Mechanism Of Human Acute Myeloid Leukemia KG-1a Cell Apoptosis Induced By Traditional Chinese Medicine Fuzheng And Quxie Therapy Based On Mitochondrial Fusion And Fission

Objective:In this study,we selected Yiqi Yangyin（supplementing Qi and nourishing Yin）Decoction and its decomposed recipes to intervene the expression of 6 kinds of mitochondrial fusion and fission proteins in human acute myeloid leukemia（AML）KG-1a cells,and explored the mechanism of traditional Chinese medicine（TCM）Fuzheng and Quxie therapy to induce the apoptosis of leukemic cells through mitochondrial pathway,to provide the theoretical basis for targeted treatment of acute leukemia,and new ideas and methods to improve the clinical efficacy of leukemia.Methods:1.AML KG-1a cells were cultured as models.2.CCK-8 assay was used to detect the inhibitory effect by different concentration and time on the growth of KG-1a cells in vitro.Thus,the IC₅₀ value and the best intervention time of Yiqi Yangyin Decoction could be determined.3.Annexin V/PI double staining flow cytometry（FCM）was used to detect the apoptosis and calculate cell apoptosis rate.PI single staining FCM was used to detect the cell cycle distribution.4.The cells’ultrastructure and morphological changes of mitochondria were observed by transmission electron microscope（TEM）.5.Western blot was used to detect the protein expression levels of Mfn-1,Mfn-2,Opa-1,Drp-1,Fis-1,and Mff in each group.6.RT-qPCR was used to detect the mRNA expression levels of Mfn-1,Mfn-2,Opa-1,Drp-1,Fis-1,and Mff in each group.Results:1.CCK-8 assay showed that Yiqi Yangyin Decoction could inhibit the proliferation of KG-1a cells（P<0.05）,and had good time-concentration dependent effect.According to the IC₅₀ value,the concentration of Yiqi Yangyin Decoction was 80 mol/L,and the intervention time was 48 h.2.After dosing for 48 h,the apoptosis rate and the G1 phase arrest rate of KG-1a cells were significantly higher than those in control group,according to the FCM detection results.The difference was statistically significant（P<0.01）.3.The results of TEM showed that the mitochondrial structural damage in Quanfang and Quxie group were more serious,while the mitochondria structures in Fuzheng and control group were basically intact.4.The results of Western-blot demonstrated that compared with control and Fuzheng group,the protein expression levels of Mfn-1,Mfn-2,Opa-1 in Quanfang and Quxie group significantly decreased,while the protein expression levels of Drp-1,Fis-1,Mff were significantly increased,and the differences were statistically significant（P<0.01）.Comparison between Quanfang and Quxie group,Fuzheng and control group,the difference was not statistically significant（P>0.05）.5.The mRNA expression levels of Mfn-1,Mfn-2,Opa-1,Drp-1,Fis-1,and Mff protein were detected by RT-qPCR.The results demonstrated that compared with control and Fuzheng group,the mRNA expression levels of Mfn-1,Mfn-2,Opa-1 in Quanfang and Quxie group were significantly decreased,while the mRNA expression levels of Drp-1,Fis-1,Mff were significantly increased,and the differences were statistically significant（P<0.01）.Compared with Quxie group,the mRNA expression levels of Mfn-1 and Drp-1were significantly different（P<0.01）,and the mRNA expression levels of in Opa-1 and Fis-1 were different（P<0.05）,while the mRNA expression levels of Mfn-2 and Mff were no difference（P>0.05）.Compared with control group,the mRNA expression levels of Mfn-2,Opa-1,Drp-1,Fis-1,and Mff in Fuzheng group were significantly different（P<0.01）,and the mRNA expression level of Mfn-1 was different（P<0.05）.Conclusion:1.The occurrence and development of leukemia could be relevant to abnormal expression of mitochondria fusion and fission proteins,including Mfn-1,Mfn-2,Opa-1,Drp-1,Fis-1,and Mff.2.In KG-1a cells,the high expression levels of mitochondrial fusion proteins Mfn-1,Mfn-2,Opa-1 and low expression of mitochondrial proteins Drp-1,Fis-1,Mff,could be one of the mechanisms of malignant proliferation of KG-1a cells.3.Yiqi Yangyin Decoction could down-regulate the expression levels of mitochondrial fusion proteins Mfn-1,Mfn-2,Opa-1 and up-regulation the expression levels of mitochondrial fission proteins Drp-1,Fis-1,Mff,thus changed the mitochondrial structure and function by intervening mitochondrial fusion and fission,to induce apoptosis of AML KG-1a cells.4.In this study,compared with Fuzheng and control group,it was demonstrated that Quanfang and Quxie group have significant difference,but there was neither significant difference between Quanfang and the Quxie group,nor Fuzheng and control group.Compared with Fuzheng group,Quxie group had more significant effects in inducing KG-1a cell apoptosis and intervening the expression levels of mitochondrial fusion and fission proteins,which could be attribute to lack of targets for regulating immunity,antioxidation and improving internal environment in vitro.Therefore,the Chinese medicine with a relatively stronger cytotoxic effect in Quxie group showed better effects.