Study On Cohort Study Data Mining And Mechanism Of Inhibiting Platelet Activation With Jianpi Huashi Tongluo Method In Treating Knee Osteoarthritis

1 Objective Using cohort study to explore the factors affecting the end point events in patients with knee osteoarthritis(KOA);using data mining techniques to explore the changes of platelet count(PLT)in patients with KOA and its relationship with immune inflammatory markers,and to explore the spleen of Chinese medicine Unit therapy improves the efficacy of KOA;changes in cytokines,platelet activation products,and VEGF/SDF-1/CXCR4 signaling pathways in KOA patients are studied by in vitro culture of peripheral blood mononuclear cells from KOA patients,based on VEGF/SDF-1/CXCR4 Signaling pathway to investigate the mechanism of Xinfeng Capsule(XFC)drug-containing serum inhibiting platelet activation in patients with KOA.2 Methods 2.1 Cohort study The KOA patients admitted to the Department of Rheumatology from June 2012 to 2016 were recruited using the case system of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine.Telephone follow-up was used to record the patient’s taking of Chinese medicine after discharge and the end event.Subsequently,Epidata 3.02 software was used to establish a database of KOA patient cohort in our hospital,and the follow-up data was compiled and uploaded.Statistical analysis was performed using SPSS 23.0 software.2.2 Data Mining Research Using SQL Server as the database software,the “Anhui Provincial Hospital of Rheumatology Data Center” was created to collect general information,course of disease,use of Chinese medicine and laboratory examination information before and after treatment.The data was processed and analyzed using the Aprior module in SPSS23.0 and Clementine 11.1.2.3 Experimental study in vitro High,medium and low doses of XFC-containing serum were prepared for use.KOA peripheral blood was collected,PBMC isolated.The optimal inhibitory concentration and optimal action time of XFC-containing serum on PBMC were detected by MTT assay.TNF-,IL-1,IL-4,IL-10,VEGFA and VEGFR2 were detected by ELISA.The expressions of SDF-1,CXCR4,VEGFA and HIF-1 were detected by RT-PCR.VEGFA and PDGF were detected by immunofluorescence.Expression of CD40L;Western-blot assay for expression of VEGFA,VEGFR2,SDF-1,CXCR4 protein.3 Results 3.1 Results of a cohort study of 1100 cases of KOA patients with endpoint events 3.1.1 End event situation During the follow-up period,the total number of four types of endpoint events was 89,including 12 all-cause deaths,45 re-admissions,12 extra-articular lesions,and 20 surgical treatments.3.1.2 Use of proprietary Chinese medicines and traditional Chinese medicines During the follow-up period,Chinese herbal medicines including Xinfeng Capsule,Huangqi Qingre Budai Capsule,Wuwei Wentong Quyu Capsule,etc.;Chinese herbal decoctions include Si Miao San,Tao Hong Si Wu Tang,Du Huo Jiu Decoction and so on.3.1.3 Logistic regression between endpoint events and each indicator TCM treatment was a protective factor for all-cause death(OR=0.552),re-entry(OR=0.552),extra-articular lesions(OR=0.519),surgical treatment(OR=0.584),age(OR= 2.652),ESR(OR=1.656)was a risk factor for all-cause death.ESR(OR=2.206),PLT count(OR=1.431),and Ig G(OR=1.509)were risk factors for re-admission,ESR(OR=1.854,OR=2.548)is a risk factor for extra-articular lesions and surgical treatment.3.2 1203 cases of KOA patients clinical data mining results 3.2.1 Changes in PLT counts in KOA patients The PLT count of 1203 patients with KOA was increased by 452(37.57%)compared with the normal value(100-300×109/L).Correlation analysis showed that PLT count was associated with ESR,hs-CRP,Ig A,Ig G,Ig M.There was a positive correlation between complement C3 and complement C4(P<0.01).3.2.2 The use of Chinese medicine 1203 patients with KOA used Chinese medicine during hospitalization,involving more than 300 flavors.The top 20 Chinese medicines with the highest frequency of use were used.The spleen and dampness categories: Coix seed,wolfberry,dried tangerine peel,Chinese yam,Alisma orientale;Qingrejiedu: Dandelion,Hedyotis diffusa,Astragalus,Anemarrhena,licorice;blood circulation and phlegm: peach kernel,safflower,spatholobus stem,salvia miltiorrhiza,Chuanxiong;hurricane dehumidification: Clematis,valerian,Eucommia,glutinous grass,solitary.Through the complex network and cluster analysis,the above drugs can also be used as a common drug for treating KOA.3.2.3 Analysis of association rules Using association rules to mine the association between KOA drugs and the association between indicators and drugs,the top three drug pairs with the highest confidence were: Fu Ling associated with Yi Yiren(96.11%),and Tao Ren associated with Yi Yiren(91.42%),Pu Gongying is associated with Yi Yiren(89.29%).The PLT count decreased and decreased with Fu Ling(95.58%),the PLT count decreased and decreased with Yi Yiren(95.18%),and the Ig M index decreased with Yi Yiren(94.38%). 3.2.4 Effect of TCM spleen unit therapy on PLT count and immune inflammatory index in patients with KOA Traditional Chinese medicine combined with XFC was superior to traditional Chinese medicine group in improving PLT count,ESR,Ig A,Ig G,Ig M and complement C3 levels(P<0.01).The traditional Chinese medicine combined with XFC group is superior to the simple Chinese medicine group in terms of random fluctuation maximum,walking positive growth rate,comprehensive evaluation index increasing rate,comprehensive improvement rate,and comprehensive evaluation index recording number,indicating patient PLT count,ESR,hs-CRP There is a long-term relationship between the improvement of indicators and the treatment of traditional Chinese medicine.The longer the intervention time of Chinese medicine,the more obvious the improvement of patient indicators.3.3 Experimental study results in vitro 3.3.1 Inhibition of PBMC by XFC-containing serum The drug-containing serum in the XFC medium dose group had the best inhibition time and dose for PBMC at 24 h.3.3.2 Effects of XFC-containing serum on cytokines and VEGFA,VEGFR2,PDGF and CD40 L in KOA patients Compared with the normal group,TNF-ɑ,IL-1β,VEGFA,VEGFR2,PDGF,CD40 L were increased(P<0.01),IL-4 and Il-10 were decreased(P<0.01).Compared with the model group,TNF-ɑ,IL-1β,VEGFA,VEGFR2,PDGF,CD40 L were decreased(P<0.01),IL-4 and Il-10 were increased in each drug intervention group(P<0.01).Compared with XFC group,XFC+AMD3100 TNF-ɑ,IL-1β,VEGFA,VEGFR2,PDGF,CD40 L decreased(P<0.01),IL-4 and Il-10 increased(P<0.01),AMD3100 group TNF-ɑ,IL-1β,VEGFA,VEGFR2 decreased(P<0.05,P<0.01),IL-4,Il-10 increased(P<0.01); compared with AMD3100 group,XFC+AMD3100 group TNF-ɑ,IL-1β,VEGFA,VEGFR2,PDGF CD40 L decreased(P<0.05,P<0.01),IL-4 and Il-10 increased(P<0.01).3.3.3 Effects of XFC-containing serum on SDF-1 m RNA,CXCR4 m RNA,VEGFA m RNA and HIF-1ɑ m RNA in KOA patients Compared with the normal group,SDF-1 m RNA,CXCR4 m RNA,VEGFA m RNA and HIF-1ɑ m RNA were increased in each model group(P<0.01).Compared with the model group,SDF-1 m RNA,CXCR4 m RNA,VEGFA m RNA and HIF-1ɑ m RNA decreased after drug intervention.(P<0.05,P<0.01);compared with XFC group,CXCR4 m RNA,VEGFA m RNA and HIF-1ɑ m RNA decreased in AMD3100 group and XFC+AMD3100 group(P<0.01);compared with AMD3100 group,XFC+AMD3100 group VEGFA m RNA,HIF-1ɑ m RNA decreased(P<0.01).3.3.4 Effect of XFC-containing serum on the expression of VEGFA,PDGF and CD40 L protein in KOA patients Under inverted fluorescence microscopy,VEGFA,PDGF,and CD40 L proteins were highly expressed in the normal group,and the expression was significantly decreased in KOA patients.After intervention with XFC,AMD3100,and XFC+AMD3100,VEGFA,PDGF,and CD40 L were increased.Protein expression.3.4.5 Effect of XFC-containing serum on the expression of SDF-1,CXCR4,VEGFA,VEGFR2 and MMP9 proteins in KOA patients Compared with the normal group,the expression of SDF-1,CXCR4,VEGFA,VEGFR2,and MMP9 protein in the model group was increased(P<0.01).Compared with the normal group,SDF-1,CXCR4,VEGFA,VEGFR2 in each group after drug intervention.The expression of MMP9 protein was decreased(P<0.01).Compared with XFC group,the expressions of SDF-1,CXCR4,VEGFA,VEGFR2 and MMP9 were decreased in AMD3100 group and XFC+AMD3100 group(P<0.01).Compared with AMD3100 group,XFC was compared.The expression of SDF-1,CXCR4,VEGFA,VEGFR2 and MMP9 protein in AMD3100 group was significantly decreased(P<0.01).4 Conclusion Patients with KOA have elevated platelet counts,which are closely related to im mune inflammation and are also risk factors for KOA endpoint events.Jianpi Hu ashi Tongluo Chinese medicine can reduce platelet and immune inflammation lev els.Platelet activation in KOA patients is associated with activation of the VEGF/SD F-1/CXCR4 signaling pathway.The mechanism by which XFC improves platelet activation in KOA patients and immune inflammation response are as follows:(1)By up-regulating anti-inflammatory factors(IL-4,IL-10)and down-regulating p ro-inflammatory factors(TNF-ɑ,I-1β),the immune inflammatory response is reduc ed,joint destruction is alleviated,and clinical symptoms are alleviated.(2)Not only can it exert a blocking agent-like effect,but synergistic blockers can better block the VEGF/SDF-1/CXCR4 signaling pathway,thereby inhibiting platelet activation and improving clinical symptoms.