1. The Changes Of Keap1-Nrf2/ARE Signaling Pathway In Hematopoietic Stem Cells Of Aging Mice. 2. The Protective Effect And Preliminary Mechanism Of Ginsenoside Rg1 On The Gastrointestinal Structure Of Aging Mice

Objective:The aging population has become one of the important social issues in the 21 st century.Cells are the basic units that constitute the structure,function and development of organisms.Therefore,cell aging is the cornerstone of the body's aging.In recent years,there are a variety of cell aging mechanism theories,but cell aging theory is until unclear.Adult stem cells play an important role in repairing damaged and aging tissue cells due to the ability to self-renewal and multi-directional differentiation.But stem cells will gradually be aging with age,which is an important basis for the occurrence and development of senile diseases.It is currently believed that the latest doctrine of body aging is the aging of its own stem cells.The previous research of our research group found that as mice age,their hematopoietic stem cells will undergo biological changes in aging.At the appropriate time,the use of certain methods to intervene in the aging process can significantly delay the senescence of hematopoietic stem cells.Ginsenoside Rg1 is an anti-aging component of the ginseng which for “invigorating qi”.Our latest research proves that it can antagonize the senescence of hematopoietic stem cells induced by oxidative senescence agents,but its mechanism is still unclear.Recently,it was discovered that Keap1-Nrf2/ARE is a defensive signaling pathway for the body to resist oxidative stress.The blockage of this pathway will lead to senescence of stem cells.Reactivating the Nrf2 pathway can effectively reverse the aging process of stem cells in patients with premature aging.By consulting the literature,we propose a research hypothesis that the mechanism of oxidative stress senescence of hematopoietic stem cells with aging may be closely related to this signaling pathway.In this paper,the above hypothesis will be verified by constructing a mouse model of aging and establishing an in vitro model of stem cell aging,aiming to lay a theoretical foundation for explaining the new mechanism of hematopoietic stem cell aging and provide an experimental basis for finding a way to delay hematopoietic stem cell aging.Methods:1.Isolation and identification of mouse hematopoietic stem cells?HSCs?: the mice were sacrificed by cervical dislocation,the femurs and tibias of the mice were aseptically removed,bone marrow mononuclear cells?BMMCs?were isolated,and separated and purified by immunomagnetic bead sorting?MACS?Sca-1⁺HSCs in bone marrow were identified by flow cytometry for cell purity.2.In vitro aging model construction and index detection of hematopoietic stem cells: the separated and purified Sca-1⁺ HSCs are divided into two groups: normal control group?conventional culture?and aging model group?D-gal,166 mmol /L,48h?.The following tests were performed: CCK-8 method to detect proliferation;SA-?-Gal staining to detect cell aging;test kit to detect SOD,CAT,MDA and GSH-Px content;q-PCR to detect Keap-Nrf2/ARE signal MRNA expression levels of pathway-related genes?GCLC,GSR,GSTM1 and GCLM?.3.Hematopoietic stem cells aging model construction and index detection: 6-8 weeks old C57BL/6J mice were randomly divided into two groups: aging model group: mice were injected intraperitoneally with D-gal?120mg/kg?,qd×42d;control group: intraperitoneal injection of isochronous saline and the same amount of saline.On the second day after the successful modeling,the whole blood and serum of the mice were collected,and the peripheral whole blood was routinely tested for the hematopoietic function of the mice;the test kit was used to detect the content of serum SOD,CAT,MDA and GSH-Px;the two groups were separated and purified mouse bone marrow HSCs,q-PCR detection of Keap-Nrf2/ARE signaling pathway related genes?GCLC,GSR,GSTM1 and GCLM?m RNA expression levels in cells.Results:1.After flow cytometry,the purity of Sca-1⁺HSCs was 85.97±5.99%.It is suggested that Sca-1⁺ HSCs obtained by immunomagnetic bead sorting have high purity.2.After D-gal induced Sca-1⁺HSCs senescence in vitro,the cell proliferation capacity was significantly reduced;the percentage of SA-?-Gal staining positive cells increased;the contents of SOD,CAT and GSH-Px all decreased,and the MDA content increased;Keap1-Nrf2/ARE signaling pathway related genes?GCLC,GSR,GSTM1 and GCLM?m RNA expression levels were significantly reduced.3.After senescence of Sca-1⁺HSCs induced by D-gal in vivo,there was no significant change in peripheral blood cell count;the content of SOD,CAT and GSH-Px in serum decreased,and the content of MDA increased;the m RNA levels of Keap1-Nrf2/ARE signaling pathway related genes?GCLC,GSR,GSTM1 and GCLM?in Sca-1⁺HSCs were significantly reduced.Conclusions:1.The oxidative decay agent D-gal can construct an in vitro aging model of Sca-1⁺HSCs.2.The oxidative decay agent D-gal can construct an in vivo aging model of Sca-1⁺HSCs.3.The aging of Sca-1⁺HSCs is related to the increase of the level of oxidative stress,and the possible mechanism is related to the reduction of the expression levels of genes related to the Keap-Nrf2/ARE signaling pathway.Aging is the organs,tissues and cells in the body gradually undergo irreversible and comprehensive structural and functional decline with age,which occurs after the organism has matured.Natural aging is not a disease in itself,but it's closely related to many senile diseases.With the increase of age,the tissue structure and physiological functions of various organs of the human body gradually decline,and the body's ability to resist disease and repair damage also decreases,so the occurrence of senile diseases is inevitable.Delaying aging,especially avoiding pathological aging is the goal pursued by modern medicine..With the acceleration of the aging process and the extension of the lifespan,we believe that promoting the study of aging mechanisms and senile diseases has an important scientific significance and social value.Ginseng contains a variety of active ingredients,including ginsenosides,fatty acids,polysaccharides and mineral oil,ginsenosides are its main medicinal ingredients,of which ginsenoside Rg1 is the most active main ingredient of ginsenosides.The previous researches have found that ginsenoside Rg1 is an important anti-aging component in ginseng,and it has the effect of "two-way regulation" and "strengthening body resistance and eliminating evil ",which means Rg1 can promote proliferation,differentiation and activate its functions for damage and aging cells,inhibit proliferation and promote apoptosis of malignant tumor cells.The latest researcheshave showed that ginsenoside Rg1 has the effect of delaying the senescence of various stem cells such as hematopoietic stem cells and mesenchymal stem cells,and its related mechanisms have been specifically described.We hypothesize that ginsenoside Rg1 can delay organ aging and antagonize the damaging effects of aging agents on organs.Degenerative changes and dysfunction in the digestive system are one of the important biological characteristics of the body's aging.The digestive and absorptive capacity of the elderly body gradually declines with age,it has a significant role to protecte the structure and function of the digestive system for delaying the body's aging.The oxidative decay agent D-galactose?D-gal?was used to establish the aging model of mice,to study the protective effect and preliminary mechanism of ginsenoside Rg1 on the digestive system of aging mice.It aims to provide theoretical and laboratory basis for the prevention and treatment of senile diseases by ginsenoside Rg1.Methods:Establishment of mouse aging model: C57BL/6J mice were randomly divided into Control group,Rg1 group,D-gal group and Rg1⁺D-gal group.D-gal group: mice were intraperitoneally injected with D-gal?120mg/kg?,qd×42d;Rg1⁺D-gal group: D-gal injection dose and time are the same as Dgal group,Rg1?40mg/kg?is injected intraperitoneally from the 15 th day,qd×28d;control group: intraperitoneal injection of normal saline?10m L/kg?,qd×42d;Rg1 group: injection of normal saline?10m L/kg?at the same time as the control group,Rg1?40mg/kg?,qd from the 15 th day,qd ×28d.On the second day after the completion of the drug injection,the gastrointestinal tract was taken for related index detection:1.Determination of gastrointestinal organ index;2.Gastrointestinal histopathology and ultrastructural observation;3.Detection of gastrointestinal senescence by ?-galactosidase staining;4.Detection of oxidative stress indexesof gastrointestinal tissues;5.Detection of Keap1-Nrf2/ARE-related proteins in gastrointestinal tissues.Results:1.The D-gal group mice gradually showed the characteristics of dull hair,poor skin elasticity,depression,and drowsiness,significantly reduced food intake and activity,and body mass slowly increase.The biological performance of aging model mice is similar to natural aging signs,while the other three groups mice have no obvious signs of aging.2.The small intestinal organ index of the D-gal group mice increased,and after intervention with Rg1,the gastrointestinal organ index of the mice decreased.3.The gastrointestinal tissue structure of the D-gal group mice was obviously damaged,the mitochondria were swollen,the nuclear membrane was dissolved,and the rough endoplasmic reticulum was expanded.After Rg1 intervention,the gastrointestinal tissue structure was improved.After injection of Rg1,the damage of gastrointestinal tissue structure wasrelieved.4.The SA-?-gal staining optical density of gastrointestinal tract tissues of aging model mice increased,suggesting that the organs show an obvious aging performance.After intervention with Rg1,the aging of gastrointestinal tract tissues in mice has relieved.5.In gastrointestinal tissues of aging model mice,the content of SOD and CAT decreased,and the content of MDA increased.After intervention with Rg1,the content of SOD and CAT increased,and the content of MDA decreased,suggesting that Rg1 enhanced antioxidant capacity of mice gastrointestinal tissue.6.In the gastrointestinal tract of the aging mice,the expression of HO-1 and Nrf2 proteins decreased and the expression of Keap1 protein increased.After Rg1 intervention,HO-1 and Nrf2 proteins expression increased and Keap1 protein expression decreased.Conclusions:1.The aging model of mice could be successfully established by injection of D-gal,and the biological behavior was consistent with that of natural aging mice.2.Rg1 can delay the aging of mice induced by D-gal,alleviate the oxidative stress state of aging mice,and improve the changes of gastrointestinal tissue structure of mice.3.Rg1 antagonizes the structural changes of gastrointestinal tract in aging mice induced by D-gal may be related to the regulation of the expression of antioxidant proteins in Keap1-Nrf2/ARE pathway,thereby to antagonize oxidative stress.Objective To investigate the effect of Angelica Sinensis polysaccharide?ASP?on proliferation,differentiation and transplantation of human leukemia stem cells.Methods By using immunomagnetic bead sorting,CD34⁺CD38^-cells isolated from the bone marrow of normal persons and myeloid leukemia patients were divided into normal CD34⁺CD38^-group,CD34⁺CD38^-LSCs group,normal CD34⁺CD38^-ASP group and CD34⁺CD38^-LSCs ASP group.The latter two groups were added ASP?terminal concentration 40 ?g/ml?on the basis of conventional culture.The purity of CD34⁺CD38^-cells was detected by flow cytometry.The viability of cells was detected by trypan blue staining.The proliferation and differentiation of CD34⁺CD38^-cells were detected by CCK-8 and CFU-Mix.The expression of aging-related gene was detected by RT-PCR.To establish CD34⁺CD38^-LSCs mice transplantation model,they were randomly divided into ASP transplantation model group?intraperitoneal injection with ASP,200mg/kg,qd × 14d?and transplantation model group?intraperitoneal injection with the same volume of saline at the same time?.On 2^nd day after drug injection was completed,leukocytes count and classification and cells morphology were observed.By using immunofluorescence cytochemistry,human CD34⁺CD38^-LSCs in mice bone marrow was detected.The percentage of SA-?-Gal staining positive cells of bone marrow mononuclear cells?BMMCs?was detected.Cell cycle distribution of BMMCs was detected by flow cytometry.The colony formation ability of BMMCs was detected by CFU-Mix.Results The purity of CD34⁺CD38^-cells purifying by immunomagnetic bead sorting was 91.14±1.02%,and the viability of cells was 95.42±3.52%.The proliferation and CFU-Mix formation ability of CD34⁺CD38^-LSCs group were significantly higher than that of normal CD34⁺CD38^-group.The proliferation and CFU-Mix formation ability of CD34⁺CD38^-LSCs ASP group were obviously lower than that of CD34⁺CD38^-LSCs group.The expression of p16^INK4a,p19^Arf,p53,p21^Cip1/Waf1 genes in CD34⁺CD38^-LSCs ASP group were increased.Human LSCs existed in the bone marrow of transplanted LSCs mice.The count of leukocytes in the transplantation model group was increased,the proportion of neutrophils was increased and lymphocytes was decreased.After injection of ASP,the count of leukocytes and the proportion of neutrophils were markedly reduced,the proportion of lymphocytes was increased.The number of BMMCs in G0/G1 phase was increased and in S phase was decreased.The percentage of SA-?-Gal positive staining cells was significantly increased;the number of CFU-Mix was decreased.Conclusion ASP can inhibit the proliferation and differentiation of CD34⁺CD38^-LSCs in vivo and in vitro.Its mechanism may be to the regulated cell senescencerelated gene expression.