The Role Of RasGRP3in The Pathogenesis Of Systemic Lupus Erythematosus

Part Ⅰ The expressions of RasGRP3in peripheral blood lymphocytes and B lymphocytes of SLE patientsObjective Systemic lupus erythematosus (SLE) is a chronic disease with diverse clinical presentations and immunological abnormalities, involving multiple tissues and organs. The immunological mechanism for lupus has not been fully classified. In order to explore the role of Ras guanine nucleotide releasing protein (RasGRP3) pathogenesis of autoimmunity in SLE, we detected the expressions of RasGRP3in peripheral blood mononuclear cells (PBMCs) of patients with SLE and further study the RasGRP3expressions in purified B cells.Methods The blood samples from patients diagnosed as SLE and healthy volunteers were collected. Human PBMCs were obtained by Ficoll method. B cells were purified from PBMCs by immunomagnetic beads separation. The purity of B cells was confirmed by FCM and the expressions of RasGRP3were detected by RT-PCR and Western Blot.Results The purity of B cell could reach more than90%by immunomagnetic beads separation. Compared with healthy volunteers, the mRNA and protein expressions of RasGRP3were elevated both in PBMCs and B cells.Conclusions The expressions of RasGRP3in both PBMC and B cells of SLE patients were much higher compared with healthy volunteers. Aberrant expressions of RasGRP3might contribute to the pathogenesis of autoimmunity in lupus. Part II The relationship between the mRNA expressions of RasGRP3in PBMCs and the clinical data of SLE patientsObjective:SLE patients manifest diverse clinical presentations and laboratory results. In order to explore whether the expression of RasGRP3is related to the clinical data in SLE patients, such as clinical signs, laboratory tests, disease activity, and so on.Methods:The peripheral blood samples from patients diagnosed as SLE were collected, as well as the detailed clinical data (basic information, complaims, symptoms, clinical signs, and laboratory test results). The PBMCs were extracted by Ficoll method, and further detected the RasGRP3mRNA expressions by semi-quantitative RT-PCR. We grouped the data by clinical features and laboratory test results, and compared the RasGRP3mRNA expressions between different two groups by Ttest.Results The mRNA expression of RasGRP3was positively correlated with SLE disease activity index (SLEDAI)(p=0.0301). The mRNA levels of RasGRP3in the patients with active disease were much higher than that in remission stage (p<0.05). In addition, the mRNA levels of RasGRP3in the patients with hematuria or proteinuria were significantly higher than that without kidney damage (p<0.05).Conclusion The mRNA expression of RasGRP3may be used as an indicator for lupus disease activity, and RasGRP3may be related to kidney damages. Due to limited sample size, further samples are needed to confirm this conclusion. Part Ⅲ Dexamethasone inhibits the expression of RasGRP3in PBMC of SLE patients both in Vitro and VivoObjective In order to observe the RasGRP3expression after dexamethasone treatment in vivo and in vitro, as well as explore the role of dexamethasone in the treatment of lupus.Methods The SLE patients who treated with moderate-dose corticosteroids (1mg/kg prednisone) were recruited in. The blood samples were acquired before and two weeks after the corticosteroids treatment to obtain PBMCs. The mRNA expression of RasGRP3was measured by RT-PCR; the protein level was examined by Western Blot. In vitro, dexamethasone of two different concentrations (1×10-5、1×10-6mol/L) was applied to co-culture with PBMC from healthy volunteers at different incubation time (Oh,12h,24h), and then we observed the RasGRP3mRNA expressions by RT-PCR to choose a concentration for better inhibition effect. We co-cultured the PBMC from untreated SLE patients with dexamethasone in this appropriated concentration and observed the mRNA expression of RasGRP3at different time points (Oh,12h,24h). In addition, the protein expressions of RasGPR3, p-ERK and p-AKT were also detected by Western Blot.Results The RasGRP3expression of SLE patients without corticosteroids treatment was much higher than that of normal people. After2weeks application of moderate-dose corticosteroids, the expression of RasGRP3was obviously inhibited. In vitro, dexamethasone could inhibit the mRNA expression of RasGRP3in normal people, and the1×10-6mol/L concentration of dexamethasone had obvious inhibited effect and did not induce cell apoptosis. We co-cultured the PBMC from SLE patients with1×10-6mol/L dexamethasone and observed the expression of RasGRP3at different time points, and found that dexamethasone showed the strongest inhibition on the expression of RasGRP3at the time points of24h;and at this time point, dexamethasone also inhibited pERK and p-AKT which were the downstream signaling pathway of RasGRP3. Conclusion:In vivo,2-week corticosteroids treatment could inhibit the RasGRP3expression in SLE patients; in vitro, dexamethasone could reduce the RasGRP3expression as well as p-ERK and p-AKT of its downstream signaling pathway of PBMCs from both healthy people and SLE patients. This may be one of the mechanisms of corticosteroids treatment of SLE.