was used to detect the expression of IL-6 and IL-12p40 in the serum.(3)RT-PCR was used to detect the expression of IL-6 and IL-12p40 in mouse peritoneal and splenic cells.Results:1.(1)High concentration of salt solution(? 40mM)weakened the activity of M1 macrophages.20 mM NaCl was considered as the appropriate high salt concentration,which did not affect cell viability with time.(2)ELISA showed that NaCl pretreatment significantly reduced the expression of IL-6 and IL-12p40,which was time and dose dependent.(3)Compared with the control grou p,NaCl pretreatment group significantly reduced the expression of IL-6 and IL-12p40,which was dose dependent.(4)Compared with the control group,flow cytometry showed that the expression of IL-12p40 was significantly reduced in the NaCl pretreatment group.(5)Compared with the control group,flow cytometry showed that the expression of MHCII and CD86 on the M1 macrophages surface was not significantly changed in the NaCl pretreatment group.2.(1)The mortality of mice was significantly lower,which were injected in BMDMs pretreated with Na Cl.(2)NaCl intervention can significantly inhibit the damage of lung and liver tissue induced by LPS.(3)In mice,which were injected in BMDMs pretreated with NaCl,M1 macrophages polarization decreased significantly,the expression of the M1 macrophages factor,IL-6 and IL-12 p40,significantly reduced.Conclusion:1.NaCl pretreatment could inhibit the polarization of M1 macrophages in vitro,the expression of IL-6 and IL-12p40,the M1 macropgahefactor,significantly decreased,and the underlying mechanism may be that NaCl reduced the expression of transcription factor IRF5.2.In mice,NaCl reduced the polarization of M1 macrophages,thereby reducing LPS induced endotoxin damage and weakening the inflammatory response induced by LPS.