基于血小板活化研究新风胶囊对佐剂性关节炎大鼠的作用机制
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摘要
1目的
以中医理论为指导,分析、总结血小板活化在类风湿关节炎(rheumatoid arthritis, RA)发病中的作用及其中医机制；通过动物实验,观察佐剂性关节炎(adjuvant arthritis, AA)大鼠血清血小板活化因子(platelet activating factor, PAF),外周血血小板参数、血小板α颗粒膜糖蛋白(granule membrane protein140, GMP-140)、血小板分化抗原40配体(cluster of differentiation40ligand, CD40L),胸腺GMP-140、CD40L、CD40L mRNA,血小板超微结构的变化及体质量、足跖肿胀度、关节炎指数(arthritis index, AI)、白细胞介素(interleukin, IL)-1、IL-6、IL-17、肿瘤坏死因子(tumor necrosis factor, TNF)-α、IL-10的变化和健脾益气中药新风胶囊(Xinfeng Capsules, XFC)对AA大鼠的疗效及上述指标的影响,并基于血小板活化探讨XFC对AA大鼠的作用机制。从而丰富RA“从脾论治,整体调节”科学内涵,为中医药治疗RA的机理研究提供新的思路。
2方法
2.1理论研究
通过文献研究,总结、分析血小板活化与中医血瘀证、RA、中医脾虚的关系。从中医理论角度,阐释RA血小板活化“从脾论治”的机制。
2.2实验研究
将40只SD (sprague dawley, SD)雄性大鼠按随机数字表分为5组：正常对照(normal control, NC)组、模型对照(model control, MC)组、雷公藤多甙片(tripterygium wilfordii polyglycoside tablet, TPT)组、甲氨喋呤(methotrexate, MTX)组和XFC组,每组8只,除NC组外,向每只大鼠右后足跖皮内注射弗氏完全佐剂(freund's complete adjuvant, FCA)0.1ml致炎,复制成AA大鼠模型,于致炎后第19天开始给予相应药物治疗,①TPT组：将TPT研成细末,加生理盐水配制混匀,按10ml/kg灌胃,每天1次。②MTX组：将MTX研成细末,加生理盐水配制混匀,按10ml/kg灌胃,每周1次；未给药的实验日,给予生理盐水灌胃,10ml/kg,每天1次。③XFC组：将XFC去除胶囊壳研成细末,加生理盐水配制混匀,按10ml/kg灌胃,每天1次。④NC组及MC组予生理盐水灌胃,10ml/kg,每天1次,连续用药30天。实验过程中观察各组大鼠的体质量、足跖肿胀度、AI。实验结束后检测大鼠各指标：采用酶联免疫吸附(enzyme-linked immunosorbent assay, ELISA)法检测各组大鼠血清PAF、IL-1、IL-6、IL-17、TNF-α、IL-10的表达,流式细胞仪检测大鼠外周血GMP-140、CD40L表达,免疫组化法检测胸腺CD40L的表达,逆转录-聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR)法检测胸腺CD40L mRNA的表达,免疫蛋白印迹(western blotting, WB)法检测胸腺GMP-140的表达,透射电镜观察血小板超微结构的变化。
3结果
3.1理论研究结果
3.1.1血小板活化在RA的发生、发展中起重要作用：血小板及其活化产物促进了RA滑膜炎症的发生与发展、滑膜组织的增生、血管翳的形成、软骨及骨破坏。
3.1.2血小板活化是中医血瘀证本质的重要表现：血瘀证本质表现之一为血小板活化,血小板活化参与了血瘀证的发生与发展,是血瘀证产生的重要生理、病理基础。
3.1.3血瘀证贯穿于RA病程的始终：血瘀在RA发病过程中既可成为主要的致病因素,又可作为主要的病理机制而贯穿于整个疾病的始终。
3.1.4脾虚是RA血瘀证产生的重要因素：脾虚引起的运化失职、化源匮乏、脾不统血、阳虚寒凝均可致瘀。
RA血小板活化的中医机制在于脾虚,应当“从脾论治”。
3.2实验研究结果
3.2.1从大鼠血小板参数、活化指标及超微结构的变化：与NC组比较,Mc组AA大鼠血清PAF,外周血PLT、PCT、GMP-140、CD40L,胸腺GMP-140、 CD40L、CD40L mRNA表达显著升高(P<0.01),血小板超微结构破坏严重。
3.2.2从大鼠体质量、足跖肿胀度、AI的变化：致炎前,各组大鼠的体质量比较,差异无统计学意义(P>0.05)；给药前1d,与Nc组相比,Mc组大鼠体质量明显减轻(P<0.05),足跖肿胀度、AI显著升高(P<0.01)。
3.2.3从大鼠细胞因子IL-1、IL-6、IL-17、TNF-α、IL-10的变化：与NC组相比,MC组大鼠IL-1、IL-6、IL-17、TNF-α明显升高(P<0.01),IL-10显著降低(P<0.01)。
3.2.4从大鼠血小板活化指标与血小板参数、足跖肿胀度、AI、IL-1、 IL-6、IL-17、TNF-α、IL-10的相关性：AA大鼠血清PAF,外周血GMP-140、 CD40L,胸腺GMP-140、CD40L、CD40L mRNA与PLT、PCT、IL-1、IL-6、 IL-17、TNF-α、足跖肿胀度、AI呈正相关,与IL-10呈负相关(P<0.01或P<0.05)。
3.2.5XFC对从大鼠血小板参数及活化指标的影响：与MC组相比,XFC可以显著降低AA大鼠血清PAF,外周血PLT、PCT、GMP-140、CD40L,胸腺GMP-140、CD40L、CD40L mRNA的表达水平(P<0.01或P<0.05)；与MTX组、TPT组相比,XFC组血清PAF、胸腺GMP-140表达水平显著降低(P<0.05),外周血GMP-140、胸腺CD40L mRNA呈下降趋势,但差异无统计学意义(P>0.05)。
3.2.6XFC对从大鼠血小板超微结构的影响：XFC组血小板表面伸出细伪足或小的突起,开放管道细,数量少,血小板α颗粒较前明显增多,线粒体大部分完好,少数肿胀,超微结构较Mc组、MTX组、TPT组有所改善。
3.2.7XFC对AA大鼠体质量、足跖肿胀度、AI的影响：XFC组大鼠的体质量显著高于Mc组、MTX组及TPT组(P<0.01或P<0.05),与Nc组无显著差异(P>0.05)；MTX组、TPT组、XFC组三个治疗组AA大鼠足跖肿胀度、AI显著低于Mc组(P<0.01),各治疗组间相比,足跖肿胀度、AI差异无统计学意义(P>0.05)。
3.2.8XFC对细胞因子IL-1、IL-6、IL-17、TNF-α、IL-10表达的影响：XFC能够降低血清IL-1、IL-6、IL-17、TNF-α表达,升高IL-10(P<0.01),与MTX组、TPT组相比,XFC组IL-10显著升高(P<0.05),IL-17呈下降趋势,但差异无统计学意义(P>0.05)。
4结论
4.1血小板活化在RA的发生、发展中发挥重要作用,其中医机制在于脾虚,应当“从脾论治”。
4.2AA大鼠存在血小板活化,表现为血清PAF,外周血PLT、PCT、GMP-140、 CD40L及胸腺GMP-140、CD40L、CD40L mRNA表达升高。
4.3AA大鼠血小板活化与炎症密切相关,表现为血小板活化指标PAF、 GMP-140、CD40L与致炎因子IL-1、IL-6、IL-17、TNF-α,抑炎因子IL-10及足跖肿胀度、AI具有相关性。
4.4XFC对AA大鼠体质量、足跖肿胀度、AI具有良好的调控作用。
5基于血小板活化XFC对AA大鼠的作用机制
5.1XFC能通过抑制AA大鼠血小板活化而调节细胞因子在炎症中的表达,从而降低足跖肿胀度、AI。
5.2XFC能通过降低AA大鼠血清PAF,外周血PLT、PCT、GMP-140、CD40L的表达,抑制血小板活化介导的炎症反应,从而降低足跖肿胀度、AI。
5.3XFC能通过降低AA大鼠胸腺GMP-140、CD40L、CD40L mRNA的表达,抑制血小板活化介导的炎症反应,从而降低足跖肿胀度、AI。
5.4XFC能通过改善AA大鼠血小板超微结构,抑制血小板活化介导的炎症反应,从而降低足跖肿胀度、AI。
1. Objective
Under 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. Methods
2.1Theory
Through 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 Study
A 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. Results
3.1Theoretical results
3.1.1Platelet 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 results
3.2.1The 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).
4Conclusions
4.1Platelet 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 activation
5.1XFC 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.
以中医理论为指导,分析、总结血小板活化在类风湿关节炎(rheumatoid arthritis, RA)发病中的作用及其中医机制；通过动物实验,观察佐剂性关节炎(adjuvant arthritis, AA)大鼠血清血小板活化因子(platelet activating factor, PAF),外周血血小板参数、血小板α颗粒膜糖蛋白(granule membrane protein140, GMP-140)、血小板分化抗原40配体(cluster of differentiation40ligand, CD40L),胸腺GMP-140、CD40L、CD40L mRNA,血小板超微结构的变化及体质量、足跖肿胀度、关节炎指数(arthritis index, AI)、白细胞介素(interleukin, IL)-1、IL-6、IL-17、肿瘤坏死因子(tumor necrosis factor, TNF)-α、IL-10的变化和健脾益气中药新风胶囊(Xinfeng Capsules, XFC)对AA大鼠的疗效及上述指标的影响,并基于血小板活化探讨XFC对AA大鼠的作用机制。从而丰富RA“从脾论治,整体调节”科学内涵,为中医药治疗RA的机理研究提供新的思路。
2方法
2.1理论研究
通过文献研究,总结、分析血小板活化与中医血瘀证、RA、中医脾虚的关系。从中医理论角度,阐释RA血小板活化“从脾论治”的机制。
2.2实验研究
将40只SD (sprague dawley, SD)雄性大鼠按随机数字表分为5组：正常对照(normal control, NC)组、模型对照(model control, MC)组、雷公藤多甙片(tripterygium wilfordii polyglycoside tablet, TPT)组、甲氨喋呤(methotrexate, MTX)组和XFC组,每组8只,除NC组外,向每只大鼠右后足跖皮内注射弗氏完全佐剂(freund's complete adjuvant, FCA)0.1ml致炎,复制成AA大鼠模型,于致炎后第19天开始给予相应药物治疗,①TPT组：将TPT研成细末,加生理盐水配制混匀,按10ml/kg灌胃,每天1次。②MTX组：将MTX研成细末,加生理盐水配制混匀,按10ml/kg灌胃,每周1次；未给药的实验日,给予生理盐水灌胃,10ml/kg,每天1次。③XFC组：将XFC去除胶囊壳研成细末,加生理盐水配制混匀,按10ml/kg灌胃,每天1次。④NC组及MC组予生理盐水灌胃,10ml/kg,每天1次,连续用药30天。实验过程中观察各组大鼠的体质量、足跖肿胀度、AI。实验结束后检测大鼠各指标：采用酶联免疫吸附(enzyme-linked immunosorbent assay, ELISA)法检测各组大鼠血清PAF、IL-1、IL-6、IL-17、TNF-α、IL-10的表达,流式细胞仪检测大鼠外周血GMP-140、CD40L表达,免疫组化法检测胸腺CD40L的表达,逆转录-聚合酶链反应(reverse transcription polymerase chain reaction, RT-PCR)法检测胸腺CD40L mRNA的表达,免疫蛋白印迹(western blotting, WB)法检测胸腺GMP-140的表达,透射电镜观察血小板超微结构的变化。
3结果
3.1理论研究结果
3.1.1血小板活化在RA的发生、发展中起重要作用：血小板及其活化产物促进了RA滑膜炎症的发生与发展、滑膜组织的增生、血管翳的形成、软骨及骨破坏。
3.1.2血小板活化是中医血瘀证本质的重要表现：血瘀证本质表现之一为血小板活化,血小板活化参与了血瘀证的发生与发展,是血瘀证产生的重要生理、病理基础。
3.1.3血瘀证贯穿于RA病程的始终：血瘀在RA发病过程中既可成为主要的致病因素,又可作为主要的病理机制而贯穿于整个疾病的始终。
3.1.4脾虚是RA血瘀证产生的重要因素：脾虚引起的运化失职、化源匮乏、脾不统血、阳虚寒凝均可致瘀。
RA血小板活化的中医机制在于脾虚,应当“从脾论治”。
3.2实验研究结果
3.2.1从大鼠血小板参数、活化指标及超微结构的变化：与NC组比较,Mc组AA大鼠血清PAF,外周血PLT、PCT、GMP-140、CD40L,胸腺GMP-140、 CD40L、CD40L mRNA表达显著升高(P<0.01),血小板超微结构破坏严重。
3.2.2从大鼠体质量、足跖肿胀度、AI的变化：致炎前,各组大鼠的体质量比较,差异无统计学意义(P>0.05)；给药前1d,与Nc组相比,Mc组大鼠体质量明显减轻(P<0.05),足跖肿胀度、AI显著升高(P<0.01)。
3.2.3从大鼠细胞因子IL-1、IL-6、IL-17、TNF-α、IL-10的变化：与NC组相比,MC组大鼠IL-1、IL-6、IL-17、TNF-α明显升高(P<0.01),IL-10显著降低(P<0.01)。
3.2.4从大鼠血小板活化指标与血小板参数、足跖肿胀度、AI、IL-1、 IL-6、IL-17、TNF-α、IL-10的相关性：AA大鼠血清PAF,外周血GMP-140、 CD40L,胸腺GMP-140、CD40L、CD40L mRNA与PLT、PCT、IL-1、IL-6、 IL-17、TNF-α、足跖肿胀度、AI呈正相关,与IL-10呈负相关(P<0.01或P<0.05)。
3.2.5XFC对从大鼠血小板参数及活化指标的影响：与MC组相比,XFC可以显著降低AA大鼠血清PAF,外周血PLT、PCT、GMP-140、CD40L,胸腺GMP-140、CD40L、CD40L mRNA的表达水平(P<0.01或P<0.05)；与MTX组、TPT组相比,XFC组血清PAF、胸腺GMP-140表达水平显著降低(P<0.05),外周血GMP-140、胸腺CD40L mRNA呈下降趋势,但差异无统计学意义(P>0.05)。
3.2.6XFC对从大鼠血小板超微结构的影响：XFC组血小板表面伸出细伪足或小的突起,开放管道细,数量少,血小板α颗粒较前明显增多,线粒体大部分完好,少数肿胀,超微结构较Mc组、MTX组、TPT组有所改善。
3.2.7XFC对AA大鼠体质量、足跖肿胀度、AI的影响：XFC组大鼠的体质量显著高于Mc组、MTX组及TPT组(P<0.01或P<0.05),与Nc组无显著差异(P>0.05)；MTX组、TPT组、XFC组三个治疗组AA大鼠足跖肿胀度、AI显著低于Mc组(P<0.01),各治疗组间相比,足跖肿胀度、AI差异无统计学意义(P>0.05)。
3.2.8XFC对细胞因子IL-1、IL-6、IL-17、TNF-α、IL-10表达的影响：XFC能够降低血清IL-1、IL-6、IL-17、TNF-α表达,升高IL-10(P<0.01),与MTX组、TPT组相比,XFC组IL-10显著升高(P<0.05),IL-17呈下降趋势,但差异无统计学意义(P>0.05)。
4结论
4.1血小板活化在RA的发生、发展中发挥重要作用,其中医机制在于脾虚,应当“从脾论治”。
4.2AA大鼠存在血小板活化,表现为血清PAF,外周血PLT、PCT、GMP-140、 CD40L及胸腺GMP-140、CD40L、CD40L mRNA表达升高。
4.3AA大鼠血小板活化与炎症密切相关,表现为血小板活化指标PAF、 GMP-140、CD40L与致炎因子IL-1、IL-6、IL-17、TNF-α,抑炎因子IL-10及足跖肿胀度、AI具有相关性。
4.4XFC对AA大鼠体质量、足跖肿胀度、AI具有良好的调控作用。
5基于血小板活化XFC对AA大鼠的作用机制
5.1XFC能通过抑制AA大鼠血小板活化而调节细胞因子在炎症中的表达,从而降低足跖肿胀度、AI。
5.2XFC能通过降低AA大鼠血清PAF,外周血PLT、PCT、GMP-140、CD40L的表达,抑制血小板活化介导的炎症反应,从而降低足跖肿胀度、AI。
5.3XFC能通过降低AA大鼠胸腺GMP-140、CD40L、CD40L mRNA的表达,抑制血小板活化介导的炎症反应,从而降低足跖肿胀度、AI。
5.4XFC能通过改善AA大鼠血小板超微结构,抑制血小板活化介导的炎症反应,从而降低足跖肿胀度、AI。
1. Objective
Under 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. Methods
2.1Theory
Through 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 Study
A 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. Results
3.1Theoretical results
3.1.1Platelet 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 results
3.2.1The 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).
4Conclusions
4.1Platelet 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 activation
5.1XFC 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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