Research On The Apoptosis Of Leukemia CEM Cells Induced By Ethanol Extract Of Pulsatilla Chinensis And Its Mechanism

Objective:The effects of ethanol extract of Pulsatilla chinensis on the growth of leukemia CEM cells were studied by in vitro experiments and its influential mechanism would be in-depth exploration.Methods:Weigh and take 150 g pieces of Pulsatilla,pulverize,soak them in 70% ethanol,heat them up and extract them by reflux,extract them by n-butanol,decompress and concentrate the extracted liquid and dry the concentrated liquid to get solid weighing.Dissolve the alcohol extract of pulsatilla chinensis with three steaming water and set aside.CCK8 assay was used to detect the inhibition rate of alcohol extracts of pulsatilla pulsatilla at different concentrations acting on CEM cells at different time periods.Light microscopy was used to observe the morphological changes of different concentrations of Pulsatilla chinensis extracts on CEM cells at different time.and DNA agarose gel electrophoresis was used to observe whether the cells had apoptosised after different concentrations of Pulsatilla chinensis acting on the it.Western blot was used to detect the protein expression of p53,Bax and Bcl-2 in leukemia CEM cells treated with different concentrations of Pulsatilla ethanol extracts for 24 h,48 h and 72 h.Results:1.Extraction rate of active ingredient(Pulsatilla saponin)of Pulsatilla150 g pulverulent pieces were ground and pulverized,soaked in ethanol,heated and refluxed,extracted with n-butanol,combined with extract,concentrated it under reduced pressure,then dried.Weighing the crude saponins of pulsatilla pulsatilla was 8.6g,and the extraction rate was 5.73%.2.Inhibition rate of ethanol extract from Pulsatilla on CEM cells growthAfter the alcohol extracts of pulsatilla pulsatilla at different concentrations were applied to CEM cells at the same time,the OD value gradually decreased with the increase of the alcohol extracts concentration(195.3125 g/ml,390.625 g/ml,781.25 g/ml,1562.5 g/ml,3125 g/ml,6250 g/ml,12500 g/ml),that is,the inhibition rate gradually increased.At the same concentration and at different time(24 h,48 h,72 h),the inhibition rate gradually increased with the extension of the action time.The IC50 values of CEM cells treated with ethanol extract of Pulsatilla at 24 h,48 h and 72 h were 1452 ?g/ml,774.2 ?g/ml and 589.6 ?g/ml,respectively.3.Cell morphology changesUnder inverted microscope,the CEM cells in the control group were observed to be in good growth condition,with round cell shape,uniform size,dense cells and agglomeration,full and bright,and good refractive index.The alcohol extracts of radix pulbatae of different concentrations were used to observe under the CEM cell microscope,and the growth density was low in different degrees,the cell size was different,the cell volume was smaller,the cell residue and debris appeared,and the refractive index was poor.After 24 h,48 h and 72 h,the changes of cell structure were observed under oil microscope.The control cells showed large and round nuclei,the nucleoplasm ratio was imbalanced and the cell membrane was intact.The cells in the drug-added group showed different degrees of cell membrane shrinkage and damage,chromatin condensation,nuclear lysis,and some of them appeared as apoptotic bodies.4.DNA agarose gel electrophoresis to observe apoptotic bandsPulsatilla alcohol extract(6250 ?g/ml,3125 ?g/ml,1562.5 ?g/ml,781.25 ?g/ml,390.625 ?g/ml)was applied to CEM cells and extracted DNA for different time periods(24 h,48 h,72 h).The results of DNA agarose gel electrophoresis showed that DNA dosing group were appeared in more than 10000 bp band,namely in 180 bp to 200 bp integer times appear banding,explain cell apoptosis;There was only one macromolecular band in the DNA of the control group,indicating that no apoptosis occurred in the cells of the control group.5.Expression of p53,Bax and Bcl-2 proteinsWestern blotting experiments showed that CEM cells were treated with ethanol extracts of Pulsatilla 390.625 ?g/ml,781.25 ?g/ml,1562.5 ?g/ml,3125 ?g/ml and 6250 ?g /ml for 24 h,48 h and 72 h,With the ?-actin strip as a reference,the results showed that the expression of p53 and Bax protein in the drug-added group was higher than that in the control group(without dosing)at same time period,and the expression of p53 and Bax protein increased with the increase of the concentration of Pulsatillae extract(P <0.05 or P<0.01);while the expression of Bcl-2 protein in the drug-added group was less than that in the control group(without dosing),and the expression of Bcl-2 protein decreased with the increase of drug concentration(P < 0.05 or P < 0.01).At same drug concentration,the expression of p53 and Bax protein increased gradually with the prolongation of time(P<0.05 or P<0.01),while the expression of Bcl-2 protein decreased gradually(P<0.05 or P<0.01).Conclusions: 1.Pulsatilla alcohol extract can inhibit the growth of leukemia CEM cells.2.The ethanol extract of Pulsatilla chinensis inhibits the growth of leukemia CEM cells by inducing apoptosis.3.The possible mechanism of the ethanol extract of Pulsatilla chinensis to induce apoptosis of leukemia CEM cells is to regulate the expression of related proteins in the mitochondrial pathway by regulating the apoptotic signaling pathway:(1)down-regulate the expression of Bcl-2 protein,up-regulate the expression of Bax protein,and make the ratio of Bcl-2/Bax decreased;(2)up-regulated the expression of p53 protein,activated the expression of downstream Bax protein,inhibit the expression of Bcl-2,and achieve the effect of promoting cell apoptosis.