| 1 Research purposes In this paper,through a large number of clinical data mining and analysis,follow-up of Chinese medicine treatment of rheumatoid arthritis patients with endpoint events,explore the changes of serum PLT and association rule cluster analysis in patients with rheumatoid arthritis and Xinfeng capsule to improve platelet activation in patients with rheumatoid arthritis And immune inflammation.In vitro experiments were conducted to observe the changes and correlation analysis of inflammatory immune index,cytokine,hypoxia-inducible factor,matrix metalloproteinase,VEGF/SDF-1/CXCR4 signaling pathway,and to explore the effect of Xinfeng Capsule on platelet activation in RA patients.The mechanism of action of immune inflammation.2 Research methods 2.1 Cohort study Using cohort study design,telephone follow-up of the first affiliated hospital of Anhui University of Traditional Chinese Medicine from January 2012 to June 2015,the first diagnosis of rheumatoid arthritis patients,the type and application time of traditional Chinese medicine decoction after discharge.And the presence or absence of recurrence of hospitalization,extra-articular lesions,surgical treatment,and all-cause death,and the use of logistic regression and other statistical analysis methods to analyze the correlation between TCM intervention and patient endpoint events in post-discharge patients.Sex.A questionnaire on the treatment of Chinese medicine and end-point events after discharge from patients with rheumatoid arthritis(CRF form)was produced.Screen out the eligible cases,call the patient after taking the Chinese medicine after discharge and the occurrence of the end point event,fill out the CRF form.Statistical methods were used to analyze the association between endpoint events and TCM applications.Establish a database of rheumatoid arthritis patients in the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine,enter patient data and other data,and export the required data to Excel 2010 and SPSS 21.0 software respectively.Comparison of multiple sets of measurement data using variance analysis,comparison of count data rates using chi-square test,logistic regression analysis method was used to analyze the correlation between endpoint events and various factors.2.2 Clinical data mining research The medical records of patients diagnosed with RA in the Department of Rheumatology and Immunology,the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine from June 2012 to December 2016 were collected and analyzed.SPSS 21.0 was used to analyze the traditional Chinese medicine and platelet parameters and immunity of Jianpi Huashi Tongluo.The relationship between changes in inflammation indicators.The core prescriptions were based on complex network technology;the frequency of oral Chinese medicines was descriptively analyzed,the frequency of drug use was analyzed,and the treatment rules and rules were summarized.The cluster analysis of the top 20 drugs using frequency was performed using SPSS 21.0;SPSS Clementine was used in this study.The Apriori module in 14.2 is an association rule mining tool to explore the relationship between oral Chinese medicine,proprietary Chinese medicine and external medicine and platelet parameters.The study group(XFC+ Chinese medicine)and the control group(Chinese medicine)were classified on the condition of whether or not to use Xinfeng Capsule(XFC).Descriptive analysis of the frequency of oral administration of Chinese medicine in two groups of patients,summed up the treatment rules and rules;using SPSS 21.0 software to analyze IgA,IgM,IgG,complement C3,complement C4,ESR,PLT,WBC,RBC,Hs-CRP before and after treatment,RF,TG,T-CHOL difference situation;using Apriori module for the excavation of Furong cream external application combined with traditional Chinese medicine oral administration and platelet parameters and immune inflammatory metabolic index;using ORACLE 10 g tool for index laboratory random walk model evaluation.2.3 In vitro experimental study Twenty patients with RA were enrolled in the study.20 patients with RA and 8 normal subjects were enrolled in 10 ml of peripheral blood.The appropriate amount of lymphocyte separation solution was added and centrifuged at 20 ° C for 30 minutes at 20 ° C for 15 minutes to separate PBMC.The cells were cultured in a DMEM medium containing 10% fetal calf serum in an incubator at 37 ° C,5% CO 2,and saturated humidity,and the cells in the log phase were subjected to an experiment.The experiment has five groups: group I(normal control group: normal healthy person PBMC and platelet co-culture),group II(model control group: RA patient PBMC and platelet co-culture),group III(XFC group: containing optimal concentration)X-ray RA patients with PBMC and platelet co-culture),group IV(XFC+AMD3100 group: PBMC with optimal concentration of XFC and AMD3100 and platelet co-culture),group V(AMD3100 group: PBMC with platelet co-culture of AMD3100-containing RA patients)).Cell proliferation was detected by MTT assay.TNF-?,IL-1?,IL-4,IL-10,VEGFA and VEGFR were detected by ELISA.SDF-1,CXCR4 and HIF were detected by real-time quantitative PCR(qT-PCR).The expressions of-1?,HIF-2? were detected by Western blot.The expressions of SDF-1,CXCR4,VEGFA,VEGFR and MMP9 were detected by immunofluorescence.The protein expressions of CD40 L,PDGF and VEGF were detected by immunofluorescence.The laboratory indicators were statistically processed using SPSS 21.0 statistical software.3 results 3.1 Cohort study 3.1.1 General situation A total of 1812 patients with rheumatoid arthritis were enrolled.1468 patients were followed up,344 patients were lost to follow-up,and the rate of loss of follow-up was 18.98%.The successful follow-up was 216 males and 1252 females.The mean follow-up age was 53.09±13.11 years.The application of Chinese medicine for a minimum of 4 days,the longest is 52 months,the average(14.28 ± 13.81)months.Among them,943 patients in the high-exposure group,137 males and 806 females,aged(52.83±13.33)years old;187 patients in the middle exposed group,17 males and 170 females,aged(53.63±11.88)years old;162 patients in the low-exposure group There were 36 males and 126 females with an age of(51.55±13.58)years old;176 patients in non-exposure group,26 males and 150 females,aged(55.22±12.51)years old.The difference was(P>0.05).There was no significant difference in the course of disease between the non-exposed group and each exposed group(P>0.05).3.1.2 Chinese patent medicines and Chinese medicine prescriptions A total of 12 Chinese patent medicines were taken after discharge from 1468 patients,with a total frequency of 3,475,and taking Chinese medicine for 0.1 to 52 months,with an average of(14.28±13.81)months.A total of 12 kinds of traditional Chinese medicine decoctions were taken,the total frequency was 3952 times,and the time of taking traditional Chinese medicine decoction was 0.1 to 53 months,with an average of(9.20±10.75)months.Most patients with rheumatoid arthritis are damp-heat and phlegm-reducing types.The main ideas of drugs such as Xinfeng Capsule,Huangqi Qingre Quyu Capsule and Si Miao San are all heat,spleen and dampness.3.1.3 Application of Chinese medicine in each exposed group According to the patient's use of traditional Chinese medicine,according to statistics,the most frequently used twelve traditional Chinese medicines are Fu Ling,salvia,tangerine peel,coix seed,safflower,clematis,dandelion,peach kernel,Hedyotis diffusa,Chuanxiong,Zihuadi,Mulberry parasitic,according to the efficacy of these twelve Chinese medicines are divided into four groups,respectively,spleen medicine(Fu Ling,Yi Yiren,Chen Pi),blood circulation medicine(danshen,peach kernel,safflower),heat medicine(White flower Hedyotis,dandelion,purple flower dices),rheumatoid medicine(Welling Xian,Chuanxiong,mulberry parasitic).3.1.4 Comparison of the occurrence of endpoints in each exposed group and non-exposed group A total of 80 endpoints occurred in the follow-up patients,including 43 re-admissions,18 extra-articular lesions,10 surgical treatments,and 9 all-cause deaths.3.1.5 Single factor analysis of end point events and multivariate logistic regression analysis The single factor ?2 test was used to analyze the correlation between gender,age,exposure intensity and other endpoint events.There was no significant difference in gender,age and end point events(P>0.05).There was significant difference between the exposure intensity and the end point event(P <0.01).Multivariate logistic regression analysis,the assignment of each factor: X1(exposure intensity is non,low,medium,and high are 0,1,2,3 respectively),X2(age < 30 years old,? 30 and < 60 years old,? 60 years old respectively 0,1,2),X3(sex male and female are 0,1 respectively),X4(spleen spleen use,unused 0,1 respectively),x5(activating blood drugs,unused 0,1 respectively)),x6(use of heat-clearing medicine,unused 0,1),x7(use of rheumatoid medicine,unused 0,1),x8(new wind capsule use,unused 0,1 respectively),x9(The use of Huangqi Qingre Quyu Capsules is 0,1);the composite end point is the dependent variable y,including all-cause death,surgical treatment,recurrence of aggravation,extra-articular lesions and other end-point events(not occurring as y = 0,occurs as y = 1).The logistic regression model of SPSS21.0 software was used for analysis.The results showed that the use of spleen,Huoxue and Xinfeng capsules is a protective factor for the occurrence of end point events,suggesting that TCM treatment of foot strength can reduce re-entry,surgical treatment,extra-articular lesions and all-cause death in patients with rheumatoid arthritis.The incidence of waiting for an endpoint event.3.2 Clinical data mining research 3.2.1 PLT parameter values of RA patients Compared with the normal reference value(125-350×109/L),the PLT value of 1951 patients with RA decreased by 252(13%),893(46%)were normal,and 806(upper).41%),compared with the normal value(9 ~ 17fL),the PDW value decreased by 33 cases(1.69%),the normal number of 1680 cases(86.11%),the increase of 238 cases(12.20%),MPV value Compared with the normal value(9 to 13 fL),there were 64 cases(3.28%,1721 cases(88.21%),and 166 cases(8.51%),PCT values and normal values(0.11%~ Compared with 0.28%,there were 17 cases(0.87%),1227 cases(62.89%),707 cases(36.24%),and P-LCR values(13%~38%).In comparison,there were 18 cases(0.92%),1414 cases(72.48%),and 519 cases(26.60%).3.2.2 Correlation analysis between serum PLT parameters and immune,inflammatory and metabolic indicators in RA patients PLT was positively correlated with PT,IGA,IGG,C4,ESR(P<0.05 or P<0.01),and negatively correlated with C3(P<0.05 or P<0.01),and had no correlation with other indicators and age and duration(P >0.05);PDW was positively correlated with TT,IGA,C4,ESR,WBC and ApoA1(P<0.05 or P<0.01),and negatively correlated with C3 and SOD(P<0.05 or P<0.01).There was no correlation with age,duration of disease and other selected laboratory parameters(P>0.05);MPV was positively correlated with CRP and HDL-C(P<0.05 or P<0.01),and negatively correlated with SOD(P<0.05 or P).<0.01),no correlation with other indicators and age,duration(P>0.05);PCT was positively correlated with IGA,C4,SOD,TG,TC(P<0.05 or P<0.01),and other indicators and age,duration There was no correlation(P>0.05);P-LCR was positively correlated with TT and CRP(P<0.05 or P<0.01),and negatively correlated with SOD(P<0.05 or P<0.01),and other indicators,age and disease duration.No correlation(P>0.05).3.2.3 Analysis of association rules between PLT parameters and laboratory indicators in RA patients Set the minimum confidence to 70% and the minimum support to 20%.According to the Aprior module,the increase in platelet count and the increase in hs-CRP,PT,rheumatoid factor,complement C4,IGM,and TG were all greater than 20%.Confidence More than 70%,increased platelet count,increased platelet width,increased hs-CRP,and increased ESR were greater than 20%,confidence was greater than 70%,platelet volume increased and ESR rose The support for high and HDL-C decline was greater than 20%,the confidence was greater than 70%,the increase in large platelet ratio and the increase in hs-CRP were greater than 20%,and the confidence was greater than 70%.3.2.4 Logistic regression analysis of PLT parameters and laboratory indicators in RA patients The results of Logistics regression analysis indicated that platelet count was positively correlated with hs-CRP,IGA,IGM,Apo-A1 and Apo-B(P<0.01 or P<0.05),and platelet count was positively correlated with SOD,EDR,IGA and TG.P<0.01 or P<0.05),platelet width was positively correlated with ESR,C4 and IGM(P<0.01 or P<0.05).The mean platelet volume was positively correlated with hs-CRP,IgA and TG(P<0.01 or P<0.05).The ratio of large platelets was positively correlated with complement C4,TG,TC,Apo-B(P<0.01 or P<0.05).3.2.5 Changes in laboratory indicators before and after treatment Platelet parameters(PLT,PDW,MPV,PCT,P-LCR),immune markers(C3,C4,IgA,IgM,IgG),inflammatory markers(ESR,hs-CRP,RF,WBC),metabolism in 1951 RA patients Indicators(SOD,TG,TC,HDL-C,LDL-C)before and after treatment,platelet parameters PLT,PCT,immune indicators complement C3,C4,IgA,IgM,IgG,erythrocyte sedimentation rate(ESR)),high-sensitivity C-reactive protein(hs-CRP),rheumatoid factor(RF)were reduced(P < 0.05).3.2.6 Analysis of the use of traditional Chinese medicine and its association with laboratory indicators The prescriptions of 1951 patients were treated with spleen and phlegm and dampness drugs such as spleen,coix seed,and yam,safflower,peach kernel,spatholobus and other blood-activating drugs,and clematis,valerian,and solo.There are four types of collaterals,dandelion,Hedyotis diffusa,Astragalus,and other antipyretic drugs.The top 5 with the highest correlation,spatholobus sinensis,sputum associated with PLT index decreased(confidence 92.34%,support degree 38.02%),spatholobus & dandelion & salvia related to hs-CRP decreased(confidence 90.36%,support Degree 29.38%),Hedyotis & Coix seed associated with ESR decline(confidence 89.98%,support 45.57%),Coix Seed & Duo activity associated with IgM decline(confidence 89.76%,support 22.61%),Fu Ling & peach The combination is associated with an increase in the PDW indicator(confidence 87.55%,support 29.84%).RA patients use the top 20 drugs,poly 1 class: peach kernel,safflower,medlar,tangerine peel,salvia,coix seed,yam,clematis,valerian,poly 2: dandelion,Hedyotis,Alisma,Spatholobus sinensis,poly 3: Zhimu,Phellodendron,Achyranthes,dog ridge,stretched grass.3.2.7 General situation of Xinfeng capsule combined with traditional Chinese medicine in the treatment of RA patients A total of 1951 patients with rheumatoid arthritis met the requirements of this study,including 782 cases in the experimental group(XFC + Chinese medicine orally)and 1169 cases in the control group(mechanical Chinese medicine alone).3.2.8 Immunological inflammation and liver and kidney function before and after treatment in two groups of RA patients PLT,PCT,IgA,IgM,IgG,C3,C4,RF,CCP-AB,Hs-CRP,and ESR decreased in the two groups of RA patients,and the difference was statistically significant(P<0.05).The experimental group was superior to the control group in reducing PLT,IgA,IgM,C3,RF,CCP-AB,Hs-CRP and ESR,.Compared with before treatment,the ALT and AST of the RA patients in the experimental group were decreased after treatment,and the difference was statistically significant(P<0.01).ALP,GGT,CREA,BUN,b-MG,MA,TRU and IgU increased in both groups after treatment,and the difference was statistically significant(P<0.01).The UA of the two groups of RA patients decreased after treatment,the difference was statistically significant(P<0.01).The experimental group was superior to the control group in reducing UA,and the difference was statistically significant(P<0.05 or P<0.01).3.2.9 Analysis of random walk model The positive rate of the experimental indicators PLT,IgM,Hs-CRP,and CCP-AB were 0.2124,0.1726,0.5289,and 0.4401,respectively.The comprehensive improvement rate for each positive walking step was 33.52%,4.23%,and 32.42%,respectively.19.35%.The positive rate of PLT,IgM,Hs-CRP and CCP-AB in the control group were 0.1526,0.1387,0.5736,and 0.5053,respectively.The comprehensive improvement rate for each positive walking step was 16.42%,6.31%,and 31.33%,respectively.21.64%.3.3 In vitro experimental study 3.3.1 Effect of XFC on cytokines and VEGF in peripheral blood mononuclear cells of patients with RA Compared with the normal group,the model group had elevated TNF-?,IL-1?,VEGF-A,and VEGFR,and decreased IL-4 and IL-10(P<0.01).Compared with the model group,after drug intervention TNF-?,IL-1?,VEGF-A,VEGFR decreased,IL-4 and IL-10 increased in each group(P<0.01).Compared with XFC group,AMD3100 group TNF-?,IL-1,VEGF and VEGFR were decreased,TNF-?,IL-1?,VEGF-A,VEGFR were decreased and IL-10 was increased in XFC+AMD3100 group(P<0.01).Compared with AMD3100 group,XFC+AMD3100 group TNF-?,IL-1,EGF-A,VEGFR decreased,IL-4,IL-10 increased(P<0.01).3.3.2 Effect of HQC on mRNA of AMPK-?1,PPAR-?,FoxO3 a and MnSOD in peripheral blood mononuclear cells of patients with RA.3.3.2 Effect of XFC on mRNA of SDF-1,CXCR4,VEGF and HIF-? in peripheral blood mononuclear cells of patients with RA Compared with the normal group,the SDF-1,CXCR4,VEGF,HIF-1?,and HIF-2? mRNAs in the model group were increased(P<0.01).Compared with the model group,each drug group SDF-1.CXCR4,VEGF,HIF-1?,HIF-2? decreased(P<0.05,P<0.01);compared with XFC group,XFC+AMD3100 group VEGF,SDF-1,CXCR4,HIF-1?,HIF-2? mRNA decreased;compared with AMD3100 group,XFC+AMD3100 group VEGF,SDF-1,CXCR4,HIF-1?,HIF-2? mRNA decreased(P<0.01).3.3.3 Effect of XFC on VEGF/SDF-1/CXCR4 signaling pathway-related proteins in peripheral blood mononuclear cells of patients with RA Compared with the normal group,the model group had elevated VEGF-A,VEGFR,SDF-1,CXCR4,and MMP9(P<0.01);compared with the model group,XFC group VEGF-A,VEGFR,SDF-1,CXCR4 The expression of MMP9 decreased(P<0.05,P<0.01),and the expression of VEGF-A,VEGFR,SDF-1,CXCR4 and MMP9 decreased in AMD3100 group(P<0.05,P<0.01),XFC The expression of VEGF-A,VEGFR,SDF-1,CXCR4 and MMP9 was decreased in the AMD3100 group(P<0.05,P<0.01).Compared with the XFC group,the AMD3100 group and the XFC+AMD3100 group were VEGF-A and VEGFR.The expression of SDF-1,CXCR4 and MMP9 protein was decreased(P<0.05,P<0.01).Compared with AMD3100 group,the expression of VEGF-A,VEGFR,SDF-1,CXCR4 and MMP9 protein in XFC+AMD3100 group Lower(P<0.05,P<0.01).3.3.4 Immunofluorescence The results of immunofluorescence showed that the cell fluorescence density was high,and the nuclear and cytoplasm of CD40L(rabbit anti-human),PDGF-A(rabbit anti-human),VEGF(mouse anti-human)showed red fluorescence,and the background was clear and non-specific.Coloring.The XFC+AMD3100 group showed the least proportion of red fluorescent cells and the weakest intensity.4 Conclusion 4.1 The application of traditional Chinese medicine can reduce the occurrence of endpoint events in RA patients.The intensity of intervention in Chinese medicine is a protective factor for endpoint events.4.2 RA patients generally have elevated platelet counts,and PLT activation has an effect on the inflammatory,metabolic,and immune indicators of RA patients.4.3 Our hospital for the treatment of RA patients with Jianpi Huashi Tongluo method,good use of Jianpi Huashi Qingre Tongluo drug compatibility,and associated with decreased immune indicators,inflammation indicators.The rheumatology department of our hospital has created the spleen unit therapy for RA.The main Chinese medicine is mainly Jianpi Huashi,Huoxuetongluo,Qufeng dehumidification and Qingrejiedu.Chinese patent medicine is Xinfeng capsule,and external application is Furong cream..The improvement of platelet parameters and immune inflammatory metabolic markers in patients treated with unit therapy is better than the simple use of traditional Chinese medicine.4.4 XFC has a VEGF/SDF-1/CXCR4 pathway blocker-like effect and can inhibit platelet activation in RA patients.The mechanism is as follows:(1)By up-regulating the proinflammatory cytokines IL-4 and IL-10,down-regulating the inflammatory factors TNF-? and IL-1?,and regulating the VEGF/SDF-1/CXCR4 pathway;(2)inhibition of platelet activation by down-regulating VEGF expression,regulating SDF-1/CXCR4 signaling pathway;(3)Regulate platelet activation by down-regulating HIF-? and regulating the VEGF/SDF-1/CXCR4 pathway. |
PDF Full Text Request