| 1. ObjectiveUnder the guidance of Chinese medicine theory, analyze and summarize the effect platelet activation in rheumatoid arthritis (RA) and Chinese mechanism; To observe the expression of platelet parameters in peripheral blood, platelet activating factor (PAF) in blood serum, platelet granule membrane protein140(GMP-140) and platelet cluster of differentiation40ligand (CD40L) in peripheral blood and thymus gland, CD40L mRNA in thymus gland, platelet ultrastructure, interleukin(IL)-1, IL-17, tumor necrosis factor (TNF)-α, IL-10in blood serum, change of body mass, voix pedis swelling, arthritis index(AI) of adjuvant arthritis (AA) rats, and the effects of Xinfeng Capsules (XFC) on the expression of them. Exploring the mechanism of Yiqi Jianpi Chinese traditional medicine XFC on AA rats based on platelet activation. Enriching the scientific connotation that the treatment of RA from "Spleen" Offering new thoughts to the mechanism study of RA treated with Chinese medicine.2. Methods2.1TheoryThrough the literature study, summarizing and analyzing the relationship between platelet activation and traditional Chinese medical blood stasis, insufficiency of the spleen, as well as RA. Explaining the main traditional Chinese medical pathogenesis of platelet activation in RA treated from the spleen.2.2Experimental StudyA total of40sprague dawley (SD) male rats were randomly divided into5groups:normal control (NC) group, model control (MC) group, methotrexate (MTX) group, tripterygium wilfordii polyglycoside tablet (TPT) group and XFC group, and there are8rats in each group; excluding NC group, the AA model was induced by intracutaneous injection of0.1ml Freund’s complete adjuvant in the right hindlimb. From the first19days after inflammation the appropriate drugs were gived for30days.①TPT group:TPT was grinded into fine powder and mixed with saline to blending. The rats were intragastric administrated with10ml/kg1time each day.②MTX group:MTX was grinded into fine powder and mixed wi th sal ine to blending. The rats were intragastric administrated with10ml/kg1time a week. They were given saline10ml/kg1time each day on the other days.③XFC group:XFC was grinded into fine powder without capsule and mixed with saline to blending. The rats were intragastric administrated with10ml/kg1times a day.④normal control group and model control group were given saline10ml/kg1times each day. The rats of each group were intragastric administrated for30d. During the experiment,body mass, voix pedis swelling and arthritis index (AI) of the rats were observated. And after the experiment platelet activation indexes were detected:the expressions of PAF, IL-1, IL-6, IL-17, TNF-α, IL-10in blood serum were measured with enzyme-linked immunosorbent assay. GMP-140and CD40L in peripheral blood were detected with flow cytometer. CD40L in thymus were detected with immunohistochemical method. CD40L mRNA in the thymus gland was detected with reverse transcription polymerase chain reaction (RT-PCR). GMP-140in thymus gland were detected with Western Blot(WB). Platelet ultrastructure was observed with transmission electron microscope (TEM).3. Results3.1Theoretical results3.1.1Platelet activation plays an important role in pathogenesis of RA:platelet activation and activation products promote the occurrence and development of joint synovitis, hyperplasia of synovial tissue, formation of pannus, damage of cartilage and bone.3.1.2Platelet activation is an important form of the essence of blood stasis syndrome:One of important forms of the essence of blood stasis syndrome is platelet activation. Platelet activation was participates in occurrence and development of blood stasis syndrome. It was the important pathophysiology foundation of blood stasis syndrome.3.1.3Blood stasis syndrome throughout the course of RA:Blood stasis was both the main pathogenic factor and pathological mechanism throughout the course of RA.3.1.4Insufficiency of the spleen was the important factor in generation of blood stasis syndrome:Transformation failure of spleen, lack the source, spleen without governing blood, Yang Xu Han Ning can cause blood stasis syndrome. the Chinese mechanism of platelet activation is insufficiency of the spleen. Platelet activation in RA should be treated from the spleen. 3.2Experimental results3.2.1The changes of platelet parameters, platelet activation indexes and platelet ultrastructure of AA rats:Compared with NC group, the expression of PLT, PCT in peripheral blood, PAF in blood serum, GMP-140and CD40L in peripheral blood and thymus gland, CD40L mRNA in thymus gland were significantly increased in MC group (P<0.01). Platelet ultrastructure was destroyed seriously.3.2.2The changes of the body mass, voix pedis swelling and AI of AA rats:The body mass of each group was no significant difference before phlegmasia (P>0.05). The body mass of model group was significantly decreased than NC group on1day before administration (P<0.05). Voix pedis swelling and AI of MC group was significantly increased than NC group on1day before administration (P<0.01).3.2.3The changes of IL-1, IL-6, IL-17, TNF-α, IL-10of AA rats: Compared with NC group, the expression of IL-1, IL-6, IL-17, TNF-a were significantly increased. And IL-10were significantly decreased in MC group (P<0.01).3.2.4The dependability between platelet activation indexes and platelet parameters, voix pedis swelling, AI, IL-1, IL-6, IL-17, TNF-α, IL-10of AA rats:PAF in blood serum, GMP-140and CD40L in peripheral blood and thymus gland, CD40L mRNA in thymus gland were positively correlation with PLT, PCT, voix pedis swelling and AI, IL-1, IL-6, IL-17, TNF-α. And they were negative correlation with IL-10(P<0.01or P<0.05).3.2.5Effect of XFC on platelet parameters, activation indexes: Compared with MC group, PLT, PCT in peripheral blood, PAF in blood serum, GMP-140and CD40L in peripheral blood and thymus gland, CD40L mRNA in thymus gland were significantly decreased in XFC(P<0.01or P<0.05). Compared with MTX-and TPT-treated groups, PAF in blood serum and GMP-140in thymus gland was significantly decreased (P<0.05). GMP-140in peripheral blood, CD40L mRNA in thymus gland showed a decreasing tendency with no significant difference (P>0.05).3.2.6Effect of XFC on platelet ultrastructure:Fine pseudopodium and small protuberance appeared on platelet surface of XFC group. The number of fine open pipe was few. Platelet alphagranule increased significantly than before. Most of mitochondrials was intact. Only a small number of it was swelling. Ultrastructure of XFC group was improved than MC, MTX and TPT groups.3.2.7Effect of XFC on the body mass, voix pedis swelling and AI: The body mass of AA rats in XFC group was significantly higher than that in MC group, MTX group and TPT group. While compared with NC group, there was no significant difference (P>0.05). Voix pedis swelling, AI of AA rats in treated groups were significantly lower than that of MC group (P<0.01or P<0.05). The treated groups showed no significant difference (P>0.05).3.2.7Effect of XFC on IL-1, IL-6, IL-17, TNF-α, IL-10:Compared with MC group, IL-1, IL-6, IL-17, TNF-α in blood serum were significantly decreased in XFC. And IL-10was significantly increased (P<0.01). Compared with MTX-treated and TPT-treated groups, IL-10was significantly increased. And IL-17showed a decreasing tendency with no significant difference (P>0.05).4Conclusions4.1Platelet activation plays an important role in the pathogenesis of RA. The Chinese medical mechanism of platelet activation is insufficiency of the spleen. Platelet activation in RA should be treated from the spleen.4.2Platelet of adjuvant arthritis rats is activating. It shows that the expression of PLT, PCT in peripheral blood, PAF in blood serum, GMP-140and CD40L in peripheral blood and thymus gland, CD40L mRNA in thymus gland were significantly increased.4.3Platelet activation has closely relation with inflammation. Platelet activation indexes PAF, GMP-140and CD40L have correlation with proinflammatory cytokine IL-1, IL-6, IL-17, TNF-α, anti-inflammatory factor IL-10, voix pedis swelling and AI.4.4XFC shows good control over general symptoms, the body mass, voix pedis swelling and AI of AA rat.5Mechanism of XFC on AA rats from platelet activation5.1XFC can decrease voix pedis swelling and AI through restraining inflammation induced by platelet activation and regulating cytokines in inflammation.5.2XFC can significantly decrease the expression of PLT, PCT in peripheral blood, PAF, GMP-140and CD40L in peripheral blood. So, it can decrease voix pedis swelling, AI through restraining inflammation induced by platelet activation.5.3XFC can significantly decrease the expression of GMP-140, CD40L and CD40L mRNA in thymus gland. So, it can decrease voix pedis swelling, AI through restraining inflammation induced by platelet activation.5.4XFC can improve platelet ultrastructure significantly. So, it can decrease voix pedis swelling, AI through restraining inflammation induced by platelet activation. |
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