Notch1-RBP-J Signal Transduction Pathway Regulates M-CSF And M-CSFR To Influence The Balance Of Macrophage Polarization In Lupus Nephritis

BackgroundSystemic lupus erythematosus(SLE)is an autoimmune disease,characterized by a chaotic immune response and complex inflammatory immune damage.SLE can develop into lupus nephritis(LN),characterized by hematuria,proteinuria and even renal failure.The pathogenesis of LN is not clear.However,current studies have shown that abnormal changes in immune cells play an important role in the development of LN.Macrophage are important infiltrating cells in the kidney of LN patients.They can be roughly polarized into two phenotypes:M1 macrophage(secreting inducible nitric oxide synthase(iNOS))and M2 macrophage(secreting Arginase-1(Arg-1)),promotes and resists inflammatory,respectively.Some studies have shown that the polarization of macrophage is regulated by Notch1-RBP-J signaling pathway and macrophage colony stimulating factor(M-CSF),but their expressions and functions in LN,and the relationship between them are all unclear.ObjectivesWe peformed an experiment to investigate the expressions of Notch1-RBP-J signaling pathway,M-CSF and M-CSFR in SLE and LN patients,and to observe the expression changes of iNOS,Arg-1,M-CSF and M-CSFR by inhibiting Notch1-RBP-J signaling pathway of peripheral blood monocytes(PBMCs)in LN.The purpose of this study was to explore whether Notch1-RBP-J signaling pathway can regulate M-CSF and its recepter,and the effect on macrophage polarization.MethodsA total of 90 participants were included in this study,including 30 SLE patients,30 LN patients and 30 healthy controls.All patients were collected from the first affiliated Hospital of Anhui Medical University and Anhui Provincial Hospital.The healthy controls were collected from the Nangang Community Health Service Center,Hefei,Anhui Province.SLE patients and healthy controls were 1:1 matched with LN patients according to the age and gender.The peripheral venous blood samples of the three groups were collected,plasma and PBMCs were extracted from blood samples.The PBMCs of LN patients was cultured,and the expression of RBP-J was interfered by small interfering RNA(siRNA),and then,the supernatant and PBMCs were all collected.In this study,Real-time Quantitative polymerase chain reaction(RT-qPCR)was used to measure the expressions of Notch1,RBP-J and M-CSFR in the three groups,and the expression of M-CSFR after siRNA interference.We also used enzyme linked immunosorbent assay(ELISA)to explore the expressions of iNOS,Arg-1,M-CSF and M-CSFR in plasma of the three groups and supernatant of the samples which successfully interfered by siRNA and matched blank group.ResultsThere was no significant difference on the expression of Notch1 between LN patients and healthy controls,but there were significant differences on the expressions of RBP-J and M-CSFR(both P<0.05),the expressions of RBP-J and M-CSFR in PBMCs of LN patients were lower than healthy controls.There were significant differences on Notch1,RBP-J and M-CSFR in PBMCs between SLE patients and healthy controls(all P<0.05),the expressions of RBP-J and M-CSFR in PBMCs of SLE patients were lower than healthy controls.There was no significant difference on the expression of Arg-1 between LN patients and healthy controls,but there were significant differences on the expressions of iNOS,M-CSF and M-CSFR(all P<0.05),the expressions of iNOS,M-CSF and M-CSFR in LN plasma were higher than healthy controls.There were no significant differences on the expressions of iNOS and Arg-1between SLE patients and healthy controls.There was significant difference on the expression of M-CSFR between siRNA interference group and blank control group(P<0.05),the expression of M-CSFR in siRNA interference group was lower than blank control group,and there was a positive correlation between the expressions of M-CSFR and RBP-J(r_s=0.527,P<0.05).There were no significant differences on the expressions of iNOS and Arg-1 of supernatant between the siRNA interference group and blank control group,but the value of iNOS/Arg-1 in the siRNA interference group was higher than blank control group(P<0.05).There were significant differences on the M-CSF and M-CSFR expressions of supernatant between siRNA interference group and blank control group(both P<0.05).The expressions of M-CSF and M-CSFR in the culture supernatant of siRNA interference group was higher than blank control group.ConclusionsCompared to healthy controls,the expressions of RBP-J and M-CSFR in PBMCs of LN patients were decreased,but the expressions of iNOS,M-CSF and M-CSFR in plasma were increased.The expressions of Notch1,RBP-J and M-CSFR in PBMCs of SLE patients were decreased,but for the expressions of iNOS,M-CSF and M-CSFR in plasma,there was no significant difference between SLE patients and healthy controls.Notch1-RBP-J signal transduction pathway could up-regulate the expressions of M-CSF and M-CSFR in LN patients,and thus,the polarization balance of M1 and M2macrophages was broken,and tended to the M1 macrophages dominant status.