| Objective:To detect the expression of inflammatory factor secretion in skin fibroblasts in vitro induced by CD4+T cells of healthy human and patients with SSc to explore the fuction transformation of CD4+T cells.To study the expression of CD4+T cells and related factors in a induced Interleukin-35(IL-35)model to explore whether IL-35 are associated with the inflammation of systemic sclerosis(SSc).To investigate the effects of IL-35 on the proliferation and differentiation of human CD4+T cells in vitro,to detect the expression of EBI3 mRNA in CD4+T cells in vitro induced by different concentrations of IL-35,therefore clarify the role of IL-35 in the chronic inflammation process of SSc.To investigate the effects of IL-35 on the proliferation,migration,inflammatory factor secretion and collagen synthesis of human healthy skin fibroblasts in vitro,and to explore its direct role in fibroblast activation,therefore clarify the role of IL-35 in the skin fibrosis of SSc,to provides a new target for the treatment of fibrosis in patients with SSc.Methods:Plasma samples from patients with SSc and healthy controls were collected.The levels of IL-35,IL-10,IL-17 A,TGF-β in plasma were measured by ELISA.Human healthy CD4+T cells were collected,primary CD4+T cells were extracted and cultured in vitro,and were stimulated by IL-35 recombinant protein.The proliferation rate of CD4+T cells was measured by CFSE method.The proliferation rate of Treg cells was measured by FACS method,Real-time Quantitative polymerase chain reaction(q PCR)were used to measure the mRNA of EBI3.Human skin fibroblasts were cultured in vitro,and were stimulated by IL-35 recombinant protein.The proliferation rate of human skin fibroblasts was measured by Cell counting kit-8(CCK-8)method,respectively.The cell scratch test was used to observe the effect of IL-35 on human skin fibroblasts migration and Western blot(WB)were used to measure the levels of collagen-related protein in human skin fibroblasts.Human skin fibroblasts were cultured in vitro and were stimulated by CD4+T cells of SSc and healthy controls.Enzymelinked immunosorbent assay(ELISA)was used to measure the level of inflammationrelated factors of human skin fibroblasts,the proliferation rate of human skin fibroblasts were detected by immunofluorescence.Results:1.The plasmas IL-35 level(86.33±58.25 vs 48.46±29.88,P=0.0005)and IL-17 A level(25.87 ± 16.87 vs 17.17 ± 4.59,P=0.0035)were significantly elevated in patients with SSc than normal controls,and IL-10 level(1.97±1.07 vs2.97±1.69,P=0.0034)were significantly decreased in patients with SSc than normal controls.There was no significant difference in serum TGF-β level(27.13±15.23 vs 40.21±21.86,P =0.0816)between SSc and normal controls.2.The results of ELISA shows that IL-6 level(0.15±0.07 vs 0.98±0.12,P<0.001)and IL-17 A level(2.09±0.08 vs 2.43±0.08,P <0.001)were significantly elevated in patients with SSc than control group,but IL-10 lever(143.52±5.41 vs 120.53±1.94,P <0.01)and TGF-β level(622.07±12.25 vs 497.87±12.43,P<0.001)and IL-35 level were significantly decreased in patients with SSc than control group.There was no significant difference in IL-10 level(120.53±1.94 vs 124.63 ± 10.41,P=0.54)and TGF-β level(622.07 ± 12.25 vs 601.8 ±48.74,P=0.52)between SSc and normal controls.3.Ki67 Immunofluorescence results shows that the viability of fibroblast was increased in SSc group(0.06 ± 0.01 vs 0.09 ± 0.01,P=0.03)than control group,And there was also significant difference between SSc group(0.09±0.01 vs 0.06±0.01,P<0.05)and normal controls.4.CFSE results shows that rh IL-35 could inhibit proliferation of CD4+T cells.Both rh IL-35 groups could significantly inhibit the proliferation of CD4+T cells(P < 0.001)except the 1 ng/ml rh IL-35 group(94.25±1.26 vs 85.61±2.87,P< 0.01).5.The results of flow cytometry shows that compared with control group 100ng/ml rh IL-35 could increase the expression of Treg cells(7.53±0.54 vs 8.77±0.44,P=0.04)while other groups shows no significant difference(P>0.05).6.The results of q PCR shows that compared with control group,the mRNA expression of EBI3 in 1 ng/ml,10 ng/ml,100 ng/ml rh IL-35 groups increased(P<0.05).7.CCK-8 result showed that IL-35 significantly inhibited proliferation of fibroblasts compared with control group and TGF-β group,all experimental groups results showed statistically significant(P<0.05).8.Compared with control group and TGF-β group,IL-35 significantly induce the production of collagen in fibroblast,α-SMA protein(0.5±0.01 vs 0.4±0.01,P<0.001)and COL-1 protein(0.63±0.03 vs 0.24±0.01,P <0.001)in fibroblast were obviously decreased.9.rh IL-35 could significantly reduce the migration of fibroblasts compared with control group(29.33±5.61 vs 14.67±4.7,P <0.05).10.CCK-8 result showed that cell culture medium include CD4+T cells which treated by rh IL-35 significantly inhibited the proliferation of fibroblasts compared with control group and TGF-β group,all experimental groups results showed statistically significant(P<0.05).Conclusion:1.IL-35 can promote the expression and release of inflammatory factors IL-6,IL-17 A,but inhibit the expression of IL-35,IL-10 and TGF-β.CD4+T cells of SSc patients can promote the proliferation of HSF compared with normal controls,We speculate that the CD4+T cells of SSc patients may have dysfunction which may be the pathogenesis of fibrosis.2.Interleukin-35 could inhibit proliferation of CD4+T cells,promot the differentiation and proliferation of Treg cells and EBI3 mRNA,showed antiinflammation property.IL-35 can significantly inhibit the roliferation and migration of human skin fibroblast,reduce the production of collagen in HSF to inhibit the process of skin fibrosis,suggesting that IL-35 plays an immunosuppressive role in the development of systemic sclerosis and immunoregulation. |
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