Study On The Efficacy And Mechanism Of Dioscorea Nipponica Saponins In The Treatment Of Aplastic Anemia Based On Notch Signaling Pathway

Objectives:To establish an aplastic anemia mouse model and observe the therapeutic effect of diosgenin from Dioscorea nipponica on aplastic anemia mice,and to explore whether diosgenin can affect Th17/Treg cell balance by regulating Notch signaling pathway in aplastic mice,thereby exerting the therapeutic effect of regulating immunity to promote hematopoiesis.Methods:1.Establishment and evaluation of aplastic anemia mice model:The model of aplastic mice was established by irradiation combined with chemical drug damage.BALB/c male mice were randomly divided into control group?n=10?and model group?n=60?.After ¹³⁷Cs irradiation,mice in model group were intraperitoneally injected with cyclophosphamide?25mg/kg/d?and chloramphenicol?62.5mg/kg/d?on the 4th,5th and 6th day.The control group received sham irradiation with lead brick shielding and intraperitoneal injection of normal saline of equal volume.On the 7th day,the blood routine and bone marrow smear of the model mice were detected to evaluate the model.The counts of peripheral blood leukocytes,hemoglobin,platelets and reticulocytes decreased to less than half of the normal hemogram,and bone marrow cell smears showed that the proliferation of bone marrow decreased or severely decreased,which was regarded as a model.2.To observe the therapeutic effect of Dioscorea paniculata saponins on AA mice:The model mice were randomly divided into six groups:model group,Dioscorea paniculata saponins low,medium and high dose group?37.44,74.88,149.76mg/kg/d?,Tripterygium wilfordii polyglycoside group?9.36mg/kg/d?,cyclosporine group?23.5 mg/kg/d?,model group and control group were given equal volume distilled water for 14 days.After the intervention,peripheral blood cell counts and bone marrow smears were measured to evaluate the proliferation of bone marrow in each group.3.Mechanisms of Dioscorea paniculata saponins regulating Notch signaling pathway in AA mice:After gastric perfusion,The ratio of Th17 and Treg to CD4⁺T cells in peripheral blood of mice in each group was detected by FCM,and the effect of diosgenin on Th17/Treg immunocyte balance in AA mice was analyzed.The expression of ROR?t,Foxp3,Notch1,Jagged1,Dll4,RBP-J?in spleen of mice in each group was detected by Q-PCR and Western-blot,and the effect of diosgenin on Notch-related signal expression in AA mice was explored.RBP-J?binds to ROR?t and Foxp3 proteins respectively,and explores whether diosgenin can interfere with the balance of Th17/Treg cells in AA mice by regulating Notch signaling pathway,thereby exerting the mechanism of promoting hematopoiesis.Results:1.WBC,Hb,PLT and RET in peripheral blood of mice in model group were significantly lower than those in control group?p<0.05?.The hemogram of mice in each treatment group had different degrees of recovery compared with that in model group,the mid-dose group of diosgenin from Dioscorea nipponica and cyclosporine group had the best effect,which was significantly different from that in model group.2.Compared with the control group,the proliferation of bone marrow in the model group was lower and the number of bone marrow cells decreased.After treatment,the number of bone marrow cells increased and the proliferation of bone marrow was more active in each group than the model group.The mid,high-dose group of diosgenin and cyclosporine group was better.3.Compared with the control group,the ratio of Th17 cells to CD4⁺T cells in peripheral blood of the model group increased significantly?p<0.05?,and Treg cells decreased,the ratio of Th17/Treg cells increased significantly?p<0.05?.Compared with the model group,there was the same trend in each treatment group,the proportion of Th17 cells decreased and Treg cells increased,mid-dose group of Dioscorea nipponica saponin decreased significantly,and the ratio of Th17/Treg cells decreased significantly?p<0.01?,but the increase of Treg was not obvious,and there was no statistical significance compared with the cyclosporine group?p>0.05?.4.Compared with the control group,the expression levels of Notch1,Foxp3,Jagged1 of model group mice spleen were significantly lower?p<0.05?,while Dll4 and ROR?t higher?p<0.05?.After treatment,the levels of Notch1,Foxp3,Jagged1mRNA and protein in spleen of mice in each group increased in varying degrees,while Dll4 and ROR?t were decreased,especially in the mid-dose group of diosgenin and cyclosporine group?p<0.05?.There was no significant difference in RBP-J?mRNA and protein expression between each groups?p>0.05?.5.Compared with the control group,the binding amount of RBP-J?to ROR?t protein increased significantly in the model group?p<0.05?,while the binding amount of RBP-J?to Foxp3 protein decreased significantly?p<0.05?.After treatment,compared with the model group,the binding amount of RBP-J?and ROR?t protein in the middle dose group and cyclosporine group decreased?p<0.05?,while the binding amount of RBP-J?and Foxp3 protein increased?p<0.05?.Conclusion:Dioscorea paniculata saponins can significantly increase and maintain the levels of WBC,Hb,PLT and RET in peripheral blood of AA mice,increase the number of bone marrow cells and improve the hematopoietic function of bone marrow.The possible mechanism is that diosgenin from Dioscorea nipponica can inhibit the number of Th17 cells and the expression of transcription factor ROR?t in AA mice,increase the number of Treg cells and the expression level of transcription factor Foxp3,promote the balance of Th17/Treg cells and the recovery of immune homeostasis.Notch signal can participate in this process.Dioscorea paniculata saponins affect downstream signal transduction by affecting the expression levels of Notch1 signal receptor and Dll4,Jagged1 ligands in AA mice.RBP-J?in Notch signal can directly bind to ROR?t and Foxp3 proteins.Dioscorea paniculata saponins may increase the binding amount of RBP-J?to Foxp3 proteins and reduce the binding amount of RBP-J?to ROR?t proteins,promote the immune balance recovery of Th17/Treg.