Experimental Study On The Effect Of Angelica Polysaccharide Combined With Astragalus Polysaccharide On The RAS-MAPK Signaling System Of Bone Marrow Hematopoietic Stem Cells In Myelosuppressed Mice

Purposes:To investigate the effect of Angelica polysaccharide combined with Astragalus Polysaccharide on Ras-MAPK signal system in bone marrow suppression rats from cell proliferation rate,hematopoietic factors,cell signaling pathway molecules,nuclear transcription factors,and so on,so as to provide basis and reference for clinical treatment.Materials and methods:(1)60 clean grade C57BL/6 mice were purchased as objects,and 10 C57BL/6 mice were randomly selected as blank control group;the remaining 50 C57BL/6 mice were used to establish mouse animal models,and the changes of diet,excretion,mental state and weight of mice were observed after modeling.Before and 4 days after modeling,5 UG blood samples were collected from the orbital vein of mice and added into the condensation tube.The red blood cells,white blood cells and hemoglobin in peripheral blood of mice were measured by automatic cell analyzer,which further confirmed the success of mouse modeling.Animals were divided into groups.After successful modeling,they were randomly divided into model control group,erythropoietin group(EPO group),angelica polysaccharide group,astragalus polysaccharide group and combination group.Ten C57BL/6 mice were randomly selected as blank control group.The remaining 50C57BL/6 mice were randomly divided into model control group,EPO group,angelica polysaccharide group and Astragalus membranaceus group Polysaccharide group and combination group,10 mice in each group;blank control group received intraperitoneal injection of the same dose of normal saline;other groups were intraperitoneally injected with cyclophosphamide 380 mg/(kg·d)for 3 consecutive days.On the fourth day,the mice in each group were killed by cutting the neck.The femur and tibia were taken and placed in 75.0%ethanol for 15 minutes.The mice were transferred to the ultra clean working table,and the femur and tibia were separated and extracted under sterile conditions.The hair,muscle and cartilage of both ends were removed to fully expose the red bone marrow cavity.1 ml sterile syringe No.6 was used to suck 1ml PBS solution,and the sterile needle sleeve was twisted and inserted into the bone marrow cavity.After washing,the bone marrow was obtained and placed in the plate for repeated washing.Most of the cells were washed out and filtered with a 300 mesh filter screen to prepare single cell suspension.Lymphocyte separation solution(equal amount)was added to the cell suspension,centrifuged for 10 minutes,and the speed was 2500rpm After finishing,remove the supernatant,add 1ml of red blood cell lysate,after 3 min standing,add 9 ml PBS solution,centrifugate for 10 min,speed 2500rpm,remove the supernatant,add 10.0%IMDM to complete the second washing,use 10.0%IMDM to complete the precipitation re suspension,adjust the cell density to 1×10~6/ml by using the counting plate,and complete the separation and culture of cells,and add them into 96/24 well plate.The cell experiment was divided into six groups:blank group,model control group,EPO group,angelica polysaccharide group,astragalus polysaccharide group and combination group,and the drugs were prepared,angelica polysaccharide,astragalus polysaccharide and drug combination.The purchased Angelica polysaccharides and astragalus polysaccharides were placed in RPMI-1640 culture medium,and the required concentrations were determined by pre experiment,i.e.angelica polysaccharide 200mg/ml,astragalus polysaccharide 200mg/ml,combined medication(Angelica polysaccharide 200mg/ml mixed with astragalus polysaccharide200mg/ml)to ensure the actual drug ratio of Angelica sinensis:Astragalus=5:1.The medicine was filtered and sterilized and placed in a refrigerator at 4?for standby.The EPO group,angelica polysaccharide group,astragalus polysaccharide group and combination group were added with equal dose of drug intervention,and the cells were cultured in 5%CO2,37?incubator;Cell culture in vitro.After labeling the type and treatment time of the cells,the cells were sterilized with alcohol and placed in the cell incubator for culture(5%CO2,temperature 37?);the morphology of the cells was observed under the inverted microscope to see if they were attached to the wall.When the culture medium turned yellow,the cells were completely changed for the first time(PBS buffer was used to clean the cells for 3times before changing the solution)The concentration of PBS was 10.0%.After80.0%-90.0%cell fusion,the cells were subcultured(the cells were in logarithmic growth phase).After centrifugation,the supernatant was removed,and the cells were resuspended with fresh complete culture medium,and 10ul of cell mixture was taken from a pipette,placed in a clean PE tube,and trypan blue staining was added 10 UL solution,gently blow and dilute,and stand for 1-2 min;take a disposable cell counting plate,take 10 ml from the re suspended cell solution,notice it in the small hole of the counting plate,insert the end of the counting plate containing the cell suspension into the hole of TC20 cell counter to complete the automatic cell counting,obtain the total number of cells and the total number of living cells in a short time,and adjust the cell density according to the results for standby.(2)Cell treatment.The bone marrow hematopoietic stem cells cultured in vitro were placed in 3 ml standard medium,mixed with 0.5 ml normal saline,and the cell concentration was adjusted to 1×105/ml.EPO group,angelica polysaccharide group,astragalus polysaccharide group and combination group were added with equal dose of drug intervention the cell proliferation rate was measured by MMT method The cell proliferation rate was measured by MMT method.Six groups of treated cells were selected,and the cell density was adjusted to 3×104 cells/ml.the cells were inoculated in 96 well plates with 100 UL per well for 48 hours.After the above operations,the culture medium was removed and 0.5 g/l was added to each well MTT solution was 100 UL,the culture was completed for 4 hours continuously,MTT was removed,and dms0200UL was added to complete the culture for 30 minutes continuously.The absorbance value(a)of each pore was determined by automatic microplate analyzer at 570nm wavelength,and the cell proliferation rate was calculated;the interleukin-2(IL-2),thrombopoietin(TPO),granulocyte macrophage colony stimulating factor(GM-CSF)were measured by enzyme-linked immunosorbent assay(ELISA)The levels of interleukin-2(IL-2),thrombopoietin(TPO),granulocyte macrophage colony stimulating factor(GM-CSF)and interferon gamma(inf-r)were determined by enzyme-linked immunosorbent assay(ELISA).The specific methods were as follows.1.Sampling.After the intervention,the cells in each group were added into the culture medium,and put into the centrifuge tube 500 ml,numbered and labeled;2.The quilt.Take six 96 well culture plates,add coating buffer solution,dilute antibody protein concentration to 10ug/ml;add 0.1ml mixed solution into the reaction hole of enzyme label plate,and place it at 4?for standby;remove the liquid in the hole after24h,and select the concentration of 15%PBS solution to wash for 3min each time,and wash three times continuously;3.Sample addition.According to the designed stimulation,they were placed on the enzyme plates of IL-2,TPO,GM-CSF and inf-?m Abs(2 for each).The supernatant of each group was put into the corresponding hole,50 UL per well,16 wells in each group.The blank control group was set,and it was cultured in a wet box for 60minutes at 37?.After the above operation,PBS was used to clean the supernatant for 3 times,each time for 3 minutes;4.Enzyme labeled antibody was added.After the above operation,biotin labeled IL-2,TPO,GM-CSF and inf-?Mc Abs(50 ul for each biotin)were added to each hole in turn,and the temperature was 37?;after the above operation,PBS was used to clean three consecutive times,each time for 3min;5.The reaction was stopped.TMB substrate solution was added into each reaction hole,the dosage was 0.1ml,and it was left standing for 15min,and the concentration of 10%sulfuric acid solution was added with the dosage of0.05ml.After the above reaction was completed,the absorbance value was determined by 450 nm enzyme labeled instrument;the standard curve was drawn on the semi logarithmic coordinate paper to obtain the concentration of IL-2,TPO,GM-CSF and inf-?.(2)the effect of Angelica polysaccharide combined with Astragalus Polysaccharide on RAS,ERK1,ERK2,p38.The expression levels of RASm RNA,ERK1m RNA,ERK2m RNA and p38 m RNA in each group were detected by real-time fluorescent PCR,and the relationship between Ras-MAPK signaling pathway and proliferation and differentiation of hematopoietic stem cells was analyzed;the effects of Angelica sinensis polysaccharide,astragalus polysaccharide and their compatibility on Ras-MAPK signal transduction pathway and proliferation and differentiation of hematopoietic stem cells were further determined.(3)The effects of Angelica polysaccharide combined with Astragalus Polysaccharide on nuclear transcription factors c-Jun,c-fos and JNK.The m RNA levels of nuclear transcription factors c-Junm RNA,c-fosm RNA and JNKm RNA were determined by real-time PCR.The relationship between cell cycle,nuclear transcription factor and cell proliferation was analyzed.The effects of Angelica polysaccharide,astragalus polysaccharide and their compatibility on nuclear transcription factor,cell cycle and hematopoietic stem cell proliferation were determined.All data in this study were processed by spss20.0software.Results:1 Effects of Angelica sinensis polysaccharide,astragalus polysaccharide and their combination on hematopoietic stem cell proliferation and hematopoietic factors in bone marrow suppression mice1.1 effects of Angelica sinensis polysaccharide,astragalus polysaccharide and their combination on the proliferation of hematopoietic stem cells in bone marrow suppressed mice1.1.1 Effects of bone marrow suppression mice on peripheral blood cells and biochemical indexes before and after modelingThe changes of biochemical indexes were not obvious in the blank control group,and the red blood cells,white blood cells and hemoglobin in other groups were significantly decreased compared with those before modeling(P<0.05)1.1.2 effects of Angelica sinensis polysaccharide,astragalus polysaccharide and their combination on the proliferation rate of hematopoietic stem cells in bone marrow suppression miceThe cells in the six groups showed an increasing trend with time.The cell rate of normal group was faster than that of normal group;Bone marrow hematopoietic stem cells were successfully isolated and cultured.After 1 day in vitro culture,the number of cells increased under the inverted microscope;3 days after the cell culture,a few cells began to form colonies,and the cell morphology was consistent,and most of them were round;after 5 days of continuous culture,the number of cells increased significantly,most of the thin colonies grew,and the cell morphology was uniform and the size was the same;after 7 days of continuous culture,the cell volume was increased The cells are evenly dispersed in the medium1.1.2.1MTTtest 24h results:there was no statistical significance in cell proliferation rate between Angelica polysaccharide group and astragalus polysaccharide group(P>0.05),but both fjsda groups were higher than EPO group(P<0.05);cell proliferation rate of combination group was higher than that of Angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).1.1.2.2MTTtest for 48h results:the cell proliferation rate of Angelica polysaccharide group and astragalus polysaccharide group was not statistically significant(P>0.05),and both groups were higher than EPO group(P<0.05);the cell proliferation rate of combination group was higher than that of Angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).1.1.2.3MTTtest for 72h results:the cell proliferation rate of Angelica polysaccharide group and astragalus polysaccharide group was not statistically significant(P>0.05),and both groups were higher than EPO group(P<0.05);the cell proliferation rate of combination group was higher than that of Angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).1.2 the effect of Angelica sinensis polysaccharide,astragalus polysaccharide and the combination of the two drugs on hematopoiesis factor in myelosuppression mice1.2.1 effect on hematopoietic factor TPOCompared with the normal group,the TPO level in the model group was significantly decreased(P<0.05),but there was no statistical significance in the comparison of Angelica polysaccharide group and astragalus polysaccharide group(P>0.05),both groups were higher than EPO group(P<0.05);the TPO level of combined medication group was higher than that of Angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).1.2.2 effect on IL-2Compared with the normal group,the level of IL-2 in the model group was significantly decreased(P<0.05),but there was no statistical significance in the comparison of Angelica polysaccharide group and astragalus polysaccharide group(P>0.05),but both groups were higher than that of EPO group(P<0.05);IL-2 in the combination group was higher than that in the angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).1.2.3 effect on hematopoietic factor GM-CSCompared with the normal group,the level of GM-CS in the model group was significantly decreased(P<0.05)but there was no statistical significance in the comparison of Angelica polysaccharide group and astragalus polysaccharide group(P>0.05),but both groups were higher than that of EPO group(P<0.05);GM-CS in the combination group was higher than that in the angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).1.2.4 effect on INF-rCompared with the normal group,the level of inf-r in the model group was significantly decreased;compared with the model group,the inf-r level of other medication groups was increased;the inf-r of Angelica polysaccharide group and astragalus polysaccharide group was not statistically significant(P>0.05),and both groups were lower than that of EPO group(P<0.05);inf-r of combined treatment group was lower than that of Angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).2.Effects of Angelica sinensis polysaccharide,astragalus polysaccharide and their combination on MAPK signal molecules of hematopoietic stem cells in bone marrow suppression mice2.1 effect on signal molecule RAS m RNACompared with the normal group,the level of Ras in the model group was significantly decreased(P<0.05),but there was no statistical significance in the comparison between Angelica polysaccharide group and astragalus polysaccharide group(P>0.05),but both groups were higher than that of EPO group(P<0.05);the RAS level of combined medication group was higher than that of Angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).2.2 effect on signal molecule ERK1/2 m RNACompared with the normal group,the level of ERK1/2 in the model group was significantly decreased(P<0.05),but there was no statistical significance between the angelica polysaccharide group and the astragalus polysaccharide group(P>0.05),but both groups were higher than that of the EPO group(P<0.05);the ERK1/2 level of the combined medication group was higher than that of the angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).2.3 effect on signal molecule p38 m RNACompared with the normal group,the level of p38 in the model group was significantly decreased(P<0.05),but there was no statistical significance in the comparison of Angelica polysaccharide group and astragalus polysaccharide group(P>0.05),but both groups were higher than that of EPO group(P<0.05);the p38 level of combined medication group was higher than that of Angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).3.The effect of Angelica polysaccharide,astragalus polysaccharide and their combination on hematopoietic stem cell nuclear transcription factor in myelosuppression mice3.1 effect on nuclear transcription factor JNK m RNACompared with the normal group,the level of JNK in the model group was significantly decreased(P<0.05),but there was no statistical significance in the comparison of Angelica polysaccharide group and astragalus polysaccharide group(P>0.05),and both groups were higher than that of EPO group(P<0.05);the JNK level of combined medication group was higher than that of Angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).3.2 effect on nuclear transcription factor c-Jun m RNACompared with the normal group,the level of c-jun in the model group was significantly decreased(P<0.05),but there was no statistical significance in the comparison of Angelica polysaccharide group and astragalus polysaccharide group(P>0.05),which were higher in the two groups than those in the EPO group(P<0.05);the c-Jun level in the combination group was higher than that in the angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).3.3effect on nuclear transcription factor c-fos m RNA Compared with the normal group,the level of c-fos in the model group was significantly decreased(P<0.05),while the c-fos level in the angelica polysaccharide group and astragalus polysaccharide group was not statistically significant(P>0.05),but higher in the two groups than in the EPO group(P<0.05);the c-fos level in the combination group was higher than that in the angelica polysaccharide group,astragalus polysaccharide group and EPO group(P<0.05).Conclusion:(1)angelica polysaccharide combined with astragalus polysaccharide can promote the proliferation of rat cells,and can play a synergistic effect of the two drugs,which is helpful to promote the levels of IL-2,TPO,GM-CSF and reduce the levels of inf-r;(2)angelica polysaccharide combined with astragalus polysaccharide can promote the expression of signal molecules RASm RNA,ERK1m RNA,ERK2m RNA,p38m RNA,and affect the levels of nuclear transcription factors c-Junm RNA,c-fosm RNA and JNK m RNA,which may affect the cells by regulating RAS signal system Proliferation and differentiation.