The Study On The Mechanism Of S1P/S1PR1-mediated Macrophage Polarization In Lupus Nephritis

Objective:1.To investigate the expression of S1 P and its receptors in lupus nephritis.2.To investigate the role of S1P/S1PR1 signaling pathway in lupus nephritis.3.To investigate the mechanisms of S1P/S1PR1 signaling pathway in the regulation of renal inflammation.4.To investigate the mechanism of S1P/S1PR1 signaling pathway in regulating M1-type macrophage polarization.Methods:Part I.The expression of S1P/S1PR1 signaling pathway in MRL/lpr mice and its mechanisms on regulating renal inflammation1.6 to 8-week-old female MRL/lpr mice were random Ly divided into saline-treated model group and FTY720 treatment group,and female BALB/c wild-type mice of the same age were used as normal controls.The urine protein of mice were tested weekly during feeding.The gavage treatment started at 10 weeks of age,FTY720 was administered at a concentration of 2 mg/kg three times a week for 12 weeks,and the urine protein test was performed every two weeks during administration.2.At the end of the intervention cycle,serum was collected to detect anti-ds-DNA antibody,S1 P concentration,creatinine and urea nitrogen;spleen was moved,and then taken for measurement and photographed;Some kidney tissues were made into tissue homogenate to detect S1 P concentration,some was embedded and sectioned for immunofluorescence,pathology and immunohistochemistry staining,and some was used to extracted total protein and RNA for western blot and Real-time PCR experiments.3.Real-time PCR was performed to detect the m RNA expression levels of renal polarized macrophage markers(M1 type: CD86,i NOS,M2 type: CD206,Arg1)and inflammatory factors IL-6,TNF-α,IL-1β,IL-18;western blot to detect the expression levels of renal NLRP3 inflammatory;immunofluorescence staining to detect the proportion of F4/80+CD86+ M1-type macrophages in renal tissue;immunohistochemical staining was used to detect renal F4/80+ macrophage infiltration,S1PR1 and NLRP3 expression levels.Part II.The vitro study on the mechanism of S1P/S1PR1 signaling pathway in M1-type macrophage polarization1.Mouse RAW264.7 macrophage cell line was cultured and stimulated with S1P(1μmol/L),and the expression levels of polarized macrophage markers(M1 type: CD86,i NOS,M2 type: CD206,Arg1)were detected by Real-time PCR;flow cytometry was used to detect the proportions of CD86+M1 and CD206+M2 type cell.2.Real-time PCR and western blot was performed to detect S1 P receptor expression on the surface of RAW264.7.After blocking S1PR1 by S1PR1 si-RNA FTY720(100nmol/L),cells was stimulated by S1 P,Real-time PCR was used to detect CD86,i NOS,CD206,Arg1;flow cytometry was used to detect CD86+M1 and CD206+M2 type cell proportions.3.Detect the expression of macrophage NLRP3 inflammatory vesicles(NLRP3,ASC)after S1 P stimulation by Real-time PCR and western blot.4.Stimulated cells by S1 P after blockade S1PR1 by FTY720(100nmol/L)or S1PR1si-RNA,then Real-time PCR and western blot was performed to detect NLRP3 inflammasome expression.5.CD86,i NOS,CD206,and Arg1 expression levels were detected by Real-time PCR after NLRP3 blockade using MCC950 under S1 P stimulation,and the proportion of CD86+M1 and CD206+M2 type cells were detected by flow cytometry.Results:1.The levels of S1 P in serum and kidney of MRL/lpr mice were elevated compared to normal control group.2.The Real-time PCR results showed the expression of S1PR1,S1PR2,S1PR3,S1PR4 in the kidney of MRL/lpr mice,and the S1PR1 expression was significantly increased compared with the normal control group.3.The results of urine protein assay showed that blockade S1PR1 by FTY720 could effectively reduce the proteinuria level and delay the disease process of MRL/lpr mice,and compared with the model group,blocking S1PR1 could reduce the serum Crea and Urea and mitigate the kidney damage.4.The pathological staining results showed that the kidney histopathological changes were reduced and inflammatory cell infiltration was decreased in the mice of FTY720-treated group.5.The transmission electron microscopy results showed that FTY720 could improve podocyte injury in mice,reduce podocyte pedicle fusion and increase the number of lacunar pores.6.The serum anti-ds-DNA antibody concentration was decreased in MRL/lpr mice in FTY720-treated group compared with the model group,and immunofluorescence assay results showed that kidney Ig G and C3 immune complex deposition decreased and overall survival rate was increased in FTY720-treated group.7.The immunohistochemical staining results showed that the renal F4/80+macrophage infiltration was reduced in FTY720-treated group mice compared with the model group,and the Real-time PCR results showed that FTY720 could inhibit the expression levels of renal inflammatory factors,including IL-6,TNF-α,and IL-18 in MRL/lpr mice.8.Real-time PCR results showed that the renal M1 macrophage markers i NOS and CD86 were significantly reduced in the FTY720-treated MRL/lpr mice,and immunofluorescence staining showed that the renal F4/80+CD86+ macrophage infiltration was reduced in the kidney of MRL/lpr mice treated with FTY720.9.Sfter the S1 P stimulation for 12 h,the expression levels of M1 and M2 type markers of macrophages were detected by Real-time PCR,and the results showed that S1 P could induce macrophages to express M1 type markers;the results of flow cytometry showed that S1 P stimulation could increase the proportion of CD86+ macrophages.10.Block S1PR1 via using FTY720(100 μmol/L)or si-RNA inhibited S1P-induced polarization of macrophages toward M1 type.11.The Real-time PCR and western blot results showed that S1 P at 1 μmol/L could induce NLRP3 inflammasome activation,which could be inhibited by blocking S1PR1 using FTY720 or S1PR1 si-RNA.12.The Real-time PCR and flow cytometry assay showed that blocking NLRP3 using MCC950 could inhibit S1P-induced polarization of macrophages toward M1 type.Conclusions:1.The expression of S1P/S1PR1 signaling pathway is elevated in MRL/lpr mice.2.Blockade of S1PR1 can improve renal function in MRL/lpr mice.3.Blockade of S1PR1 can improve autoimmunity condition in MRL/lpr mice.4.Blockade of S1PR1 can inhibit renal M1-type macrophage infiltration in MRL/lpr mice and reduce renal inflammation.5.S1P/S1PR1 signaling pathway promotes macrophage polarization toward M1 type.6.S1P/S1PR1 signaling pathway induces macrophage polarization toward M1 type through activation of NLRP3 inflammasome.