Study On Shikon In Inhibits Of Skin Allograft Rejection In Mice And Its Immunomodulatory Mechanism

ObjectiveOrgan transplantation as an effective treatment for patients undergoing end-stage organ diseases,is widely used in clinic.However,almost all transplantation patients require continuous treatment with immunosuppressive drugs to prevent allograft rejection.Thus,immunosuppressive agents play an essential role in maintaining allograft survival.However,continuous global immunosuppression may cause severe side effects,including nephrotoxicity,tumors and infections.Consequently,invention of novel immunosuppressive molecules with better mode of action and minimum side-effects are urgently desired for patients undergoing long-term treatment.Shikonin is a bioactive naphthoquinone pigment,an ingredient originally extracted from the root of Lithospermum erythrorhizon.Recent studies have also revealed that shikonin can exert anti-inflammatory,anticancer and antimicrobial effects.In particular,it can ameliorate arthritis in animal models and suppress human T lymphocyte activation.However,it is unclear whether shikonin inhibits alloimmunity or allograft rejection.In this study,a mouse skin transplantation model was established to investigate the effect of shikonin on allograft rejection and to clarify the mechanism of immune regulation of shikonin.Methods1.Effect of shikonin on skin allograft rejection in miceSkin donors were wild-type BALB/c mice while skin graft recipients were C57BL/6 mice.Mice were randomly grouped into control groups,and experimental groups that were administrated with shikonin(p.o.20 mg/kg or 40 mg/kg body weight)or cyclosporine(CsA,i.p.20 mg/kg)for a period of two consecutive weeks or until graft rejection/sample collection,investigate the effect of shikonin on the survival time of the grafts;HE staining,immunohistochemistry and immunofluorescence were used to determine the inflammatory cell infiltration or Foxp3 and IDO expression in the graft;Frequencies of CD4+Foxp3+Treg,CD8+CD44high CD62Llow effector T cells and CD11c+CD80+/CD11c+CD86+mature DCs in draining lymph nodes and spleen cells analyzed using a flow cytometer;The mRNA expressions of IFN-γ,TNF-α,IL-6,IL-10,IL-17A,FoxP3,IDO and TGF-β1 in skin allografts were determined by quantitative real-time reverse transcription PCR.2.Effect of shikonin combined with anti-CD25 antibody on skin allograft rejection in miceIn this study used a skin transplantation model in mice.C57BL/6 mice were randomly divided into five groups,namely,allogeneic skin transplantation group(Control),cyclosporine A group,cyclosporine A combined with anti-CD25 antibody group,shikonin group and shikonin combined with anti-CD25 antibody group.To deplete Tregs,recipient mice were administrated i.p.with anti-CD25 Ab at 0.2 mg on days 0,4,and 8 post-transplantation.Skin allograft rejection was monitored daily and defined as graft necrosis greater than 90%,the survival time of the skin graft was recorded and indicated by median survival time(MST).3.The study of shikonin in vitroFACS-sorted CD3+T cells derived from C57BL/6 mice,using CCK-8 assays detected T cell cytotoxicity.FACS-sorted CD3+T cells were labeled with 3 μM CFSE,cell proliferation was measured using a FACSCalibur and the levels of IFN-y,IL-10,TGF-β1 and IL-17A in the supernatant were measured using ELISA according to the manufacturer’s instructions.The protein expressions of phosphorylated p70S6K(P-p70S6K),p70S6K,P-mTOR and mTOR in T cells were measured using western blotting analysis two days after T cells were stimulated in vitro with anti-CD3/CD28 Abs in the absence or presence of shikonin.FACS-sorted CD4+CD25-T cells from C57BL/6 mice were co-stimulated with anti-CD3/CD28(2.5 μg/mL)and IL-2(10 ng/mL)in the absence or presence of TGF-β1 or shikonin for four days,the frequencies of CD4+ Foxp3+ Tregs were determined using a flow cytometer.Bone marrow-derived DCs were co-stimulated with rmGM-CSF(20 ng/mL)and rmIL-4(10 ng/mL)cultured in the absence or presence of shikonin for two days,IDO protein expression by DCs was then determined via Western Blotting.Results1.Effect of shikonin on skin allograft rejection in miceBoth low and high doses of shikonin significantly prolonged skin allograft survival compared to the control group[median survival time(MST)=16(low dose)vs.12 days and 22(high dose)vs.12 days,both.P<0.05 or P<0.01].Interestingly,treatment with high doses of shikonin was nearly as effective as CsA(20mg/kg)in prolongation of skin allograft survival(MST=22 vs.26 days,P>0.05).On the other hand,the skin allografts were analyzed via H&E or immunohistochemical(IHC)staining 10 days post-transplantation.We found that treatment with either shikonin or CsA obviously reduced overall cellular infiltration as well as CD3+T-cell infiltration in the skin allografts compared to the control group(P<0.05 or P<0.01).Shikonin(20mg/kg and 40mg/kg),but not CsA,augmented Foxp3 expression in skin allografts(P<0.01).Furthermore,it also increased IDO expression in the grafts while IDO was mainly expressed by DCs,as demonstrated by the double stainings of both IDO and CD11c(P<0.01).In the secondary lymphatic organs of recipient mice,shikonin significantly increased the frequencies of CD4+Foxp3+ Tregs in draining lymph nodes compared with the control group while CsA did the opposite(P<0.05 or P<0.01).In contrast,shikonin at high doses,but not low doses,significantly augmented the frequencies of splenic Tregs post-transplantation(P<0.05).Either CsA or shikonin significantly reduced the percentage of CD11c+ CD86 mature DCs in both the spleen and lymph nodes(P<0.05 or P<0.01).As for CDllc CD80 cells,another subset of mature DCs,shikonin only decreased their frequency in the draining lymph nodes,but not the spleen(P<0.01).Shikonin,at either low or high doses,significantly reduced the percentage of CD8+CD44high CD62Llow effector T cells in both lymph nodes and spleens of the recipients compared with the control group(P<0.01).In skin allografts,the mRNA expressions of these proinflammatory cytokines in skin allografts were significantly downregulated after the treatment with either CsA or shikonin(P<0.05 or P<0.01),especially at high doses,whereas shikonin,but not CsA,upregulated IL-10 and Foxp3 mRNA levels compared with control group(P<0.05 or P<0.01).On the other hand,both CsA and high doses of shikonin augmented TGF-β1 gene expression while either low or high doses of shikonin,but not CsA,increased IDO gene expression(P<0.05 or P<0.01).2.Effect of shikonin combined with anti-CD25 antibody on skin allograft rejection in miceThe experiment results show that treatment with shikonin significantly reduced skin allograft survival compared to the control group(MST=16 vs.25 days,P<0.05).However,there was no significant difference between the CsA+anti-CD25 group and CsA group.we found that depleting CD25+Tregs mostly reversed skin allograft survival prolonged by shikonin but not CsA.3.The study of shikonin in vitroAt 24h 48h and 96h after administration of different concentrations of shikonin(0.1,0.25,0.5,1.0,2.0 μM),cell viability was not compromised when up to 1.0 μM shikonin was used in the cell culture,suggesting that suppression of T cell proliferation and activation by shikonin was not attributed to its cytotoxicity(P<0.01).Shikonin,at both low and high concentrations,significantly suppressed T cell proliferation.As expected,CsA did the same(P<0.01).On the other hand,shikonin moderately promoted IL-10 secretion in the supernatant but dramatically inhibited production of IFN-y(P<0.05 or P<0.01).Interestingly,CsA did not significantly alter IL-10 secretion although it largely blocked IFN-y production(P<0.01).In contrast,shikonin at a high,but not low,concentration significantly reduced IL-17 level compared with control group(P<0.05).Further,CsA or high concentration of shikonin increased TGF-β1 level(P<0.05 or P<0.01).shikonin,at both low and high concentrations,significantly increased the percentages of Foxp3+ Tregs in vitro,and so did TGFβ1,a positive control.However,CsA did not augment the Treg frequency(P<0.05 or P<0.01).Shikonin significantly inhibited the expression of phospho-p70S6K(both low and high concentrations:p<0.01)and phospho-mTOR(high concentration:p<0.01)compared to the control group.As a positive control,rapamycin largely blocked expressions of both phospho-p70S6K and phospho-mTOR.Shikonin,but not CsA,also enhanced IDO protein expression in DCs in vitro(P<0.05 or P<0.01).ConclusionsOur data revealed a novel role of shikonin in inducing Tregs/IDO and suppressing allograft rejection.shikonin significantly prolonged skin allograft survival in a mouse model of skin allotransplantation by promoting CD4+Foxp3+Treg differentiation.It also suppressed T cell proliferation in vitro while reducing their mTOR signaling.Furthermore,shikonin inhibited expressions of proinflammatory cytokines while increasing expression of immunosuppressive cytokines and IDO in skin allografts.These findings indicate that shikonin,an originally natural product,may be utilized as a potent immunosuppressive drug for the prevention of human transplant rejection in the future.