| 1.ObjectiveTo research changes of the coagulation fibrinolytic indexes, quality of life,inflammatory/suppression of inflammatory cytokines, high coagulation state, the NF-κB signaling pathways, miR-155 and related laboratory parameters in patients with rheumatoid arthritis(RA) from the perspective of clinical and experiment in vitro cell. And evaluate the protective effect of traditional Chinese medicine(TCM)Xinfeng Capsule. Investigate the mechanism for improving the high coagulation state in RA patients.2 Methods2.1 Theoretical researchThrough the Chinese hownet database and Wan Fang database to retrieve a large number of literature, then we analysis the the relationship between blood stasis and spleen deficient in RA patients, summarize the medical etiology and pathogenesis and clinical curative effect of RA patients, elaborated the theory basis of blood stasis syndrome treated from the spleen in RA patients.2.2 Clinical Research60RA patients which choosed from Department of Rheumatology, Anhui Provincial Hospital of Chinese Medicine were randomly divided into observer group(XFC, 30cases), control group(LEF, 30 cases) and 20 healthy volunteers from anhui university first affiliated hospital of traditional Chinese medicine health physical examination center, as normol group.Interleukin-10(IL-10),IL-4, IL-17, IL-6, NF-κB activator 1(Act1), p50, p65, IκBα,platelet activating factor(PAF), platelet activating factor-platelet-activating factor acetylhydrolase(PAF-AH), TXB2, 6-keto-PGF1 a and anti-cyclic citrullinated peptide(CCP) were detected using ELISA. The number of platelet(PLT) was detected using Sysmex XT-2000 i automated hematology analyzer. D-dimer(D-D), thrombin time(TT), prothrombin time(PT), partial thromboplastin time(APTT) and fibrinogen(FBG) levels were detected using Sysmex CA-1500 automatic coagulation analyzer.Erythrocyte sedimentation rate(ESR) was detected using Westergren method.C-reactive protein and rheumatoid factor(RF) were detected using Hitachi 7060 automatic biochemical analyzer. Meanwhile, the m RNA expresions of Act1,p65,p50,IκBα and IκB kinase α(IKKα) were detected using semi-quantitative reverse transcription PCR. The m RNA expresion of Act1,p50 and p65 and miR-155 were detected using real time fluorescent quantitative PCR; Act1, p65, p50 and IκBαprotins were examined using Western blotting. Observe the quality of life and anxiety depression of RA patients by the questionnaire scale.2.3 Experimental Research in vitroCollected 10 ml peripheral blood of 15 RA patients which choosed from the selected 60 RA patients. Put in suitable amount of lymphocyte separation medium.And put into the high speed cylindrical centrifuge with 20℃,3000r/min, centrifuged15 minutes.Then add 1640 completely nutrient solution to culture.And add the LPS to stimulate for 24 hours, then add transfection reagent, miR-155 mimic or miR-155 mimic cntrol, XFC to cultrue.Then extracted RNA from PBMC. miR-155 were detected using real time fluorescent quantitative PCR; At the same time, collected the cell supernatant, IL-17,IL-6 were detected using ELISA.3 Results3.1 Theoretical researchThere is closely relationship between spleen deficient and the syndrome of blood stasis, Such as according to spleen deficient, the production of qi and blood decrease,and don’t transport the body fluid, as well as the functions of governing and warming, then results in the syndrome of blood stasis condition. And there is a inseparable relationship between clinical symptoms and the syndrome of blood stasis in RA patients, such as weight, swelling, joint pain, numbness, inconvenient flexion,joint around the nodules, subcutaneous ecchymosis, etc. The syndrome of blood stasisgo along with the course of the RA disease. If the disease can’t be cured for a long time,it can resrult in spleen deficient. So spleen deficient is an important factor for the productin of RA blood stasis.So protecting the spleen and stomach is very important for the treatment of RA.3.2 Clinical studies3.2.1 The coagulation index levels change in the peripheral blood.Detection of coagulation index levels in 60 cases of patients with RA and 20 cases of normal persons.Compared with the normal group, D-D, FBG and PLT were significantly increased in the peripheral blood with 60 RA patients( P<0.05).Abnormal cases were respectively 51, 34 and 24, respectively, accounting for 85%,56.67% and 40%. But TT, APTT and PT were not significantly different between the two groups( P>0.05).However, a difference was apparent; abnormal cases were respectively 2, 8, and 4, respectively, accounting for 3.33%, 13.33%, and 6.67%.3.2.2 The coagulation/anticoagulation factors change in the peripheral blood.Detection of coagulation/anticoagulation factors levels in 60 cases of patients with RA and 20 cases of normal persons. Compared with the normal group, PAF and TXB2 were significantly increased(P<0.05), while PAF-AH and 6-keto-PGF1 a were significantly decreased(P<0.05),in the peripheral blood with RA patients.3.2.3The proinflammatory/anti-inflammatory cytokines change in the peripheral blood.Detection of proinflammatory/suppression of inflammatory cytokines in 60 cases of patients with RA and 20 cases of normal persons. Compared with the normal group,IL-17 and IL-6 were significantly increased(P<0.05), while IL-10 and IL-6 were significantly decreased(P<0.05),in the peripheral blood with RA patients.3.2.4 Analysis of NF-κB signaling molecule m RNA in the peripheral blood.Compared with the normal group, m RNA encoding Act1, p65, p50, IκBa and Iκκ a levels were significantly increased in RA patient samples,the difference was statistically significantly(P <0.05).3.2.5 Analysis of NF-κB signaling pathway related proteins in the peripheral blood.Compared with the normal group, Act1, p50, p65 and IκBa related proteins were significantly increased in RA patient samples(P <0.05).3.2.6 The miR-155 changes in the peripheral blood.Compared with the normal group, miR-155 levels was significantly increased in RA patient samples(P <0.05).3.2.7 The coagulation indexes and coagulation/anticoagulation factors-related researchCorrelation analysis showed that, D-D positively correlated with PAF, DAS28,ESR, IL-6, IL-17, P65, joint pain, joint tenderness, morning stiffness, loss of appetite,joint weight, total integral of blood stasis, tongue and subcutaneous ecchymosis(P<0.05), negatively correlated with IL-10, PAF-AH(P<0.05). FBG positively correlated with TXB2, CCP, IL-6, joint weight and pulse condition(P<0.05),negatively correlated with IL-10(P<0.05). PLT positively correlated with PAF, DAS28,IL-6, P65, morning stiffness and total integral of blood stasis(P<0.05). APTT negatively correlated with ESR, CRP, CCP and subcutaneous ecchymosis(P<0.05).PT negatively correlated with total integral of blood stasis(P<0.05), positively correlated with IκBα(P<0.05). TT negatively correlated with CCP, RF, P50 and total integral of blood stasis(P<0.05). PAF positively correlated with D-D, PLT, IL-6, IL-17,P65, Act1, loose stool and total integral of blood stasis(P<0.05), negatively correlated with IL-4(P<0.05). PAF-AH negatively correlated with D-D, P65 and morning stiffness(P<0.05), positively correlated with IL-4 and IκBα(P<0.05). 6-keto-PGF1 a negatively correlated with ESR, joint pain and tongue(P<0.05). TXB2 positively correlated with FBG, DAS28 and IL-6(P<0.05).3.2.8 The Clinical therapeutic effect of XFC in RA patientsACR20 of LEF group and XFC group after treatment for fourth weeks, eighth weeks, Twelfth weeks were 15% and 26.7%, 41.7% and 58.3%, 85% and 85%,ACR50 were 3.3% and 3.3%, 11.7% and 18.3%, 41.7% and66.7%, ACR70 were 0%and 0%, 3.3% and 3.3%, 15% and 21.7%, chi square test the P value of >0.05,explains the difference between the two groups has no statistical significance between the ACR20/50/70.3.2.9The effect of XFC on the coagulation indexes and coagulation/anticoagulation factors.Compared with before treatment, D-D, FBG, PLT, PAF and TXB2 were significantly decreased, PAF-AH and 6-keto-PGF1 a were significantly increased after XFC treatment(P<0.05;P <0.05). D-D, FBG,and TXB2 were significantly decreased,6-keto-PGF1 a was significantly increased after LEF treatment, PLT,PAF and PAF-AH weren’t statistical difference(P >0.05). While APTT,PT and TT weren’t obviously changed in the tow groups(P >0.05). Compared with after LEF treatment,D-D, PLT, PAF and PAF-AH were significantly inproved(P <0.05).3.2.10 The effect of XFC on proinflammatory/anti-inflammatory cytokines.Compared with before treatment, IL-4 and IL-10 were significantly increased,IL-17 and IL-6 were significantly decreased after XFC treatment(P<0.05;P <0.05).Compared with after LEF treatment, IL-17 were significantly decreased in XFC group(P <0.05).3.2.11 The effect of XFC on NF-κB signaling molecule m RNA in the peripheral blood.Compared with before treatment, m RNA encoding Act1, p65, p50, IκBa and Iκκalevels were significantly decreased in RA patient samples(P <0.05). Compared with after LEF treatment, m RNA encoding p65 were significantly decreased in XFC group(P <0.05).3.2.12 The effect of XFC on NF-κB signaling pathway related proteins in the peripheral blood.Compared with before treatment, Act1, p50, p65 and IκBa protein levels were significantly decreased after XFC treatment(P<0.05). Compared with after LEF treatment, p50 and p65 related proteins were significantly decreased in XFC group(P<0.05).3.2.13 The effect of XFC on miR-155 levels in the peripheral blood.Compared with before treatment, miR-155 levels was significantly improved in tow groups in RA patient samples(P <0.05). But compared with after LEF treatment,there was no statistically significant difference(P >0.05).3.2.14 The effect of XFC on activity indexes in RA patients.Compared with before treatment, ESR, RF, CRP, CCP, DAS28 and Ig G were significantly decreased after XFC treatment(P<0.05). Compared with after LEF treatment, ESR and CRP were significantly decreased after XFC treatment(P<0.05).While compared with after XFC treatment, RF were significantly decreased after LEF treatment(P <0.05).3.2.15 The effect of XFC on blood stasis symptoms and signs in RA patients.Compared with before treatment, blood stasis symptoms and signs was significantly improved after XFC treatment(P <0.05);Compared with after LEF treatment, Joint tingling, tongue, subcutaneous ecchymosis and total integral of blood stasis were significantly decreased after XFC treatment(P <0.05), there was no statistically significant difference(P >0.05).3.2.16 The effect of XFC on quality of life in RA patients.Compared with before treatment, the quality of life with RA patients was significantly improved after XFC treatment( P<0.05).Compared with after LEF treatment, the quality of life total points, social function and healthy self-awareness ability were significantly improved(P<0.05).3.2.17 The effect of XFC on symptoms of TCM in RA patients.Compared with before treatment, joint pain, joint swelling, joint tenderness and morning stiffness were significantly improved after XFC treatment(P <0.05).And there were no difference between LEF group and XFC group after treatment(P>0.05).3.2.18 The effect of XFC on Symptomatic of spleen deficiency in RA patients.Symptomatic spleen deficiency was significantly improved after XFC treatment(P <0.05). But Loss of appetite, less gas lazy words were not improvement after LEF treatment(P >0.05). Compared with after LEF treatment, loss of appetite, lessgas lazy words, languid, stool thin pond were more obviously improved after XFC treatment(P <0.05).3.3 Experimental study in vitroXFC can restrain the mononuclear cells to secrete IL-6 and IL-17, and even have ability to inhibit the expression of miR-155 in monocytes, according to down-regulate the expression of miR-155 levels and inhibit the expression of IL-6 and IL-17 levels so as to inhibit mononuclear cells promote inflammation.4 Conclusion4.1 There exists a state of blood stasis, and its specific performance embodies in the blood stasis symptoms and signs of higher levels and the disorder of coagulation indexes in RA patients.4.2 Observe 60 cases of patients with RA,and find that blood coagulation indexes are increased, and it is significantly increased,comparing with the normal group.4.3 XFC improves the clinical curative effect of RA patients,and at the same time,it can improve the state of blood stasis, and it is better than the control group.4.4 XFC improved the state of blood stasis from the following tips:(1) XFC can adjust the balance of cytokine network level so as to improve the symptoms of blood stasis, and it is better than the control group.(2) XFC can adjust the level of the protein of NF-κB so as to improve the symptoms of blood stasis, and it is better than the control group.(3) XFC can down-regulate the level of miR-155 so as to improve the levels of cytokines and then improve the symptoms of blood stasis, and it is better than the control group.(4) XFC can adjust the level of blood stasis indicators so as to renew it, improve the symptoms of blood stasis, and it is better than the control group. |
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