Clinical Observation And The Mechanism Of Regulating HIF-1α On The Treatment Of Myelodysplastic Syndrome With Arsenic-Containing Herbal Compound

In a previous study,we found the efficacy and safety of Qinghuang Powder(QHP)Combined with Bushen Jianpi Formula(BJF),that consist of arsenic-containing herbal combination(AHC)in the treatment of Myelodysplastic Syndrome(MDS),and found that QHP may regulate HIF-la levels,promote hematopoiesis and contain malignant clones in the treatment of MDS.HIF-la is a prognostic risk factor for MDS.In order to further explore the clinical observation of AHC for the treatment of MDS,and the expression of HIF-la in MDS patients and its effect with the treatment time,as well as the mechanism of HIF-la regulation by QHP,this study was conducted from the initial investigation of clinical study assay,and then combined with MDS cellular experiment for mechanism exploration.Part 1Clinical observation of arsenic-containing herbal compound for the treatment of MDS patients and the effect on HIF-la expressionObjective:To analyze the efficacy and characteristics of AHC for the treatment of MDS,as well as the level of HIF-1α in bone marrow cells of MDS patients,and to explore the correlation between HIF-la and clinical characteristics of patients.Methods:Patients receiving treatment for MDS at the Department of Hematology,xiyuan hospital,from January 2022 to September 2022 were enrolled according to the Inclusion and exclusion criteria,observed for 2 courses and efficacy assessment,and followed up regularly 1 year after receiving treatment to record blood routine changes.According to the time of treatment(before treatment,after 6 months of treatment and after 1 year of treatment),detected the expression level of HIF-1α in the bone marrow of MDS patients in each group through QRCR and ELISA.Analysis the relative expression of HIF-la in the bone marrow of MDS patients in relation to bone marrow proliferation,karyotype,blood routine,IPSS-R and other clinical indexes.Results:(1)A total of 28 MDS patients were included,and the total effective rate was 64.3%after 2 courses of treatment with arsenic-containing herbal compound.The effective rate of MDS-LB(60%)was similar to MDS-IB1(66.7%)according to the 2022 MDS diagnostic typing criteria.And it was found that the PLT of MDS treated with arsenic-containing herbal compound would be significantly rebounded after 6-12 months.(2)HIF-1α mRNA levels in bone marrow single nuclei of MDS patients were significantly lower before treatment(P=0.0023),after 6 months of treatment(p<0.0001),and after 1 year of treatment(p=0.0001),which compared with healthy controls.and there was a decreasing trend with treatment time;HIF-1α protein levels in bone marrow plasma of MDS patient group and healthy controls did not differ;(3)there was indeed a moderate positive correlation between mRNA and protein of HIF1α in the bone marrow of MDS patients(r=0.4923,p=0.0380).mRNA of HIF-la in bone marrow mononuclear cells of MDS patients was positively correlated with the degree of bone marrow hyperplasia(r=0.3839,p=0.0481);and with the percentage of bone marrow granulocytes(r=0.5596,p=0.0024);negative correlation with routine MCV(r=-0.4060,p=0.0356);positive correlation between the relative expression of HIF-1α protein in bone marrow plasma and the degree of myeloproliferation in MDS patients(r=0.5628,p=0.0121);and significant positive correlation with routine WBC(r=0.6038,p=0.0062).Conclusion:1.The total effective rate of AHC for the treatment of MDS was 64.3%after 2 courses.The peripheral blood improvement time of MDS treated with AHC is around 6-12 months,and the hemogram becomes more stable with the increase of treatment course;2.the treatment regimen based on AHC may silence the expression level of HIF-1α in bone marrow single nuclei of MDS patients;and it has some correlation with clinical characteristics.3.Bone marrow cell HIF-1α levels in MDS patients were positively correlated with the degree of myeloproliferation and granulocytes,with some association with peripheral blood.Part 2Study on the mechanism of HIF-1α regulation by QHP intervention in MDS cell line SKM-1Objective:In order to clarify the effect and role of QHP in regulating HIF-1α,as well as the regulatory effect of drug concentration target genes,this study was conducted to investigate the in vitro molecular effects of Qing Dai and Xiong Huang,Indirubin and As2S2,the main components of QHP,by intervening in SKM-1 cells of MDS cell line.Methods:(1)CCK-8 assay was used to analyze the survival rate of Indirubin and As2S2 on SKM-1 cells and to calculate the semi-inhibitory drug concentration;(2)a concentration gradient was set according to the IC50 of the drug to intervene in the cells,and the mRNA expression levels of HIF-1α were analyzed by qRCR for single drug and combined effects on SKM-1 cells,and the drug ratios were set to simulate different concentrations of Qing Huang San;(3)To explore the molecular mechanism of the effect of QHP on the regulation of HIF-1α by MDS through QRCR.Western blot,and LC-MS/MS techniques.Results:(1)As2S2 could down-regulate the mRNA expression level of HIF1α,and the level of HIF1α decreased significantly when the drug pre-concentration was 1,2 and 4 μmonl/L(P<0.05);QHP could significantly interfere with the protein expression of HIF-1α,which was significantly different from the blank control group(P<0.0001),Inhibition of HIF-1α protein expression was more pronounced in the high concentration group of QHP(P<0.01);(2)Both QHP could significantly upregulate PHD1 mRNA expression level and up-regulate PHD1 protein level,and could down-regulate PHD2 mRNA level and up-regulate PHD2 protein level,and the effect was significant in the high concentration group(P<0.001);down-regulate PHD3 protein expression level;(3)QHP can affect the down-regulation of TCA intermediate metabolites α-KG,α-HG,and succinic acid,and the low concentration group of QHP can significantly down-regulate α-KG(P<0.01),and the high concentration group has no difference;both groups of QHP can significantly inhibit the production of α-HG(P<0.0001),and the high concentration group has significant effect;both groups can down-regulate the intermediate metabolites succinic acid level(P<0.01),but there is no difference between different concentration interventions(P>0.05).(4)QHP could down-regulate the relative expression of VHL mRNA(P<0.05),but had no effect on the protein expression level of VHL;the high concentration group could significantly up-regulate the relative expression of pVHL protein,and there was no significant difference between the interventions of low concentration group compared with the control.Conclusion:1.QHP significantly down-regulated the expression level of HIF-1αprotein in MDS cell lines,and the effect of high concentration of QHP molecules was more obvious;2.QHP up-regulated the activity of PHD2 through regulating the expression of mitochondrial intermediate metabolites α-HG,succinate and α-KG,which in turn promoted the hydroxylation of HIF-1α;3.QHP promotes the recognition and ubiquitous degradation of HIF-1α by up-regulating pVHL protein levels.