| Objective:Acute myeloid leukemia(AML)is a kind of malignant tumor disease originating from hematopoietic stem/progenitor cells that seriously endangers human health.In this study,we explored the changes of mitochondrial related genes of AML disease and the regulatory effect of Qilian Yisui Jiedu Granule on mitochondrial fusion/division during the occurrence and development of AML,and explained the mechanism of the effect of Yi Qi Yang Yin Jie Du traditional Chinese medicine on inducing apoptosis of AML cells by balancing mitochondrial fusion/division pathway,and lay a theoretical basis for the clinical treatment of AML with traditional Chinese medicine.Methods:The up-regulation and down-regulation genes related to the regulation network of mitochondrial related pathways in bone marrow samples from AML patients and healthy donors were screened by PCR Array,and analyzed by Ingenity Pathway Analysis(IPA),and verify the differential genes.First of all,the NOD-SCID IL-2Receptor gamma null-AML(NSG-AML)model was constructed by THP-1 cell line,and the effect of traditional Chinese medicine on the morphology,histopathological changes and mitochondrial ultrastructure of bone marrow cells of NSG-AML mice was observed through the intervention of traditional Chinese medicine,which was used to replenish qi,nourish yin and detoxify,and the effect of traditional Chinese medicine on the expression of mitochondrial fusion/mitosis-related factors and apoptosis-related molecules in NSG-AML mice leukemia cells was analyzed and discussed.At the same time,the ROS level and mitochondrial membrane potential of mice in each group were detected to reveal the mechanism of Yi Qi Yang Yin Jie Du traditional Chinese medicine in the treatment of AML.In addition,the effects of Yi Qi Yang Yin Jie Du traditional Chinese medicine on bone marrow cell morphology,histopathological changes and mitochondrial ultrastructure of mice in each group were observed by constructing Opa-1overexpression and Drp-1 knockdown models.Detect the changes of mitochondrial fusion/division and apoptosis-related factors of AML leukemia cells treated with Yi Qi Yang Yin Jie Du traditional Chinese medicine,and detect the mitochondrial membrane potential,reactive oxygen species(ROS)level and apoptosis level of cells in each group,and analyze the influence of Yi Qi Yang Yin Jie Du traditional Chinese medicine on AML cell apoptosis after interfering with mitochondrial fusion/division related molecules.Results:In the clinical study,13 up-regulated and down-regulated genes were screened by detecting mitochondrial related pathway genes in bone marrow of AML patients and healthy control samples with PCR Array.The results of classical pathway enrichment by IPA showed that the occurrence and development of AML disease were related to classic pathways such as mitochondrial dysfunction,necrosis signal pathway and HIV1-induced apoptosis,among which mitochondrial dysfunction was significantly related to AML.Through deep excavation,it was found that the up-regulation of Mfn-2 located in the nucleus can activate mitochondrial fusion in the mechanism of mitochondrial dysfunction.Opa-1 and Mfn-1/2 located on the outer membrane of mitochondria can activate mitochondrial fusion through the membrane gap,while DNM1L(Drp-1)and Fis-1 located on the outer membrane of mitochondria can participate in the process of mitochondrial fragmentation,suggesting that both are closely related to mitochondrial division.The enrichment results showed that it was mainly related to mitochondrial division,mitochondrial morphogenesis,mitochondrial fusion,mitochondrial fragmentation,mitochondrial dysfunction and other diseases and biological functions.Mfn-2,Opa-1 and Drp-1,as upstream regulators of mitochondrial fusion and mitosis,regulate the function of downstream mitochondrial fusion and mitosis.Through the construction of molecular network and regulatory mechanism network between mitochondrial related differential genes,it was further found that Mfn-1,Mfn-2 and Opa-1 can positively regulate mitochondrial fusion,but play a negative role in mitochondrial division.On the contrary,Drp-1negatively regulates mitochondrial fusion and promotes mitochondrial division.Fis-1,as the upstream regulatory molecule of Drp-1,mediates mitochondrial division.Fis-1can directly promote mitochondrial division function,and affect the activation of mitochondrial division function by regulating the expression of Drp-1.Finally,the role of mitochondrial fusion/division imbalance in AML was further verified by Western blot(WB)detection of clinical samples,suggesting that there is disruption of mitochondrial fusion/division balance in the development of AML.In the animal experiment,firstly constructs NSG-AML model through THP-1cell line,and carries out Chinese medicine intervention through Qilian Yisui Qingdu Granule.It is found that Qilian Yisui Qingdu Granule has the therapeutic effect of reducing toxicity and increasing efficiency on AML mice,and the infiltration of leukemia cells in the bone marrow of mice in QLYS group is reduced,which proves that Qilian Yisui Qingdu Granule has significant improvement and treatment effect on AML disease.The ultrastructural observation of the electron microscope showed that the morphology of mitochondria in the bone marrow of mice in the AML model group was oval,the shape of the nucleus was irregular,the volume was increased,and the cristae were slightly expanded,suggesting that the mitochondria were in a fusion state and enhanced.After treatment with Qilian Yisui Granule,many lysosomes and autophages can be seen in the bone marrow of mice,the volume of mitochondria is reduced,the cristae are not clear and arranged orderly,a few cristae are slightly dissolved,and the matrix is uneven.It is suggested that Qilian Yisui Qingdu Granule can change the morphology of mitochondria in bone marrow of leukemia mice,destroy the ultrastructure of mitochondria,and promote mitochondrial division.After treatment with traditional Chinese medicine,the levels of Opa-1,Mfn-1,Mfn-2 and Bcl-2 in QLYS group mice were significantly decreased,and the levels of Drp-1,Fis-1,Mff and Bax were significantly increased,suggesting that the dynamic imbalance of mitochondrial fusion/division was closely related to the occurrence and development of AML.The traditional Chinese medicine Qilian Yisui Qingdu Granule could regulate the balance of mitochondrial fusion/division in the bone marrow of AML mice,and promote the apoptosis of leukemia cells in the bone marrow of AML mice.In the second part of the study,we analyzed the effect of traditional Chinese medicine for supplementing qi,nourishing yin and detoxicating on the expression of apoptosis-related molecules in NSG-AML mice after interfering with mitochondrial fusion/mitosis-related molecules by constructing the mouse model of Opa-1overexpression and sh RNA-Drp-1 transfection.After the intervention of Qilian Yisui Qingdu Granule,the leukemic cells in the bone marrow smears of mice in QLYS group,Opa-1 overexpression control group and sh RNA-Drp-1 control group decreased significantly,the expression level of mitochondrial fusion and anti-apoptotic factor decreased significantly,the expression level of mitochondrial mitosis and proapoptotic factor increased significantly,the level of ROS increased significantly,and the level of mitochondrial membrane potential decreased significantly,It further shows that the Yi Qi Yang Yin Jie Du traditional Chinese medicine can promote the apoptosis of leukemia cells by regulating the balance of mitochondrial fusion/division.In addition,after the intervention of mitochondrial fusion/mitosis-related molecules,there was no significant difference in the level of apoptosis of leukemia cells in NSG-AML mice after the intervention of traditional Chinese medicine.Further verification revealed that the Yi Qi Yang Yin Jie Du traditional Chinese medicine promoted the molecular mechanism of apoptosis of leukemia cells by regulating mitochondrial fusion/mitosis,providing a theoretical basis for the therapeutic effect of the Chinese medicine for supplementing qi,nourishing yin and detoxicating on AML.In the vitro experiment,the changes of mitochondrion fusion/mitosis-related factors,apoptosis-related molecules expression and their effects on apoptosis and mitochondrial membrane potential were observed by interfering THP-1 cell line and Opa-1 overexpression and sh RNA-Drp-1 cell line stably transfection with the serum containing Chinese herbs of supplementing qi,nourishing yin and detoxicating.After the intervention of drug-containing serum,the level of apoptosis in leukemic cells in QLYS group,Opa-1 overexpression control group and sh RNA-Drp-1 control group increased significantly,and the level of mitochondrial membrane potential decreased,indicating that the drug-containing serum of Qilian Yisui Qingdu Granule can significantly induce the apoptosis of leukemic cells and promote the reduction of mitochondrial membrane potential.This part explains the relationship between the apoptosis of leukemic cells induced by traditional Chinese medicine and the regulation of mitochondrial fusion/division by analyzing the influence of Yi Qi Yang Yin Jie Du traditional Chinese medicine on the expression of apoptosis-related molecules in AML cells after the intervention of mitochondrial fusion/division related molecules,and further proves the mechanism of Chinese medicine for supplementing qi,nourishing yin and detoxicating promoting the apoptosis of leukemic cells by regulating mitochondrial fusion/division,Furthermore,the role of Yi Qi Yang Yin Jie Du traditional Chinese medicine in the treatment of AML is clarified,which provides a theoretical basis for the treatment of AML by traditional Chinese medicine.Conclusions:The pathological state of leukemia is related to the dynamic imbalance of mitochondrial fusion/division.Yi Qi Yang Yin Jie Du traditional Chinese medicine can regulate the imbalance of mitochondrial fusion/division,induce apoptosis of leukemia cells,and play a therapeutic role in AML.Clinical studies have found that the occurrence and development of AML are significantly related to mitochondrial dysfunction.Mfn-1,Mfn-2 and Opa-1 can positively regulate mitochondrial fusion and negatively regulate mitochondrial division.On the contrary,Fis-1 and Drp-1 can negatively regulate mitochondrial fusion and promote mitochondrial division.After the expression of Opa-1 and knockdown of Drp-1 were verified in vivo and in vitro,there was no significant difference in the level of apoptosis of leukemia cells after the intervention of Yi Qi Yang Yin Jie Du traditional Chinese medicine.Further verification revealed that Yi Qi Yang Yin Jie Du traditional Chinese medicine promote the molecular mechanism of apoptosis of leukemia cells by regulating mitochondrial fusion/division pathway.This study laid a theoretical foundation for the clinical application of Yi Qi Yang Yin Jie Du traditional Chinese medicine to treat AML. |
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