| BackgroundMyelodysplastic syndromes(MDS)is a group of highly heterogeneous clonal diseases that originates from hematopoietic stem cells,and are characterized by hematopoietic and ineffective hematopoiesis with one or more lines of hematopoietic cells,and high risk of transformation to acute leukemia.The chronic pathogenesis of MDS is characterized by anemia,hemorrhage,and severe infections,which are the main clinical features of the MDS.In Chinese medicine,MDS belongs to the category of syndromes such as "deficiency labor","blood syndrome",and "internal injury fever".Through long-term clinical observation,the clinical syndromes of MDS are complicated.Most of the patients had fatigue and weakness,palpitation and shortness of breath,blushing in the afternoon,skin ecchymosis and petechiae,pale tongue,less moss,fine pulse and other "qi and yin deficiency,blood stasis resistance"symptoms.Therefore,"Qi and Yin deficiency,blood stasis internal resistance" is the main pathogenesis throughout the pathogenesis of myelodysplastic syndrome,so the principle of clinical treatment can be "Supplementing qi,nourishing yin and activating blood".The pathogenesis of MDS is extremely complex,involving multiple stages and multiple factors.The abnormal activation of Wnt/β-catenin signaling pathway is closely related to the occurrence,development and prognosis of MDS.The study found that hypermethylation of the Wnt signaling pathway inhibitor gene in patients with MDS is an important cause of abnormal activation of the Wnt signaling pathway.Although the efficacy of demethylating drugs for the treatment of MDS has been recognized,and the overall clinical response rate has been significantly improved compared with conventional therapy,but there is no obvious advantage in overall survival time and also obvious adverse reactions.In addition,the clinical practice in the early stage of the research group found that patients with MDS were treated with "Supplementing qi,nourishing yin and activating blood" drugs,and the clinical symptoms of the patients were significantly improved,and adverse reactions caused by demethylation drugs were significantly reduced.Therefore,according to the clinical features of MDS,the research group proposed the therapeutic principle of "Supplementing qi,nourishing yin and activating blood".We will take the abnormality of methylation of MDS as the core and Wnt signaling pathway as the clue,and we expect that by using subcutaneous tumor-bearing nude mice in SKM-1 cells as the research object.We strive to clarify the potential mechanism of Yisui Granules in the treatment of MDS.ObjectBy injecting cyclophosphamide intraperitoneally,and subcutaneous implantation of SKM-1 cells in nude mice to establish a model of MDS.Yisui granule was used as an intervention drug,and set up different experimental groups to observe changes in Wnt signaling pathway related genes and proteins after drug intervention.To explore the potential mechanism of Yisui Granules in the treatment of MDS.MethodBy injecting cyclophosphamide intraperitoneally,and subcutaneous implantation of SKM-1 cells in nude mice to establish a model of MDS.We set model group,a positive drug group,a high-dose and a middle-dose group and a low-dose group of Yisui Granules.After grouping,except that the model group was not treated with drugs,the positive drug group was given intraperitoneal injection of decitabine at a dose of 0.5mg/kg/d x 5d,and the high,middle,and low doses of Yisui Granules were given at 0.2 ml/10g/d volume of drug gavage for 14d.After the 15th day of the experiment,the xenografts of nude mice were excised,and the tumor inhibition rate and survival time were calculated.The expression of Wnt signaling pathway-related genes or proteins was detected by BSAS,Real-time PCR,Western blot and immunofluorescence.The test results shown as(x±s).One-way analysis of variance or Kruskal-Wallis test was used for statistical analysis.Statistical significance was indicated by P<0.05.ResultThe results of above groups is listed below:(1)Flow cytometric immunoassay results:The CD33+:90.3%,CD13+:53.1%,CD11b+:53.6%,CD4+:74.5%,HLA-DR:56.0%were similar to the cell line SKM-1.(2)①Compared with the model group,there was no significant difference in the body weight of the nude mice in Yisui Granules(P>0.05),but there was a statistically significant difference between the positive drug group and the other groups(P<0.05).②There was no significant difference in the volume of the transplanted tumors between the Yisui Granules group and model group(P>0.05),while there was a statistically significant difference between the positive drug group and model group(P<0.05).③The inhibitory rate was 56.64%in the positive drug group,50.691%in the high-dose group,26.97%in the middle-dose group,and 23.78%in the low-dose group,and the tumor weights in the high-dose group and the positive-dose group were significantly different from in the model group(P<0.05).(3)The average survival time of nude mice in the high-dose group was 36.71d,and the life-extension rate was 45.2%.There was a statistically significant difference compared with the model group and the positive drug group(P<0.05).(4)The results of BSAS showed that the average methylation of SFRP5 gene in the positive drug group,the high dose and middle dose of Yisui Granule group was lower than that in the model group(P<0.01),and the average methylation of SFRP5 gene in the positive drug group,high dose and middle dose of Yisui Granule group was lower than that in the low dose of Yisui Granule group(P<0.05).(5)The results of Real-time RCR showed that:①The expression of DNMT1 mRNA in the positive drug group,the high,middle,and low doses of Yisui granules was lower than that in the model group(P<0.05).②The expression of SFRP5 mRNA in the positive drug group,the high and low doses of Yisui granules group was higher than that in the model group(P<0.05).③The expression of β-catenin mRNA in the positive drug group and the high-dose Yisui granules group was lower than that in the model group(P<0.05).④The expression of C-myc mRNA in the positive drug group and the high-dose Yisui granules group was lower than that in the model group(P<0.05).⑤The expression of CyclinD1 mRNA was lower in the positive drug group,the high,middle,and low doses of Yisui granules was lower than that in the model group(P<0.05).(6)The results of Western blot showed that:①The expression of DNMT1 protein in the positive drug group,the high and middle doses of Yisui granules was lower than that in the model group(P<0.05).②The expression of SFRP5 protein in the positive drug group,the high and middle doses of Yisui granules group was higher than that in the model group(P<0.05).③The expression of Wnt3a protein in the positive drug group,the high,middle,and low doses of Yisui granules was lower than that in the model group(P<0.05).The ability of high-dose Yisui granules group to inhibit Wnt3a protein expression was higher than that of low-dose Yisui granules group.④ The expression of β-catenin protein in the positive drug group,the high and middle doses Yisui granules group was lower than that in the model group(P<0.05).There was a statistically significant difference in the the high,middle,and low doses of Yisui granules.⑤The expression of C-myc and CyclinD1 protein in the positive drug group and the high-dose of Yisui granules was lower than that in the model group(P<0.05).The ability of the positive drug group group to inhibit C-myc and CyclinD1 protein expression was higher than that of the middle-dose Yisui granules group.(7)The results of Immunofluorescence showed that:The expression of Wnt3a protein in the positive drug group,the high,middle and lower doses of Yisui granules were all lower than that in the model group.The expression of β-catenin protein in the positive drug group,the high,middle and doses Yisui granules group were all lower than that in the model group.ConclusionBy injecting cyclophosphamide intraperitoneally,and subcutaneous implantation of SKM-1 cells in nude mice to establish a model of MDS.This method of modeling has high tumorigenesis rate and long animal survival time.It can be used for the study of the mechanism of drug treatment of myelodysplastic syndrome.Yisui Granules can inhibit the proliferation of subcutaneously transplanted tumors of SKM-1 cells and extend the life span of tumor-bearing nude mice.Yisui Granules can be used for the treatment of MDS by regulating DNA methylation and Wnt/β-catenin signaling pathways. |
PDF Full Text Request