| 1 ObjectiveTo research changes of platelet microparticles(PMPs)、Cytokine and PI3 K / AKT signaling pathway in Rheumatoid arthritis(RA) patients and rats, evaluate the protective effect of XFC, investigate the mechanism for reducing PMPs in RA.2 Methods2.1 Experimental research72 male SD rats were randomly divided into those six groups: normal control group(NC), model control group(MC), Xinfeng Capsule High Dose group(XFCH),Xinfeng Capsule Medium Dose group(XFCM), Xinfeng Capsule Low Dose group(XFCL), Methotrexate group(MTX), with 12 in each group. Except the NC group, each rat was injected in the right rear paw intradermally with Freund’s complete adjuvant to induce inflammation and thus obtain AA model. The rats were given the dose from the19 th days after inflammation, and the dosage was equivalent to 10 times the clinical dosage. all the rats were continuously dosed once per day for 30 days.To observe the activity state and behavior of AA rats, measure body mass, paw swelling and arthritic index, PMPs, cytokines(IL-6、VEGF、IL-10), expression of PI3 K and Akt1/2/3m in abdominal aorta tissues; electron microscope observation of PLT; observing rat synovium tissue pathological changes were observed under the microscope. To analyze the effects of XFC on AA rats.2.2 Clinical Research60 cases of RA patients which choosed from Anhui Provincial Hospital of Chinese Medicine were randomly divided into XFC group(20 cases),MTX group(20cases),XFC+MTX group(20 cases) and normal group(20 cases). To observe changes of age, gender, course, quality of life, mental health and other laboratory indicators of 60 cases RA patients. Using the biochemical analyzer instrument to detect RA patients’basic biochemical indexes, ELISA detect IL-1、IL-4、IL-6、IL-10、TNF-α、VEGF,CD61 and CD41(the markers of PMPs)detected by flow cytometry.To analyze the improvement of PMPs in RA,compare DAS28, ACR20\50\70, SF36, SAS, SDS scores,and related laboratory parameters(ESR, etc.) before and after treatment. Using statistical software of SPSS17.0 to analyze the relationship between those indexes.3 Results3.1 Experimental results3.1.1 The change of AA Rat quality, paw swelling and AI&the impact of XFCBody weights of rats in each group before modeling, paw swelling and AI have no statistical significance. The first 19 administration, compared with the NC, MC,XFCL, XFCM, XFCH, MTX group rat paw swelling and AI were significantly increased after inflammation(P <0.01), There was no difference(P> 0.05) between MC group and each treatment group. Continuous administration of 30 days, compared with the NC group, MC group and each rat paw swelling and AI treatment group remained elevated, weight loss,(P <0.01 or P <0.05); Compared with MC group, the treatment group rat paw swelling and AI significantly reduced weight gaining,(P <0.05 or P<0.01); compared with the MTX group, XFCM group volume increased toe swelling and AI decreased, but the difference was not statistically significance.3.1.2 AA rats articular pathology’s change and the effect of XFC on itObserving toe joints HE staining biopsy AA rats in each group by light microscope:NC rats synovial tissue structure is clear, no proliferation, projection, no inflammatory cell infiltration, cell arrangement is regularly, less hierarchical, articular cartilage and bone structure is intact; MC group rats’ synovial seen a large number of inflammatory cell infiltration, obvious synovial tissue and synovial pannus, synovial lining cells increased stratification, blurred articular surface, partial articular cartilage is rough surface or absent; XFCM group rats ’synovial joint&blood vessel wall can be seen a small amount of inflammatory cell infiltration, Compared with MC group,synovial tissue structure is significantly improved, the degree of proliferation is reducing, the articular surface is orderly; Compared with XFCM group,the MTX group rats’ synovial lining cells is thickening, part of the articular cartilage can see pannus shaping, articular surface is less structured; XFCL and XFCH groups’ joint can see the infiltration of inflammatory cells in the synovium and surrounding tissue, pannus synovial hyperplasia, synovial lining cells thickening, and the obscure articular surface,destruction of cartilage and bone.3.1.3 AA rat serum PMPs’ change and XFC’s effect on itCompared with NC group, MC, MTX, XFCL, XFCH groups’ PMPs were significantly increased, And the difference is statistically significant(P <0.01), XFCM group’s PMPs increased slightly, but there is no statistically significant; Compared with MC group, MTX, XFCL, XFCM, XFCH groups’ PMPs were significantly decreased(P<0.01); Compared with the MTX group, XFCM group’s PMPs is reducing(P <0.05),However there is no significant difference between XFCL and XFCH group.3.1.4 Effect of serum cytokines IL-6, IL-10, VEGF in AA rats after treated by XFC capsuleCompared with NC group, MC, XFCL, XFCH, MTX group, IL-6, VEGF were significantly increased, IL-10 was significantly reduced(P <0.01), XFCM group’s IL-6 was increased, IL-10 reduced(P <0.01), VEGF was increased, but the difference has no statistically significant; compared with MC group, XFCL, XFCM, XFCH, MTX group’s IL-6, VEGF was obviously decreased, IL-10 was significantly increased(P<0.05 or P < 0.01); Compared with the MTX group, XFCM group’s VEGF was significantly reduced(P <0.05), and the difference is no statistically significant.3.1.5 The change of PI3 K, AKT AA rat abdominal aorta and XFC’s effectIn rat aortic tissue, compared with the NC group, MC, MTX, XFCL, XFCM,XFCH groups’ PI3 K, AKT was significantly increased(P <0.01); Compared with MC group, MTX, XFCL, XFCM, XFCH groups’ PI3 K, AKT was significantly reduced(P<0.01); Compared with the MTX group, XFCL, XFCM, XFCH groups’ PI3 K was significantly increased(P <0.01), AKT also increased(P <0.01 or P <0.05).3.1.6 AA rat platelet ultrastructure change and XFC’s impactElectron microscope: NC platelet is oval, surface smooth, rare pseudopodia,scattered α particles in the cytoplasm, mitochondria and vesicular open piping system; MC platelet extend pseudopodia, some of it shedded shaping vesicular PMPs,the cytoplasmic significant damaged, α particles in the cytoplasm, mitochondria decreased; Compared with MC group, MTX group’s projections platelet pseudopodia is reduced, α particles&mitochondria is increased, partly fat swelling and vacuolar degeneration; XFCL have more projections platelet pseudopodia, α particles,mitochondria increased comparing with MC group, but is less than MTX group, portion swelling, vacuolar degeneration; XFCM has no platelet pseudopodia, basic saved oval structure, α particles, mitochondria significantly is higher than the MTX group, few vacuoles; XFCH has few platelet pseudopodia, α particles&mitochondria were increased significantly.3.2 Clinical studies3.2.1 PMPs and cytokines changes in RA patientsDetection of PMPs and cytokines(IL-1、IL-4、IL-6、IL-10、TNF-α、VEGF) in20 cases of normal persons and 60 cases of patients with RA.Compared with the normal group, the amount of RA group’s PMPs、IL-1、IL-6、TNF-αand VEGF were higher than the normal range group,and the amount of RA group’s IL-4 、 IL-10 were decreased significantly(P<0.01).3.2.2 Study on the relationship between the PMPs and cytokines in the RA patientsCorrelation analysis showed that the negative correlation between PMPs and IL-4、IL-10(P<0.05).PMPs and IL-1,IL-6,TNF- α,VEGF, PLT, CRP, ESR were positively correlated. Joint pain, Joint swelling, joint tenderness were positively correlated with PMPs、IL-1、IL-6、TNF-α,and negatively correlated with IL-4,IL-10; morning stiffness were positively correlated with PMPs,IL-1,IL-6; Quality of life integral scores and PMPs positively correlated;DAS28 and PMPs were positively correlated.3.2.3 Clinical therapeutic effect of three groups in the treatment of RAACR50 of 12 weeks was P<0.05, MTX+XFC group was higher than XFC group and MTX group.3.2.4 Comparison of three groups to the PMPs and cytokines in the RA patientsPMPs, IL-1, IL-6, TNF-α, VEGF in three groups after drug treatment were significantly reduced, and IL-4、IL-10 improved. After treatment, Compared with MTX group, MTX+XFC group’s PMPs、IL-6、TNF-α reduced(P<0.01),IL-4、I L-10increased(P<0.05 或(P<0.01); and XFC group’s IL-1、VEGF increased(P<0.05).3.2.5 Changes of laboratory indexes of three RA groupsAfter treatment Three groups of ESR, CRP, PLT decreased significantly. TCM symptoms and signs of the integral and total integral, the integral quality of life; HAQ,DAS28, SAS, SDS were significantly reduced(P <0.05 or P <0.01);and after treatment MTX + XFC group’s ESR, CRP and PLT decreased more(P<0.05).Meanwhile,Joint pain, swelling, less gas lazy words, loss of appetite were improved significantly(P<0.05). AQT, social function, mental function, healthy self-awareness ability were also improved(P <0.05 or P <0.01),HAQ, DAS28, SDS was significantly reduced r(P<0.05); XFC group’s less gas lazy words, loss of appetite decreased too(P <0.05).4 Conclusion4.1 AA rats appear weight lossing and the similar to RA’s structure of joint inflammation XFC can improve the paw edema, reduce arthritis index, Reducing synovial inflammation, pannus proliferation inhibition, and no adverse effects on the growth of rat4.2 Platelet activation and PMPs’ releasing existed in AA rat,XFC can reduce the expression of PMPs and inhibit the activation state of platelet.4.3 XFC can significantly improve the levels of Anti-inflammatory factors and reduce the levels of proinflammatory factors and Vasoactive factors.4.4 XFC can effectively regulate AA rat aortic tissue PI3 K / Akt signaling pathway level, and superior to MTX.4.5 PMPs can promote the formation of joint synovitis and synovial pannus reaction,and play an important role in the development of RA by promoting.4.6 By downing PI3 K / Akt pathway,XFC can reduce PMPs positive expression rate, maintain the balance of cytokine network, thereby improving RA synovitis and pannus proliferation, achieve the purpose of treatment of RA.4.7 In RA patients,PMPs was significantly higher than normal group,the rise of proinflammatory factors and Vasoactive factors and the decrease of Anti-inflammatory factors relevant with the release of PMPs.4.8 PMPs disease activity in patients with RA and the joint symptoms was directly proportional.4.9 XFC can significantly reduce the expression of PMPs in RA patients.4.10 In RA patients,XFC can significantly improve the level of Anti-inflammatory factors and reduce the expression of proinflammatory factors and Vasoactive factors.4.11 XFC can significantly improve local and systemic symptoms, quality of life and mental state of patients with RA and improving the less gas lazy words, loss of appetite aspects is better than MTX. |
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