| Objective:1)To evaluate the clinical efficacy of Invigorating Spleen,Tonifying Kidney and Detoxifying Formula in the treatment of different risk myelodysplastic syndrome(MDS)in Chinese and Western medicine,and to clarify the distribution pattern and clinical significance of Bregs by comparing the percentage change of regulatory B cells(Bregs).2)By observing the proportions of Th1,Th2,Th17 and Tregs of polarized Naive CD4~+T cells after co-culture with Bregs in vitro,and the expression levels of corresponding cytokine profiles,as well as the changes in the above indexes of the Invigorating Spleen,Tonifying Kidney and Detoxifying Formula,to explore the immunomodulatory mechanism of Bregs in the progress of MDS and the possible mechanism of Invigorating Spleen,Tonifying Kidney and Detoxifying Formula in the treatment of MDS.3)With transcriptome technology as the support,by analyzing the changes and differential gene expression of Bregs and hematopoietic stem/progenitor cells in the co-culture system before and after the intervention of Invigorating Spleen,Tonifying Kidney and Detoxifying Formula,combined with bioinformatics,the effect of Bregs on hematopoietic function and the intervention mechanism of Invigorating Spleen,Tonifying Kidney and Detoxifying Formula were preliminarily explored.Method:1.Clinical effect of Invigorating Spleen,Tonifying Kidney and Detoxifying Formula and its effect on Bregs1)A total of 75 outpatients and inpatients were collected from March 2019 to January2021 in Yueyang Hospital of Integrated Traditional Chinese and Western Medicine,Shanghai University of Traditional Chinese,including 50 patients in the treatment group and 25 patients in the control group.The control group was treated with conventional treatment of MDS of different risk,and the treatment group was additionally treated with Invigorating Spleen,Tonifying Kidney and Detoxifying Formula on the basis of the control group.The clinical efficacy of western medicine,curative effect of TCM syndromes,peripheral blood images,blood transfusion volume,infection rate and adverse reactions were analyzed and compared between the two groups before and after treatment.2)On the basis of the above groups,another 25 cases of normal control group were set up.Peripheral blood samples were collected from the above 3 groups.Flow cytometry was used to detect the percentage of CD19~+CD25~+Bregs between MDS patients and normal control group,treatment group and control group before and after treatment,and correlation analysis was made with clinical indicators.2.The effect of Bregs on T cell subset function in MDS patients and Invigorating Spleen,Tonifying Kidney and Detoxifying Formula1)The experiment was divided into four groups:(1)Group A:normal control Naive CD4~+T cells;(2)Group B:CD19~+CD25~+Bregs of normal control group and Naive CD4~+T cells of normal control group;(3)Group C:CD19~+CD25~+Breg cells in MDS patients were Naive CD4~+T cells(positive deficiency and toxic accumulation type+positive deficiency and toxic accumulation type).(4)Group D:on the basis of group A,add Invigorating Spleen,Tonifying Kidney and Detoxifying Formula(positive deficiency and toxic accumulation type+toxic Sheng and positive deficiency type).2)CD19~+CD25~+Breg cells and Naive CD4+T cells were selected by immunomagnetic beads for in vitro co-culture and induction differentiation of Naive CD4~+T cells.Flow cytometry was used to detect the percentages of CD3~+CD4~+IFN-γ~+Th1,CD3~+CD4~+IL-4~+Th2,CD3~+CD4~+IL-17A~+Th17,and CD4~+CD25~+Foxp3~+Treg cells in each group.3)The expression levels of IFN-γ,TNF,IL-2,IL-4,IL-6,IL-10,and IL-17A in Th1 cytokine spectrum were detected by CBA.3.The effect of Bregs on hematopoietic stem/progenitor cell function in MDS patients and the intervention effect of Invigorating Spleen,Tonifying Kidney and Detoxifying Formula1)The experiment was divided into 4 groups.(1)Group A:CD34~+CD38~+hematopoietic stem/progenitor cells of normal control group;(2)Group B:CD19~+CD25~+Breg cells of normal control group and CD34~+CD38~+hematopoietic stem/progenitor cells of normal control group;(3)Group C:CD19~+CD25~+Breg cells of MDS patients and CD34~+CD38~+hematopoietic stem/progenitor cells of normal controls(positive deficiency and toxic accumulation type+positive deficiency and toxic accumulation type).(4)Group D:on the basis of group A,add Invigorating Spleen,Tonifying Kidney and Detoxifying Formula(positive deficiency and toxic accumulation type+toxic Sheng and positive deficiency type).2)CD19~+CD25~+Breg cells and CD34~+CD38~-hematopoietic stem/progenitor cells were selected by immunomagnetic beads,and then co-culture with Transwell technology was used to detect CD34~+CD38~-hematopoietic stem/progenitor cells by colony forming unit(CFU-HSPC).3)The transcriptome sequencing analysis was performed on group B and group C to screen out significantly different genes.Cluster analysis and function enrichment of differential genes were performed by bioinformatics methods,and the core genes and key signaling pathways were further screened by building protein interaction networks.Result:1.Clinical effect of Invigorating Spleen,Tonifying Kidney and Detoxifying Formula and its effect on Bregs1)A total of 83 patients were collected,including 55 cases in the treatment group(28cases of positive deficiency and toxic accumulation type,27 cases of toxic accumulation type,and 28 cases in the control group(17 cases of positive deficiency and toxic accumulation type,11 cases of toxic accumulation and positive deficiency type).A total of 75 cases were actually completed,including 50 cases in the treatment group(26 cases of positive deficiency and toxic accumulation type,24 cases of toxic accumulation and positive deficiency type).The control group had 25 cases(14 cases with positive deficiency and toxic accumulation type,11 cases with positive deficiency and toxic accumulation type).2)General background information:there was no statistical significance in the comparison of baseline data such as gender,age and disease risk among all groups(P>0.05).3)Clinical efficacy of western medicine:the total effective rate of the treatment group was 86.00%,and the total effective rate of the control group was 76.00%.Comparison of the total effective rate between the two groups showed statistical difference(P<0.05),indicating that the curative effect of the treatment group was significantly better than that of the control group.Among them,the total effective rate of positive deficiency and toxic accumulation type in the treatment group was 84.61%,and the total effective rate of positive deficiency and toxic accumulation type was 87.50%in the treatment group.There was no statistical difference in the total effective rate(P>0.05),so it could not be considered that there was difference in the curative effect between positive deficiency and toxic accumulation type in the treatment group and toxic accumulation and toxic accumulation type.4)TCM syndrome quantification scores:Compared with the control group before and after treatment,TCM syndrome scores in the treatment group were decreased(P<0.05);Compared with the control group,the decrease of TCM syndrome score in the treatment group was more obvious(P<0.05),and the treatment group was more able to improve TCM clinical symptoms than the control group.After treatment,the TCM syndrome scores of the positive deficiency and toxic accumulation type and the positive deficiency and toxic accumulation type decreased compared with before treatment(P<0.05),and the decrease was more obvious in the positive deficiency and toxic accumulation type.5)Efficacy of TCM syndromes:the total effective rate of the treatment group was92.00%,and the total effective rate of the control group was 64.00%.The improvement of TCM syndromes efficacy in the treatment group was better than that in the control group,with statistically significant difference(P<0.05).The total effective rate of positive deficiency and toxic accumulation was 92.31%,while that of positive deficiency and toxic accumulation was 91.66%,and there was no statistical difference between the two groups(P>0.05).6)Peripheral blood patterns:(1)Compared between the treatment group and the control group,there were no significant differences in WBC,Neut,Hb and PLT between the two groups before treatment(P>0.05).Compared with before and after treatment,WBC,Hb and PLT levels were higher after treatment than before(P<0.05),and there was no statistical difference in NEUT(P>0.05).The levels of WBC,Hb and PLT in the treatment group were higher than those in the control group(P<0.05),and there was no statistical difference in Neut between the two groups(P>0.05).(2)Compared between positive deficiency and toxic accumulation type and positive deficiency type,there was no statistical significance in WBC,Neut,HB and PLT between the two syndrome types before treatment(P>0.05).Before and after treatment,the levels of WBC,Hb and PLT in the two syndrome types were higher than before treatment(P<0.05),and there was no statistical difference in Neut(P>0.05).The levels of Hb and PLT in the syndrome type were higher than before treatment(P<0.05),and there was no statistical difference in WBC and Neut(P>0.05).The WBC,HB and PLT levels of positive deficiency and toxic accumulation type were higher than those of positive deficiency and toxic accumulation type(P<0.05),and there was no statistical difference between Neut and positive deficiency type(P>0.05).7)The amount of blood transfusion:the amount of platelet and red blood cell transfusions in the treatment group was lower than that in the control group(P<0.05).8)Infection rate:the infection rate in the treatment group was significantly lower than that in the control group(P<0.05).9)Adside reactions:the incidence of digestive system symptoms,nervous system symptoms,skin and mucosal reactions,abnormal liver and kidney function in the treatment group was lower than that in the control group(P<0.05).10)Analysis of the proportion of Breg cells and the correlation with clinical indicators:(1)The expression of CD19~+CD25~+Bregs in MDS patients was higher than that in normal control group(P<0.05).The expression of CD19~+CD25~+Breg in MDS patients was higher than that in the positive deficiency type and the positive deficiency type(P<0.05).The proportion of CD19~+CD25~+Breg cells in peripheral blood of MDS treatment group was lower than that before treatment(P<0.05),but there was no significant change in the proportion of CD19~+CD25~+Breg cells in MDS control group before and after treatment(P>0.05).The proportion of Breg cells in the type of positive deficiency and toxic accumulation decreased significantly after treatment(P<0.05),while there was no significant difference in the type of positive deficiency and toxic accumulation before and after treatment(P>0.05).(2)CD19~+CD25~+Bregs expression in MDS patients was positively correlated with WBC,Hb and PLT(P<0.05),but had no significant correlation with NEUT(P>0.05).2.The effect of Bregs on T cell subset function in MDS patients and the intervention effect of Invigorating Spleen,Tonifying Kidney and Detoxifying Formula1)Ratio of Th1,Th2,Th17 and Treg cells:(1)Compared with group A,the proportions of Treg and Th2 in groups B,C and D were all increased(P<0.01),suggesting that in vitro Bregs could promote the polarization of Naive CD4~+T cells to Treg and Th2.Compared with group B,the proportion of group C was higher(P<0.01),suggesting that Bregs in MDS patients could promote Treg and Th2 polarization more than that in normal control group.Compared with group C,the proportion of group D was decreased(P<0.01),suggesting that Invigorating Spleen,Tonifying Kidney and Detoxifying Formula could inhibit the promoting effects of Bregs on Treg and Th2 polarization in MDS patients.There was no statistically significant difference in the proportion of Th1 cells between group A and group B(P>0.05).There was no significant difference between group B and group C(P>0.05)and between group C and group D(P>0.05).Compared with group A,the proportion of Th17 in group B,C and D was significantly lower(P<0.01),suggesting that Bregs can inhibit the polarization of Naive CD4~+T cells to Th17 in vitro.Compared with group B,the proportion of group C was decreased,and the difference was statistically significant(P<0.01),suggesting that Bregs in MDS patients had stronger inhibitory effect on Th17polarization than that in normal control group.Compared with group C,the proportion of group D was increased,and the difference was statistically significant(P<0.05),suggesting that the prescription of invigorating spleen,nourishing kidney and jiedu could enhance the inhibitory effect of Bregs on Th17 polarization in MDS patients.(2)As for the proportion of Treg and Th2 in different syndromes,the proportion of Treg and Th2 cells in group C was higher than that in group C(P<0.01),suggesting that with the increase of disease risk,the polarization expression of Treg and Th2 by Bregs in MDS patients was enhanced.Breg of positive deficiency and toxic accumulation type in group D and positive deficiency and toxic accumulation type in group D and positive deficiency and toxic accumulation type in group D and positive deficiency and toxic accumulation type in group C were both weakened in promoting Treg polarization(P<0.01),suggesting that Breg of Invigorating Spleen,Tonifying Kidney and Detoxifying Formula could inhibit the promoting effect of Breg of different risk MDS on Treg polarization.There was no statistically significant difference between positive deficiency and toxic accumulation type of group D and positive deficiency and toxic accumulation type of group C in the proportion of Th2(P>0.05).Compared with positive deficiency and toxic accumulation type of group D and positive deficiency type of group C,Breg promoted the decrease of Th2 polarization ability,with statistical significance(P<0.05).It is suggested that Invigorating Spleen,Tonifying Kidney and Detoxifying Formula has an intervention effect on the promotion of Th2 polarization by Bregs in high-risk MDS,while it does not have an intervention effect on low-risk MDS.The proportion of Th17 in group C was lower than that in group C(P<0.01),suggesting that with the increase of disease risk,the ability of Bregs to inhibit the polarization expression of Th17 in MDS patients was enhanced.Compared with positive deficiency and toxic accumulation type in group D and positive deficiency and toxic accumulation type in group C and positive deficiency type in group D and positive deficiency type in group D and positive deficiency type in group C,the ability of Breg to inhibit Th17 polarization was weakened(P<0.05),suggesting that Invigorating Spleen,Tonifying Kidney and Detoxifying Formula has intervention efficacy on both low risk and high risk MDS Breg’s inhibition of Th17 polarization.2)Expression level of cytokine profile:(1)In the expression of Th1 cytokine profile,there were no significant differences in the levels of IFN-γ,TNF and IL-2 between group A and group B(P>0.05).There was no significant difference between group B and group C(P>0.05)and between group C and group D(P>0.05).In the expression profile of Th2 cytokines,the expression levels of IL-4,IL-6 and IL-8 in group A were lower than those in group B(P<0.01).The levels of IL-4,IL-6 and IL-10 in group C were higher than those in group B(P<0.05),and the levels of IL-6 and IL-10 in group D were lower than those in group C(P<0.01).There was no difference in the expression of IL-4 between group D and group C(P>0.05).There was no significant difference in the expression of Ail-17A in Th17cytokine profile between group B and group B(P>0.05).There was no statistical difference between group B and group C(P>0.05)and between group C and group D(P>0.05).(2)As for the expression profile of Th2 cytokines,the expression levels of IL-4,IL-6and IL-10 in group C were lower than those in group D(P<0.01).There was no significant difference in the expression levels of IL-4 in group C and group D(P>0.05),and the expression levels of IL-6 and IL-10 were both decreased(P<0.05).There was no statistical difference in the expression of IL-4 between group C and D(P=0.910>0.05),and the expression of IL-6 and IL-10 were decreased(P<0.05).3.The effect of Bregs on hematopoietic stem/progenitor cell function in MDS patients and the intervention effect of Invigorating Spleen,Tonifying Kidney and Detoxifying Formula1)Detection of CD34~+CD38~-hematopoietic stem/progenitor cell colony formation unit:(1)Compared with group A,CFU-E,BFU-E and CFU-GM in groups B,C and D were all decreased,and the differences were statistically significant(P<0.05),suggesting that Bregs could inhibit the formation of CD34~+CD38~-hematopoietic stem/progenitor cell colony formation in vitro.Compared with group B,CFU-E,BFU-E and CFU-GM were decreased in group C,and the differences were statistically significant(<0.01),suggesting that Bregs in MDS patients had a stronger inhibitory effect on CD34~+CD38~-hematopoietic stem/progenitor cell colony formation than that in normal control group.Compared with group C,the levels of CFU-E,BFU-E and CFU-GM in group D were increased,and the differences were statistically significant(P<0.05),suggesting that Invigorating Spleen,Tonifying Kidney and Detoxifying Formula could enhance the inhibitory effect of BREG on the formation of CD34~+CD38~-hematopoietic stem/progenitor cell colony in MDS patients.(2)Comparison of different syndrome types of MDS in group C and group D showed that CFU-E,BFU-E and CFU-GM in group C were higher than those in group C,with statistical significance(P<0.01),suggesting that with the increase of disease risk,the inhibitory effect of Bregs on the formation of hematopoietic stem/progenitor cell colonies in MDS patients was enhanced.Compared with positive deficiency and toxic accumulation type in group D and positive deficiency and toxic accumulation type in group C and positive deficiency type in group D and positive deficiency type in group C,the ability of Breg to inhibit the formation of hematopoietic stem/progenitor cell was weakened(P<0.05),suggesting that Invigorating Spleen,Tonifying Kidney and Detoxifying Formula had intervention efficacy on the inhibition of the formation of hematopoietic stem/progenitor cell in both low risk and high risk MDS Breg.2)Transcriptome sequencing:(1)The results of gene expression profile showed that a total of 2909 differentially expressed genes were obtained,among which 1367 genes were up-regulated and 1542 genes were down-regulated.(2)GO enrichment analysis of differential genes showed that the biological processes involved in significant enrichment included the positive regulation of IL-6 biosynthesis and Th17differentiation,etc.(3)KEGG enrichment analysis of differential genes showed that significant enrichment pathways included antigen processing and presentation related signaling pathways,cell cycle related signaling pathways,Th17 cell differentiation related signaling pathways,DNA replication related signaling pathways,hematopoietic cell pedigree related signaling pathways,etc.(4)Key core genes such as FPR2,LILRB2,TYROBP,PTAFR and HLA-E were further screened out by protein interaction network.Conclusion:1)Invigorating Spleen,Tonifying Kidney and Detoxifying Formula has good efficacy in the treatment of MDS at different risk levels.Compared with western medicine alone,it has more advantages in improving the efficacy of western medicine,the efficacy of traditional Chinese medicine,and the improvement of TCM syndromes,and has better effect for the low-risk group.2)The increased proportion of Bregs in MDS causes the negative regulation of immune dysfunction,thus participating in the immune pathogenesis of MDS.In addition,the proportion of Bregs with positive deficiency and positive deficiency was more significantly unbalanced than that with positive deficiency and toxic accumulation,indicating that the balance disorder of Bregs is closely related to the progression of MDS disease,and establishing the material basis of the theory of"Lao-Toxin-inducing disease"in MDS.3)Increased Bregs ratio of MDS can induce the polarization of Naive CD4~+T cells to Treg and Th2,inhibit Th17 polarization,disrupt the balance axis of Th1/Th2 and Treg/Th17,and with the increase of risk and progression of MDS,Bregs enhanced the polarization induction or inhibition of Naive CD4~+T cell subsets to varying degrees.4)The increased proportion of Bregs in MDS can inhibit the formation of hematopoietic stem/progenitor cell colonies in vitro,and it is speculated that Bregs affect the function of hematopoietic stem/progenitor cell through bone marrow microenvironment,and is related to the changes of core genes such as FPR2,LILRB2,Tyrobp,PTAFR and HLA-E.5)The effective mechanism of Invigorating Spleen,Tonifying Kidney and Detoxifying Formula in the treatment of MDS is that it interferes with the polarization of Bregs on Th1,Th2,Th17 and Treg cells by reducing the secretion level of Bregs and inhibiting the negative immune regulation function of Bregs,so as to restore the balance of Th1/Th2 and Treg/Th17 and inhibit immune escape.Therefore,it is speculated that Bregs may be a potential target of i Invigorating Spleen,Tonifying Kidney and Detoxifying Formula in the treatment of MDS. |
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