Research On The Inhibitory Effect And Mechanism Of Chelidonium Majus Alkaloids On The Proliferation Of Leukemia CEM Cells

Objective:To study the inhibitory effect of the alkaloids extract from Chelidonium majus on the proliferation of leukemia CEM cells,and to explore the mechanism of its inhibition on the proliferation of CEM cells.Methods:100g of celandine hay decoction pieces were crushed by Chinese medicine highspeed centrifugal grinder,and weighed out 50 g.After ethanol reflux extraction,chloroform ex traction,rotary evaporation,and drying the extract,the solid was obtained.MTT colorimetric experiment detects the OD value of the control group after the alkaloids extract from Chelidonium majus is treated at different drug concentrations for 24 h,48h,72 h and the corresponding time period,and calculates the inhibition rate and the half inhibitory concentration;optical microscope observes the control group and elastin The morphological and structural differences of CEM cells under different drug concentrations of alkaloids extract from Chelidonium majus at the corresponding time;DNA agarose gel electrophoresis verifies the formation of DNA Ladder of different concentrations of alkaloids extract from Chelidonium majus on CEM cells at different time periods;RT-qPCR detection different drug concentrations of alkaloids extract from Chelidonium majus acted on CEM cells,at 24 h and 48 h.apoptosisregulating genes Bax,Bcl-2 and downstream apoptosis initiating gene Caspase9 and apoptosis executive gene Caspase3 gene expression of the mitochondrial apoptosis pathway;Western blot It was detected that the different drug concentrations of alkaloids extract from Chelidonium majus acted on CEM cells,the expression of apoptosis-initiating protein Caspase9 and apoptosis-executing protein Caspase3 in leukemia CEM cells at 24 h and 48 h.Results:1.The extraction rate of celandine active ingredients(Chelidonium majus alkaloids)50 g hay powder of Chelidonium majus,extract with ethanol,Chloroform extraction,Concentrate and dry under reduced pressure to obtain an alkaloid extract from Chelidonium majus0.126 g,extraction rate of 0.525%.2.The inhibitory effect of ethanol extract of Chelidonium majus on the growth of CEM cellsIn the same time of action,the alkaloids extract from Chelidonium majus can inhibit the the proliferation of leukemia CEM cells in different drug concentrations(0.64 μg/ml,3.2 μg/mL,16 μg/mL 80 μg/mL)and through increasing concentration of alkaloids extract from Chelidonium majus,enhancement means that the inhibition rate is positively correlated with the drug concentration at the same time of action;at the same drug concentration,the OD values of 24 h,48 h,and 72 h was decreased if increases the time of the action and drug concentration,that is means,the longer time of action,the higher the inhibition rate by alkaloids extract from Chelidonium majus.The IC50 at 24 h,48 h,and 72 h were 27.62 μg/mL 11.45 μg/mL and 8.65μg/mL,respectively.3.Cell morphology changesUnder an inverted microscope(100×),it was observed that the CEM cells in the control group grew well,with uniform cell distribution,regular morphology,complete edges,uniform size,obvious scoliosis,and translucent refraction;after treatment with ethanol extract of Chelidonium majus in different drug concentrations,the number of cells was decreased,the size of the cells was different,the refractive index became poor,the edges were rough,and there were cell debris.Observed under the oil microscope(1000×),it was found that the alkaloids extract from Chelidonium majus drug group cells stained with nuclei Shrinkage,marginalized or fragmented nuclei,uneven staining in the nucleus,and aggregation of chromatin,while the control group cells are stained evenly,the edges of the nucleus are intact,the size of the nucleus is the same,and the boundary between the nucleus and the cytoplasm is clear.4.Observe apoptosis bands by DNA agarose gel electrophoresisAt corresponding action time(24 h,48 h,72 h)of ethanol extract of Chelidonium majus in different drug concentrations(0.64 μg/mL,3.2 μg/mL 16 μg/mL,80 μg/mL)acting on CEM cells,extract Cell DNA.From the results of DNA electrophoresis,compared with the control group,DNA Ladder appeared in different drug concentrations of the drug group,especially in the electrophoresis graphs of 24 h and 48 h,It is clearly visible in the range of integer multiples of 180 bp～200 bp the DNA Ladder of the different drug concentration of alkaloids extract from Chelidonium majus;in the three time periods,the control area did not show obvious DNA band imprinting,it means the cell DNA is intact and there is no apoptosis.5.RT-qPCR detect the transcription of Bcl-2,Bax,Caspase9,Caspase3 geneThe real-time fluorescent quantitative PCR experiment verified the transcription level of apoptosis-related genes in the mitochondrial pathway.From the results we can know the transcription of Bcl-2 gene was significantly different from control group.Compared with the control group.The transcription of Bcl-2 gene in cells with alkaloids extract from Chelidonium majus(there were 4 μg/mL 8 μg/mL and 16 μg/mL)was significantly inhibited after 24 h and 48 h of drug treatment(P<0.05 or P<0.01);the transcription of Bax gene was increased that paired with Bcl-2.after 24 h and 48 h with the effect by drug,The transcription of Bax gene was significantly up-regulated(P<0.05 or P<0.01);the apoptosis promoter gene Caspase9 on the mitochondrial pathway was more transcripted than the control group(P<0.05 or P<0.01)and was concentration-dependent;apoptosis execution The transcription of gene Caspase3 was increased compared with the control group(P<0.05 or P<0.01).6.Western blot detect the expression of Caspase9 and Caspase3 proteinThe results of Western blot experiments proved that the alkaloids extract from Chelidonium majus at the concentration of 4 μg/mL,8 μg/mL,16 μg/mL,and the action time of 24 h and 48 h,respectively,can up-regulate the expression of Caspase9 and Caspase3 of CEM cells(P<0.05 or P<0.01).Although there was no significant difference in expression of Caspase3 between the drug group and the control group at the drug concentration of 4 μg/mL and 24 h of drug treatment,the expression of Caspase3 was significantly increased when the action time was48h(P<0.01).conclusions:1.The ethanol extract of Chelidonium majus can inhibit the proliferation of leukemia CEM cells,and it is concentration and time dependent.2.The inhibitory effect of ethanol extract of Chelidonium majus on the proliferation of leukemia CEM cells is related to drug-induced cell apoptosis.3.The related mechanism of alkaloids extract from Chelidonium majus inducing apoptosis of leukemia CEM cells can inhibit the transcription of Bcl-2 gene in the mitochondrial-mediated apoptosis pathway,promote the transcription of Bax gene,and make the ratio of Bcl-2/Bax gene transcription decrease and promote the transcription of Caspase9 and Caspase3 genes;at the same time,increase the expression of Caspase9 and Caspase3 proteins,thereby promoting cell apoptosis.