The Mechanisms Of SIRT6/NF-?B Signaling Pathway In The Regulation Of Ginsenoside Rg1 On Acute Myeloblastic Leukemia

ObjectiveLeukemia is a malignant tumor of hematopoietic system caused by the uncontrolled proliferation and differentiation of hematopoietic stem cells,with high malignancy,rapid progress and poor prognosis.At present,the treatment of leukemia by targeted therapy drugs,although it has made remarkable achievements,but in the cytotoxicity of chemotherapeutic drugs also killing normal human cells and the leukemic cells to chemotherapy drug resistance is a major problem faced by the leukemia relapse and treatment.Finding effective and low-toxic effective treatment is the focus of leukemia treatment research.Ginseng is the sovereign medicine of"Invigorating vital and generating blood"in Chinese medicine.Ginsenoside Rg1 is the main active ingredients of ginseng which can promote blood cell function,anti-tumor,anti-aging,anti radiation effects.Our previous studies have demonstrated that Rg1 can delay the aging of hematopoietic stem/progenitor cells,promote the proliferation and differentiation,and inhibit proliferation of K562 cells.However,it is not clear that the role and mechanism of ginsenoside Rg1 in the regulation of leukemia in vivo is still unclear.An acute myeloid leukemia model of transplanted NOD/SCID mice was established in this study,Ginsenoside Rg1 was applied to this model to explore the effect and molecular mechanism of ginsenoside Rg1 on leukemia mice,so as to provide experimental evidence for drug therapy of leukemia.Methods1.The replication and identification of NOD/SCID immunodeficiency mice leukemia model:12 NOD/SCID mice were randomly divided into two groups:the control group and the model group,with 6 rats in each group.Two days before cell transplantation,NOD/SCIDmiceweregivenintraperitonealinjectionof cyclophosphamide 2 mg,for 2 consecutive days.Model group:on the day of transplantation,under aseptic conditions of tail vein transplantation of 2×10⁷ CD34⁺CD38^-LSCs.Control group:on the day of transplantation,intravenous injection of sterile PBS equivalent time.To observe the change of mass in general and abdominal mice,peripheral blood changes of blood cell detection,liver pathological changes were observed in mice,analysis of bone marrow cell cycle phase change of flow cytometry,bone marrow cell detection ability of proliferation of CCK-8 cells by immunofluorescence staining method to identify the source of bone marrow cells.2.The effect of ginsenoside Rg1 on acute myeloid leukemia mouse model:determine the success of the NOD/SCID mouse leukemia model after experiment.Groups:the control group,the model group,the positive control group and the ginsenoside Rg1 group.The positive control group:after modeling,on the basis of the control group,intraperitoneal injection of ginsenoside Rg1,200mg/kg,for 25consecutive days.Ginsenoside Rg1 group:on the basis of the model group,intraperitoneal injection of ginsenoside Rg1,200mg/kg,for 25 consecutive days.The control group and model group were given intraperitoneal injection of the same amount of sterile PBS,for 25 consecutive days.To observe the change of mass in general and abdomen of mice,to detect the changes of blood cells of peripheral blood,liver pathological changes were observed in mice,analysis of bone marrow cell cycle phase change of flow cytometry,bone marrow cells to detect CCK-8 value ability,immunofluorescence staining method to identify the source of bone marrow cells.To evaluate the effect of ginsenoside Rg1 in the treatment of transplanted acute myeloid leukemia mice.3.The mechanism of SIRT6/NF-?B signaling pathway in the regulation of ginsenoside Rg1 on leukemic mouse model:the control group,model group,positive control group,ginsenoside Rg1 group bone marrow cells were collected.?1?The expression of SIRT6 and NF-?B protein was detected by Western Blotting.?2?The expression of SIRT6 and NF-?B m RNA were detected by fluorescence quantitative PCR.Results1.Compared with the control group,model group mice showed deprementia,unstable gait,rough fur messy,abdominal mass is obvious,the average body weight was significantly lower than the control group?P<0.05?.The total number of white blood cells was significantly higher than the control group?P<0.05?,the number of red blood cell,hemoglobin,platelet count was significantly lower than the control group?P<0.05?.The basic structure of hepatic lobule was destroyed,the existence of a large number of white blood cells in the liver tissue,liver cells appeared vacuolar degeneration.The cell ratio of G₀/G₁ was lower than that of control group?P<0.05?,the proportion of S is higher than that of control group?P<0.05?.Bone marrow cell proliferationwashigherthanthatofthecontrolgroup?P<0.05?.The immunofluorescence showed that a large number of human leukemia cells were derived from the bone marrow cells of the model group.2.Compared with the model group,ginsenoside Rg1 group spirited in good condition,abdominal mass was less than the model group,the average body weight was higher than in the model group?P<0.05?.The total number of white blood cells than in the model group?P<0.05?,the number of red blood cell,hemoglobin,platelet count was higher than in the model group?P<0.05?.Structure of liver no lobule damage model group obviously.the proportion of G₀/G₁ phase was higher than in the model group?P<0.05?,S phase was lower than that of model group?P<0.05?.Bone marrow cells proliferation was lower than that of model group?P<0.05?.The immunofluorescence indicated that the model group and ginsenoside Rg1 group of mice bone marrow cells in human leukemia cell.3.Compared with the control group,the expression of SIRT6 mRNA and protein in the model group was down regulated,and the expression of NF-?B mRNA and protein was up-regulated.Compared with the model group,the expression of SIRT6 mRNA and protein in the ginsenoside Rg1 group was down regulated,and the expression of NF-?B mRNA and protein was up-regulated.Conclusions1.The model of acute myeloid leukemia in NOD/SCID mice could be successfully constructed by transplantation of CD34⁺CD38^-LSCs.2.Ginsenoside Rg1 has the effect of alleviating and treating leukemia symptoms in leukemia mice.3.Regulation of SIRT6/NF-?B signal transduction pathway was one of the mechanisms of ginsenoside Rg1 in alleviating and treating leukemia symptoms in leukemia mice.