Clinical Study On The Treatment Of Myelodysplastic Syndrome With Arsenic-containing Traditional Chinese Medicine Compound Qinghuangsan And Its Regulation Of M6

ObjectiveMyelodysplastic syndrome(MDS)is a malignant clonal disease with high heterogeneity,which originates from hematopoietic stem cells.MDS is characterized by decreased numbers of peripheral blood cells and ineffective hematopoiesis,with a high risk of transformation to acute myeloid leukemia(AML).Clinically,demethylated drugs such as decitabine are mainly used in the treatment of high-risk MDS patients,and decitabine is also used in the treatment of low-risk MDS patients with blood transfusion dependence.However,decitabine treatment is often accompanied by adverse reactions such as hemocytopenia and infection.The aim of this research is to compare the clinical efficacy,overall survival and adverse reactions of arsenic containing traditional Chinese medicine compound Qinghuang powder and decitabine in the treatment of MDS,so as to provide more powerful evidence for compound Qinghuang powder in the treatment of MDS.At the same time,the mechanism of compound Qinghuang powder in the treatment of MDS was discussed based on m6A methylation regulation.Methods1.Compare the clinical efficacy and adverse reactions of compound Qinghuang powder and decitabine in the treatment of MDS patients.A total of 93 patients with MDS were included in the Department of Hematology,Xiyuan Hospital,China Academy of Chinese Medical Sciences from January 2016 to June 2021.According to the treatment,the patients were divided into compound Qinghuang powder group and dexitabine group.The dosage of compound Qinghuang powder group was 3 capsules each time,3 times a day,every 3 months as a course of treatment.The clinical effect was evaluated after 2 courses of treatment;The dose of decitabine group was 20 mg/m2,dl-5,a course of treatment every 28 days.The clinical effect was evaluated after≥ 4 courses of treatment.The clinical effects and adverse reactions of the two groups were compared by evaluating the changes of peripheral blood cell count and bone marrow morphology before and after treatment.At the same time,the overall survival time of the two groups was compared and the factors affecting the survival time were analyzed.2.Analyze the role of m6A methylation regulation in the pathogenesis of MDS and its impact on the prognosis of MDS patients.Based on GEO public database(159 MDS patients and 17 healthy samples),the expression of m6A-related genes in bone marrow mononuclear cells in healthy samples and MDS patients was compared by bioinformatics analysis.At the same time,the samples of 60 patients with MDS and 10 patients in the control group(patients with non malignant hematological diseases)from September 2019 to June 2021 were used to verify the expression differences of m6A-related differential genes at the mRNA and protein levels.The relationship between the expression of m6A-related differential genes and the prognosis of MDS patients was analyzed.3.To explore the regulation mechanism of arsenic containing traditional Chinese medicine compound Qinghuang powder on m6A methylation in patients with MDS.The mRNA and protein expression levels of m6A methylation regulatory gene in bone marrow mononuclear cells of 30 patients with MDS before and after treatment were detected by fluorescence quantitative PCR and Western blot.4.To explore the mechanism of arsenic disulfide(As2S2),the main component of realgar in compound Qinghuang powder,on the methylation regulation of MDS cell line SKM-1 m6A.CCK-8 method was used to determine the administration time and concentration of cell experiment.The MDS cell line SKM-1 was intervened with As2S2,the main component of realgar in compound Qinghuang powder,for 48 hours.The mRNA and protein expression levels of m6A methylation regulatory genes in control group(0μmol/L)and As2S2(4 μmol/L)group were detected by fluorescence quantitative PCR and Western blot.Results1.The Clinical efficacy,adverse reactions of compound Qinghuang powder and decitabine in the treatment of MDS patients,and the analysis of factors affecting prognosis(1)Clinical efficacy:Both compound Qinghuang powder and decitabine can significantly improve the level of hemoglobin(HGB)in patients with MDS.The overall response rate(ORR)and overall improvement rate of compound Qinghuang powder group were 10.7%(6/56)and 35.7%(20/56),respectively.The ORR and overall improvement rate of decitabine group were 18.9%(7/37)and 45.9%(17/37),respectively.There were no significant differences in ORR and overall improvement rate between the two groups(P=0.264;P=0.324).The proportion of patients with stable disease(SD)in compound Qinghuang powder group was significantly higher than that in decitabine group(P<0.05).(2)Overall survival time and annual survival rate:The median survival time ofcompound Qinghuang powder group was 58 months and that of decitabine group was 30 months.The median survival time of compound Qinghuang powder group was significantly longer than that of decitabine group(P=0.035).The 1-year,2-year,3-year and 5-year survival rates of compound Qinghuang powder group were significantly higher than those of decitabine group(1-year P=0.015;2-year,3-year and 5-year P<0.001).(3)Adverse reactions:No hematological adverse reactions and treatment-relatedinfections were observed in the compound Qinghuang powder group,and the patients were well tolerated.The most common adverse reaction in decitabine group was hematological adverse reaction.The incidence of≥ grade 3 hemocytopenia was 89.2%(33/37),and the incidence of≥ grade 3 infection was 59.5%(22/37).(4)The analysis of factors affecting prognosis:Univariate analysis showed thatage,proportion of bone marrow blasts,chromosome prognosis,IPSS-R risk category and curative effect were correlated with the overall survival of patients with MDS(P<0.1).Multivariate analysis showed that age,proportion of bone marrow blasts and curative effect were independent factors affecting the overall survival of patients with MDS(P<0.05).The survival rate of MDS patients aged>60 years,the proportion of bone marrow blasts≥5%and the progression after treatment were significantly reduced and the risk of death was significantly increased.2.The role of m6A methylation regulation in the pathogenesis of MDS and its impact on the prognosis of patients with MDSBased on the data in GEO public database,there were differences in the expression of m6A-related genes HNRNPC,METTL14 and ALKBH5 between healthy samples and MDS patients.Among them,the expression of HNRNPC in MDS patients was significantly lower than that in healthy samples,and the expressions of METTL14 and ALKBH5 were significantly higher than those in healthy samples(P<0.05).Based on the results of q-PCR and Western blot of clinical samples,there were differences in the expression of HNRNPC,METTL14 and ALKBH5 mRNA and protein between patients with MDS and patients with non malignant hematological diseases.Among them,the expression of HNRNPC mRNA and protein in patients with MDS was significantly lower than that in patients with non malignant hematological diseases(P<0.05),and the expressions of METTL14 and ALKBH5 mRNA and protein were significantly higher than those in patients with non malignant hematological diseases(P<0.05).The expressions of HNRNPC,METTL14 and ALKBH5 were closely related to the prognosis of MDS patients.3.Regulatory effect of compound Qinghuang powder on m6A methylation in patients with MDSCompared with that before treatment,the expression levels of m6A-related gene HNRNPC mRNA and protein in bone marrow mononuclear cells of MDS patients after treatment with compound Qinghuang powder increased significantly,and the expression levels of METTL14 and ALKBH5 mRNA and protein decreased significantly.4.Effect of As2S2,the main component of realgar in compound Qinghuang powder,on methylation regulation of MDS cell line SKM-1 m6AThe half maximal inhibitory concentration(IC50)of As2S2 on SKM-1 cells for 48 hours was 4.82 μmol/L.Compared with the control group(As2S2 0mol/L),the expression levels of m6A-related gene HNRNPC mRNA and protein in As2S2 4μmol/L group increased significantly,and the expression levels of METTL14,ALKBH5 mRNA and protein decreased significantly.Conclusions1.Compound Qinghuang powder can improve the level of HGB in peripheral blood of patients with MDS.Compared with decitabine,compound Qinghuang powder has a higher proportion of patients with SD after treatment,and can significantly prolong the overall survival time of patients,with no hematological adverse reactions and treatment-related infections,and better tolerance.2.m6A methylation regulation may be involved in the pathogenesis of MDS.The expression of m6A-related genes is closely related to the prognosis of MDS patients.3.Compound Qinghuang powder can regulate m6A methylation in MDS patients,and As2S2,the main component of realgar,can regulate m6A methylation in MDS cell line SKM-1.Compound Qinghuang powder may play a therapeutic role by regulating m6A methylation in patients with MDS.