基于TGF-β1/Smads和ERK通路cross-talk研究新风胶囊改善AA大鼠肺功能的机理
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摘要
1目的
在中医“肺脾相关”理论指导下，以“肺痹”为切入点，分析、总结类风湿关节炎（RA）肺功能损伤的中医学病机，从动物实验角度系统观察佐剂关节炎（AA）大鼠足跖肿胀度、关节炎指数（AI）动态变化和肺功能变化特点，检测AA大鼠滑膜、肺组织TGF-β1/Smads、ERK信号通路及细胞因子变化，评价中药新风胶囊（XFC）对其影响，探讨XFC改善AA大鼠肺功能的作用机制。
2方法
2.1理论研究
系统搜集和整理RA肺病变相关文献，筛选和总结引起RA肺病变发病机制的国内外研究现状，评价和分析转化生长因子β1（TGF-β1）诱导Smads、 ERK通路激活途径，从理论角度研究RA肺功能降低与TGF-β1/Smads、ERK信号通路cross-talk的关系。依从中医基础理论阐述中医“肺”与“脾”之间关系，从生理病理及病因病机角度探讨“肺脾”的联系，探寻健脾化湿通络法治疗RA肺功能降低的机制。
2.2实验研究
将大鼠随机分为正常对照（NC）组，模型对照（MC）组，甲氨喋呤（MTX）组，雷公藤多苷片（TPT）组和XFC组，每组10只。除NC组外，其余大鼠右后足跖皮内注射弗氏完全佐剂（CFA）0.1ml致炎造模，第15d在尾根部注射CFA0.05ml加强免疫，第19d给药。各组的给药量如下：XFC组0.034g/1ml/100g，TPT组0.57mg/1ml/100g，MTX组0.095mg/1ml/100g，NC、MC组予生理盐水9mg/1ml/100g；XFC、TPT、NC、MC组一天一次，MTX组一周一次，各组连续用药30d。观察各组大鼠体质量、足跖肿胀度、关节炎指数（AI）和肺功能变化，光镜、电镜观察滑膜、肺组织形态学变化，酶联免疫吸附法检测细胞因子TGF-β1、白介素（IL）-1β、IL-4、IL-10、γ干扰素（IFN-γ）、结缔组织生长因子（CTGF）、成纤维细胞生长因子（FGF）表达，PCR、免疫组化、免疫印迹法检测滑膜、肺组织Smads/ERK通路的变化。
3结果
3.1理论研究结果
3.1.1TGF-β1/Smads通路的激活促使RA肺功能降低：TGF-β1首先与其受体TβRⅡ、TβRI结合形成三聚体复合物，进一步磷酸化Smad2/3，p-Smad2/3与TβRI受体分离，即与Smad4结合形成复合体，携带信号从胞浆进入胞核激活转录，促进肺病变；而Smads7是抑制分子，Smad7抑制能力降低，不能抑制Smads信号的转录，失去对RA肺部病变的抑制作用，导致肺功能降低。
3.1.2ERK信号通路诱导RA肺功能降低：ERK通路激活后可介导细胞生长、分化、增殖，促进成纤维细胞分泌和细胞外基质（ECM）在肺间质和肺泡中过度积聚，使得肺泡距离增加形成间质肺，肺毛细血管床和血流量的减少，血液流变学改变，出现肺弥散功能降低，氧和二氧化碳的交换作用减弱，同时ERK通路激活能调节炎症相关基因表达增加，导致肺功能降低。
3.1.3Smads和ERK通路cross-talk是RA肺功能下降的重要因素：TGF-β1/Smad信号通路的激活过程也受ERK通路的调控，Smad2/3含有ERK磷酸化的位点，可被ERK磷酸化，促进p-Smad2/3与Smad4的结合并转录。同时，ERK通路受Smads通路调控，Smad4可调控ERK通路相关细胞因子或激酶的转录，从而激活ERK信号传导通路，Smads和ERK通路cross-talk共同导致肺功能降低。
3.1.4“脾虚”是RA肺功能降低的发病基础：脾虚致气血亏虚，脾土不能生养肺金，日久导致肺气失养，肺宣肃功能失调，出现咳嗽、自汗、气短等；脾虚运化功能失常，水液停滞，湿停中焦，痰湿内生，聚而生痰成饮，上干于肺，肺失治节，可见痰多，胸满憋闷，喉中痰鸣有声、胸背痛，迁延不愈，甚而喘促；脾虚致痰瘀互结，肺络阻滞，多见肺纹理紊乱、结节、间质纤维增生等。
3.1.5“健脾化湿通络法”能改善RA肺功能水平：根据RA肺功能降低的中医病机呈现“脾胃虚弱、气血不足、湿浊内生、痰瘀互结”和“虚实夹杂”的特点，临床上拟用“扶正祛邪”治则，采用“健脾化湿通络”治法，应用XFC治疗RA及肺功能损伤，取得了良好效果，XFC通过改善关节和全身症状体征，从而进一步改善肺部症状体征，提高弥散功能和通气功能。
3.2实验研究结果
3.2.1AA大鼠肺功能变化及XFC对其影响
与NC组比较，MC组大鼠肺功能FEV1、FEF50、FEF75、MMF、PEF降低（P <0.01）。
与MC组比较，XFC组FEV1、FEF50、FEF75、MMF、PEF升高（P<0.05或P <0.01）。
与MTX组比较，XFC组FEF50、FEF75、MMF升高（P <0.05）；与TPT组比较，XFC组肺功能参数FEF75、MMF升高（P <0.05或P <0.01）。
3.2.2AA大鼠足趾肿胀度、AI、体质量变化及XFC对其影响
相关分析显示：AA大鼠肺功能FEF75与足跖肿胀度呈负相关，FEF50、FEF75与AI呈负相关（P <0.01或P <0.05）。
给药前1d，与NC组相比，MC组大鼠体质量降低，足跖肿胀度、AI显著升高（P <0.01）。
给药后30d，与NC组比较，MC组大鼠足趾肿胀度、AI升高；与MC组比较，各治疗组大鼠足跖肿胀度、AI降低；与MTX组比较，XFC组足趾肿胀度差值降低（P <0.01或P <0.05）。
3.2.3AA大鼠关节形态学变化及XFC对其影响
光镜观察：与NC组比较，MC组大鼠滑膜及其附属组织可见大量炎性细胞浸润，血管增多，滑膜分层增多，呈绒毛状突起嵌入关节腔内，关节软骨表面剥脱或缺如。电镜观察：MC组滑膜细胞变形，线粒体肿胀，粗面内质网减少、破坏，核膜不完整，染色质分布不均，嵴突排列不规整或部分消失；
XFC治疗后，大鼠滑膜可见少量中性粒细胞等炎细胞浸润，部分滑膜嵌入关节腔内，关节面较规整；滑膜细胞线粒体少量变形、肿胀，核膜边界较清楚，染色质较均匀，大部分嵴突排列完整。与MTX组比较，XFC组滑膜细胞线粒体肿胀减轻；XFC组TPT组比较无明显差异。
3.2.4AA大鼠肺组织形态学变化及XFC对其影响
光镜观察显示：与NC组比较，MC组大鼠肺泡结构不规整，肺泡腔萎缩或消失，部分呈肺实质性改变，肺间隔增宽，间质中可见大量炎细胞浸润，肺泡上皮细胞增生；电镜观察Ⅱ型肺泡上皮细胞数量减少，胞膜结构不完整，线粒体肿胀、变性，板层小体减少呈排空状态，肺间质内成纤维细胞增生。
XFC治疗组大鼠肺泡结构较规整，部分肺泡腔萎缩、变形，间质中可见少量中性粒细胞等炎细胞浸润；Ⅱ型肺泡上皮细胞结构基本完整，粗面内质网较丰富，线粒体大部分完好，偶见板层小体排空现象；与MTX组比较，XFC组肺组织炎细胞浸润和肺泡上皮细胞减少；与TPT组比较无显著差异。
3.2.5AA大鼠血清细胞因子的变化及XFC对其影响
相关分析显示：AA大鼠肺功能FEV1、FEF75与IL-1β、IL-4呈负相关，FEF25、FEF50、FEF75、MMF与TGF-β1、CTGF、FGF呈负相关；PEF与IL-10呈正相关，FEV1、FEF50与Th1/Th2呈正相关，FEF50、FEF75与IFN-γ呈正相关（P <0.01或P <0.05）。
与NC组比较，MC组血清细胞因子IL-1β、IL-4、TGF-β1、CTGF升高，IFN-γ、IL-10、Th1/Th2细胞、FGF降低（P <0.01或P <0.05）。
与MC组比较，XFC组IL-1β、IL-4、TGF-β1、CTGF降低，IFN-γ、IL-10、Th1/Th2、FGF表达升高（P <0.01或P <0.05）。
与MTX组比较，XFC组CTGF降低，IFN-γ、IL-10、Th1/Th2表达量升高（P <0.05）；与TPT组比较，XFC组TGF-β1降低（P <0.05）。
3.2.6AA大鼠滑膜、肺组织Smads通路变化及XFC对其影响
相关分析显示：AA大鼠肺功能FEV1与TGF-β1mRNA、蛋白呈负相关，FEF25与Smad4蛋白呈负相关，FEF50与TβRⅡ蛋白表达呈负相关，FEF75与TGF-β1、Smad3mRNA、p-Smad2/3蛋白呈负相关，PEF与TGF-β1蛋白呈负相关（P <0.05）；FEF50、MMF与Smad7mRNA呈正相关（P<0.05）。
与NC组比较，MC组大鼠滑膜、肺组织TGF-β1、Smad2/3/4mRNA和TGF-β1、TβRI、TβRⅡ、Smad2/3、p-Smad2/3、Smad4蛋白表达升高，Smad7mRNA、蛋白表达降低（P <0.01或P <0.05）。
与MC组比较，XFC组大鼠滑膜、肺组织Smad2、Smad3、Smad4mRNA和TGF-β1、TβRI、TβRⅡ、Smad2/3、p-Smad2/3蛋白表达降低， Smad7mRNA、蛋白表达升高（P <0.01或P <0.05）。
与MTX组比较，XFC组滑膜、肺组织TGF-β1、Smad3mRNA和TGF-β1、Smad2/3蛋白表达降低，肺Smad4mRNA、TβRI蛋白降低，滑膜、肺组织Smad7mRNA升高（P <0.05）；与TPT组比较，XFC组滑膜Smad3mRNA和TGF-β1、p-Smad2/3、Smad4蛋白降低，Smad7蛋白升高（P <0.05），肺组织p-Smad2/3降低，Smad7mRNA升高（P <0.05）。
3.2.7AA大鼠滑膜、肺组织ERK1/2变化及XFC对其影响
相关分析显示：AA大鼠肺功能FEV1与ERK2mRNA呈负相关，FEF25与p-ERK1/2蛋白呈负相关，FEF75、MMF分别与ERK1/2、p-ERK1/2蛋白呈负相关（P <0.05）。肺组织ERK1mRNA与Smad3mRNA、TβRⅡ蛋白呈正相关，ERK2mRNA与p-Smad2/3呈正相关，ERK1/2蛋白与Smad2mRNA、Smad4蛋白呈正相关，p-ERK1/2蛋白与Smad4mRNA、p-Smad2/3蛋白呈正相关（P <0.05）；p-ERK1/2蛋白与Smad7蛋白呈负相关（P <0.05）。
与NC组比较，MC组大鼠滑膜组织ERK2mRNA、p-ERK1/2蛋白表达升高，肺组织ERK1、ERK2mRNA、ERK1/2、p-ERK1/2蛋白表达升高（P <0.01或P <0.05）。
与MC组比较，XFC组大鼠滑膜组织ERK2mRNA、p-ERK1/2蛋白表达降低，肺组织ERK1、ERK2mRNA、ERK1/2、p-ERK1/2蛋白降低（P<0.01或P <0.05）。
与MTX组比较，XFC组肺组织ERK1、ERK2mRNA表达降低（P<0.05）。
4结论
4.1AA大鼠肺功能的变化
4.1.1AA大鼠肺功能损伤特点：AA大鼠存在肺功能损伤，肺功能变化以参数FEV1、MMF、PEF、FEF50、FEF75降低为主，主要表现特征为通气功能障碍，并伴有一定程度的小气道功能损伤。
4.1.2Smads和ERK通路cross-talk导致肺功能降低：Smads和ERK通路在启动子TGF-β1诱导下，逐渐被磷酸化激活，进而进入细胞核参与转录活动，导致AA肺部病变发生，出现肺功能降低。
4.1.3RA肺功能降低中医病机呈“脾虚湿盛”特征：“脾虚”在RA肺功能损伤发生、发展中起重要作用，脾气亏虚，正气不足，肺气虚弱；脾气亏虚，痰湿内生，肺失治节；脾气亏虚，痰瘀互结，肺络阻滞。
4.2“健脾化湿通络”中药XFC对AA大鼠的疗效
4.2.1XFC能明显抑制AA大鼠关节炎症反应：XFC能降低AA大鼠足趾肿胀度和AI，减少炎细胞对关节滑膜的浸润，减轻局部充血和肿胀，抑制血管翳的形成，改善关节功能。
4.2.2XFC能明显改善AA大鼠肺功能水平：XFC通过提高肺功能参数FEV1、FEF50、FEF75、MMF、PEF表达，改善AA大鼠肺部通气功能和小气道功能，增强肺部气体交换率，提高肺功能水平。
4.2.3XFC能明显提高AA大鼠体质量：XFC通过健脾益气、化湿通络的功效，能明显提高AA大鼠食欲及饮水量，增加体质量水平，改善全身症状和机能活动。
4.2.4XFC能明显调节AA大鼠肺组织形态学：XFC能减轻炎性细胞浸润程度，通过抑制炎性介质对肺组织的刺激，降低AA大鼠肺间质纤维化程度，维持Ⅱ型肺泡细胞细胞器的功能和形态。
4.3XFC改善AA大鼠肺功能的机制
4.3.1XFC可降低AA大鼠关节炎症反应提高肺功能：XFC通过抑制滑膜组织炎细胞浸润，降低全身炎症反应，减少炎症介质渗出及对肺组织器官的刺激，抑制肺间质病变的发生，改善肺功能水平。
4.3.2XFC可减轻AA大鼠肺部炎症反应提高肺功能：XFC通过抗炎作用，减少炎性介质对肺泡的直接刺激，促进中性粒细胞等炎细胞的吸收或清除，降低肺组织炎症反应，改善肺功能水平。
4.3.3XFC可调节AA大鼠细胞因子平衡提高肺功能：XFC通过上调IL-10、IFN-γ、FGF表达，下调IL-1β、IL-4、TGF-β1、CTGF表达，抑制AA大鼠肺间质纤维化的发生，提高肺功能水平。
4.3.4XFC可改善AA大鼠肺组织形态学提高肺功能：XFC通过减少炎性渗出物对肺组织损伤，保持肺组织病理形态学，维持肺泡Ⅱ型上皮细胞结构，增加肺泡的通气功能和弥散功能，改善肺功能。
4.3.5XFC可调节AA大鼠TGF-β1/Smads通路提高肺功能：XFC通过提高Smad7表达，抑制p-Smad2/3与Smad4的结合及阻止Smad2/3磷酸化，延缓AA大鼠肺纤维化的发生，改善肺功能。
4.3.6XFC可抑制AA大鼠ERK1/2通路表达提高肺功能：XFC通过抑制ERK1/2磷酸化过程，阻止信号通路的激活，减少致纤维化因子对肺组织的损伤，降低AA大鼠肺病变程度，改善肺功能。
4.3.7XFC可调节AA大鼠Smads和ERK通路cross-talk提高肺功能：XFC通过调节信号通路cross-talk，降低信号传导与DNA的转录活动，抑制AA大鼠肺间质纤维化形成，改善肺功能。
1Objective
Based on the Traditional Chinese Medicine theory of "correlationbetween lung and spleen ",“Lung Paralysis" as the breakthroughpoint,this paper analyzes and summarizes medical pathogenesis of lungfunction damage in rheumatoid arthritis (RA). From the perspective ofanimal experiment to observe the dynamic changes of paw swelling,arthritis index (AI) and lung function changes in adjuvant arthritis (AA)rat, detects changes of TGF-β1/Smads, ERK signaling pathways andcytokine in AA rats’ synovial and lung tissue, evaluates influence ofChinese medicine Xinfeng Capsule (XFC), and explores the mechanismof XFC improves lung function in AA rats.
2Methods
2.1Theoretical study
Collecting and organizing the relevant literature of RA lung disease,screening and summarize the research status of the RA lung diseasepathogenesis,evaluation and analysis of transforming growth factor beta1(TGF-beta1) induced by Smads, ERK pathway activation pathways.Studied from a theoretical point of the relationship between lung functionreduce in RA and the TGF-β1/Smads, ERK signaling pathway cross-talk. This article based on the basic theories of TCM, discussed therelationship between TCM "lung" and "spleen", by studying from theperspective of physiological pathology and etiology pathogenesis, andexplained the mechanism of “jianpihuashi Tongluo therapy” in thetreatment of RA decline in lung function.
2.2Experimental Study
50Wistar rats rats were randomly divided into five groups:normalcontrol (NC) group, model control (MC) group, methotrexate (MTX)group, Tripterygium glycosides piece (TPT) and XFC group, n=10. Inaddition to the NC group, right rear foot plantar skin of the rest ratsinjected Freund's complete adjuvant (CFA)0.1ml of proinflammatorymodeling, strengthen the immune by injection in the base of the tail ofCFA0.05ml at the15d, dosage at the19d. Dosage groups are asfollows: the XFC group:0.034g/1ml/100g, TPT group:0.57mg/1ml/100g,MTX group0.095mg/1ml/100g, NC、MC group:physiological saline9mg/1ml/100g; XFC,TPT,NC,MC group once a day, MTX group once aweek, each group continuous medication for30d. Observed the rats’body mass, paw swelling, arthritis index (AI) and pulmonary functionchanges, morphological changes of lung tissue, synovium by lightmicroscopy and electron microscopy, tested the expression of cytokineTGF-β1in interleukin the prime (IL)-1β, IL-4, IL-10, gamma interferon(IFN-γ), connective tissue growth factor (CTGF), fibroblast growth factor(FGF) by enzyme-linked immunosorbent assay, detected changes ofSmads/ERK pathway in synovial membranes and lung tissue by PCR,immunohistochemistry and Western blot.
3Results
3.1The theoretical research results
3.1.1TGF-β1/Smads pathway activated prompted RA lung function decreased.TGF-β1can first combined TβR Ⅱ (its receptor) to form atrimer complex TβRI, than further phosphorylated Smad2/3, p-Smad2/3to separate from TβRI, which than combined to form complexes withSmad4, carried signals from the cytoplasm into the nucleus to activatetranscription, promote lung lesions; whereas Smads7is inhibitorymolecules, if reduceing Smad7inhibition ability, inhibition oftranscription of Smads signals cannot be lost, the inhibitory effect on RAlung lesions, resulting in decreased lung function.
3.1.2ERK signaling pathway induced by RA lung function decreased:the activation of the ERK pathway may mediates cell growth,differentiation, proliferation, promotes the secretion of fibroblasts andexcessive accumulation of extracellular matrix (ECM) in pulmonaryinterstitial and alveolar, makes the alveolar distance increased to forminterstitial lung, decreases in pulmonary capillary bed and blood flow,changes in blood rheology, reduces pulmonary diffusion, weakens theexchange of oxygen and carbon dioxide, while the activation of ERKpathway can regulate the expression of inflammation-related genes,leading to lung function decreased.
3.1.3Smads and ERK pathway cross-talk is an important factor in thedecline in RA lung function: the activation process of TGF-β1/Smadsignaling pathway is also affected by the regulation of the ERK pathway,Smad2/3contains ERK phosphorylation sites,which can be phosphoryl-ated by ERK, promote p-Smad2/3and Smad4in binding andtranscription. Meanwhile, ERK pathway is regulated by Smads pathway,Smad4regulates transcription of ERK pathway related cytokines orkinase, which activates ERK signaling pathway, Smads and ERKpathway cross-talk common led to the decline in lung function.
3.1.4The "Spleen asthenia" is the pathological basis of RA decline in lung function: spleen asthenia caused by Qi and blood deficiency,spleen can not bear the lung,lead to deficiency of lung-qi and yin,andalso lead to failure of pulmonary qi to disperse and descend，resulting insuch syndrome like: cough, shortness of breath, spontaneousperspiration,etc; asthenia of splenic qi,failure of transportation andtransformation, which fails to distribute fluid and leads to attack of fluidretention on the lung, then lung fails to its Regulatory functions,manifeasting in such syndrome like: cough with profuse whitish sputum,even asthmatic breath with sputum rale, chest pain, prolonged andincurable; lung collaterals block, see more lung texture disorder,nodules, interstitial fibrosis were frequent in spleen deficiency caused byphlegm and blood stasis.
3.1.5The “Jianpihuashitang Tongluo Therapy” makes significantimprovement in RA lung function: According to the TCM pathogenesis ofRA decline in lung function “asthenia of spleen and stomach, asthenia ofqi and blood, accumulation of fluid and dampness, phlegm and bloodstasis " and “inclusion of asthenia and sthenia”characteristics, clinicallydefered to "eliminating pathogen and strengthening vital qi " principle oftreatment, using the " Jianpihuashitang Tongluo Therapy ", applyedXFC treatment of RA and pulmonary dysfunction, and achieved goodresults, XFC could further improve pulmonary symptoms and signs, toimprove the dispersion function and ventilation function by improvingjoint and systemic symptoms and signs.3.2The experimental study results
3.2.1Changes of lung function in rats and the impact of XFCCompared with NC group, the MC rats lung function FEV1, FEF50, FEF75,MMF, PEF decreased (P <0.01).Compared with the MC group, FEV1, FEF50, FEF75, MMF, PEF were increased in the XFC group (P <0.05or P <0.01).Compared with the MTX group, the level of FEF50, FEF75MMF wereincreased in the XFC group (P <0.05); compared with TPT group,pulmonary function parameters FEF75, MMF were increased in the XFCgroup (P <0.05or P <0.01).
3.2.2Changes of paw swelling, AI in rats and the impact of XFCCorrelation analysis showed the AA rat lung function FEF75, pawswelling were negative correlation, FEF50, FEF75and the AI werenegatively correlated (P <0.05or P <0.01).Prior to administration, compared with the NC group, body weight wasreduced in MC rats, and paw swelling degree, AI were significantlyhigher than those the MC rats (P <0.01).30days after administration, compared with the NC group, toe swelling,AI were elevated in MC rats; compared with the MC group, rat pawedema degrees and AI were lower in each treatment group; comparedwith the MTX group (P <0.05or P <0.01).
3.2.3Changes of joint morphological in rats and the impact of XFCLight microscopic observation: Compared with NC group, the MC ratssynovium and its affiliated organizations visible to a large number ofinflammatory cell infiltration increased vascular increased synoviallayered, showed villous projections embedded in the intra-articularsurface of the articular cartilage exfoliative or missing such as. Electronmicroscopy: the MC group synovial cells deformation, swelling ofmitochondria, rough endoplasmic reticulum was reduced, damaged,incomplete nuclear membrane, the uneven distribution of chromatin,irregular or partial disappearance of cristae arranged.The XFC treatment, the rat synovial visible small number of neutrophilsand other inflammatory cell infiltration, embedded in the intra-articular part of the synovial tissue, the articular surface was orderly; synovialcells the mitochondrial small amount of deformation, swelling, nuclearmembrane boundary more clearly, chromatin more uniform, most of thecristae arranged complete. Compared with the MTX group the XFCgroup synovial cells reduce mitochondrial swelling; the XFC group TPTgroup no significant difference.
3.2.4Changes of lung tissue morphologic in rats and the impact of XFCLight microscopy showed that: compared with the NC group, MC grouprat alveolar structure was irregular, the alveolar cavity atrophy ordisappear, was part of the lung substantive changes in lung intervalswidened visible in the interstitial infiltration of inflammatory cells, alveolarepithelial cell proliferation; reduce the number of electron microscopy Ⅱalveolar epithelial cells, the membrane structure was not completemitochondrial swelling, degeneration, reduced lamellar bodies wereemptying state, interstitial lung fibroblasts.XFC treated rats compared with regular alveolar structure, part of thealveolar space atrophy, deformation, seen in a small number ofneutrophils and other inflammatory cells in the interstitial infiltration;basic integrity of the structure of type Ⅱ alveolar epithelial cells,abundant rough endoplasmic reticulum, mitochondria and large partiallyintact, occasionally lamellar bodies emptying phenomenon; comparedwith the MTX group, the the XFC group of lung tissue inflammatory cellinfiltration and decrease in alveolar epithelial cells; the TPT groupshowed no significant differences.
3.2.5Changes of serum cytokine in rats and the impact of XFCCorrelation analysis showed AA the rats lung function FEV1, FEF75IL-1β, IL-4was negatively correlated FEF25, FEF50, FEF75, MMF withTGF-β1, CTGF, FGF negatively correlated; PEF and IL-10were positively correlated, FEV1, FEF50were positively correlated withTh1/Th2,FEF50, FEF75and IFN-γ were positively correlated (P <0.05orP <0.01).
Compared with the NC group, the level of IL-1β, IL-4, TGF-β1, CTGF ofserum were increased in MC, IFN-γ, IL-10, Th1/Th2cells, FGF werereduced (P <0.05or P <0.01).
Compared with the MC group, the expression of IL-1β, IL-4, TGF-β1,CTGF were reduce, and the expression of IFN-γ, IL-10, Th1/Th2, FGFwere increased in the XFC group (P <0.05or P <0.01).Compared with the MTX group, CTGF was decreased, and IFN-γ,expression of IL-10, Th1/Th2were increased in XFC group (P <0.05);Compared with TPT group, TGF-β1were decreased in the XFC group(P <0.05).
3.2.6Changes of Smads pathway in rats and the impact of XFCCorrelation analysis showed FEV1and TGF-β1mRNA, protein werenegatively correlated, FEF25and Smad4protein were negativelycorrelated, FEF50and TβRⅡ protein expression were negativelycorrelated with FEF75and TGF-β1, Smad3mRNA, p-Smad2/3proteinwas negatively correlated with PEF and TGF-β1protein were negativelycorrelated (P <0.05); FEF50, MMF Smad7mRNA were positivelycorrelated(P <0.05).
Compared with NC group, the level of TGF-β1, Smad2/3/4mRNA,TGF-β1, TβRI, TβRⅡ, Smad2/3, p-Smad2/3, Smad4protein wereincreased, expression of Smad7mRNA, protein of synovial lung tissuewere decreased in MC group (P <0.05or P <0.01).Compared with the MC group, expression of Smad2, Smad3, Smad4mRNA and TGF-β1, TβRI, TβRⅡ, Smad2/3, p-Smad2/3protein weredecreased, expression of Smad7mRNA and protein of lung tissue were elevated in XFC group (P <0.05or P <0.01).
Compared with the MTX group, expression of TGF-β1,Smad3mRNAand TGF-β1, Smad2/3protein were decreased, Smad4mRNA TβRIprotein were reduced, Smad7mRNA was increased in XFC group (P<0.05); Compared with TPT group, Smad3mRNA, TGF-β1, p-Smad2/3, Smad4protein were reduced, Smad7protein was increased in theXFC group (P <0.05), p-Smad2/3of lung tissue was lowered, Smad7mRNA was increased in the XFC group (P <0.05).
3.2.7Changes of ERK1/2in rats and the impact of XFC
Correlation analysis showed pulmonary function FEV1and ERK2mRNAwere negatively correlated, FEF25and p-ERK1/2protein werenegatively correlated, FEF75, MMF and ERK1/2, p-ERK1/2proteinwere negatively correlated (P <0.05). ERK1mRNA, Smad3mRNA inTβRⅡ protein of lung tissue were positively correlated, ERK2mRNA andp-Smad2/3was positively correlated, ERK1/2proteins, Smad2mRNA,Smad4protein were positively correlated, p-ERK1/2proteins andSmad4mRNA, p--Smad2/3protein were positively related (P <0.05);p-ERK1/2protein and Smad7were negatively correlated (P <0.05).Compared with the MC group, expression of ERK2mRNA, p-ERK1/2protein of lung tissue were decreased, ERK1, ERK2mRNA, ERK1/2,p-ERK1/2protein were decreased in the XFC group (P <0.05or P<0.01).
Compared with the MTX group, expression of ERK1, ERK2mRNA oflung tissue were decreased in the XFC group (P <0.05).
4Conclusions
4.1Changes of lung function in AA rats
4.1.1Pulmonary dysfunction features: changes of pulmonary functionparameter FEV1, MMF, PEF, FEF50, FEF75were reduced in AA rats, which mainly characterized ventilatory dysfunction, and accompanied bya certain degree of small airway function damage.
4.1.2Smads and ERK pathway cross-talk lead to reduce lung function:Smads and ERK pathway can inducte TGF-β1, which graduallyactivated by phosphorylation, and then enter the nucleus involved intranscriptional activity, resulting in AA lung lesions occur, pulmonaryThe function was reduced.
4.1.3TCM pathogenesis characteristics:"Spleen" in RA lung functionoccurred, plays an important role in the development, temper deficiency,lack of righteousness, lung weakness; temper deficiency sputum Thewet endogenous governance section of lung failure; temper deficiency,phlegm, pulmonary collaterals block.
4.2Efficacy of XFC on AA rats
4.2.1XFC can inhibit joint inflammation reaction of AA rats: XFC toreduce the the AA rat paw swelling and AI, to reduce the infiltration ofinflammatory cells of the synovial alleviate local congestion and swelling,inhibit the formation of pannus, improve joint function.
4.2.2XFC can improve the level of AA rats lung function by improvinglung function parameters FEV1, FEF50, FEF75, MMF, PEF expression:XFC improve the AA rat pulmonary ventilation function and small airwayfunction, enhanced pulmonary gas exchange rate and improve lungfunction level.
4.2.3XFC can improve the quality of the AA rats: XFC Jianpiyiqi thedampness Tongluo the efficacy, can significantly improve the the AArats appetite and water intake, increase the level of body mass, improvesystemic symptoms and functional activity.
4.2.4XFC can adjust the AA rat lung tissue morphology significantly:XFC can reduce the degree of infiltration of inflammatory cells, inflammatory mediators by inhibiting lung tissue stimulation, reduce theAA rat pulmonary interstitial fibrosis, maintain alveolar type Ⅱ cellfunction and morphology of the organelle.
4.3Mechanisms of XFC improve lung function
4.3.1XFC can reduce the AA rats joint inflammation reaction to improvelung function: XFC seeping through the inhibition of inflammatory cellinfiltration of synovial tissue, reduce the systemic inflammatoryresponse, reducing inflammatory mediators and the stimulation of lungtissues and organs, inhibition of interstitial lung disease occur, improvelung function level.
4.3.2XFC can reduce the AA rats lung inflammation to improve lungfunction: XFC by the anti-inflammatory effect, reducing the directstimulation of inflammatory mediators of alveolar promote absorption ofneutrophils and other inflammatory cells or clear, to reduce lunginflammation reaction, improve lung function level.
4.3.3XFC can adjust the AA rats cytokine balance to improve lungfunction: XFC FGF expression by up-regulating IL-10, IFN-γ, loweredIL-1β, IL-4, TGF-β1, CTGF expression, inhibition of AA rats Theincidence of pulmonary fibrosis to improve lung function level.
4.3.4XFC can improve the AA rat lung tissue morphology to improvelung function by reducing the inflammatory exudate lung tissue damage,keep the lung tissue pathomorphological maintain alveolar type Ⅱepithelial cell structure, increased alveolar ventilation function: XFCimprovement in lung function and diffusion capacity.
4.3.5XFC can adjust AA rats TGF-β1/Smads pathway to improve lungfunction: XFC increase Smad7expression, inhibition of p-Smad2/3with Smad4binding and prevents Smad2/3phosphorylation, delayingthe the AA rat lung fibrosis the occurrence, and improve lung function.
4.3.6XFC can inhibit the AA rats ERK1/2pathway expression toimprove lung function by inhibition of ERK1/2phosphorylation process,preventing the activation of the signaling pathway, reducing fibrogenicfactors on lung tissue damage.
4.3.7XFC can adjust Smads and ERK pathway cross-talk to improvelung function: XFC reduce signaling and DNA transcription activity,inhibiting the formation of the AA rat pulmonary interstitial fibrosis byregulating signaling pathways cross-talk to improve lung function.
在中医“肺脾相关”理论指导下，以“肺痹”为切入点，分析、总结类风湿关节炎（RA）肺功能损伤的中医学病机，从动物实验角度系统观察佐剂关节炎（AA）大鼠足跖肿胀度、关节炎指数（AI）动态变化和肺功能变化特点，检测AA大鼠滑膜、肺组织TGF-β1/Smads、ERK信号通路及细胞因子变化，评价中药新风胶囊（XFC）对其影响，探讨XFC改善AA大鼠肺功能的作用机制。
2方法
2.1理论研究
系统搜集和整理RA肺病变相关文献，筛选和总结引起RA肺病变发病机制的国内外研究现状，评价和分析转化生长因子β1（TGF-β1）诱导Smads、 ERK通路激活途径，从理论角度研究RA肺功能降低与TGF-β1/Smads、ERK信号通路cross-talk的关系。依从中医基础理论阐述中医“肺”与“脾”之间关系，从生理病理及病因病机角度探讨“肺脾”的联系，探寻健脾化湿通络法治疗RA肺功能降低的机制。
2.2实验研究
将大鼠随机分为正常对照（NC）组，模型对照（MC）组，甲氨喋呤（MTX）组，雷公藤多苷片（TPT）组和XFC组，每组10只。除NC组外，其余大鼠右后足跖皮内注射弗氏完全佐剂（CFA）0.1ml致炎造模，第15d在尾根部注射CFA0.05ml加强免疫，第19d给药。各组的给药量如下：XFC组0.034g/1ml/100g，TPT组0.57mg/1ml/100g，MTX组0.095mg/1ml/100g，NC、MC组予生理盐水9mg/1ml/100g；XFC、TPT、NC、MC组一天一次，MTX组一周一次，各组连续用药30d。观察各组大鼠体质量、足跖肿胀度、关节炎指数（AI）和肺功能变化，光镜、电镜观察滑膜、肺组织形态学变化，酶联免疫吸附法检测细胞因子TGF-β1、白介素（IL）-1β、IL-4、IL-10、γ干扰素（IFN-γ）、结缔组织生长因子（CTGF）、成纤维细胞生长因子（FGF）表达，PCR、免疫组化、免疫印迹法检测滑膜、肺组织Smads/ERK通路的变化。
3结果
3.1理论研究结果
3.1.1TGF-β1/Smads通路的激活促使RA肺功能降低：TGF-β1首先与其受体TβRⅡ、TβRI结合形成三聚体复合物，进一步磷酸化Smad2/3，p-Smad2/3与TβRI受体分离，即与Smad4结合形成复合体，携带信号从胞浆进入胞核激活转录，促进肺病变；而Smads7是抑制分子，Smad7抑制能力降低，不能抑制Smads信号的转录，失去对RA肺部病变的抑制作用，导致肺功能降低。
3.1.2ERK信号通路诱导RA肺功能降低：ERK通路激活后可介导细胞生长、分化、增殖，促进成纤维细胞分泌和细胞外基质（ECM）在肺间质和肺泡中过度积聚，使得肺泡距离增加形成间质肺，肺毛细血管床和血流量的减少，血液流变学改变，出现肺弥散功能降低，氧和二氧化碳的交换作用减弱，同时ERK通路激活能调节炎症相关基因表达增加，导致肺功能降低。
3.1.3Smads和ERK通路cross-talk是RA肺功能下降的重要因素：TGF-β1/Smad信号通路的激活过程也受ERK通路的调控，Smad2/3含有ERK磷酸化的位点，可被ERK磷酸化，促进p-Smad2/3与Smad4的结合并转录。同时，ERK通路受Smads通路调控，Smad4可调控ERK通路相关细胞因子或激酶的转录，从而激活ERK信号传导通路，Smads和ERK通路cross-talk共同导致肺功能降低。
3.1.4“脾虚”是RA肺功能降低的发病基础：脾虚致气血亏虚，脾土不能生养肺金，日久导致肺气失养，肺宣肃功能失调，出现咳嗽、自汗、气短等；脾虚运化功能失常，水液停滞，湿停中焦，痰湿内生，聚而生痰成饮，上干于肺，肺失治节，可见痰多，胸满憋闷，喉中痰鸣有声、胸背痛，迁延不愈，甚而喘促；脾虚致痰瘀互结，肺络阻滞，多见肺纹理紊乱、结节、间质纤维增生等。
3.1.5“健脾化湿通络法”能改善RA肺功能水平：根据RA肺功能降低的中医病机呈现“脾胃虚弱、气血不足、湿浊内生、痰瘀互结”和“虚实夹杂”的特点，临床上拟用“扶正祛邪”治则，采用“健脾化湿通络”治法，应用XFC治疗RA及肺功能损伤，取得了良好效果，XFC通过改善关节和全身症状体征，从而进一步改善肺部症状体征，提高弥散功能和通气功能。
3.2实验研究结果
3.2.1AA大鼠肺功能变化及XFC对其影响
与NC组比较，MC组大鼠肺功能FEV1、FEF50、FEF75、MMF、PEF降低（P <0.01）。
与MC组比较，XFC组FEV1、FEF50、FEF75、MMF、PEF升高（P<0.05或P <0.01）。
与MTX组比较，XFC组FEF50、FEF75、MMF升高（P <0.05）；与TPT组比较，XFC组肺功能参数FEF75、MMF升高（P <0.05或P <0.01）。
3.2.2AA大鼠足趾肿胀度、AI、体质量变化及XFC对其影响
相关分析显示：AA大鼠肺功能FEF75与足跖肿胀度呈负相关，FEF50、FEF75与AI呈负相关（P <0.01或P <0.05）。
给药前1d，与NC组相比，MC组大鼠体质量降低，足跖肿胀度、AI显著升高（P <0.01）。
给药后30d，与NC组比较，MC组大鼠足趾肿胀度、AI升高；与MC组比较，各治疗组大鼠足跖肿胀度、AI降低；与MTX组比较，XFC组足趾肿胀度差值降低（P <0.01或P <0.05）。
3.2.3AA大鼠关节形态学变化及XFC对其影响
光镜观察：与NC组比较，MC组大鼠滑膜及其附属组织可见大量炎性细胞浸润，血管增多，滑膜分层增多，呈绒毛状突起嵌入关节腔内，关节软骨表面剥脱或缺如。电镜观察：MC组滑膜细胞变形，线粒体肿胀，粗面内质网减少、破坏，核膜不完整，染色质分布不均，嵴突排列不规整或部分消失；
XFC治疗后，大鼠滑膜可见少量中性粒细胞等炎细胞浸润，部分滑膜嵌入关节腔内，关节面较规整；滑膜细胞线粒体少量变形、肿胀，核膜边界较清楚，染色质较均匀，大部分嵴突排列完整。与MTX组比较，XFC组滑膜细胞线粒体肿胀减轻；XFC组TPT组比较无明显差异。
3.2.4AA大鼠肺组织形态学变化及XFC对其影响
光镜观察显示：与NC组比较，MC组大鼠肺泡结构不规整，肺泡腔萎缩或消失，部分呈肺实质性改变，肺间隔增宽，间质中可见大量炎细胞浸润，肺泡上皮细胞增生；电镜观察Ⅱ型肺泡上皮细胞数量减少，胞膜结构不完整，线粒体肿胀、变性，板层小体减少呈排空状态，肺间质内成纤维细胞增生。
XFC治疗组大鼠肺泡结构较规整，部分肺泡腔萎缩、变形，间质中可见少量中性粒细胞等炎细胞浸润；Ⅱ型肺泡上皮细胞结构基本完整，粗面内质网较丰富，线粒体大部分完好，偶见板层小体排空现象；与MTX组比较，XFC组肺组织炎细胞浸润和肺泡上皮细胞减少；与TPT组比较无显著差异。
3.2.5AA大鼠血清细胞因子的变化及XFC对其影响
相关分析显示：AA大鼠肺功能FEV1、FEF75与IL-1β、IL-4呈负相关，FEF25、FEF50、FEF75、MMF与TGF-β1、CTGF、FGF呈负相关；PEF与IL-10呈正相关，FEV1、FEF50与Th1/Th2呈正相关，FEF50、FEF75与IFN-γ呈正相关（P <0.01或P <0.05）。
与NC组比较，MC组血清细胞因子IL-1β、IL-4、TGF-β1、CTGF升高，IFN-γ、IL-10、Th1/Th2细胞、FGF降低（P <0.01或P <0.05）。
与MC组比较，XFC组IL-1β、IL-4、TGF-β1、CTGF降低，IFN-γ、IL-10、Th1/Th2、FGF表达升高（P <0.01或P <0.05）。
与MTX组比较，XFC组CTGF降低，IFN-γ、IL-10、Th1/Th2表达量升高（P <0.05）；与TPT组比较，XFC组TGF-β1降低（P <0.05）。
3.2.6AA大鼠滑膜、肺组织Smads通路变化及XFC对其影响
相关分析显示：AA大鼠肺功能FEV1与TGF-β1mRNA、蛋白呈负相关，FEF25与Smad4蛋白呈负相关，FEF50与TβRⅡ蛋白表达呈负相关，FEF75与TGF-β1、Smad3mRNA、p-Smad2/3蛋白呈负相关，PEF与TGF-β1蛋白呈负相关（P <0.05）；FEF50、MMF与Smad7mRNA呈正相关（P<0.05）。
与NC组比较，MC组大鼠滑膜、肺组织TGF-β1、Smad2/3/4mRNA和TGF-β1、TβRI、TβRⅡ、Smad2/3、p-Smad2/3、Smad4蛋白表达升高，Smad7mRNA、蛋白表达降低（P <0.01或P <0.05）。
与MC组比较，XFC组大鼠滑膜、肺组织Smad2、Smad3、Smad4mRNA和TGF-β1、TβRI、TβRⅡ、Smad2/3、p-Smad2/3蛋白表达降低， Smad7mRNA、蛋白表达升高（P <0.01或P <0.05）。
与MTX组比较，XFC组滑膜、肺组织TGF-β1、Smad3mRNA和TGF-β1、Smad2/3蛋白表达降低，肺Smad4mRNA、TβRI蛋白降低，滑膜、肺组织Smad7mRNA升高（P <0.05）；与TPT组比较，XFC组滑膜Smad3mRNA和TGF-β1、p-Smad2/3、Smad4蛋白降低，Smad7蛋白升高（P <0.05），肺组织p-Smad2/3降低，Smad7mRNA升高（P <0.05）。
3.2.7AA大鼠滑膜、肺组织ERK1/2变化及XFC对其影响
相关分析显示：AA大鼠肺功能FEV1与ERK2mRNA呈负相关，FEF25与p-ERK1/2蛋白呈负相关，FEF75、MMF分别与ERK1/2、p-ERK1/2蛋白呈负相关（P <0.05）。肺组织ERK1mRNA与Smad3mRNA、TβRⅡ蛋白呈正相关，ERK2mRNA与p-Smad2/3呈正相关，ERK1/2蛋白与Smad2mRNA、Smad4蛋白呈正相关，p-ERK1/2蛋白与Smad4mRNA、p-Smad2/3蛋白呈正相关（P <0.05）；p-ERK1/2蛋白与Smad7蛋白呈负相关（P <0.05）。
与NC组比较，MC组大鼠滑膜组织ERK2mRNA、p-ERK1/2蛋白表达升高，肺组织ERK1、ERK2mRNA、ERK1/2、p-ERK1/2蛋白表达升高（P <0.01或P <0.05）。
与MC组比较，XFC组大鼠滑膜组织ERK2mRNA、p-ERK1/2蛋白表达降低，肺组织ERK1、ERK2mRNA、ERK1/2、p-ERK1/2蛋白降低（P<0.01或P <0.05）。
与MTX组比较，XFC组肺组织ERK1、ERK2mRNA表达降低（P<0.05）。
4结论
4.1AA大鼠肺功能的变化
4.1.1AA大鼠肺功能损伤特点：AA大鼠存在肺功能损伤，肺功能变化以参数FEV1、MMF、PEF、FEF50、FEF75降低为主，主要表现特征为通气功能障碍，并伴有一定程度的小气道功能损伤。
4.1.2Smads和ERK通路cross-talk导致肺功能降低：Smads和ERK通路在启动子TGF-β1诱导下，逐渐被磷酸化激活，进而进入细胞核参与转录活动，导致AA肺部病变发生，出现肺功能降低。
4.1.3RA肺功能降低中医病机呈“脾虚湿盛”特征：“脾虚”在RA肺功能损伤发生、发展中起重要作用，脾气亏虚，正气不足，肺气虚弱；脾气亏虚，痰湿内生，肺失治节；脾气亏虚，痰瘀互结，肺络阻滞。
4.2“健脾化湿通络”中药XFC对AA大鼠的疗效
4.2.1XFC能明显抑制AA大鼠关节炎症反应：XFC能降低AA大鼠足趾肿胀度和AI，减少炎细胞对关节滑膜的浸润，减轻局部充血和肿胀，抑制血管翳的形成，改善关节功能。
4.2.2XFC能明显改善AA大鼠肺功能水平：XFC通过提高肺功能参数FEV1、FEF50、FEF75、MMF、PEF表达，改善AA大鼠肺部通气功能和小气道功能，增强肺部气体交换率，提高肺功能水平。
4.2.3XFC能明显提高AA大鼠体质量：XFC通过健脾益气、化湿通络的功效，能明显提高AA大鼠食欲及饮水量，增加体质量水平，改善全身症状和机能活动。
4.2.4XFC能明显调节AA大鼠肺组织形态学：XFC能减轻炎性细胞浸润程度，通过抑制炎性介质对肺组织的刺激，降低AA大鼠肺间质纤维化程度，维持Ⅱ型肺泡细胞细胞器的功能和形态。
4.3XFC改善AA大鼠肺功能的机制
4.3.1XFC可降低AA大鼠关节炎症反应提高肺功能：XFC通过抑制滑膜组织炎细胞浸润，降低全身炎症反应，减少炎症介质渗出及对肺组织器官的刺激，抑制肺间质病变的发生，改善肺功能水平。
4.3.2XFC可减轻AA大鼠肺部炎症反应提高肺功能：XFC通过抗炎作用，减少炎性介质对肺泡的直接刺激，促进中性粒细胞等炎细胞的吸收或清除，降低肺组织炎症反应，改善肺功能水平。
4.3.3XFC可调节AA大鼠细胞因子平衡提高肺功能：XFC通过上调IL-10、IFN-γ、FGF表达，下调IL-1β、IL-4、TGF-β1、CTGF表达，抑制AA大鼠肺间质纤维化的发生，提高肺功能水平。
4.3.4XFC可改善AA大鼠肺组织形态学提高肺功能：XFC通过减少炎性渗出物对肺组织损伤，保持肺组织病理形态学，维持肺泡Ⅱ型上皮细胞结构，增加肺泡的通气功能和弥散功能，改善肺功能。
4.3.5XFC可调节AA大鼠TGF-β1/Smads通路提高肺功能：XFC通过提高Smad7表达，抑制p-Smad2/3与Smad4的结合及阻止Smad2/3磷酸化，延缓AA大鼠肺纤维化的发生，改善肺功能。
4.3.6XFC可抑制AA大鼠ERK1/2通路表达提高肺功能：XFC通过抑制ERK1/2磷酸化过程，阻止信号通路的激活，减少致纤维化因子对肺组织的损伤，降低AA大鼠肺病变程度，改善肺功能。
4.3.7XFC可调节AA大鼠Smads和ERK通路cross-talk提高肺功能：XFC通过调节信号通路cross-talk，降低信号传导与DNA的转录活动，抑制AA大鼠肺间质纤维化形成，改善肺功能。
1Objective
Based on the Traditional Chinese Medicine theory of "correlationbetween lung and spleen ",“Lung Paralysis" as the breakthroughpoint,this paper analyzes and summarizes medical pathogenesis of lungfunction damage in rheumatoid arthritis (RA). From the perspective ofanimal experiment to observe the dynamic changes of paw swelling,arthritis index (AI) and lung function changes in adjuvant arthritis (AA)rat, detects changes of TGF-β1/Smads, ERK signaling pathways andcytokine in AA rats’ synovial and lung tissue, evaluates influence ofChinese medicine Xinfeng Capsule (XFC), and explores the mechanismof XFC improves lung function in AA rats.
2Methods
2.1Theoretical study
Collecting and organizing the relevant literature of RA lung disease,screening and summarize the research status of the RA lung diseasepathogenesis,evaluation and analysis of transforming growth factor beta1(TGF-beta1) induced by Smads, ERK pathway activation pathways.Studied from a theoretical point of the relationship between lung functionreduce in RA and the TGF-β1/Smads, ERK signaling pathway cross-talk. This article based on the basic theories of TCM, discussed therelationship between TCM "lung" and "spleen", by studying from theperspective of physiological pathology and etiology pathogenesis, andexplained the mechanism of “jianpihuashi Tongluo therapy” in thetreatment of RA decline in lung function.
2.2Experimental Study
50Wistar rats rats were randomly divided into five groups:normalcontrol (NC) group, model control (MC) group, methotrexate (MTX)group, Tripterygium glycosides piece (TPT) and XFC group, n=10. Inaddition to the NC group, right rear foot plantar skin of the rest ratsinjected Freund's complete adjuvant (CFA)0.1ml of proinflammatorymodeling, strengthen the immune by injection in the base of the tail ofCFA0.05ml at the15d, dosage at the19d. Dosage groups are asfollows: the XFC group:0.034g/1ml/100g, TPT group:0.57mg/1ml/100g,MTX group0.095mg/1ml/100g, NC、MC group:physiological saline9mg/1ml/100g; XFC,TPT,NC,MC group once a day, MTX group once aweek, each group continuous medication for30d. Observed the rats’body mass, paw swelling, arthritis index (AI) and pulmonary functionchanges, morphological changes of lung tissue, synovium by lightmicroscopy and electron microscopy, tested the expression of cytokineTGF-β1in interleukin the prime (IL)-1β, IL-4, IL-10, gamma interferon(IFN-γ), connective tissue growth factor (CTGF), fibroblast growth factor(FGF) by enzyme-linked immunosorbent assay, detected changes ofSmads/ERK pathway in synovial membranes and lung tissue by PCR,immunohistochemistry and Western blot.
3Results
3.1The theoretical research results
3.1.1TGF-β1/Smads pathway activated prompted RA lung function decreased.TGF-β1can first combined TβR Ⅱ (its receptor) to form atrimer complex TβRI, than further phosphorylated Smad2/3, p-Smad2/3to separate from TβRI, which than combined to form complexes withSmad4, carried signals from the cytoplasm into the nucleus to activatetranscription, promote lung lesions; whereas Smads7is inhibitorymolecules, if reduceing Smad7inhibition ability, inhibition oftranscription of Smads signals cannot be lost, the inhibitory effect on RAlung lesions, resulting in decreased lung function.
3.1.2ERK signaling pathway induced by RA lung function decreased:the activation of the ERK pathway may mediates cell growth,differentiation, proliferation, promotes the secretion of fibroblasts andexcessive accumulation of extracellular matrix (ECM) in pulmonaryinterstitial and alveolar, makes the alveolar distance increased to forminterstitial lung, decreases in pulmonary capillary bed and blood flow,changes in blood rheology, reduces pulmonary diffusion, weakens theexchange of oxygen and carbon dioxide, while the activation of ERKpathway can regulate the expression of inflammation-related genes,leading to lung function decreased.
3.1.3Smads and ERK pathway cross-talk is an important factor in thedecline in RA lung function: the activation process of TGF-β1/Smadsignaling pathway is also affected by the regulation of the ERK pathway,Smad2/3contains ERK phosphorylation sites,which can be phosphoryl-ated by ERK, promote p-Smad2/3and Smad4in binding andtranscription. Meanwhile, ERK pathway is regulated by Smads pathway,Smad4regulates transcription of ERK pathway related cytokines orkinase, which activates ERK signaling pathway, Smads and ERKpathway cross-talk common led to the decline in lung function.
3.1.4The "Spleen asthenia" is the pathological basis of RA decline in lung function: spleen asthenia caused by Qi and blood deficiency,spleen can not bear the lung,lead to deficiency of lung-qi and yin,andalso lead to failure of pulmonary qi to disperse and descend，resulting insuch syndrome like: cough, shortness of breath, spontaneousperspiration,etc; asthenia of splenic qi,failure of transportation andtransformation, which fails to distribute fluid and leads to attack of fluidretention on the lung, then lung fails to its Regulatory functions,manifeasting in such syndrome like: cough with profuse whitish sputum,even asthmatic breath with sputum rale, chest pain, prolonged andincurable; lung collaterals block, see more lung texture disorder,nodules, interstitial fibrosis were frequent in spleen deficiency caused byphlegm and blood stasis.
3.1.5The “Jianpihuashitang Tongluo Therapy” makes significantimprovement in RA lung function: According to the TCM pathogenesis ofRA decline in lung function “asthenia of spleen and stomach, asthenia ofqi and blood, accumulation of fluid and dampness, phlegm and bloodstasis " and “inclusion of asthenia and sthenia”characteristics, clinicallydefered to "eliminating pathogen and strengthening vital qi " principle oftreatment, using the " Jianpihuashitang Tongluo Therapy ", applyedXFC treatment of RA and pulmonary dysfunction, and achieved goodresults, XFC could further improve pulmonary symptoms and signs, toimprove the dispersion function and ventilation function by improvingjoint and systemic symptoms and signs.3.2The experimental study results
3.2.1Changes of lung function in rats and the impact of XFCCompared with NC group, the MC rats lung function FEV1, FEF50, FEF75,MMF, PEF decreased (P <0.01).Compared with the MC group, FEV1, FEF50, FEF75, MMF, PEF were increased in the XFC group (P <0.05or P <0.01).Compared with the MTX group, the level of FEF50, FEF75MMF wereincreased in the XFC group (P <0.05); compared with TPT group,pulmonary function parameters FEF75, MMF were increased in the XFCgroup (P <0.05or P <0.01).
3.2.2Changes of paw swelling, AI in rats and the impact of XFCCorrelation analysis showed the AA rat lung function FEF75, pawswelling were negative correlation, FEF50, FEF75and the AI werenegatively correlated (P <0.05or P <0.01).Prior to administration, compared with the NC group, body weight wasreduced in MC rats, and paw swelling degree, AI were significantlyhigher than those the MC rats (P <0.01).30days after administration, compared with the NC group, toe swelling,AI were elevated in MC rats; compared with the MC group, rat pawedema degrees and AI were lower in each treatment group; comparedwith the MTX group (P <0.05or P <0.01).
3.2.3Changes of joint morphological in rats and the impact of XFCLight microscopic observation: Compared with NC group, the MC ratssynovium and its affiliated organizations visible to a large number ofinflammatory cell infiltration increased vascular increased synoviallayered, showed villous projections embedded in the intra-articularsurface of the articular cartilage exfoliative or missing such as. Electronmicroscopy: the MC group synovial cells deformation, swelling ofmitochondria, rough endoplasmic reticulum was reduced, damaged,incomplete nuclear membrane, the uneven distribution of chromatin,irregular or partial disappearance of cristae arranged.The XFC treatment, the rat synovial visible small number of neutrophilsand other inflammatory cell infiltration, embedded in the intra-articular part of the synovial tissue, the articular surface was orderly; synovialcells the mitochondrial small amount of deformation, swelling, nuclearmembrane boundary more clearly, chromatin more uniform, most of thecristae arranged complete. Compared with the MTX group the XFCgroup synovial cells reduce mitochondrial swelling; the XFC group TPTgroup no significant difference.
3.2.4Changes of lung tissue morphologic in rats and the impact of XFCLight microscopy showed that: compared with the NC group, MC grouprat alveolar structure was irregular, the alveolar cavity atrophy ordisappear, was part of the lung substantive changes in lung intervalswidened visible in the interstitial infiltration of inflammatory cells, alveolarepithelial cell proliferation; reduce the number of electron microscopy Ⅱalveolar epithelial cells, the membrane structure was not completemitochondrial swelling, degeneration, reduced lamellar bodies wereemptying state, interstitial lung fibroblasts.XFC treated rats compared with regular alveolar structure, part of thealveolar space atrophy, deformation, seen in a small number ofneutrophils and other inflammatory cells in the interstitial infiltration;basic integrity of the structure of type Ⅱ alveolar epithelial cells,abundant rough endoplasmic reticulum, mitochondria and large partiallyintact, occasionally lamellar bodies emptying phenomenon; comparedwith the MTX group, the the XFC group of lung tissue inflammatory cellinfiltration and decrease in alveolar epithelial cells; the TPT groupshowed no significant differences.
3.2.5Changes of serum cytokine in rats and the impact of XFCCorrelation analysis showed AA the rats lung function FEV1, FEF75IL-1β, IL-4was negatively correlated FEF25, FEF50, FEF75, MMF withTGF-β1, CTGF, FGF negatively correlated; PEF and IL-10were positively correlated, FEV1, FEF50were positively correlated withTh1/Th2,FEF50, FEF75and IFN-γ were positively correlated (P <0.05orP <0.01).
Compared with the NC group, the level of IL-1β, IL-4, TGF-β1, CTGF ofserum were increased in MC, IFN-γ, IL-10, Th1/Th2cells, FGF werereduced (P <0.05or P <0.01).
Compared with the MC group, the expression of IL-1β, IL-4, TGF-β1,CTGF were reduce, and the expression of IFN-γ, IL-10, Th1/Th2, FGFwere increased in the XFC group (P <0.05or P <0.01).Compared with the MTX group, CTGF was decreased, and IFN-γ,expression of IL-10, Th1/Th2were increased in XFC group (P <0.05);Compared with TPT group, TGF-β1were decreased in the XFC group(P <0.05).
3.2.6Changes of Smads pathway in rats and the impact of XFCCorrelation analysis showed FEV1and TGF-β1mRNA, protein werenegatively correlated, FEF25and Smad4protein were negativelycorrelated, FEF50and TβRⅡ protein expression were negativelycorrelated with FEF75and TGF-β1, Smad3mRNA, p-Smad2/3proteinwas negatively correlated with PEF and TGF-β1protein were negativelycorrelated (P <0.05); FEF50, MMF Smad7mRNA were positivelycorrelated(P <0.05).
Compared with NC group, the level of TGF-β1, Smad2/3/4mRNA,TGF-β1, TβRI, TβRⅡ, Smad2/3, p-Smad2/3, Smad4protein wereincreased, expression of Smad7mRNA, protein of synovial lung tissuewere decreased in MC group (P <0.05or P <0.01).Compared with the MC group, expression of Smad2, Smad3, Smad4mRNA and TGF-β1, TβRI, TβRⅡ, Smad2/3, p-Smad2/3protein weredecreased, expression of Smad7mRNA and protein of lung tissue were elevated in XFC group (P <0.05or P <0.01).
Compared with the MTX group, expression of TGF-β1,Smad3mRNAand TGF-β1, Smad2/3protein were decreased, Smad4mRNA TβRIprotein were reduced, Smad7mRNA was increased in XFC group (P<0.05); Compared with TPT group, Smad3mRNA, TGF-β1, p-Smad2/3, Smad4protein were reduced, Smad7protein was increased in theXFC group (P <0.05), p-Smad2/3of lung tissue was lowered, Smad7mRNA was increased in the XFC group (P <0.05).
3.2.7Changes of ERK1/2in rats and the impact of XFC
Correlation analysis showed pulmonary function FEV1and ERK2mRNAwere negatively correlated, FEF25and p-ERK1/2protein werenegatively correlated, FEF75, MMF and ERK1/2, p-ERK1/2proteinwere negatively correlated (P <0.05). ERK1mRNA, Smad3mRNA inTβRⅡ protein of lung tissue were positively correlated, ERK2mRNA andp-Smad2/3was positively correlated, ERK1/2proteins, Smad2mRNA,Smad4protein were positively correlated, p-ERK1/2proteins andSmad4mRNA, p--Smad2/3protein were positively related (P <0.05);p-ERK1/2protein and Smad7were negatively correlated (P <0.05).Compared with the MC group, expression of ERK2mRNA, p-ERK1/2protein of lung tissue were decreased, ERK1, ERK2mRNA, ERK1/2,p-ERK1/2protein were decreased in the XFC group (P <0.05or P<0.01).
Compared with the MTX group, expression of ERK1, ERK2mRNA oflung tissue were decreased in the XFC group (P <0.05).
4Conclusions
4.1Changes of lung function in AA rats
4.1.1Pulmonary dysfunction features: changes of pulmonary functionparameter FEV1, MMF, PEF, FEF50, FEF75were reduced in AA rats, which mainly characterized ventilatory dysfunction, and accompanied bya certain degree of small airway function damage.
4.1.2Smads and ERK pathway cross-talk lead to reduce lung function:Smads and ERK pathway can inducte TGF-β1, which graduallyactivated by phosphorylation, and then enter the nucleus involved intranscriptional activity, resulting in AA lung lesions occur, pulmonaryThe function was reduced.
4.1.3TCM pathogenesis characteristics:"Spleen" in RA lung functionoccurred, plays an important role in the development, temper deficiency,lack of righteousness, lung weakness; temper deficiency sputum Thewet endogenous governance section of lung failure; temper deficiency,phlegm, pulmonary collaterals block.
4.2Efficacy of XFC on AA rats
4.2.1XFC can inhibit joint inflammation reaction of AA rats: XFC toreduce the the AA rat paw swelling and AI, to reduce the infiltration ofinflammatory cells of the synovial alleviate local congestion and swelling,inhibit the formation of pannus, improve joint function.
4.2.2XFC can improve the level of AA rats lung function by improvinglung function parameters FEV1, FEF50, FEF75, MMF, PEF expression:XFC improve the AA rat pulmonary ventilation function and small airwayfunction, enhanced pulmonary gas exchange rate and improve lungfunction level.
4.2.3XFC can improve the quality of the AA rats: XFC Jianpiyiqi thedampness Tongluo the efficacy, can significantly improve the the AArats appetite and water intake, increase the level of body mass, improvesystemic symptoms and functional activity.
4.2.4XFC can adjust the AA rat lung tissue morphology significantly:XFC can reduce the degree of infiltration of inflammatory cells, inflammatory mediators by inhibiting lung tissue stimulation, reduce theAA rat pulmonary interstitial fibrosis, maintain alveolar type Ⅱ cellfunction and morphology of the organelle.
4.3Mechanisms of XFC improve lung function
4.3.1XFC can reduce the AA rats joint inflammation reaction to improvelung function: XFC seeping through the inhibition of inflammatory cellinfiltration of synovial tissue, reduce the systemic inflammatoryresponse, reducing inflammatory mediators and the stimulation of lungtissues and organs, inhibition of interstitial lung disease occur, improvelung function level.
4.3.2XFC can reduce the AA rats lung inflammation to improve lungfunction: XFC by the anti-inflammatory effect, reducing the directstimulation of inflammatory mediators of alveolar promote absorption ofneutrophils and other inflammatory cells or clear, to reduce lunginflammation reaction, improve lung function level.
4.3.3XFC can adjust the AA rats cytokine balance to improve lungfunction: XFC FGF expression by up-regulating IL-10, IFN-γ, loweredIL-1β, IL-4, TGF-β1, CTGF expression, inhibition of AA rats Theincidence of pulmonary fibrosis to improve lung function level.
4.3.4XFC can improve the AA rat lung tissue morphology to improvelung function by reducing the inflammatory exudate lung tissue damage,keep the lung tissue pathomorphological maintain alveolar type Ⅱepithelial cell structure, increased alveolar ventilation function: XFCimprovement in lung function and diffusion capacity.
4.3.5XFC can adjust AA rats TGF-β1/Smads pathway to improve lungfunction: XFC increase Smad7expression, inhibition of p-Smad2/3with Smad4binding and prevents Smad2/3phosphorylation, delayingthe the AA rat lung fibrosis the occurrence, and improve lung function.
4.3.6XFC can inhibit the AA rats ERK1/2pathway expression toimprove lung function by inhibition of ERK1/2phosphorylation process,preventing the activation of the signaling pathway, reducing fibrogenicfactors on lung tissue damage.
4.3.7XFC can adjust Smads and ERK pathway cross-talk to improvelung function: XFC reduce signaling and DNA transcription activity,inhibiting the formation of the AA rat pulmonary interstitial fibrosis byregulating signaling pathways cross-talk to improve lung function.
引文
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