Treatment Of Ankylosing Spnodylitis With Jianpi Huashi Tongluo Therapy Queue Study Data Mining And Inhibition Of Platelet Activation Mechanism Study

1 Objective Through clinical cohort study,data mining research and in vitro experiments,the changes of drugs,platelet parameters,inflammatory immune response and Xinfeng Capsule(XFC)in the treatment of ankylosing spondylitis(AS)in Anhui Provincial Hospital of Traditional Chinese Medicine were observed in 3 parts to improve platelet parameters and inflammation.Immune indicators and explore the mechanism by which XFC improves platelet activation and inflammation through the SDF-1/CXCR4-VEGFA pathway.2 Methods 2.1 Clinical Cohort Study Telephone follow-up from January 2012 to June 2016,the hospitalized patients in the hospital,the time and type of taking Chinese patent medicine and Chinese medicine decoction after discharge,enter the data.The exposure group was defined according to the time of administration.Data were collected from AS patients,and the use of oral Chinese patent medicines and traditional Chinese medicine decoctions,as well as the occurrence of end points in each exposure group,were analyzed.Chi-square test and multiple regression analysis were used to analyze the relationship between each factor and the end event.2.2 Data Mining Research From the Clinical Data Center of Anhui Provincial Hospital of Traditional Chinese Medicine,a total of 1110 electronic medical records were obtained from AS patients who were hospitalized in the rheumatology department of Anhui Provincial Hospital of Traditional Chinese Medicine from June 2012 to July 2017.Collect the general situation of inpatients,the use of the first 20 flavors of Chinese medicine and Xinfeng capsules,platelet parameters and immune inflammation indicators.Patients were divided into spleen(Chinese medicine + XFC+ Hibiscus cream)group and simple(Chinese medicine)group according to whether or not to use XFC.The data was analyzed using SPSS 21.0 and SPSS Clementine 11.1 software.2.3 Experimental study in vitro 2.3.1 Preparation of XFC-containing serum Thirty-five male Sprague-Dawley rats were randomly divided into control group,negative control group,high,medium and low doses(different according to 20 times,10 time,5 times XFC clinical dose).After continuous gavage for 3 days,the blood was removed and used for further use.The MTT assay detects the optimal XFC dose.2.3.2 Isolation and culture of monocytes(PBMCs)from clinical AS patients and healthy individuals 24 patients with AS peripheral blood and 6 healthy subjects were collected and divided into normal group,model group,XFC group,XFC+AMD3100 group and AMD3100 group.PBMC were isolated according to the procedure and cultured in medium containing high glucose.In the incubator,cultured for 24 hours,the optimal concentration of XFC-containing serum,XFC+AMD3100,AMD3100 was added to the XFC group,XFC+AMD3100 group and AMD3100 group.Place again in a standard incubator and collect cells 24 hours later.The expressions of CD62 p,CD40L and PDGFA were detected by immunofluorescence.The expressions of IL-1?,TNF-?,IL-4,IL-10,CD62 p,CD40L,PDGFA,VEGFA and VEGFR were detected by ELISA.SDF-1 mRNA was detected by qRT-PCR.,CXCR4 mRNA,VEGFA mRNA expression;WB detection of SDF-1,CXCR4,VEGFA,VEGFR protein expression.3 Results 3.1 AS patient clinical cohort study results 3.1.1 Use of Jianpi Huashi Yishen Tongluo Recipe The total number of adult medicines in 323 patients was 215,including 10 species,Duzhong Zhuanggu Pills 70 times(32.56%);Xinfeng Capsules 50 times(23.26%);Huangqi Qingre Decoction Capsules 19 times(8.83%);Lei Gong Rattan glycosides tablets 15 times(6.98%);cervical vertebrae blood circulation capsules 10 times(4.65%);Xinshuang tablets 10 times(4.65%);other classes 41 times(19.07%).The time for taking Chinese patent medicines was 0.5 to 50 months,with an average of(10.12 � 12.71)months.The total frequency of oral Chinese herbal decoction in 323 patients with ankylosing spondylitis was 556,including 13 traditional Chinese medicine decoctions,152 times(27.34%)of Si Miao San;92 times(16.55%)of Duhuo parasitic soup;Times(11.87%);Taohong Siwu Decoction 53 times(9.53%);Xuanyuan Decoction 23 times(4.14%);Anti-Wind Decoction 20 times(3.60%);Erchen Decoction 11 times(1.98%);Other Classes 139 times(25.00%).The application time of traditional Chinese medicine decoction was 0.25~50 months,with an average of(7.93�11.88)months.3.1.2 Frequency of application of Jianpi Huashi Yishen Tongluo Drug in each exposed group A total of 716 patients with AS were treated with spleen and phlegm drugs,including 116 in the non-exposed group,159 in the low-exposure group,90 in the middle-exposure group,and 351 in the high-exposure group.The exposure group was 69 times,the low-exposure group was 83 times,the middle-exposure group was 48 times,and the high-exposure group was 205 times;the Huoxuetongluo drug was 726 times,including 125 non-exposed groups,156 low-exposure groups,and 101 medium-exposure groups.High exposure group 344 times.3.1.3 Correlation between end point events and treatment of Jianpi Huashi Yishen Tongluo Recipe Four patients were followed up in the follow-up group,including 2 in the non-exposed group,1 in the low-exposure group,1 in the middle-exposed group,1 in the non-exposed group,and 1 in the low-exposure group..After 2 tests,the difference between exposure intensity and end point event was statistically significant(P<0.023).Logistic regression model showed that the exposure intensity,tangerine peel,mulberry parasitization and clematis factor were P<0.05,B<0 and OR value<1.3.2 AS patient data mining research results3.2.1 Core prescription for the treatment of AS Through the analysis of complex network technology,the core prescriptions for treating AS are: coix seed,medlar,yam,tangerine peel,licorice,clematis,valerian,Alisma,salvia,spatholobus,peach kernel,safflower,dandelion,white flower snake Tongcao,Eucommia,dog ridge.3.2.2 Treatment of traditional Chinese medicine use of AS The first 20 Chinese medicines used in the treatment of AS patients can be divided into the following four categories: Jianpi Huashi(31.61%),Huoxuetongluo(26.69%),Hurricane Dehumidification(20.09%),Yishen Heat medicine(21.61%).3.2.3 Cluster Analysis of the Compatibility of Traditional Chinese Medicine in Treating AS When the European distance is 20,the common Chinese medicine compatibility is 4 groups: the first group is peach kernel,safflower,salvia,tangerine peel,coix seed,clematis,yam,dandelion;the second group includes alfalfa,Alisma,and alfalfa.The third group has Eucommia ulmoides and dog ridges;the fourth group contains Hedyotis diffusa,live alone,stretched grass,and spatholobus.3.2.4 AS patients with platelet parameters Compared with the normal reference value,PLT increased by 579(52.16%),normal 505(45.5%),and decreased by 26(2.34%).Compared with the normal reference value,PCT increased by 643 cases(57.93%),normal 454 cases(40.9%),and decreased by 13 cases(1.17%).Compared with normal values,PDW increased by 102 cases(9.19%),normal 788 cases(70.99%),and decreased by 220 cases(19.82%).Compared with the normal value,MPV increased by 208 cases(18.74%),normal 645 cases(58.11%),and decreased by 257 cases(23.15%).3.2.5 Correlation analysis between platelet parameters and inflammatory immune markers in AS patients Correlation analysis showed that PLT was positively correlated with ESR;PLT was positively correlated with CRP and C4;PCT was positively correlated with ESR and IgG.The results of regression analysis of PLT showed that P<0.01 of CRP,OR>1;P<0.05 of C4,OR>1;regression analysis of PCT showed: P<0.05 of ESR,OR>1;P<0.01 of IgG,OR>1.3.2.6 Association analysis between platelet parameters and inflammatory immune markers The correlation with PLT is higher(confidence >60%,support >20%,and lift >1).The five indicators are CRP,C4,ESR,IgG,and C3.The correlation with the PCT is higher(confidence >60%,support >20%,and lift >1).The five indicators are ESR,CRP,C4,IgG,and C3.3.2.7 General condition of AS patients According to whether the treatment with spleen unit therapy(Chinese medicine + XFC+ Hibiscus cream),1110 patients with stable AS were divided into 435 patients with spleen unit,including 287 males and 148 females with an average age of 35.79�10.99 years.The average length of hospital stay was 17.56�10.42 days;490 patients in traditional Chinese medicine group,302 males and 188 females;the average age was 34.80�10.19 years old,and the average length of hospital stay was 16.75�8.15 days.3.2.8 The situation of laboratory indicators before and after treatment in the spleen group and the simple group After treatment,the PLT,ESR,CRP,IgG,C3,and C4 indexes of the spleen group decreased significantly,and the PCT and IgA indexes decreased.The PLT,ESR,CRP,and C3 indexes of the simple group decreased significantly,and the PCT,IgA,IgG,and C4 indexes decreased.After treatment,the PLT,PCT,ESR,CRP,and C3 decreased significantly in the spleen group.3.2.9 Association analysis between drugs and indicators of spleen unit therapy Confidence >60%,support >20% and lift >1.Xinfeng Capsule & Fuling & Chenpi associated with PLT decline,Xinfeng Capsule & Fuling & Salvia associated with CRP decline,Xinfeng Capsule & Dandelion & Clematis associated with ESR decline,Xinfeng Capsule & Fuling & Coix Seed associated with PCT decline,Xinfeng Capsule &fuling& Salvia associated with C4 decline,Xinfeng Capsule & Eucommia & Yam associated with MPV rise,Yam & Dandelion & Peach Kernel associated with PDW rise,Alisma & Clematis & Eucommia associated with IgA decline.3.2.10 Random walk evaluation of laboratory indicators after treatment in two groups The random walk model was used to evaluate the effects of platelet parameters PLT,PCT,and immunoinflammatory markers C4,ESR,and CRP in the spleen group and the simple group AS.For each group of patients,the PLT needs to walk 5.89 and 10.36 steps for each point of improvement.For each point of improvement of PCT,it is necessary to walk 5.36 and 6.79 steps.For each point of C4 improvement,it is necessary to walk 1.77 and 1.88 steps.For each improvement of ESR,it is necessary to walk.2.95,step 3.01;CRP needs to walk 2.37,2.83 for each point of improvement;or PLT comprehensive improvement rate is 16.99%,9.66% for each forward step;PCT comprehensive improvement rate is 18.64%,14.72%;C4 The overall improvement rate was 56.59% and 53.19%,respectively;the comprehensive improvement rate of ESR was 33.85% and 33.20%,respectively;the comprehensive improvement rate of CRP was 42.24% and 35.28%,respectively.3.3 Experimental results in vitro 3.3.1 General conditions of AS patients and healthy people 24 cases of peripheral blood samples from AS patients in the Department of Rheumatology and Immunology of Anhui Provincial Hospital of Traditional Chinese Medicine.There were 12 males and females;the average age was 31.66�8.76 years.Peripheral blood samples from the normal group were from 6 normal physical examinations in the hospital,3 in males and females;the average age was 29.79�9.69 years.3.3.2 MTT experiment: the effect of XFC on the inhibition of PBMCs Compared with the blank control group,the absorbance(A)of the middle and high dose XFC group decreased,and the middle dose XFC group A decreased significantly(P<0.01,P<0.05).The inhibitory effect of each dose of XFC group on PBMC was cultured for 24 hours.The inhibitory effect gradually increased,and at 48 h,the inhibitory effect decreased compared with 24 h.3.3.3 Correlation between platelet activation index and SDF-1/CXCR4-VEGFA pathway and cytokines Platelet activation index CD62 p was positively correlated with IL-1?,TNF-?,VEGFA,VEGFR,SDF-1 and CXCR4,and negatively correlated with IL-10.CD40 L was positively correlated with IL-1?,TNF-?,VEGFA,VEGFR,and SDF-1,and negatively correlated with IL-10;PDGFA and IL-1? TNF-?,VEGFA,VEGFR,SDF-1,CXCR4 were positively correlated with IL-10.(P<0.01)3.3.4 Expression of CD62 p,CD40L and PDGFA in each cell Compared with the normal group,the expression of CD62 p,CD40L and PDGFA in the model group increased.Compared with the model group,the expression of CD62 p,CD40L and PDGFA in the XFC group decreased.Compared with XFC group,CD62 p,CD40L and PDGFA were significantly decreased in AMD3100 group and XFC+AMD3100 group.Compared with AMD3100 group,CD62 p,CD40L and PDGFA decreased more significantly in XFC+AMD3100 group.(P <0.05,P<0.01)3.3.5 Expression of cytokines in each group Compared with the normal group,IL-1? and TNF-? in the peripheral blood mononuclear cells of the model group were significantly increased,IL-4 and IL-10 were significantly decreased;compared with the model group,IL-1?,TNF-? decreased,IL-4 and IL-10 increased in XFC group,AMD3100 group and XFC+AMD3100 group.Compared with XFC group,AMD3100 group,XFC+AMD3100 The levels of IL-1? and TNF-? were significantly decreased,and IL-10 was significantly increased.Compared with AMD3100 group,the decrease of IL-1? and TNF-? in XFC+AMD3100 group was more obvious,IL-4,IL-10 increased more significantly.(P <0.05,P <0.01)3.3.6 Platelet activation index and vascular endothelial growth factor expression in each group Compared with the normal group,CD62 p,CD40L,PDGFA,VEGFA and VEGFR were significantly increased in the model group.Compared with the model group,XFC group,AMD3100 group,XFC+AMD3100 group CD62 p,CD40L,PDGFA,VEGFA and VEGFR were decreased.Compared with XFC group,CD62 p,CD40L,PDGFA,VEGFA and VEGFR were significantly decreased in AMD3100 group and XFC+AMD3100 group.Compared with AMD3100 group,the decrease of CD62 p,CD40L,VEGFA and VEGFR in XFC+AMD3100 group was more significant.3.3.7 Expression of SDF-1,CXCR4 and VEGFA gene in each group Compared with the normal group,the expression levels of SDF-1,CXCR4 and VEGFA mRNA in the model group were significantly increased.Compared with the model group,XFC group,AMD3100 group and XFC+AMD3100 The mRNA levels of SDF-1,CXCR4 and VEGFA were decreased.Compared with XFC group,SDF-1,CXCR4 and VEGFA mRNA in AMD3100 group and XFC+AMD3100 group.The decrease was significant.Compared with AMD3100 group,the mRNA of SDF-1,CXCR4 and VEGFA was significantly decreased in XFC+AMD3100 group.(P<0.05,P<0.01)3.3.8 Expression of SDF-1?CXCR4?VEGFA and VEGFR proteins in each group Compared with the normal group,the expression levels of SDF-1,CXCR4,VEGFA and VEGFR in the model group were significantly increased.Compared with the model group,XFC group,AMD3100 group and XFC+AMD3100 The expressions of SDF-1,CXCR4,VEGFA and VEGFR protein were decreased.Compared with XFC group,AMD3100 group,XFC+AMD3100 group SDF-1,CXCR4,VEGFA,VEGFR,The expression of MMP9 protein was significantly decreased.Compared with AMD3100 group,the expression of SDF-1,CXCR4,VEGFA and VEGFR in XFC+AMD3100 group was significantly lower.(P<0.05,P<0.01)4 Conclusions(1)Jianpi Huashi Yishen Tongluo Recipe can reduce the risk of end-point events in patients,and long-term use can improve the prognosis of AS.(2)The core drugs for the treatment of AS in our hospital can be classified as Jianpi Huashi,Huoxuetongluo,Qufeng Dehumidification,and Yishen Qingre.(3)Patients with AS have platelet activation and are closely related to inflammatory immune responses.(4)XFC plays an important role in the improvement of platelet activation and inflammatory response in AS patients.The mechanism is to block SDF-1/CXCR4-VEGFA pathway,improve platelet activation,down-regulate IL-1?,TNF-?,up-regulate IL-4,IL-10,thereby reducing the inflammatory response.(5)XFC has a similar effect to the blocker AMD3100.