Research On Nrf2 Antioxidant Pathway, Regulatory B Cells And Immuno-inflammatory Responses Of CGVHD Murine

Objective To construct the chronic graft versus host disease(c GVHD) murine model after allogeneic hematopoietic stem cell transplantation(allo-HSCT). To detect Nrf2 antioxidant pathway, oxidative activity, regulatory B cells(Bregs) and cytokines levels of c GVHD mice. And to preliminary explore their roles in the pathogenesis of the c GVHD.Method To construct the c GVHD murine model, irradiated female CB6F1 recipients were transplanted with spleen cells(6-7×107) plus bone marrow cells(8×106) from male BALB/c donors. All mice were randomly allocated into control(wild type), total body irradiation(TBI), syn BMT(non-c GVHD) and allo BMT(c GVHD) group. In syn BMT group, donors were changed to be male CB6F1 mice.All mice were sacrificedon day 27-33 post BMT. Clinical c GVHD score and histologic analysis(H&E and Masson) were used to evaluate c GVHD. Oxidative activity in PBMCs was assessed by intracellular reactive oxygen species(ROS)production. BM and liver samples were collected to explore the expression of Nrf2,NQO1 and HO-1 by western blot and real-time RT-PCR.Bregs(CD19+CD5+CD1d+ B cells) in peripheral blood were measured by flow cytometry.Inflammation inhibiting factor(IL-10) and fiber generates related factor(TGF-?1)levels in serum were detected by ELISA. All measurement data are analyzed by two-tailed Student's t-test, shown as mean±SEM. And all count data are analyzed withChi-Square Tests. A difference was considered statistically significant when P<0.05.Results(1)Establishment of the c GVHD murine model: clinical c GVHD score and histologic analysis consistently indicated that c GVHD developed in allo BMT group while non-c GVHD developed in syn BMT group;(2)ROS levels in c GVHD group was significantly higher than that in non-c GVHD group(P<0.001);(3)Nrf2, NQO1 and HO-1 protein levels measured by western blot in bone marrow and liver tissues of c GVHD group significantly diminished than those in non-c GVHD group(P<0.05);(4)Nrf2, NQO1 and HO-1 m RNA levels measured by real time RT-PCR in bone marrow and liver tissues of c GVHD group also significantly decreased than those of non-c GVHD group(P<0.05);(5)Bregs(CD19+CD5+CD1d+B cells) in peripheral blood measured by flow cytometry of c GVHD group were significantly lower than those of non-c GVHD group(P<0.05);(6)The IL-10 level in c GVHD group measured by ELISA was significantly lower than that in non-c GVHD group(P<0.001),however TGF-?1 level in c GVHD group was not significantly different from that in non-c GVHD group.Conclusion Oxidative activity increased in c GVHD mice undergoing allo-HSCT,while Nrf2 antioxidant pathway was down regulated. This study firstly provided the evidence of down-regulation of Nrf2 antioxidant signaling in c GVHD murine model,which may induce c GVHD. What is more, Bregs level significantly reduced inc GVHD mice, and IL-10 levelalso significantly decreased in c GVHD mice. Bregs may present immunological inhibitory activity though producing IL-10, which suggests that deficiency of Bregs and IL-10 may contribute to development of c GVHD. It deserves further study that whether Nrf2 and Bregs can be the novel target against c GVHD.