| Objective:Primary immune thrombocytopenia(ITP)is an autoimmune hemorrhagic disease with reduced platelet destruction caused by abnormal humoral and cellular immune abnormalities,accounting for about 1/3 of the total number of bleeding disorders.The proliferation-inducing ligand APRIL is a member of the tumor necrosis factor superfamily and is structurally and functionally similar to the B cell activating factor BAFF.APRIL is secreted as a soluble factor by various cells,such as non-activated B cells,T cells,monocytes,dendritic cells,megakaryocytes,and platelets.APRIL not only plays a role in the normal immune response,but also plays an important role in the establishment and maintenance of autoimmune and inflammatory diseases,especially in the proliferation of B cells,mediating the conversion of immunoglobulin classes,and Affects the response of T cells.lt has been previously reported that the mRNA level of APRIL is significantly up-regulated in peripheral mononuclear cells of patients with ITP,and the content of soluble APRIL in plasma of patients with ITP is also significantly increased.Many studies have found that in the autoimmune diseases such as rheumatoid arthritis,systemic lupus erythematosus and primary systemic sclerosis,the expression level of APRIL is also significantly increased,and its targeted drug belimumab and Acesep has been used in the treatment of rheumatoid arthritis.Based on the role of APRIL in promoting proliferation and regulating immune function,we hypothesize that APRIL may also be involved in the development and progression of ITP immune disorders.This study will determine whether APRIL is expressed on the surface of platelets and whether there is abnormal expression,and further clarify whether APRIL mediates the reverse regulation of platelet on ITP autoimmunity and provides a theoretical basis for the development of new therapeutic interventions.Methods:(1)A total of 50 ITP patients with a clear diagnosis were enrolled,and 25 healthy controls(age vs.gender)were selected at the same time.Peripheral venous blood was collected and plasma and peripheral blood mononuclear cells(PBMC)were isolated.(2)Plasma is separated by high-speed centrifugation.(3)Flow cytometry to detect the surface membrane type APRIL of platelets in ITP patients and normal controls.(4)Analysis of the level of platelet surface membrane-bound APRIL in patients with ITP and clinical indicators of patients(platelet count,platelet autoantibodies,efficacy))The relationship between.(5)Flow cytometry was used to detect the proportion of CD19+CD24hi CD38hi Bregs cell subsets in peripheral mononuclear cells of patients with ITP,and compared with healthy controls.(6)ELISA was used to detect the level of soluble APRIL in the plasma of ITP patients and normal controls.(7)APRIL was used to stimulate peripheral blood mononuclear cells in ITP and HC groups in vitro,and B10 cell content was detected by flow cytometry.At the same time,changes in B10 cell content after addition of APRIL blocker were observed.(8)APRIL in vitro stimulation and BLys stimulation of peripheral blood mononuclear cells in ITP and HC groups were performed in vitro,and the changes of B10 cells by different stimulating factors were detected by flow cytometry.Results:(1)The results of flow cytometry showed that the expression of APRIL on the platelet surface of the newly diagnosed and chronic ITP patients was significantly lower than that of the healthy control group(1120.0400±241.4837),(p<0.001).In addition,the newly diagnosed ITP group was more chronic than the ITP group.Lower MFI(626.1200±93.6978 VS 806.8400 ± 198.9369,p=0.001),the difference was statistically significant.Moreover,platelet count was positively correlated with APRIL fluorescence intensity(r=0.396;p=0.015),and the difference was statistically significant.The higher the platelet count in patients with ITP,the higher the fluorescence intensity of APRIL on the surface.(2)The fluorescence intensity of platelet surface APRIL in the specific anti-platelet membrane protein antibody positive group was higher than that in the specific anti-platelet membrane protein antibody negative group MFI(958.7143±221.4511 VS 693.9091±128.3156)(p=0.05),the difference was statistically significant.The fluorescence intensity of APRIL on the surface of platelets in 10 patients with complete remission group was significantly higher than that before treatment(M.361.3000±134.0481 VS 599.6000±70.32338,p=0.02).The difference was statistically significant.However,in 8 patients with ITP in the treatment-ineffective group,there was no significant change in the fluorescence intensity of platelet surface APRIL before and after treatment(872.6667±225.53935 vs 846.0000±204.91624,p=0.81).(3)The levels of soluble APRIL in the plasma of the initial treatment group and the chronic group of ITP patients were significantly higher than those of the normal control group(6.8770±2.9066pg/ml)(p=0.04 VS p=0.06).There was no significant difference in plasma soluble APRIL levels between the initial and chronic groups of ITP patients(10.4665± 5.2198 pg/ml VS 10.6993±5.0411 pg/ml,p=0.856).We then analyzed the correlation between plasma soluble APRIL levels and clinical parameters,and we found a statistically negative correlation between plasma soluble APRIL and platelet count(r=-0.3443,p=0.029).(4)Compared with normal controls,the content of Bregs in ITP patients was significantly lower,and the difference was statistically significant.The content of Bregs was positively correlated with platelet count,and the difference was statistically significant.(5)The content of B10 cells in the APRIL-stimulated group was significantly higher than that in the APRIL+inh-APRIL stimulated group and the control group(12.1250±5.4494%vs 3.7370±3.475%vs 3.4860±3.1804%)(p<0.001).The difference was statistically significant.APRIL factor stimulation produced more B10 cells,which was significantly inhibited after the addition of APRIL inhibitors.The B10 cells in the APRIL group and the B lymphocyte stimulating factor group were all increased,while the B10 cells in the APRIL group were significantly higher than those in the B lymphocyte stimulating factor group and the control group(p=0.001 VS p<0.001),and the difference was statistically significant.Conclusion:Abnormal expression of APRIL is involved in the autoimmune response of ITP.We hypothesize that APRIL inhibits the pathogenesis of ITP rather than promoting immune-mediated inflammatory responses.Our current observations support that the immunomodulatory effects of APRIL may be due,at least in part,to stimulation of IL-10 producing B cells. |
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