Alleviation Of Murine Sclerodermatous Chronic Graft Versus Host Disease By The Nrf2 Activator Mangiferin

ObjectiveIn this study,we set up an established murine sclerodermatous chronic graft versus host disease（scl-cGVHD）model,treated the mice with 20mg/kg,50mg/kg,and 100mg/kg of mangiferin（MF）to investigate the effect and mechanism of MF,the Nrf2 activator,on scl-cGVHD in vivo.Methods10-to 12-week-old CB6F1 female mice as recipients were randomly divided into control,syngeneic hematopoietic stem cell transplantation（synHSCT）,allogeneic hematopoietic stem cell transplantation（allo HSCT）+vehicle,allo HSCT+20mg/kgMF,allo HSCT+50mg/kg MF,allo HSCT+100mg/kg MF（Five mice in each group）.Irradiated recipients were transplanted with 8×10⁶male BALB/c bone marrow cells and 7×10⁷ male BALB/c spleen cells（allogeneic）or 8×10⁶female CB6F1 bone marrow cells and 7×10⁷ female CB6F1spleen cells（syngeneic）.20,50,100mg/kg body weight MF and equal amount of cmc-Na vehicle solution were respectively administrated to the allo HSCT+20mg/kg MF group,allo HSCT+50mg/kg MF group,allo HSCT+100mg/kg MF group,and allo HSCT+vehicle group by gavage once daily starting from day 1 to day 14 after HSCT.All mice were sacrificed on day 33 after HSCT.Clinical cGVHD score（body weight,skin lesion,hunch）and target organs（skin,liver,spleen,ileum）histologic analysis were used to evaluate cGVHD severity.Oxidative activity in peripheral blood mononuclear cells was assessed by intracellular reactive oxygen species production.The m RNA levels and protein levels of Nrf2,NQO1,HO-1 in liver tissues and bone marrow cells were respectively examined by real time RT-PCR and western blot.Levels of CD19⁺B cells and CD19⁺CD5⁺CD1d^hiBregs in splenic mononuclear cells were measured by flow cytometry.Levels of interleukin-6 and interleukin-17A,which were both the pro-inflammatory and pro-fibrogenic factors,in serum were detected by Luminex.All measurement data are analyzed by two-tailed student’s t-test,shown as mean±sem.All count data are analyzed with Chi-Square Tests.A difference was considered statistically significant when P<0.05.Results1.MF significantly reduced the clinical symptoms of scl-cGVHD in mice（1）Body weight change:+18-+33d（cGVHD phase）,the body weight of mice in the control group and the synHSCT group increased steadily and slowly.The body weight of mice in the allo HSCT+vehicle group constantly dropped,which was always significantly lower than those of mice in the control group and the synHSCT group（P<0.05）.MF significantly gained the body weight of scl-cGVHD mice in a dose-dependent manner（P<0.05）.（2）Skin leision:Since entering the cGVHD phase,skin leision was scored in each group according to the internationally accepted cGVHD skin score standard.The scores of control group and synHSCT group were 0.Mice in the allo HSCT+vehicle group showed typical skin lesions（alopecia and scab）,and the score was significantly higher than those of the control group and the synHSCT group（P<0.05）.MF significantly reduced skin leision scores of scl-cGVHD mice in a dose-dependent manner（P<0.05）.（3）Hunch:Since entering the cGVHD phase,there was no mouse hunched in control group and synHSCT group;4 mice’s movement were affected by hunch in the allo HSCT+vehicle group,3 mice’s movement were affected by hunch in the allo HSCT+20mg/kg MF group.1 mice’s movement was affected by hunch in the allo HSCT+50mg/kg MF group.None of mice’s movement was affected by hunch in the allo HSCT+100mg/kg MF group.（4）Clinical score:Since entering the cGVHD phase,cGVHD clinical scores were obtained according to the above three clinical manifestations.The scores of control group and synHSCT group were0.The score of allo HSCT+vehicle group was significantly higher than those of control group and synHSCT group（P<0.01）.MF significantly decreased the cGVHD clinical scores of scl-cGVHD mice in a dose-dependent manner（P<0.05）.2.MF significantly reduced the damage of scl-cGVHD target organs:（1）H&E staining:The morphology and structure of target organs were normal in the control group and the synHSCT group,there were no significant pathological changes.The target organs of mice in the allo HSCT+vehicle group showed chronic inflammatory changes:Both the dermis and the tunica muscularis of ileum wall were thickened,the morphology and structure of liver and spleen tissues were severely destroyed,and mononuclear cells infiltration appeared in all target organs.In the allo HSCT+MF group,the thickness of the dermis and the tunica muscularis of ileum wall were similar to those of the control group and the synHSCT group,the liver and spleen tissues remained normal structures,and there were no apparent mononuclear cells infiltration in all target organs.（2）Masson-trichrome staining:There were no fibrosis histopathological changes in the target organs of mice in the control group and the synHSCT group.Significant fibrosis histopathological changes were found in all target organs of mice in the allo HSCT+vehicle group（P<0.05）.MF significantly reduced the degree of fibrosis in all target organs of scl-cGVHD mice（P<0.05）.3.MF significantly reduced ROS levels in PBMCs of scl-cGVHD mice（1）Compared with the synHSCT group,the ROS level of mice in the allo HSCT+vehicle group was significantly increased（P<0.0001）.（2）MF significantly reduced ROS levels in scl-cGVHD mice（P<0.001）.4.MF activated the expression of Nrf2 antioxidant pathway in liver and bone marrow of scl-cGVHD mice（1）Expression level:In the liver tissues,compared with the synHSCT group,the expression levels of Nrf2,NQO1 and HO-1 proteins in the allo HSCT+vehicle group were significantly reduced（P<0.05）.MF significantly upregulated the expression levels of Nrf2,NQO1 and HO-1 proteins in liver tissues in a dose-dependent manner（P<0.05）.In the bone marrow cells,compared with the synHSCT group,the expression levels of Nrf2,NQO1 and HO-1 proteins in the allo HSCT+vehicle group were significantly reduced（P<0.05）.MF significantly upregulated the expression levels of Nrf2,NQO1 and HO-1 in bone marrow cells in a dose-dependent manner（P<0.05）.（2）Transcription level:In the liver tissues,the m RNA levels of Nrf2,NQO1 and HO-1 in the allo HSCT+vehicle group were significantly lower than those in the synHSCT group（P<0.01）.MF significantly upregulated the m RNA levels of Nrf2,NQO1 and HO-1 in liver tissues in a dose-dependent manner（P<0.05）.In the bone marrow,Nrf2,NQO1 and HO-1 m RNA levels in the allo HSCT+vehicle group were significantly lower than those in the synHSCT group（P<0.01）.MF significantly upregulated the m RNA levels of Nrf2,NQO1 and HO-1 in bone marrow cells in a dose-dependent manner（P<0.05）.5.MF significantly increased Bregs level in SMNCs of scl-cGVHD mice（1）Compared with the control group and the synHSCT group,there was no significant difference in the level of CD19⁺B cells in the SMNCs of allo HSCT+vehicle group（P>0.05）.MF had no effect on the level of CD19⁺B cells in SMNCs of scl-cGVHD mice（P>0.05）.（2）Compared with the control group,the levels of CD19+CD5+CD1dhi Bregs cells in CD19⁺B of the synHSCT group and allo HSCT+vehicle group were significantly decreased（P<0.01）.MF significantly upregulated the level of CD19⁺CD5⁺CD1d^hiBregs cells in CD19⁺B of scl-cGVHD mice in a dose-dependent manner（P<0.05）.6.MF significantly downregulated plasma IL-6 and IL-17A expression of scl-cGVHD mice（1）Compared with the control group and the synHSCT group,the plasma IL-6 level of the allo HSCT+vehicle group significantly increased（P<0.01）.MF significantly downregulated plasma IL-6 expression in scl-cGVHD mice in a dose-dependent manner（P<0.05）.（2）Compared with the control group and the synHSCT group,the plasma IL-17A level of the allo HSCT+vehicle group significantly increased by P（P<0.05）.MF significantly downregulated plasma IL-17A expression in scl-cGVHD mice in a dose-dependent manner（P<0.05）.ConclusionMF attenuates the development of murine scl-cGVHD by dose-dependently activating Nrf2 antioxidant pathway,increasing the frequencies of Bregs and suppressing the production of the IL-6 and IL-17A.In conclusion,our findings suggest MF could be a promising agent for the treatment or prevention of cGVHD after allo-HSCT,which deserves further research.