| Hematopoietic microenvironment participates in the maintenance,self-renewal and directed differentiation of hematopoietic stem cells.Bone marrow stromal cells are the core components of the hematopoietic microenvironment.They regulate the self-renewal,proliferation,and differentiation of hematopoietic stem cells by directly contacting with hematopoietic cells,secreting hematopoietic regulatory factors,and synthesizing extracellular matrix.The literatures indicated that a large number of chemotherapy drugs have toxic side effects on bone marrow,and bone marrow stromal cell injury is one of the main causes of myelosuppression,which is the main side effect of chemotherapy.The damage effect of chemotherapy on bone marrow stromal cells is dose-dependent.Even conventional doses of chemotherapeutic drugs can cause the death of bone marrow stromal cells,thereby induce myelosuppression,hematopoietic dysfunction,hematopoietic reconstitution disorders,and drug resistance.Our previous work confirmed that 5-fluorouracil can cause oxidative damage of bone marrow stromal cells,alter bioactive substance produced by bone marrow stromal cells,and induce oxidative stress premature aging of hematopoietic cells.However,the specific mechanism of 5-FU-induced bone marrow stromal cells injury is still unclear.To explore its related mechanisms to reduce the side effects of chemotherapy drugs and to screen protection drugs during chemotherapy has important value for scientific inquiry and clinical guiding significance.In recent years,it has been found that Wnt/?-catenin signaling pathway can affect bone marrow stromal cell function extensively,participate in bone marrow stromal cell proliferation,regulate oxidative stress,and regulate hematopoietic stem cell self-renewal through stroma-dependent manner.Angelica is a traditional Chinese medicine for "blood enrichment and blood circulation".Angelica Sinensis Polysaccharides(ASP)are major effective ingredients of Angelica,which have a variety of effects including anti-oxidation,promoting hematopoiesis,delaying senescence of hematopoietic stem cells and bone marrow stromal cells,etc.Our previous work confirmed that ASP can delay the oxidative stress premature aging of hematopoietic cells by alleviating the oxidative damage of 5-FU on bone marrow stromal cells.However,the specific molecular mechanism of ASP that protects bone marrow stromal cells is still unclear and needs further study.Therefore,this study focused on whether the mechanism of 5-FU-induced bone marrow stromal cell injury is related to Wnt/?-catenin signaling.Whether the mechanism of ASP that protects bone marrow stromal cells is related to the regulation of Wnt/?-catenin signaling.In this study,human bone marrow stromal cell HS-5 was used as the research object to investigate the relationship between the inhibitory effect on the proliferation of bone marrow stromal cell induced by 5-FU and Wnt/?-catenin signaling pathway and its possible mechanism.And the relationship between the effects of ASP,such as alleviating the damage of bone marrow stromal cells induced by 5-FU,protecting the proliferation of bone marrow stromal cells,and the regulation of Wnt/?-catenin signaling pathway.The aim of this study is to elucidate the possible mechanism of chemotherapeutic which would damage bone marrow stromal cells,to provide new therapeutic targets for the prevention and treatment of chemotherapy side effects,and to provide new strategies for drug screening which could help increase the possibility of prevention of chemotherapy injury and bone marrow protection.Methods :1.To observe the effect of 5-FU on the growth of human bone marrow stromal cell HS-5: HS-5 cells were cultured in vitro and added with 12.5?g/mL,25?g/mL,50?g/mL,100?g/mL concentrations of 5-FU,CCK-8 detects the inhibition rate of HS-5 cells.2.To observe the effect of ASP on the growth of HS-5 cells after 5-FU treatment and its relationship with Wnt signaling: positive control group was established with Wnt pathway activator LiCl.The experiment was divided into control group,5-FU group,5-FU + ASP group and 5-FU+ LiCl group.The control group was routinely cultured;the 5-FU group was treated by 5-FU on the concentration of 25?g/mL;the 5-FU+ ASP group was pretreated with ASP on the concentration of 100 ? g/mL,and 25 ?g/mL 5-FU was added after 6 hours;the 5-FU+ LiCl group was pretreated with LiCl on the concentration of 10 mmol/L,and 25 ?g/mL 5-FU was added after 6 hours,each group was cultured for 48 h.EdU detects the viability of cell proliferation.The negative control group was set up with the Wnt pathway inhibitor Dkk1.The experimental groups were: control group;5-FU group;Dkk1 group: control group was added with a concentration of 50 ng/mL Dkk1 for 48 hours;ASP+5-FU group;Dkk1+ASP+ 5-FU group: control group was pretreated with Dkk1 on the concentration of 50 ng/mL,after 1 hour,100 ?g/mL of ASP was added,and after 6 hours,25 ?g/mL 5-FU was added.CCK-8 detects the viability rate of HS-5 cells.3.To investigate the effect of ASP on the expression of Wnt/?-catenin pathway-related signaling proteins in HS-5 cells after 5-FU treatment: the experiment was divided into control group,5-FU group,5-FU + ASP group and 5-FU+ LiCl group,same as above.The expression level of ?-catenin,a key effector protein of Wnt pathway,was detected by immunofluorescence,and the expression of ?-catenin,GSK-3?,p-GSK-3?,Lef-1,Cyclin D1,and C-myc proteins were detected by Western Blot.4.To observe the effect of ASP on oxidative stress of HS-5 cells after 5-FU treatment: the experiment was divided into control group,5-FU group,5-FU + ASP group and 5-FU+ LiCl group,same as above.Cellular reactive oxygen species(ROS)content was detected by DCFH-DA fluorescence;malondialdehyde(MDA)content was detected by TBA method;superoxide dismutase(SOD)activity was detected by WST-1 method;catalase(CAT)activity was detected by the visible light method.5.To investigate the effect of ASP on the oxidative stress transcription factor FoxO1 in HS-5 cells after 5-FU treatment: the experiment was divided into control group,5-FU group,5-FU + ASP group and 5-FU+ LiCl group,same as above.Western Blot was used to detect the expression of FoxO1,p-FoxO1,P27 Kip1,Bim,Bax,Bcl-2 and caspase-3 proteins.6.To investigate the effect of ASP on HS-5 cell injury after 5-FU treatment: the experiment was divided into control group,5-FU group,5-FU + ASP group and 5-FU+ LiCl group,same as above.Flow cytometry detects apoptosis and cell cycle.Senescence-specific ?-galactosidase staining was used to detect cellular senescence.Results:1.5-FU inhibits the growth of bone marrow stromal cells in a time and dose dependent manner.25?g/mL 5-FU treating cells for 48 h can inhibit half of the cell proliferation.Subsequent experiments were carried out at this concentration and time.2.The results of EdU showed that the proportion of proliferating cells after 5-FU treatment was significantly lower than the control group;the proportion of proliferating cells after ASP pretreatment was significantly higher than the 5-FU group.The results of CCK-8 showed that the survival rate of HS-5 cells decreased significantly after 5-FU treatment;pretreatment of ASP partially reversed the 5-FU-induced decrease of cell viability rate,compared to the 5-FU group.3.The results of immunofluorescence showed that the expression of ?-catenin protein was significantly decreased after 5-FU treatment,and the expression of protein in the nucleus was significantly decreased.Pretreatment with ASP can increase the expression of ?-catenin protein in cells and significantly increase the translocation of nucleus protein.The results of Western Blot showed that in 5-FU group the total protein level of GSK-3? was unchanged;the expression levels of p-GSK-3?,total ?-catenin,nuclear ?-catenin,Lef-1,Cyclin D1,and C-myc proteins were down-regulated.Pretreatment with ASP,the total protein level of GSK-3? was unchanged,and the expression levels of p-GSK-3?,total ?-catenin,nuclear ?-catenin,Lef-1,Cyclin D1,and C-myc were higher than those of in 5-FU group.4.5-FU weakens the antioxidant capacity of cells,decreases the activity of SOD and CAT,leads to an increase in intracellular ROS and MDA levels,and an increase in cellular oxidative stress.Pretreatment with ASP,compared with the 5-FU group,the antioxidant capacity of HS-5 cells increased,the activities of SOD and CAT increased,and intracellular ROS and MDA content decreased.5.Western Blot results showed that the expression levels of FoxO1,P27 Kip1,Bim,Bax,and caspase-3 were increased and the expression levels of p-FoxO1 and Bcl-2 were decreased in 5-FU group compared with the control group.ASP could down-regulate the activity of FoxO1 in cells after 5-FU treatment,and the expression of P27 Kip1,Bim,Bax,and caspase-3 protein was significantly decreased,and the expression of p-FoxO1 and Bcl-2 proteins were significantly increased.6.5-FU damaged the bone marrow stromal cells.Compared with the control group,the proportion of apoptotic cells was increased,the cell cycle was arrested in G1 phase,and the proportion of senescent cells increased significantly.ASP can reduce the proportion of apoptosis and senescent cells after 5-FU treatment.Conclusion:1.5-FU down-regulates Wnt/?-catenin signaling pathway to inhibit the proliferation of bone marrow stromal cells.2.5-FU induces oxidative stress of bone marrow stromal cells and activates the oxidative stress transcription factor FoxO1,which leads to oxidative damage,apoptosis or senescence.3.ASP can attenuate the proliferation inhibition of bone marrow stromal cells induced by 5-FU by activating the Wnt/?-catenin signaling pathway.4.ASP down-regulates FoxO1 by directly antagonizing oxidative stress or indirectly activating Wnt/?-catenin signaling,thereby reducing apoptosis,restoring the cell cycle and inhibiting the cell senescence induced by 5-FU. |
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