Study On The Mechanism Of Treatment Of Aplastic Anemia Through The HIF-1α/NF-κB Pathway By Dioscorea Nipponica Makino

Onjetive:To establish a mouse model of aplastic anemia（AA for aplastic anemia）by improvingγ-irradiation combined with cyclophosphamide and chloramphenicol,and to intervene with Dioscorea nipponica Makino.The number of peripheral blood cells,the number of bone marrow nucleated cells and the proliferation of bone marrow in mice were used to determine the therapeutic effect of Dioscorea nipponica Makino on AA.The detection and analysis of HIF-1α,NF-κB and STAT3 in mouse bone marrow were observed.The changes of HIF-1α/NF-κB pathway in AA mouse model,explored the possible mechanism of the action of Dioscorea nipponica Makino in the treatment of AA model mice,and provided the corresponding theoretical and animal experimental basis for clinical treatment of aplastic anemia..Methods:1.Establishment and evaluation of AA mouse model:γ-ray irradiation combined with cyclophosphamide and chloramphenicol intraperitoneal injection modeling method,mice（n=90）were given different doses ofγ-rays at d1（3Gy,4Gy,5Gy）irradiation,d4,d5,d6 were given different doses of cyclophosphamide（25mg/（kg·d）,50mg/（kg·d））,and the same dose of chloramphenicol（62.5mg/（kg·d））)Intraperitoneal injection,6 groups of AA mouse models with different dose combinations were made.Peripheral blood cell counts of AA mice,bone marrow nucleated cell counts,and bone marrow pathological sections were examined at d7,d14,d21,and d28 to establish a stable AA mouse model.2.Pharmacodynamic study of Astragalus saponins on AA mouse model:AA mouse model was established（n=60）,and AA model mice were randomly divided into model group,Dioscorea nipponica Makino group and Chinese medicine on d8 days.The control group（triptoside polyglycoside）,the western medicine control group（Cyclosporin A）（n=15）,the normal control group（n=10）,each treatment group was treated with drug for 2 weeks,and each group was tested.Peripheral blood cell count,bone marrow nucleated cell count,and bone marrow pathological sections were used for pharmacodynamic evaluation.3.Study on the mechanism of treatment of aplastic anemia through the HIF-1α/NF-kB pathway by Dioscorea nipponica Makino:The expression of HIF-1α,NF-κB,STAT3 mRNA and protein in bone marrow was detected by qPCR and Western blot.The expression levels of IL-1βand TNF-αin peripheral blood serum were detected,and the possible mechanism of the action of Chuanlong diosgenin in the treatment of AA model mice was discussed.Results:1.Establishment of AA model:Compared with the normal control group,after the intraperitoneal injection of ¹³⁷Csγ-ray irradiation combined with cyclophosphamide and chloramphenicol,the three lines of peripheral blood cells were rapidly reduced,among them,white blood cells and platelets.The reticulocyte reduction was more significant（P<0.01）,the number of bone marrow nucleated cells decreased significantly（P<0.05）,the difference was statistically significant,the degree of myeloproliferation was reduced or severely reduced,bone marrow pathological sections showed severe reduction of hematopoietic cells,non-hematopoietic cells such as adipocytes Increased,consistent with the pathological features of AA model mice,¹³⁷Csγ-ray（3Gy）irradiation combined with cyclophosphamide 25 mg/（kg·d）,chloramphenicol 62.5 mg/（kg·d）intraperitoneal injection of AA mice The model is better.2.Pharmacodynamic study of Dioscorea nipponica Makino in the treatment of AA model mice:1)Compared with the model group,the peripheral blood leukocytes of the AA mice in each treatment group increased in different degrees after treatment,and the d15 the Dioscorea nipponica Makino group and Cyclosporin group A were significantly different from the model group（P<0.01,P<0.05）.2)The peripheral blood hemoglobin in the model group and each treatment group showed a trend of decreasing first and then rising.d15 was the lowest,which was significantly different from the normal control group（P<0.01）.Compared with the model group,the Dioscorea nipponica Makino group and Cyclosporin A group and the model group were significant difference between the d19,d22,（P<0.01）.Compared with the model group,the Tripterygium glycosides group was different from the model group（P<0.01）.After treatment,the hemoglobin of the d22 Dioscorea nipponica Makino group and the Cyclosporin A group increased significantly,but it still had some differences with the normal control group（P<0.05）,the difference was statistically significant;3)Peripheral blood platelets of each group of AA mice showed different degrees of rebound after d7,and the treatment groups were significantly increased compared with the model group（P<0.01）,the Dioscorea nipponica Makino group and Cyclosporin A were returned to normal level at d15;4)femur bone of each treatment group Pathology and sternum bone marrow smears compared to the untreated group showed a marked proliferation of bone marrow activity,the number of nucleated femoral bone marrow cells as compared to the model group increased significantly（P<0.01）.3.Study on the mechanism of treatment of aplastic anemia through the HIF-1α/NF-kB pathway by Dioscorea nipponica Makino:1)Compared with normal control group,HIF-1α,NF-kB,STAT3 mRNA in bone marrow of AA model group mice The expression of protein was enhanced（P<0.01,P<0.05）the difference was statistically significant.After the intervention of each group of drugs,the expression levels of HIF-1αand NF-kB mRNA and protein in the bone marrow of the Dioscorea nipponica Makino group and the Cyclosporin A group AA mice decreased significantly compared with the model group（P<0.01,P<0.05）,where in the expression levels of NF-kB mRNA and protein were still different compared with the normal control group（P<0.05）,the difference was statistically significant,and the expression of HIF-1αmRNA and protein was not significantly different from that of the normal control group;The expression of STAT3 mRNA and protein in each group was not significantly different from that in the model group.2)The content of IL-1βin peripheral blood of mice in AA model group was significantly lower than that in normal control group（P<0.01）,and the content of TNF-αwas significantly increased（P<0.01）;After intervention with the Dioscorea nipponica Makino and Cyclosporin A and Tripterygium glycosides,the content of IL-1βwas significantly increased compared with the model group（P<0.01,P<0.05）,the content of TNF-αwas significantly decreased（P<0.05）.Conclusion:1.The modified AA mouse model accords with the basic pathological features of aplastic anemia.The combination of 3Gy ¹³⁷Csγ-ray combined with25mg/（kg·d）cyclophosphamide and 62.5mg/（kg·d）chloramphenicol is injected intraperitoneally.Higher mold rate and lower mortality.2.Dioscorea nipponica Makino can effectively increase the number of peripheral blood cells in AA mice,increase the number of nucleated cells in the bone marrow of AA mice,improve the proliferation of bone marrow,and promote bone marrow hematopoiesis.3.The mechanism of improving the bone marrow hematopoiesis in AA mice by Dioscorea nipponica Makino is inhibiting the secretion of HIF-1αand NF-kB in the bone marrow of AA mice,thereby increasing the secretion of related cytokine IL-1βand reducing the release of TNF-α.It can improve bone marrow inflammation,promote the recovery of bone marrow hematopoietic function,and produce positive therapeutic effects on AA mice.