Study Of Effects And Mechanism Of Jianpi Huashi Qingre Tongluo Therapy On Oxidative Stress In Ankylosing Spondylitis

1 Objective To analyze and summarize the etiology and pathogenesis of ankylosing spondylitis(AS)based on Xin'an medical theory of ‘spleen deficiency to causing arthralgia syndrome'.To analyze the effect of Chinese medicine on immune inflammatory biomarkers and superoxide dismutase(SOD)in treating ankylosing spondylitis(AS)patients based on data mining technology and association rules analysis.To research changes of oxidative Stress indexes,inflammatory/suppression of inflammatory cytokines,high coagulation state,AMPK-FOXO3 a and related laboratory parameters in patients with AS from the clinical research and in vitro research.And evaluate the protective effect of Traditional Chinese Medicine(TCM)Hungqin Qingre Chubi Capsule(HQC).Investigate the mechanism for improving oxidative stress state in AS patients.2 Methods2.1 Data mining research AS hospitalized patients were retrospectively studied from June 2012 to June 2016 in department of rheumatology,the first affiliated hospital of Anhui University of Chinese Medicine based on clinical data mining technology.The technology for complex networks was integrated to mine the core prescription.The frequency and the association rules of the prescription patterns were analyzed using the SPSS 21.0 and Aprior module in SPSS Clementine 12.0,The effect on immune inflammatory biomarkers and superoxide dismutase(SOD)were estimated by the association rules and random walk model.2.2 Clinical research AS patients were enrolled in the study and divided into HQC group and SASP group according to the random digits table.HQC group were treated with Hungqin Qingre Chubi Capsule(three capsules each time,three times a day)and associated with Fu Rong Ointment treatment for 12 weeks.SASP group were taken Sulfasalazine(four tablets each time,two times a day)and associated with Fu Rong Ointment treatment for12 weeks.30 healthy volunteers from health physical examination center,as normol group.Interleukin(IL)-1?,IL-4,IL-10,Tumor necrosis factor-alpha(TNF-?),superoxide dismutase(SOD),total antioxidant capacity(TAOC),Malondialdehyde(MDA),Lipid peroxidatio(LPO),peroxi some proliferator-activated receptor gamma(PPAR?),AMP-activated protein kinase(AMPK),Forkhead box O3a(FOXO3a),manganese superoxide dismutase(Mn SOD),CD36 were tested by enzyme-linked immunosorbent method(ELISA).The level of CD36 is detected by flow cytometry.the mRNA expresions of PPAR?,AMPK-?,FOXO3 a were detected by real-time fluorescence quantitative(Q-PCR).PPAR?,AMPK-?,FOXO3 a,P-FOXO3 a,Mn SOD protins were examined by Western blotting.Erythrocyte sedimentation rate(ESR)was detected by Instrument method.C-reactive protein were detected by Hitachi 7060 automatic biochemical analyzer.Observe the clinical symptoms and signs of AS patients by the questionnaire scale.The relationship between those indexes analyzed by using statistical software of SPSS 21.0.2.3 In vitro research Peripheral blood mononuclear cells(PBMCs)derived from AS patients and divided into model group,Compound C group,HQC+Compound C group,and HQC group,cultured in vitro.Control group derived from healthy people.IL-1?,IL-4,SOD,TAOC,MDA,LPO of culture supernatants were tested by ELISA.the mRNA expresions of AMPK-?,FOXO3 a,Mn SOD were detected by Q-PCR.AMPK-?,P-AMPK-?,FOXO3 a,P-FOXO3 a,Mn SOD protins were examined by Western blotting.The relationship between those indexes analyzed by using statistical software of SPSS 21.0.3 Results3.1 Dating mining research results3.1.1 SOD changes and its relationship with related indexes in AS patients Compared with the normal range [(129-216)mmol/L],SOD were lower and there are132 patients,accounting for 53.23% in AS patients.SOD were negatively correlated with ESR,CRP,Ig A,C3,C4(P<0.05).3.1.2 Chinese medicine used by our department of rheumatology By analyzing the herbal prescription,the core herbs of prescription were found as: Coix Seed,Rhizoma Cibotii,Pericarpium Citri Reticulatae,Cortex Eucommiae,Glycyrrhizae Radix et Rhizoma,Poria Cocos,Radix Clematidis,Semen Pruni Persicae,Salvia Miltiorrhiza,Herba Taraxaci,Oldenlandia,Rhizoma Alismatis,Rhizoma Dioscoreae,Flos Carthami,Herba Siegesbeckiae,The most commonly used in treatment were spleen invigorating herbals,which mainly distributed to spleen,stomach channel.Spleen invigorating herbals were high-related to herbs of promoting circulation and removing stasis herbs,clearing heat and removing toxicity and dispelling wind and eliminating dampness(the confidence coefficient were 95.78%,95.24%,94.50%).3.1.3 The association rules of herbals and immune inflammatory biomarkers Scutellaria baicalensis was high-related to hs-CRP,ESR and C4(the confidence coefficients were 78%,86.49% and 87.5%),Anemarrhena asphodeloides Bungeradix was high-related to Ig G and Ig M(the confidence coefficients were 85.71% and 70.59%);Scutellaria baicalensis was high-related to hs-CRP,C4 when compatibility with Rhizoma Dioscoreae and Salvia Miltiorrhiza(the confidence coefficients were 79.17%and 90%);Compatibility of Semen Pruni Persicae and Cortex Phellodendri Chinensis was associated with ESR(the confidence coefficients was 87.18%),and compatibility of Heracleumhemsleyanum and herba siegesbeckiae was associated with WBC(the confidence coefficients was 75.44%).Compatibilities of Anemarrhena asphodeloides Bunge with Poria Cocos and Cortex Eucommiae were high-related to Ig G and Ig M respectively(the confidence coefficients were 90% and 81.82%).3.1.4 The association rules of herbals and SOD The five high-related herbs to SOD were Coix Seed,Heracleumhemsleyanum,Rhizoma Dioscoreae,Clematis chinensis Osbeck,and herba siegesbeckiae.(the confidence coefficients were 92.31%,90%,84.62%,82.5% and 81.82%);The five high-related couplet herbs to SOD were found as: Coix Seed paired with Rhizoma Dioscoreae,Heracleumhemsleyanum paired with herba siegesbeckia,Coix Seed paired with Cortex Eucommiae,Coix Seed paired with Clematis chinensis Osbeck,Rhizoma Dioscoreae paired with Heracleumhemsleyanum(the confidence coefficients were100%,100%,95%,94.73% and 91.30%).3.1.5 Evaluation index of random walk model of hs-CRP and ESR in two groups The growth rate,improvement rate and improvement index of hs-CRP in research group were 0.28,28.04% and 0.49;The growth rate,improvement rate and improvement index of ESR in research group were 0.44,25.19% and 0.44,were higher than control group.3.2 Clinical research3.2.1 The oxidative Stress indexes and cytokines levels change in AS patients Detection of oxidative Stress index levels in 60 AS patients and 30 cases of normal persons.Compared with the normal group,TAOC,SOD,IL-4,IL-10 were significantly decreased and MDA,LPO,TNF-?,IL-1? were significantly increased in the AS patients(P<0.05).3.2.2 The related indexes of AMPK-FOXO3 a signaling pathway change in AS patients Detection of AMPK-FOXO3 a signaling pathway index levels in 60 AS patients and 30 cases of normal persons.Compared with the normal group,PPAR-?,AMPK,FOXO3 a,P-FOXO3 a,Mn SOD were significantly increased in the AS patients(P<0.05);Compared with the normal group,PPAR-?,AMPK-?,FOXO3 a mRNA were significantly decreased in the AS patients(P<0.05);Compared with the normal group,PPAR-?,AMPK-?,FOXO3 a proteins were significantly decreased in the AS patients(P<0.05).3.2.3 The CD36 changes in AS patients Compared with the normal group,CD36 was significantly decreased in the AS patients(P<0.05).3.2.4 Correlation analysis among oxidative Stress index in AS patients Correlation analysis showed that,SOD negatively correlated with BASDAI,BAS-G,VAS,ESR,hs-CRP and TNF-a,positively correlated with IL-4,PPAR-?,FOXO3 a and Mn SOD;TAOC negatively correlated with TNF-a,BASDAI,BAS-G,VAS and ESR,positively correlated with AMPK and CD36;LPO negatively correlated with IL-10,P-FOXO3 a and CD36,positively correlated with IL-1?;MDA negatively correlated with IL-4,PPAR-? and P-FOXO3 a,positively correlated with BASDAI,VAS,ESR,hs-CRP,TNF-a and IL-1?.(P<0.05)3.2.5 The Clinical therapeutic effect of HQC in AS patients HQC could significantly decrease BASDAI,BASFI,BAS-G,BASMI,VAS,ESR,hs-CRP of AS patients.The ASAS20 and BASDAI50 rates of reaching the standard were 83.33%(25/30),80.00%(24/30),BASDAI50 was significantly higher than that in SASP group(P<0.05)3.2.6 The effect of HQC on oxidative Stress indexes and cytokines in the AS patients Compared with before treatment,TAOC,SOD,IL-4,IL-10 were significantly increased,MDA,LPO,TNF-?,IL-1? were significantly decreased after HQC treatment(P<0.05).Compared with after SASP group,SOD and IL-10 was more significantly increased and MDA decreased in HQC group(P <0.05).3.2.7 The effect of HQC on AMPK-FOXO3 a signaling pathway indexes levels in the AS patients Compared with before treatment,PPAR-?,AMPK,FOXO3 a,P-FOXO3 a and Mn-SOD were significantly increased after HQC treatment(P<0.05).Compared with after SASP group,P-FOXO3 a and Mn-SOD was more significantly increased in HQC group(P<0.05);Compared with before treatment,mRNA encoding PPAR-?,AMPK-? and FOXO3 levels were more significantly increased after HQC treatment(P<0.05);Compared with before treatment,PPAR?,AMPK-?,FOXO3 a,P-FOXO3 a,Mn SOD protein levels were more significantly increased after HQC treatment(P<0.05);3.2.8 The effect of HQC on CD36 levels in the AS patients Compared with before treatment,CD36 was significantly increased after HQC treatment(P<0.05).3.3 In vitro research3.3.1 The effect of HQC containing serum on oxidative Stress indexes?cytokines in the AS patients PBMCs Compared with control group,IL-1?,MDA and LPO were significantly increased,IL-4,SOD and TAOC were significantly decreased in model group(P<0.01).Compared with model group,IL-1?,MDA and LPO were significantly increased,IL-4,SOD and TAOC were significantly decreased in Compound C group(P<0.05).Compared with model group,IL-1?,MDA and LPO were significantly decreased,IL-4,SOD and TAOC were significantly increased in HQC group(P<0.01).3.3.2 The effect of HQC containing serum on AMPK-FOXO3 amRNA in the AS patients PBMCs Compared with control group,AMPK,FOXO3 a,Mn SOD mRNA were significantly decreased in model group(P<0.01).Compared with model group,AMPK,FOXO3 a,Mn SOD mRNA were significantly decreased in Compound C group(P<0.05).Compared with model group,AMPK,FOXO3 a,Mn SOD mRNA were significantly increased in HQC group(P <0.01)3.3.3 The effect of HQC containing serum on AMPK-FOXO3 a proteins in the AS patients PBMCs Compared with control group,AMPK,P-AMPK,FOXO3 a,P-FOXO3 a,Mn SOD proteins were significantly decreased(P<0.01).Compared with model group,AMPK?P-AMPK,FOXO3 a,P-FOXO3 a,Mn SOD proteins were significantly decreased in Compound C group.Compared with model group(P<0.01),AMPK,P-AMPK,FOXO3 a,P-FOXO3 a,Mn SOD proteins were significantly increased in HQC group(P<0.01)4 Conclusions4.1 Dating mining research According to the analysis of the proportion of each herbal,our department proposed that the treatment of active type AS should mainly concentrate on regulating the spleen,and also effect on dispelling wind and eliminating dampness,clearing heat and removing toxicity,promoting circulation and removing stasis.Scutellaria baicalensis was high-related to hs-CRP,ESR,Coix Seed was high-related to SOD.Internal medicine coordinated with Furong ointment have random long-range connections,which have better curative effect than only internal medicine.4.2 There exists oxidative stress injury in the active AS patients4.2.1 The characteristics of oxidative stress injury in the active AS patients The biomarkers of oxidative stress injury MDA and LPO are significantly increased,while the antioxidase TAOC and SOD are significantly decreased.4.2.2 Inhibition of AMPK-FOXO3 a signaling pathway may cause oxidative stress injury in the active AS patients Inhibition of AMPK-FOXO3 a signaling pathway may cause the decrease of phosphorylation reaction,conceal the nuclear locating signals of Fox O3 a,inhibit transcriptional activity,and induce the accumulation reactive oxygen species and cell apoptosis,and finally increase oxidative stress injury.4.3 Effect of HQC on active AS patients HQC could reduce oxidative stress injury of the active AS patients and improving clinical symptoms and laboratory indexes,The BASDAI50 rates of reaching the standard are higher than SASP group.4.4 The potential mechanisms of HQC improving oxidative stress injury in AS patients4.4.1 HQC can improve the level of anti-inflammatory cytokines(IL-4,IL-10),reduce the level of inflammatory cytokines(IL-1?,TNF-?),regulate inflammatory reaction and improve oxidative stress injury,so as to improve the symptoms of AS patients;4.4.2 HQC can activate the PPAR? expression and adjust the level of CD36,regulate inflammatory reaction and improve oxidative stress injury,so as to improve the symptoms of AS patients;4.4.3 HQC can activate the PPAR? expression and increase the AMPK expression,inducing phosphorylation reaction of FOXO3 a,and increase the Mn SOD expression,regulate inflammatory reaction and improve oxidative stress injury,so as to improve the symptoms of AS patients.