Effects And Mechanism Reaserch Of Xinfeng Capsule On Inproving Blood Stasis Of Sjogren’s Syndrome Based On The Signal Pathway Of MiR-155/NF-κB

1 ObjectiveTo analysis and summarize the TCM etiology and pathogenesis of the formation of blood stasis in Sjogren’s syndrome(SS) patients under the guidance of Chinese traditional medicine theory about “theatment of the Bizheng from the spleen”. A clinical trial observed the changes of coagulation parameters, blood stasis symptoms, signs and symptoms, quality of life, anxiety and depression, peripheral cytokines, NF-кB signaling pathway proteins and related laboratory indexes in SS patients, to evaluate the influence of Chinese patent medicine Xinfeng capsule(XFC) on them, so as to explore the mechanism for improving blood stasis in patients with SS. 2 Methods 2.1 Theoretical studyThrough the study of a large number of ancient physicians’ about literatures,the relationship between “spleen-qi deficient”and the formation of blood stasis in SS patients was analyzed.The TCM etiology and pathogenesis of the formation of blood stasis was summed up,and the TCM theoretical basis about theatment of the formation of blood stasis in SS patients from the spleen was interpreted. 2.2 Clinical study66 cases of SS patients treated in the Department of Rheumatology, Anhui Province Hospital of traditional Chinese Medicine in March 2014 to April 2015 were randomly divided into Xinfeng capsule group(treatment group) and hydroxychloroquine group(control group), each group of 33 cases.In addition, 20 cases which exclude autoimmune diseases through physical examination and immunological tests, physical health, no organic diseases was selected as normal control group.Sysmex Corporation CA7000 automatic coagulation analyzer is used to detect the coagulation parameters, including prothrombin time(PT), activated partial thromboplastin time(APTT), fibrinogen(FIB), thrombin time(TT) and D-dimer(D-D). Tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-10(IL-10), interleukin-4(IL-4), nuclear factor-кBp65(NF-кB p65), nuclear factor-кB p50(NF-кB p50), nuclear factor-κB inhibitor protein(IкBα) are detected by using ELISA kits.The expression level of NF-кB p65, NF-κB p50, Iкα m RNA were measured by real-time quantitative(RT-PCR),and the level of Micro RNA-155(miR-155) was determined by one-step fluorescence quantitative PCR.Erythrocyte Sedimentation Rate(ESR) was evaluated by westergren method.Using Hitachi 7060 automatic biochemical analyzer to determine Hypersensitive c-reactive protein(hs-CRP).Questionnaire was used to observe the score of blood stasis symptom, symptom, quality of life, anxiety, depression and other points. The above indexes were used the statistical software SPSS17.0 for analysis. 3 Results 3.1 Theoretical study results 3.1.1 Sjogren’s syndrome is closely related with blood stasisRelationship between SS and blood stasis is very close, blood stasis can lead to the occurrence of Sjogren’s syndrome, Sjogren’s syndrome can also lead to the formation of blood stasis, the two reinforce each other, cementation is difficult to distinguish. 3.1.2 Spleen-qi deficient is the basis of the incidence of Sjogren’s syndromeSS is closely related to spleen, lung and kidney, and the relationship with the spleen is the most critical,as the spleen can transport of water cereal essence for the source of blood and qi, and the spleen-qi can spread this nutrients to various parts of the body. Meanwhile, the spleen "opens into the mouth", the essence of the spleen can be transported up to the mouth by way of the Spleen Meridian of Foot-Taiyin,leading salivary fluid, thereby promoting secretion of the salivary glands. 3.1.3 Spleen-qi deficient is the key pathogenesis of the formation of blood stasis in patients with SSAlthough the formation of blood stasis in SS patients for various reasons, such as weakness, dryness, depression, long duration, and affected by many factors, but the spleen-qi plays the most important role, spleen-qi deficient is the key pathogenesis of the formation of blood stasis in patients with SS. Spleen-qi deficient, lack of source, blood is not sufficient,stagnant blood flow.Spleen-qi deficient, shipped wet dereliction of duty, clustered into phlegm, hinder the movement of Qi.Spleen-qi deficient, loss of control,not popular along the blood vessels,overflow extravascular.Spleen-qi deficient, middle energizer cold blood lack warmth, flowing slowly, eventually leading to the formation of blood stasis state. 3.2 Clinical study results 3.2.1 Changes of the coagulation parameters in patients with SSIn patients with SS, at least one abnormal coagulation parameters of a total of 47 cases, accounting for 71.21% of the 66 cases of tested SS patients.Among them, the highest abnormal rate is D-D(33 cases, 50.00%), then followed by FIB(21 cases, 31.82%), TT(9 cases, 13.64%), APTT(3 cases, 4.55%), PT(2 cases, 3.03%).Two parameters were abnormal at the same time is 14 cases, three cases were abnormal at the same time is 14 cases, four and five parameters were abnormal at the same time are 0 case.Compared with the normal control group,D-D,FIB were significantly increased in SS group,while TT,PT,APTT were not found obviously different(P<0.05 or P<0.01). 3.2.2 Changes of peripheral blood cytokines, NF-kappa B pathway protein, inflammation indexes in patients with SSCompared with normal control group, the level of peripheral blood cytokines IL-4,IL-10 was significantly decreased,cytokines IL-1β,TNF-α,inflammation indexes like hs-CRP,ESR and pathway protein P50,P65,IкBα were significantly increased in SS group(P<0.05 or P<0.01). 3.2.3 Correlation research of the coagulation parameters with ankylosing spondylitis in patients with SSCorrelation analysis showed as follows:Coagulation parameter PT was negatively correlated with the score of tongue nature;FIB was positively correlated with the score of joint tingling,lip color, tongue nature,pulse conditions;D-D was positively correlated with the score of lip color, tongue nature,pulse conditions(P<0.05 or P<0.01).Coagulation parameter D-D was significantly positively correlated with the course of disease(P<0.01).Coagulation parameters FIB was negatively correlated with the static salivary flow rate, while positively correlated with the ESSDAI score;and coagulation parameters D-D was positively correlated with the ESSDAI score(P<0.05 or P<0.01).Coagulation parameters FIB was positively correlated with the score of dry eyes, palpitations score; and coagulation parameters D-D was positively correlated with the score of dry eyes, and sallow complexion(P<0.05 or P<0.01).Coagulation parameter APTT was positively correlated with physical pain score;and coagulation parameters FIB was positively correlated with anxiety scale score,while negatively correlated with the score of general health, social function score;and coagulation parameter D-D was positively correlated with anxiety scale score and depression scale score,while negatively correlated with the score of general health, emotional function score(P<0.05 or P<0.01).Coagulation parameter APTT was negatively correlated with P65;and FIB was positively correlated with TNF-α,P50,P65,ESR,hs-CRP, while negatively correlated with IL-4; and TT was negatively correlated with TNF-α;and D-D was positively correlated with TNF-α,IL-1β,P65,ESR,hs-CRP, while negatively correlated with IL-4,IL-10(P<0.05 or P<0.01). 3.2.4 Effect of XFC in patients with SSAfter treatment,compared with control group,the clinical cure rate, significant effect rate and total effective rat were no obvious difference(P>0.05),but the effective rate was significantly higher in XFC group than that in control group(P<0.05). 3.2.5 Effect of XFC on the coagulation parameters in patients with SSCompared with before treatment, the level of FIB,D-D were significantly decreased in the two groups after treatment(P<0.05 or P<0.01);and the effect on reducing the level of D-D of XFC group is significantly better than that in HCQ group(P<0.05). 3.2.6 Effect of XFC on the score of blood-stasis symptom in patients with SSCompared with before treatment, the score of blood-stasis were significantly decreased to varying degrees in the two groups after treatment.The total integral of blood-stasis symptom and the score of scaly dry skin, lip color, the substance of the tongue and pulse conditions in XFC group were significantly reduced(P<0.05 or P<0.01);and the effect of XFC group on reducing the total integral of blood-stasis symptom and the score of lip color, the substance of the tongue and pulse conditions are significantly better than that in control group(P<0.05 or P<0.01). 3.2.7 Effect of XFC on the clinical signs and the TCM symptomg scores in patients with SSCompared with before treatment, the score of TCM symptoms,like dry mouth and throat, dry eyes, loose tooth, tired body fatigue, sallow complexion, anorexia, shortness of breath were significantly reduced in HCQ group,while the static salivary flow rate was increased,and the score of dry mouth and throat, dry eyes, loose tooth, tired body fatigue, sallow complexion, anorexia, palpitation, shortness of breath, loose stool wese significantly reduced in XFCgroup;and the effect on increasing the static salivary flow rate, reducing the score of dry mouth and throat, dry eyes, loose tooth, tired body fatigue, sallow complexion, anorexia are significantly better than that in HCQ group(P<0.05 or P<0.01). 3.2.8 Effect of XFC on the scores of SF-36,SAS,SDS in patients with SSCompared with before treatment, the score of each dimension of SF-36 were increased to varying degrees in the two groups after treatment,while the score of SAS,SDS wese reduced; and the effect of XFC group is better than that of HCQ group(P<0.05 or P<0.01). 3.2.9 Effect of XFC on the inflammation indxes in patients with SSCompared with before treatment, the level of the inflammation indxes like ESR, hs-CRP and the score of ESSDAI wese reduced in the two groups after treatment;and the effect on reducing the level of ESR,hs-CRP and the score of ESSDAI were significantly better than that in HCQ group(P<0.05 or P<0.01). 3.2.10 Effect of XFC on peripheral miR-155 in patients with SSCompared with before treatment, the lev of peripheral miR-155 was reduced in the two groups after treatment(P<0.01),and the curative effect of XFC group is better than that of HCQ group(P<0.05). 3.2.11 Effect of XFC on peripheral cytokines in patients with SSCompared with before treatment, the level of IL-1β,TNF-a were significantly increased,while the level of IL-4, IL-10 were significantly improved in the two groups after treatment(P<0.05 or P<0.01),and the effect on reducing the level of TNF-a, improving the level of IL-10 are significantly better than that in HCQ group(P<0.05 or P<0.01). 3.2.12 Effect of XFC on NF- kappa B singling pathway in patients with SSResults with ELISA showed: compared with before treatment, the level of peripheral P50, P65, IκBα were significantly reduced in the two groups after treatment(P<0.05 or P<0.01), and the effect of XFC group on reducing the level of P50, P65 is better than that of HCQ group(P<0.05 or P<0.01).Results with RT-PCR showed: compared with before treatment, the expression frequency of P50, P65, IκBα m RNA were significantly reduced in the two groups after treatment(P<0.05 or P<0.01), and in the aspect of reducing the expression frequency of P50, P65, IκBα m RNA, the effect of XFC group is better than that of HCQ group(P<0.05 or P<0.01).Results with Western Blotting showed: compared with before treatment, the expression frequency of NF-κB singling pathway proteins like P50,P65 were significantly reduced in the two groups after treatment,while the expression frequency of SOCS-1 protein was increased(P<0.05 or P<0.01),and the effect of XFC group on reducing the expression frequency of P50,P65,and increasing the expression frequency of SOCS-1 were better than that of HCQ group(P<0.05 or P<0.01). 4 Conclusion 4.1 Blood-stasis is impaired in patients with SS,and the spleen-qi deficient has important consequences for the formation of blood-stasis. 4.2 The formation of blood stasis in patients with SS may be related to the abnormal activation of miR-155/NF- kappa B signaling pathway 4.3 XFC can significantly improve the blood-stasis and clinical symptoms of patients with SS. 4.4 The mechanisms of XFC on improving the blood stasis and the clinical symptoms of patients with SS may be: 4.4.1 Reduce inflammation indexes,relieve the deposition of acute proteins and immune complexes in blood vessels,and improv the blood-stasis. 4.4.2 Increase anti-inflammatory cytokines, reduce pro-inflammatory cytokines, reducing the injury on vascular endothelial cell,there maintaining the stability of bloog coagulation/fibrinolysis system,improve blood-stasis. 4.4.3 Inhibit the NF-kappa B signaling pathway directly, and keep the balance of inflammation- cytokines- blood-stasis network, reduce the continuously damage on vascular endothelial cell, improve blood-stasis. 4.4.4 Reduce the expression of miR-155, elevate the expression of SOCS-1,further inhibit the excessive activation of NF-kappa B signaling pathway, improve blood-stasis.