| Objective:1.To observe the clinical efficacy of the Invigorating Spleen,Tonifying th Kidney and Detoxifying formula(ISTKDF)in the treatment of patients with myelodysplastic syndrome(MDS).2.By observing the regulatory B cells(Breg),cytokines of IL-10,TGF-β1,IL-4,IFN-γ,killer cell immunoglobulin-like receptor(KIR),to explore the pathogenic role of Breg/KIR immune abnormalities in the pathogenesis of MDS and the immunological connotation of"Lao-Toxin-inducing disease".3.To explore the possible mechanism of ISTKDF in treating MDS by observing the changes of cytokines IL-10,TGF-β1,IL-4,IFN-γexpression and the correlation between killer cell immunoglobulin-like receptor(KIR)and therapeutic effect before and after treatment with ISTKDF and Western medicine.Method:1.Clinical observation:45 patients with MDS were enrolled according to inclusion,exclusion criteria.According to the digital table method they were randomly divided into 30 cases in the treatment group treated with integrated traditional Chinese Medicine and western medicine,and 15 cases in the control group treated with western medicine.In the treatment group,there were 17 cases with“intrinsic deficiency and toxin accumulation”,13 cases with“flourishing toxin and intrinsic deficiency”type.The control group was treated with routine western medicine,while the treatment group was treated with ISTKDF on basis of the control group.All patients were treated for 3months as a course of treatment.Clinical evaluation was carried out after 2 courses of treatment.Blood routine cell count,clinical efficacy of Western medicine,TCM clinical scores,improvement of TCM syndromes,complications and adverse reactions such as infection and bleeding were observed before and after treatment.2.Experimental detection:(1)Peripheral blood was collected from 45 patients with MDS(26 with intrinsic deficiency and toxin accumulation type,19 with flourishing toxin and intrinsic deficiency type)and 15 healthy controls.Mononuclear cells labeled with CD25+CD86+as Breg were isolated.The percentage of CD25+CD86+cells in peripheral blood was detected by flow cytometry.(2)The levels of IL-10,TGF-β1,IL-4 and IFN-γin peripheral blood were measured by ELISA in 45 MDS patients(26 in intrinsic deficiency and toxin accumulation type,19in flourishing toxin and intrinsic deficiency type)and 15 healthy controls.(3)45 patients with MDS were randomly divided into the treatment group of 30 cases and the control group of 15 cases.The treatment group was further divided into 17 cases of intrinsic deficiency and toxin accumulation type and 13 cases of flourishing toxin and intrinsic deficiency type.After 6 months of treatment,the levels of IL-10,TGF-β1,IL-4and IFN-γin peripheral blood were detected by ELISA before and after treatment.(4)45 patients with MDS(26 in intrinsic deficiency and toxin accumulation type,19 in flourishing toxin and intrinsic deficiency type)and 15 healthy controls were enrolled.Anticoagulant venous blood was taken to extract DNA,and KIR genotyping was detected by sequence specific primer polymerase chain reaction.Result:1.Clinical efficacy:1)Hemogram changes:(1)Comparison of therapeutic effect between the treatment group and the control group:After treatment,the white blood cell count in the treatment group increased from 2.67±2.09×10~9/L to 3.73+3.65×10~9/L)(P<0.05).The concentration of hemoglobin increased from 61.59±22.70 g/L to 84.10±27.51 g/L(P<0.05).The platelet count increased from 74.83±96.22×10 ~9/L)to 112.70±136.68×10~9/L)(P<0.05).There was no statistically significant difference in blood routine indexes in the control group after treatment(P>0.05).(2)Comparison of curative effect of different syndrome types:In the intrinsic deficiency and toxin accumulation type of treatment group,after treatment,the white blood cell count of MDS patients increased from 3.08±2.37×10~9/L)to 4.23±3.44×10~9/L)(P<0.05);the platelet count increased from 105.41±118.71×10~9/L to 141.76±163.76×10~9/L(P<0.05);the concentration of hemoglobin increased from 61.49±22.18 g/L to 83.28±28.25 g/L(P<0.05).After treatment,the concentration of hemoglobin in flourishing toxin and intrinsic deficiency type increased from 61.72±24.28 g/L to 85.17±27.60 g/L(P<0.05).There was no significant difference in hemoglobin difference before and after treatment between different syndrome types of MDS treated with integrated traditional Chinese and Western medicine(P>0.05).2)Western medical effect:The total effective rate of the treatment group was 86.67%,and that of the control group was 73.33%,there was no significant difference between the two groups(P>0.05).The total effective rate in the intrinsic deficiency and toxin accumulation type of the treatment group was 94.12%and that of the flourishing toxin and intrinsic deficiency type was 76.92%respectively.There was no statistical difference in the western medicine efficacy in different syndrome types of MDS in the treatment group.3)TCM efficacy:(1)TCM syndrome score:after treatment,the TCM syndrome score of the treatment group decreased from 9.10±2.29 points to 2.93±2.59 points(P<0.01),while that of the control group decreased from 8.40±2.80 points to 5.80±3.42 points(P<0.05).After MDS treatment,the TCM syndrome score of intrinsic deficiency and toxin accumulation type decreased from 8.71±2.49 to 2.06±1.66(P<0.05),while that of the flourishing toxin and intrinsic deficiency type decreased from 9.62±1.98 to 4.08±3.15(P<0.05).(2)Therapeutic effect of TCM syndromes:After treatment,the total response rate of the treatment group was 93.33%,with 4 cases cured,11 cases markedly effective,13 cases effective and 2 cases ineffective.Among the 15 patients in the western medicine control group,there were 0 clinical cured cases,3 markedly effective cases,6 effective cases and 6 ineffective cases,with a total response rate of 62.50%.The efficacy of TCM syndromes in the treatment group was significantly better than that in the western medicine control group(P<0.05).There were 3 cases of clinical cure,7 cases of marked effect,7 cases of effective effect and 0 cases of ineffective in intrinsic deficiency and toxin accumulation type groups.The total response rate was 100%.There were 1 case of clinical cure,4 cases of marked effect,6 cases of effective and 2cases of ineffective in flourishing toxin and intrinsic deficiency type groups.The total response rate was 84.62%.There was no significant difference between the two different syndrome type groups(P>0.05).4)Observation of complications:the incidence of infection in the treatment group was23.33%,significantly lower than that of 73.33%in the control group(P<0.05);the improvement rate of bleeding grade in the treatment group was 40.00%,significantly higher than that in the control group(20.00%(P<0.05).5)Observation of adverse reactions:The incidence of adverse reactions such as gastrointestinal symptoms,skin and mucosal reactions in the treatment group was significantly lower than that in the control group(P<0.05),suggesting that the safety of the treatment group was better than that of the control group.2.Experimental results:1)Breg,cytokines and KIR in MDS patients and normal controls(1)The proportion of CD25+CD86+phenotype Breg was 0.1904(0.0400,2.5200)%in MDS group higher than that of 0.0214(0.0000,0.1056)%in normal control group(P<0.05.The proportion of CD25+CD86+phenotype Breg was 0.1630(0.0199,2.8264)%in intrinsic deficiency and toxin accumulation type group and 0.2869(0.1019,2.5399)%in flourishing toxin and intrinsic deficiency type group respectively.There was no significant difference between the two different type syndrome groups(P>0.05).(2)Cytokines of IL-10,TGF-β1,IL-4 and IFN-γin peripheral blood of MDS group were 52.65(38.64,104.35)pg/ml,210.60(153.70,429.97)pg/ml,235.73(148.04,497.21)pg/ml and 99.30(71.06,255.54)pg/ml,respectively.In the health control group,IL-10,TGF-β1,IL-4 and IFN-γconcentrations were 30.05(22.98,41.12)pg/ml,124.42(105.17,139.48)pg/ml,116.74(96.05,139.51)pg/ml and 44.68(40.06,65.72)pg/ml,respectively.The levels of IL-10,TGF-β1,IL-4 and IFN-γin MDS patients were significantly higher than those in the control group(P<0.01).The levels of IL-10,TGF-β1,IL-4 and IFN-γincreased with the increase of MDS risk.The levels of IL-10,TGF-β1,IL-4 and IFN-γin intrinsic deficiency and toxin accumulation type group were 48.13(36.52,76.24)pg/ml,190.36(148.68,281.28),190.21(116.48,296.10)pg/ml and 85.68(61.18,115.04)pg/ml,respectively.The corresponding values in flourishing toxin and intrinsic deficiency type group were 87.98(46.00,215.44)pg/ml,374.60(163.74,946.08)pg/ml,319.54(172.62,956.92)pg/ml and 266.11(76.62,419.05)pg/ml,respectively.The levels of TGF-β1,IL-4 and IFN-γin flourishing toxin and intrinsic deficiency type group were significantly higher than those in intrinsic deficiency and toxin accumulation type group<0.05.(3)comparison of KIR gene frequency between MDS patients and the normal group:Compared with the normal control group,2DS3 and 2DS4 in MDS patients were significantly higher(P<0.01).Compared with the normal group,the gene frequency of2DS3 in intrinsic deficiency and toxin accumulation type of MDS was significantly higher(P<0.01),while the 2DS4 was significantly higher and 2DS1 was significantly lower in the flourishing toxin and intrinsic deficiency type MDS group(P<0.01).Compared with the MDS patients in flourishing toxin and intrinsic deficiency type group,2DS1 in intrinsic deficiency and toxin accumulation type group was significantly higher(P<0.01).2)Analysis of the correlation between cytokine change,KIR gene type and therapeutic effect in treatment group and control group after treatment(1)Comparison of peripheral blood cytokines before and after treatment in 45 patients with MDS:After treatment,in treatment group IL-10 decreased from 69.06(47.07,119.77)pg/ml to 48.41(30.66,103.49)pg/ml(P<0.05),IL-4 decreased from 238.77(166.20,531.26)pg/ml to 180.25(133.86,318.50)pg/ml(P<0.05),IFN-γdecreased from 116.91(79.12,274.90)pg/ml to 84.34(53.45,199.76)pg/ml(P<0.05).However,after treatment,the levels of IL-10,TGF-β1,IL-4 and IFN-γin MDS patients were still significantly higher than those in the normal control group(P<0.05).There was no significant change in TGF-β1 before and after treatment in the treatment group(P>0.05).There were no significant changes in IL-10,TGF-β1,IL-4 and IFN-γin the control group before and after treatment(P>0.05).(2)Correlation analysis between KIR and curative effect:after 6 months of treatment.37 of the 45 MDS patients had therapeutic response(the curative effect was higher than HI),and 8 patients had no response(the curative effect was PD).The frequency of2DS1 gene in the response group was significantly higher than that in the no response group(P<0.05,OR>1).In the treatment group,2DS1 gene was significantly higher in26 patients with response than in 4 patients with non-response(P<0.05,OR>1).Conclusion:1)ISTKDF has good curative effect on MDS.ISTKDF Combined with basic treatment of Western medicine can effectively improve clinical symptoms,control infections,improve bleeding and other complications,reduce the incidence of adverse reactions,obviously superior to basic treatment of Western medicine alone.This,to a certain extent,verifies the theory and its application value of"Lao-Toxin-inducing disease"of MDS.2)Immunosuppressive Bregs are involved in the negative regulation mechanism of MDS.Increased number of CD25+CD86+phenotype Bregs in MDS patients may contribute to trigger immune escape of abnormal cloned hematopoietic cells.3)The negative regulation of Bregs can increase the release of cytokines such as IL-10,TGF-β1,IL-4 and IFNγ,and lead to ineffective hematopoiesis and excessive apoptosis of hematopoietic cells.The levels of IL-10,IL-4,TGF-β1 and INF-γin peripheral blood of MDS patients with flourishing toxin and intrinsic deficiency type were significantly higher than those of patients with intrinsic deficiency and toxin accumulation type,reflecting the risk and prognosis of the disease to a certain extent.4)The polymorphism of KIR gene is related to the incidence of MDS,and the high frequency of activated KIR genes of 2DS3 and 2DS4 may easily lead to the abnormal transmission of activation signals to NK and T cells.This may be an important factor in the ineffective hematopoiesis of MDS hematopoietic stem cells and one of the material bases of"Lao-Toxin-inducing disease".5)One of the mechanisms of ISTKDF in treating MDS may be to regulate the number and activation and inhibition of NK cells by reducing the release of cytokines such as IL-10,IL-4 and IFNγ,and to exert the immunomodulatory effect,which is beneficial to restoring normal hematopoiesis and eliminating malignant clones of hematopoietic stem cells. |
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