Mechanism Of The Inhibition Of Erythroleukemia By Limonoid Compounds Extracted From Melia Azedarach

Objective:A class of limonoid compounds extracted from the Chinese medicinal plant melia azedarach and a similar structure of cedrelone,its inhibitory effect on erythroleukemia cells in vitro,and its therapeutic effect on erythroleukemia mice in vivo,and carry out research on its specific mechanism.Methods:In vitro:1.Screening compounds:A class of compounds with anti-leukemia activity was screened by MTT in a drug library.2.Detecting cell viability:HEL(human erythroleukemia cells)and CB7(mouse erythroleukemia cells)were seeded in a 96-well plate by 10~4 per well,and treated with the compounds for 0,24,48,and 72 hours.The MTT was cultured for 4 hours,and then the absorbance value was measured with a microplate reader,and a cell growth curve was drawn.3.Detection of apoptosis:Both cell line was treated with compound for 24 hours and then cell apoptosis was analyzed by a flow cytometer.4.Cell cycle and cyclin expression:Cycle arrest was detected by flow cytometry,and cyclin changes were further verified by Western Blot.5.Cell differentiation and changes in genes involved differentiation:The direction of cell differentiation was detected by flow cytometry and verified by Q-PCR.6.Molecular docking and MAPK/ERK pathway:The binding sites of compounds to proteins were analyzed by Autodock software to find the potential targets of compounds in erythroleukemia.The expression of key proteins in MAPK/ERK pathway was detected by Western Blot.n vivo:1.Establishment and grouping of animal models:Friend virus was intraperitoneally injected to newly born Balb/c mouse,and an animal model of erythroleukemia was established.After 4-5weeks,the female and male mice were separated and randomly divided into groups(DMSO control group and experimental group).2.Drug treatment:One week after the completion of the grouping,the mice were intraperitoneally injected with compound every other day(3 mg/kg)for a total of 6 injections.3.Take the material and draw the survival curve:About 2 weeks after the completion of the administration,a part of the mice were sacrificed to take the spleen?heart blood and marrow.And the values of measurements were recorded.The ratio of bone marrow used to detect hematopoietic stem cells;the other mice continued to observe,and the death time was recorded after the death of the mice,and the survival curve was drawn.Results:In vitro experiments:1.Screen out three limonoid compounds:12-hydroxyamoorastin,1-deacetylsendanin and 29-iso-butylsendanin(number:A1541,A1542,A1543).2.Cell growth curve showed that A1541,A1542,A1543 and cedrelone could significantly inhibit the growth of erythroleukemia cells.3.Flow cytometry results showed that compounds can cause apoptosis.4.Compounds can cause cycle arrest,A1541,A1542,and A1543 cause G2 arrest,but cedrelone is S-phase block;but both can down-regulate CDK2 expression.5.These four compounds cause different differentiation directions depending on the cells.A1541and A1543 cause megakaryocyte differentiation and erythroid differentiation of HEL cells.A1541,A1543 and cedrelone cause only erythroid differentiation of CB7 cells.The results at the RNA level are consistent.6.Molecular docking results show that the potential targets of these four compounds for erythroleukemia are ERK;in the MAPK/ERK pathway,A1541,A1542,and A1543 up-regulate both P-ERK and P-MEK while cedrelone down-regulated.In vivo experiments:1.Compared with the DMSO group,the survival time of the experimental group was prolonged.2.The spleen size of the mice did not change significantly.3.The hematocrit(Hct)of the experimental group increased.4.Increased proportion of hematopoietic stem cells.Conclusion:Limonoid compounds can inhibit the growth of erythroleukemia cells,and the target of MAPK/ERK pathway is ERK1/2,which causes apoptosis,cycle arrest and cell differentiation of HEL and CB7 cells,which ultimately leads to cell death.This study provides a potential theoretical basis for further clinical applications and treatment of other tumors.