| Myelosuppression and osteoporosis,characterized as bone loss,are common side effects of chemotherapy.Stem cell populations in bone marrow stromal cells(BMSCs)have multidirectional differentiation potential,which can differentiate into both osteo-lineage cells and adipocytes.The balance of osteogenesis and adipogenesis is indispensable to osteoblastic niche homeostasis in hematopoietic microenvironment,and the osteoblastic niche plays a pivotal role in maintaining positive hematopoiesis.Previous studies demonstrated that oxidative stress may facilitate adipogenesis at the loss of osteogenesis of BMSCs.The number of osteoblasts and their precursor cells in the bone marrow was decreased with adiposis of the bone marrow cavity.The underlying mechanism may be that oxidative stress activates FOXO transcription,promotes FOXO binding to ?-catenin,inhibits downstream osteogenic-related factor transcription,and enhances lipogenic-related factor transcription.Angelica sinensis Polysaccharides(ASP),an active component of traditional Chinese medicine Angelica sinensis,has the functions of antioxidation and promoting hematopoiesis.Previous studies of our group have confirmed that ASP can alleviate BMSCs oxidative damage caused by chemotherapeutic drug 5-fluorouracil(5-FU),thus delay the oxidative stress induced premature of hematopoietic cells.Whether the protective mechanism of ASP on hematopoiesis is related to its protective role on osteoblastic niche is still unknown.The current study focused on ASP.An in vitro drug model of human bone marrow stromal cells and an in vivo chemotherapeutic drug model of mouse were established respectively to observe the reversal effect of ASP on myelosuppression after chemotherapy.Furthermore,we investigated whether the protective effect of ASP with antioxidant properties on hematopoiesis was related to its role in inhibiting oxidative stress,maintaining bone marrow stromal cell differentiation balance and stabilizing osteoblast niche,and the mechanism was related to ASP regulation FOXO/Wnt-?-catenin signaling.This study will provide theoretical and experimental basis for the further investigation for pharmacological effects of ASP and reducing the damage of chemotherapy.Methods: 1.Human bone marrow stromal cells(HS-5),a cell line with multidirectional differentiation potential,was used to establish an in vitro drug model after treatment of 5-FU,ASP and Li Cl to investigate the role of ASP in protecting osteogenic differentiation potential of bone marrow stromal cells after 5-FU chemotherapeutic injury,inhibiting adipogenic differentiation ability and regulating ?-catenin signaling molecules.CCK-8 was used to detect the inhibition rate of 5-FU on HS-5 cells,and 25 ?g/m L 5-FU treating HS-5 cells for 48 h was selected in the subsequent experiments.The experiment was divided into control group: routine culture;ASP group: 100 ?g/m L ASP was added on the basis of routine culture;5-FU group: 25 ?g/m L 5-FU was added on the basis of routine culture;5-FU+ASP group: 100 ?g/m L ASP was pretreated on the basis of conventional culture for 6 h,then 25 ?g/m L 5-FU was added;5-FU+Li Cl group: 10 mmol/L of Li Cl pretreated for 6 h then 25 ?g/m L was added.Each group was cultured for 48 h at 37?.HS-5 cells apoptosis was detected by flow cytometry.After differentiation culture,Alizarin red was used to detect osteogenic nodules to determine osteogenesis ability;Oil red O was used to detect lipid droplets and adipogenesis ability;Western blot was used to detect the expression of ?-catenin,Runx2 and PPAR? protein in HS-5 cells;RT-q PCR was used to detect the expression of ?-catenin,Runx2,Osterix,OCN,BMP-2 and PPAR? genes.2.The chemotherapeutic drug 5-FU was used to establish an in vivo myelosuppression model of mice to study the protective effect and its mechanism of angelica polysaccharide(ASP)pretreatment on myelosuppression and bone marrow microenvironment damage in chemotherapy mice.C57 mice were randomly divided into control group,ASP group,5-FU group and 5-FU+ASP group.In the control group,10 m L/kg/d saline was injected intraperitoneally for 7 days.In the ASP group,100 mg/kg/d ASP was injected intraperitoneally for 7 days.In the 5-FU group,10 m L/kg saline was injected intraperitoneally for 6 days,then 150 mg/kg 5-FU was injected intraperitoneally on the 7th day.In the 5-FU+ASP group,100 mg/kg/d ASP was injected intraperitoneally into mice for 7 days,150 mg/kg 5-FU was also injected intraperitoneally on the 7th day.Blood samples were collected using a blood cell count analyzer for routine blood examination.The bilateral femurs and tibiae of C57 mice were taken under sterile conditions,and the whole bone marrow cell suspension was extracted and counted.The femurs were fixed by 4% paraformaldehyde and decalcified by 0.5% nitric acid to make paraffin sections.Histomorphology of femurs was observed under light microscope,also the expression of SDF-1 factor in osteoblastic cells in the bone marrow was detected by immunohistochemistry.BMSCs were isolated by whole bone marrow adherent culture.The level of ROS in BMSCs was detected by flow cytometry and DCFH-DA fluorescence;MDA content,Mn SOD and CAT activity of BMSCs were detected by TBA method,WST-8 method and visible light method.Western blot and RT-q PCR assays were used to detect the related factors of osteogenic differentiation and adipogenic differentiation as well as key signaling molecules in FOXO and Wnt pathways in BMSCs.3.An in vitro drug model of mouse BMSCs was established to detect the osteogenic and adipogenic differentiation ability.BMSCs were isolated from femur and tibia of C57 mice by whole bone marrow adherent culture.When BMSCs were subcultured to the third generation,the cells were divided into 4 groups.Control group: routine culture;ASP group: 100 ?g/m L ASP was added into the routine culture;5-FU group: 25 ?g/m L 5-FU was added on into the routine culture;5-FU+ASP group: 100 ?g/m L ASP was pretreated on the basis of routine culture for 6 h,then 25 ?g/m L 5-FU was added.Each group was cultured for 48 h at 37? with 5% carbon dioxide and followed with osteogenic and adipogenic differentiation culture respectively to test the ability of osteogenesis and adipogenesis.Result: 1.ASP reduced the apoptosis rate of HS-5 cells after 5-FU induction.ASP reversed inhibition on osteogenic differentiation potential of HS-5 cells induced by 5-FU.Osteogenic nodules were increased significantly after osteogenic induced differentiation.ASP promoted the expression of osteogenic differentiation factor Runx2 protein,also facilitated the transcription of Runx2,Osterix,OCN and BMP-2 osteogenic differentiation genes.ASP alleviated the promoting effect of 5-FU on adipogenesis of HS-5 cells.Lipid droplets were significantly reduced after adipogenic differentiation culture,also the protein and gene expression of adipogenic differentiation factor PPAR? were reduced significantly.ASP attenuated 5-FU inhibition of ?-catenin expression and upregulated the expression of ?-catenin.2.ASP reduced myelosuppression induced by 5-FU in mice.ASP improved the number of erythrocytes,leukocytes and platelets after 5-FU treatment,and accelerated the recovery process of peripheral blood.ASP alleviated the decrease in the BM cellularity,and inhibited adipogenesis of the bone marrow.ASP prevented against 5-FU induced damages including thinner and shorter femoral trabecular bones with enlarged space,and promoted the length and width recovery of the femoral trabecular bone.ASP reversed the number of osteoblasts and the expression of hematopoietic chemokine SDF-1 in the bone marrow.3.ASP antagonized the impairment of antioxidative capacity of BMSCs in mice after 5-FU treatment,enhanced cellular antioxidative capacity such as Mn SOD and CAT activity,and reduce intracellular reactive oxygen species(ROS)and lipid oxidation marker MDA.4.In the in vitro drug model of BMSCs,ASP pretreatment reversed the imbalance of BMSC differentiation after 5-FU treatment.ASP increased osteogenic nodules and decreased lipid droplets significantly in the differentiation cultures.ASP up-regulated the osteogenic differentiation ability and down-regulated adipogenesis of BMSCs.The expression of osteoblast-related factors Runx2,OCN,BMP-2 and Osterix were increased,whereas the expression of PPAR? was decreased.5.The in vivo experiment of BMSCs demonstrated that ASP inhibited the activation of FOXO1 after 5-FU treatment detected by Western blot assay.ASP promoted the phosphorylation of FOXO1,inhibited the expression of FOXO1 and its downstream Bim and P27-Kip1.ASP reversed 5-FU-mediated FOXO1 competitive combination with ?-catenin,the key protein of Wnt pathway,thus up-regulated the expression of ?-catenin,pGSK-3? and Lef-1 protein,and inhibited GSK-3? expression.Conclusion: 1.ASP may reverse the myelosuppression induced by 5-FU and promote the recovery of hematopoiesis in bone marrow.2.ASP may alleviate oxidative stress of BMSCs induced by 5-FU,enhance their osteogenic differentiation ability at the loss of adipogenesis.ASP may maintain osteoblastic niche homeostasis via keeping the balance of osteogenic/adipogenic differentiation in BMSCs,thus promote hematopoiesis.3.The mechanism of ASP maintaining the balance of BMSCs may be related to its protective role against oxidative stress to down-regulate FOXO1,thus to activate Wnt/?-catenin signals and activate its downstream osteogenic transcription factors whereas inhibit adipogenic transcription factors.Cellular senescence of hepatocytes is involved in the progression of a wide spectrum of parenchymal liver diseases.Aging is accompanied by the occurrence of non alcoholic fatty liver disease(NAFLD).It is important to understand the underlying mechanisms of aging-driven NAFLD and to find effective treatments to limit chronic liver disease.Ginseng is a traditional Chinese medicine widely used in the treatment of various diseases,including liver diseases.it has antioxidant and anti-aging properties.Rg1 is the most effective component of ginseng.This experiment explores the potential protective effect and related mechanisms of Rg1 on aging driving chronic liver disease.Methods: 1.The mice were randomly divided into four groups: Control group,Rg1 group,the Rg1+D-gal group,and D-gal group.In the D-gal group,Dgal(120mg/kg/day)was injected intraperitoneally into mice for 42 d.In the Rg1+D-gal group,since the 16 th day of D-gal injection,ginsenoside Rg1(40mg/kg/day)was injected intraperitoneally daily for 27 d concomitantly.In the Rg1 group,ginsenoside Rg1(40mg/kg/day)was intraperitoneally injected for 42 d.All control animals were given 10 ml/kg body weight normal saline intraperitoneally.The liver index of mice in each group was detected,serum was collected and serum ALT and AST content were detected by microplate method.Liver homogenates were prepared to detect MDA?SOD and CAT content.2.The frozen sections were prepared and detected SA-?-gal activity.The ultrastructure of the liver was observed by transmission electron microscopy.Histopathological examinations were stained with hematoxylineosin(HE),periodic acid Schiff(PAS)and colorimetric TUNEL apoptosis assay kit.The oil red O staining of frozen sections was used to observe lipid.3.The expression of inflammatory factor IL-1?,IL-6 and MCP-1 in liver homogenate was detected by ELISA method.The expression of FOXO1 was detected by immunohistochemistry.The protein expression of P53,P21,FOXO1,p-FOXO1,Bcl-2,Bax and Bim was detected by Western blot.Result: 1.Rg1 down-regulates the liver index,and reverses the abnormal increase of serum ALT and AST induced by D-gal.2.Rg1 plays an anti-aging role in reversing D-galactose induced increase in senescence-associated SA-?-gal staining and P53,P21 protein in hepatocytes of mice.Rg1 protects livers from D-galactose caused hepatic steatosis,reduction in hepatic glucose production,hydrogenic degeneration,inflammatory phenomena including senescence-associated secretory phenotype(SASP)IL-1?,IL-6,MCP-1 elevation and lymphocyte infiltration.3.Rg1 suppresses drastic elevation in FOXO1 phosphorylation resulting in maintaining FOXO1 protein level in the liver after D-galactose treatment,followed by FOXO1 targeted antioxidase superoxide dismutase(SOD)and catalase(CAT)significant up-regulation concurrent with marked decrease in lipid peroxidation marker malondialdehyde(MDA).Conclusion: 1.Rg1 exerts pharmaceutic effects of maintaining FOXO1 activity in liver,which enhances anti-oxidation potential of Rg1 to ameliorate SASP and to inhibit inflammation.2.Rg1 promotes metabolic homeostasis,and thus protects livers from senescence induced fatty liver disease. |
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