| 1ObjectiveTo observe the expressions of related indexes including blood stasis, tumor necrosis factor-alpha(TNF-α), interleukin(IL)-4,IL-10,IL-17, nuclear factor kappa B(NF-κB) signal pathway, and micro RNA155(mi RNA155) in patients with active ankylosing spondylitis(AS). To observe the effect of Xin Feng Capsule(XFC) and explore potential mechanism of XFC improving blood stasis in active ankylosing spondylitis.2Methods2.1Theoretical ResearchThrough the study of ancient classical literature, we analyzed the relationship among blood stasis, spleen deficiency and kidney deficiency. To clarify the basic pathogenesis of blood stasis, and provide theoretical basis for the treatment of blood stasis based on spleen and kidney in active AS.2.2Clinical researchSixty patients were enrolled in the study and randomly divided into Xin Feng capsule group(XFC group)(n=30) and Sulfasalazine group(SASP group)(n=30) in active AS.All cases were treated with Xinfeng capsule three capsules each time three times a day for twelve consecutive weeks in XFC group.The others were taken Sulfasalazine four tablets each time two times daily for twelve consecutive weeks. Platelet(PLT) and coagulation routine were detected by Sysmex K-4500 automatic blood analyzer.Thromboxane B2(TXB2), 6- ketone-prostaglandin F1( 6-keto-PGF1), platelet granular membrane protein140(GMP140), platelet activation factor(PAF), vascular endothelial growth factor(VEGF), TNF-α, IL-4, IL-10, IL-17, nuclear factor activating agent-1(Act1), nuclear factor kappa B inhibitor protein-alpha(IκBα), nuclear factor kappa B kinase beta(IKKβ), nuclear factor protein 65(NF-κBp65),and nuclear factor protein 50(NF-κBp50) were detected by enzyme-linked immunosorbent method(ELISA). The m RNA expressions including Act1, IκBα, IKKβ, NF-κBp65, and NF-κBp50 were detected by real time polymerase chain reaction(RT-PCR).And the protein expressions of NF-κBp65 and NF-κBp50 were detected by western blotting. The expression of mi RNA155 was detected by one-step real-time fluorescence quantitative PCR detection kit. Erythrocyte sedimentation rate(ESR) was determinated by westergren method, and C reactive protein(CRP) was determinated by Hitachi 7060 automatic biochemistry analyzer.At the same time,observe clinical symptoms, scores of Traditional Chinese Medicine(TCM) symptom, scores of blood stasis syndrome, and quality of life in active AS. SPSS19.0 software was used to compare all dataes in different groups.3Results3.1Theoretical research results3.1.1 Theory study on blood stasis in ASBlood stasis was one of important pathogenic factors in the process of Bi zheng.Therefore, there was blood stasis before the process of Bi zheng. No blood stasis, no bi zheng. And blood stasis for a long time could cause Bi zheng.Pathogenic factors, like cold, wet, heat and so on,could cause Bi zheng. "There was blood stasis in Bi zheng ".Ankylosing spondylitis, owing to chronic and progressive diseases, had a longer course.And pathogenic factors easily came into AS. In summary, the pathogenic factors of blood stasis in AS were not only cold, wet, heat and so on, but also closely related to spleen and kidney.Modern researches showed that the formation of atherosclerosis and blood stasis mainly related to microcirculation disturbance, abnormal blood rheology study,disequilibrium of coagulation-fibrinolysis system, vascular skin cells and so on.3.1.2Treating blood stasis had clinical effect based on the spleen and kidney in active AS"Bi zheng" and "blood stasis" have an important influence with each other, which were closely related with spleen and kidney. In view of this, treating blood stasis in active AS should pay more attention to strengthen spleen and kidney.It could fill in kidney essence, and support healthy in eliminating pathogenic factors.On the other hand,it could help promot blood circulation to remove blood stasis and meridian obstruction.A lot of studies found that treatments of blood stasis were effective based on the spleen and kidney in AS.3.2Clinical research results3.2.1 Changes of PLT and coagulation routines in active ASCompared with the normal range [(125-300)×109/L], PLT was higher and there were45 cases(75%) in active AS.FBG was higher than the normal range [(2-4) g/L], and there were 40 cases(66.7%) in active AS. Compared to the normal range[(0-0.55)mg/L], D-D were higher and there were 37 cases(61.7%) in active AS.The average abnormal rate of them was 67.7%. According to the above method, the abnormal rates of PT, APTT and TT respectively were 8.3%, 6.7% and 5% in active AS.And the average abnormal rate of them was 6.7%.3.2.2 Changes of related indexes including coagulation-fibrinolysis system, and NF-κB signal pathway in active ASCompared with the normal group, TXB2,GMP140, PAF,VEGF,TNF-α,IL-17,Act1, IκBα, IKKβ, NF-κBp65,and NF-κBp50 were significantly increased, but 6-keto-PGF1, IL-4 and IL-10 were significantly decreased in active AS.Compared with the normal group, the m RNA expressions including IκBα, NF-κBp65, and NF-κBp50 were significantly increased in active AS.Compared with the normal group, the protein expressions of NF-κBp65 and NF-κBp50 were significantly higher in active AS.3.2.3Expressions of mi RNA155 and relationship with related indexes in active ASCompared with the normal group, expressions of mi RNA155 were higher in active AS.There had positively correlated with TNF-α, ESR, and NF-κBp65.3.2.4Correlation analysis among coagulation-fibrinolysis system, NF-κB signal pathway, and mi RNA155 in active ASPLT was positively correlated with TNF-α,IL-17, and NF-κBp65.FBG was positively correlated with NF-κBp50. D-D was positively correlated with mi RNA155.TXB2 was positively correlated with IL-17 and NF-κBp65. GMP140 was positively correlated with NF-κBp65.VEGF was positively correlated with mi RNA155.3.2.5Correlation analysis among coagulation-fibrinolysis system, diseases activities and clinical symptoms in active ASPLT and D-D were positively correlated with ESR, and BASDAI.TXB2 was positively correlated with BASDAI.GMP140 was positively correlated with ESR.VEGF was positively correlated with VAS and BASDAI.3.2.6Correlation analysis between coagulation-fibrinolysis system and scores of TCM symptom in active ASPLT was positively associated with body pain, spinal restriction, and total scores of TCM symptom. FBG was positively correlated with body pain. D-D was positively associated with soreness and weakness of waist and knees, and total scores of TCM symptom.GMP140 was positively associated with spinal restriction, and body trapped heavy. VEGF was positively associated with total scores of TCM symptom.3.2.7Correlation analysis between coagulation-fibrinolysis system and scores of blood stasis in active ASPLT was positively correlated with joint sting and total scores of blood stasis. FBG,was positively correlated with total scores of blood stasis.3.3Clinical efficacy of Xin Feng capsuleBASDAI50 in XFC group was significantly higher than that in SASP group. The total effective rate of TCM symptom in XFC group was 83.3%, which was significantly higher than that in SASP group.3.4Xin Feng capsule had the following effects in active ASXin Feng capsule not only significantly decreased PLT, FBG, D-D, TXB2, GMP140,PAF, VEGF, but also increased 6-keto-PGF1 to improve blood stasis in active AS.Xin Feng Capsule not only significantly decreased IL-17, IκBα,NF-κBp65, NF-κBp50, the m RNA expressions inclduing Act1, IκBα, NF-κBp65, and NF-κBp50, the protein expressions of NF-κBp65 and NF-κBp50, but also significantly increased IL-4 and IL-10. So Xin Feng Capsule could inhibit activation of NF-κB signal pathway, and adjust imbalance of cytokines and coagulation-fibrinolysis system.Xinfeng Capsule significantly decreased mi RNA155 expression.Xin Feng capsule could significantly decrease BASDAI, BASFI, and BAS-G to improve clinical symptoms in active AS.Xin Feng capsule could significantly reduce body fatigue, spinal restriction, morning stiffness, soreness and weakness of waist and knees, body trapped heavy, less words and lazy, eating less and anorexia, abdominal distension, and total scores of TCM symptom to improve TCM symptom in active AS.Xin Feng capsule could significantly reduce joints sting, lips color, subcutaneous ecchymosis and total scores of blood stasis syndrome to improve blood stasis.Xin Feng capsule could significantly increase the score of role limitation due to physical problems(RP), society function(SF), body pain(BP), vitality(VT), and mental health(MH) to improve quality of life in active AS.4Conclusion4.1Manifestation of blood stasis in active ASCompared with the normal range, the abnormal rates of PLT, FBG, and D-D respectively were 75%, 66.7%, and 61.7% in active AS patients;There was blood stasis in active AS, which mainly manifested by the abnormal increase of PLT, FBG and D-D.4.2Effect of XFC in active AS patientsXinfeng capsule were similar to SASP in ASAS20, and improving clinical symptoms and laboratory indexes. But XFC could significantly improve total scores of TCM symptoms, total scores of blood stasis, and mental health(MH).4.3The potential mechanism of Xin Feng capsule improving blood stasis in active ASXin Feng capsule regulated imbalance of cytokines and coagulation-fibrinolysis system.Xin Feng capsule could significantly inhibit activation of NF-κB signal pathway, and reduce the expression of mi RNA155 to futher regulate imbalance of cytokines and coagulation-fibrinolysis system. |
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