【作者】 马艳苗；

【导师】 周然；

【作者基本信息】 湖北中医药大学 ， 方剂学， 2013， 博士

【摘要】 类风湿关节炎(rheumatoid arthritis, RA)是一种以滑膜炎症、骨骼和软骨破坏及滑膜组织增殖为特征的自身免疫性疾病。RA的发病机制不仅涉及T淋巴细胞活化,而且与机体的氧化应激状态及线粒体功能失活参与调控细胞凋亡进程密切相关。近年来的研究表明活性氧(Reactive oxygen species,ROS)在RA的防治中发挥着重要的作用。在许多组织中,ROS引发的氧化应激破坏线粒体呼吸链,损伤线粒体并导致细胞衰老和死亡。而对于RA类肿瘤样增生的滑膜组织而言,诱导ROS表达或生物活性的发挥,能够上调促凋亡蛋白的表达,促进线粒体通透性转运孔的开放,激活天冬氨酸特异的半胱氨酸酶(caspase),诱导过度增殖的滑膜细胞走向凋亡,这无疑将成为遏制RA病情发展的一个重要环节,有利于RA的防治。风湿宁胶囊(以下简称风湿宁,FSN)主要用于治疗寒湿兼瘀血阻络型痹症,经十余年临床应用证实其对RA疗效显著。前期已开展的实验研究表明,FSN具有抗炎、镇痛、免疫调节等药理作用,在抗RA方面具有良好的应用前景,但其发挥抗炎、免疫调节的分子机制尚未明了,特别是其抗RA的机制是否与诱导过度增殖的滑膜细胞凋亡有关目前尚未明确。为此,我们针对这一问题进行了一系列研究,探讨了FSN对免疫功能的调节及其促进滑膜细胞凋亡的分子机理,结果显示,FSN可调节炎症因子的分泌和恢复Th1/Th2、CD4+/CD8+平衡,而且能够通过升高体内活性氧的表达来诱导过度增殖的滑膜细胞经线粒体途径实现凋亡,而后者很可能是FSN抗RA的一个新机制。目的：以牛II型胶原诱导的大鼠为模型,从CIA大鼠的足肿胀度、关节指数、病理组织形态学改变、细胞因子、T淋巴细胞亚群等方面考察FSN的抗炎及免疫调节作用,阐明其可能的分子机制。观察风湿宁胶囊对CIA大鼠滑膜细胞是否具有促凋亡作用并从活性氧的生成、线粒体功能、Bcl-2、caspase等角度探讨其可能的作用机制。方法：大鼠足跖皮内注射弗氏完全佐剂诱发CIA模型；以FSN0.33.0.66、1.32g-kg-ig给药,持续30d,0.03g·kg-1雷公藤多苷为阳性对照。采用足容积法测量足肿胀度；利用关节炎指数对大鼠关节损伤程度进行评价；称重法测定大鼠体重、脾、胸腺的重量；光学显微镜下观察关节组织病理学变化；拍摄关节X线片；ELISA法检测血清TNF-α、IL-1、IFN-γ、IL-4、IL-10含量；流式细胞术检测T淋巴细胞亚群水平。透射电镜下观察大鼠滑膜细胞的超微结构；Fe2++H2O2系统检测羟自由基含量；紫外分光光度法检测滑膜ATP含量、caspase-3、caspase-9活性；ELISA法检测滑膜上清液中Cytc的水平；免疫组化法检测滑膜组织VEGF、Bcl-2、Bax的表达；RT-PCR检测凋亡相关基因caspase-3、caspase-9mRNA的表达水平；Bradford蛋白浓度法测定滑膜蛋白的浓度；Western blot法检测滑膜caspase-3、caspase-9p35、VDAC1、AIF蛋白表达变化。结果：1、FSN对CIA大鼠具有治疗作用,其抗炎及免疫调节作用与降低Thl细胞分泌的IL-1、TNF-α、IFN-γ等炎性细胞因子水平,升高Th2细胞分泌的抗炎细胞因子IL-4、IL-10含量及恢复CD4+/CD8+平衡密切相关。此外,在血管微环境方面,证实了CIA大鼠高表达的VEGF蛋白在FSN用药干预后表现出不同程度地降低趋势,血管翳的形成和滑膜细胞的生长得到抑制。FSN (0.66、1.32g-kg-1)能够明显抑制CIA大鼠足肿胀、减轻关节疼痛、多发性关节炎等症状,减缓CIA大鼠体重的下降；光镜下观察到关节滑膜组织轻度增生、炎细胞浸润及滑膜组织新生血管减少、血管翳减轻、偶见少量纤维增生、骨及软骨受损程度减轻；FSN能够不同程度地下调Thl细胞分泌的IL-1、TNF-α、IFN-γ水平,上调Th2细胞分泌的IL-4、IL-6、IL-10水平,并降低CIA大鼠的胸腺指数；流式结果也表明,FSN能恢复CIA大鼠CD4+/CD8+比值,降低CD4+T淋巴细胞的水平。2、FSN上调CIA大鼠滑膜组织ROS的表达CIA大鼠滑膜组织中ROS含量降低,FSN给药(0.33、0.66、1.32g·kg-1,ig,d30)后ROS的表达增高,而FSN升高CIA大鼠ROS水平是其调节线粒体膜通透性转运孔开放、滑膜细胞内ATP水平及线粒体跨膜电位、影响滑膜细胞凋亡进程、抑制免疫亢进、发挥其治疗作用的特点之一。3、FSN通过影响信号转导途径诱导滑膜细胞凋亡FSN体内给药后CLA大鼠滑膜细胞基本结构恢复正常且能够抑制间质胶原纤维的异常增生,改善线粒体的肿胀或空泡变性状态；Western-blot结果显示,FSN可促进VDAC1蛋白表达量升高；紫外分光光度法结果表明FSN用药组的ATP含量显著降低；而在滑膜组织匀浆的上清液中检测到FSN (0.66g-kg-1)的Cytc释放量明显增多；免疫组化结果表明,随着剂量的增大,FSN给药组滑膜细胞的Bcl-2阳性表达逐渐减少,呈下降趋势,Bax日性细胞的表达明显增多；经RT-PCR、Western-blot证实,FSN干预后的CIA滑膜组织中,caspase-3、caspase-9p35、AIF的表达明显上调；caspase酶活性试验数据也表明,caspase-3、caspase-9活性在FSN给药前后有显著差异。结论：1、FSN对CIA大鼠多发性关节炎症具有明显的改善作用。FSN抑制CIA大鼠体内炎性细胞因子IL-1、TNF-α、IFN-γ的分泌,促进抗炎细胞因子IL-4、IL-6、IL-10的生成,降低CD4+/CD8+及抑制VEGF蛋白表达,是其发挥调节CIA大鼠亢进的免疫功能效应的关键。2、FSN通过调节体内ROS的生成影响滑膜线粒体功能及Th细胞的分化,促进经caspase依赖性途径、AIF途径的细胞凋亡,抑制滑膜的过度增殖,从而发挥其治疗作用。3、线粒体功能改变导致滑膜细胞凋亡参与了FSN防治RA的病理生理改变,表现为：线粒体在ROS的刺激下,促进VDAC1通道开放,影响MPTP的开放及ATP等能量物质的逸出,造成线粒体呼吸链抑制、膜电位丧失,释放Cytc和AIF,引发细胞凋亡的两条途径：一条需要Bcl-2家族和caspase酶依赖性参与,激活caspase-9和下游的caspase-3,导致细胞凋亡；另一条是需要AIF调节的多聚聚合酶途径的参与。在这一过程中,抗凋亡因子Bcl-2和促凋亡因子Bax蛋白通过与VDAC1的结合,调节凋亡进程。因此,线粒体功能改变可能在滑膜细胞凋亡中发挥了重要作用。更多还原

【Abstract】 Rheumatoid arthritis (RA) is a autoimmune disease characterized by inflammation and proliferation of synovial tissue resulting in bone and cartilage destruction.The pathogenesis of RA involving the activation of t-lymphocytes and is closed related with oxidative stress, deactivated mitochondrial function which regulated the apoptosis process.In recent years, studies have shown that reactive oxygen species (ROS) played an important role in the prevention and treatment of RA. In many organizations, ROS-induced oxidative stress could damage mitochondria respiratory chain, inducing mitochondrial injury and cell apoptosis and death.But it is known that for tumor-like proliferation of synovial tissue, inducing the expression or biological activity of ROS, will become an important pathological aspects that curbing the excessive proliferation of RA synovial cell, which will also be in favour of prevention and treatment of RA. Fengshining capsule (FSN) is mainly for the treatment of rheumatoid arthritis casued by cold-dampness and blood stasis blocking collaterals, has been confirmed its significantly effect through more than10years clinical application. Early experimental studies have shown that FSN had a wide range of pharmacological effects such as anti-inflammatory, analgesic and immune regulation.Although FSN has a good anti-RA prospects, its mechanisms on anti-inflammatory, molecular of immune regulation are not yet cleared, especially if its anti-RA mechanism has some relationship of inducing the excessive proliferation of synovial cell apoptosis, so far is not yet clear. Therefore, a series of studies were focused on the subject to explore the FSN in the regulating immune function and promoting the molecular mechanism of synovial cells apoptosis. Results proved that FSN could regulate the secretion of inflammatory factors and reinstate Th1/Th2, CD4+/CD8+balance.Meanwhile, it could increase expression of reactive oxygen species in inducing apoptosis of synovial cells via mitochondrial pathway, which probably is a new resistance mechanisms of FSN for RA.Objective:To enunciate the FSN possible molecular mechanism of the anti-inflammatory and immune regulation effects from the foot swelling, bone of CIA rat index and pathologic histological changes and cell factors and t-lymphocyte subsets. Bovine type Ⅱ collagen-induced rats model were prepared. The apoptosis-promoting effects of the FSN on CIA rat synovial cell was observated and its possible mechanism was investigaged on generation of reactive oxygen species, mitochondria function, Bcl-2, caspase.Method:The CIA model in rats was induced by Freund’s complete adjuvant. Therapeutic treatment of interagastric administration FSN (0.33,0.66,1.32g·kg-1,30d), tripterygium glycosides (0.03g·kg-1) were given as positive control. Foot swelling of CIA rats was measured with foot volume method. Arthritis index was used to evaluate the degree of joint damage of rat arthritis. Body weight, spleen and thymus weights were measured. Histopathological changes of joints were observed under the light microscope. Joint X ray films were taken. ELIS A method was used for detecting the content of serum TNF-a, IL-1, IFN-y, IL-4and IL-10. The level of t-lymphocyte subsets were detected with flow cytometry. Transmission electron microscopic was used to observate the ultrastructure of rat synovial cells. Hydroxyl free radicals were detected with Fe2++H2O2system.Synovial ATP content, activity of caspase3and caspase-9were assayed using UV spectrophotometric detection.ELISA was used to assay synovial Cytc levels in the supernatant. VEGF, Bcl-2, Bax expression in synovial tissues were detected by immunohistochemical assay. Apoptosis-related gene expression levels of caspase-3, caspase-9mRNA were detected by RT-PCR assay.Bradford protein concentration assay was used to detect synovial membrane protein concentration. The expression changes of synovial caspase-3, caspase-9p35, VDAC1, AIF were detected by Western blot assay.Results:1. FSN played a treatment role on CIA rats. Its anti-inflammatory and immunomodulatory effects were related with reducing levels of Inflammatory Cytokines(IL-1, TNF-a, IFN-y)secreted by Th1cell, increasing the content of anti-inflammatory cytokine (IL-4, IL-10) secreted by Th2cells and restoring CD4+/CD8+balance. In addition, in vascular micro-environment, it was confirmed that high expression VEGF protein in CIA rats were decreased on varying degrees after FSN medication intervention, which further inhibited the growth of synovial cells.FSN (0.66,1.32g· kg-1) could significantly suppress the CIA rat foot swelling and relieve joint pain, multiple arthritis symptom, as well as eased the decreased weight of CIA rats. Articular mild hyperplasia, inflammatory cell infiltration in the synovium and synovial tissue reduction of neovascularization, vascular proliferation of associated mitigation, occasionally a small amount of fiber, bone and cartilage damage mitigation were observed under light microscopic observation. FSN could reduce IL-1, TNF-a, IFN-y levels secreted by Thl cells on varying degrees and increase IL-4, IL-6, IL-10levels secreted by Th2cells. FSN was proved to lower CIA rat thymus index. Streaming results also showed that FSN could restore CD4+/CD8+ratio in CIA rats, reducing the level of CD4+T lymphocyte.2. FSN raised the level of ROS expression in CIA rats synovial tissueROS content of synovial tissue were reduced in CIA rats. The increasing ROS expression of FSN (0.33,0.66,1.32g· kg-1, ig,d30) in drug delivery was indicated that FSN risen ROS level of CIA rat was one of its features on regulating mitochondrial membrane permeability transition pore open, ATP levels in synovial cells, mitochondrial transmembrane potential, affecting the synovial apoptosis process, suppressing immune hyperthyroidism and playing treatment role.3. FSN induced the synovial cells apoptosis by affecting the signal transduction pathwaysThe basic structure of synovial cells in CIA rats is returned to normal after FSN in vivo delivery and FSN was able to contain abnormal hyperplasia of interstitial collagen fibers, as well as improved mitochondrial swelling or voided denatured state. The result of Western-blot analysis demonstrated that FSN could promote the expression of VDAC1rising.The result of ultraviolet spectrophotometry analysis showed that FSN significantly decreased the ATP content in drug group. The supernatant of Synovial tissue homogenate was detected that FSN (0.66g·kg-1) Cytc emissions were clearly increased.Immunohistochemical results showed that as the dose increasing, Bcl-2positive expression of synovial cells in FSN drug delivery decreased, had a downward trend.Meanwhile,Bax expression of positive cells significantly increased.The result of RT-PCR, Western-blot analysis confirmed that the expression of caspase-3, caspase-9p35, AIF markedly increased in the CIA synovial tissue of FSN intervention group. The caspase enzymes experimental data also stated that caspase-3, caspase-9activity in FSN had a notable difference between before and after medication.Conclusion:1.FSN has significantly improve effect on inflammation of multiple joints in CIA rats.FSN might inhibit proinflammatory cytokines IL-1, CIA rats TNF-α, IFN-γ secreted, promoting anti-inflammatory cytokines(IL-4, IL-6, IL-10) formated, reducing CD4+/CD8+and suppressing the expression of VEGF protein.That may be the key which FSN regulate hyperthyroidism immune function in CIA rats.2. FSN could promote the apoptosis via caspase-dependent, AIF pathway and curb the excessive proliferation of synovium by regulating ROS generation in vivo to affect synovial mitochondrial function, the Th cell differentiation to play its therapeutic effect.3. Mitochondrial features change led to synovial cells apoptosis participating RA pathology physiological change in FSN control group,such as:Under the stimulation of ROS, mitochondrial promote VDAC1channel open, affecting the opening of MPTP and the escaping of ATP energy matter, resulting in inhibition of the mitochondrial respiratory chain, loss of membrane potential, release of Cytc and AIF,which triggered apoptosis in two ways:one is a Bcl-2family and Caspase enzymes dependented participation way, activating caspase-9and the downstream of caspase-3, leading to apoptosis. Another is multiplex polymerase pathway regulating by AIF. The anti-apoptotic factor Bcl-2and apoptosis factor Bax protein through combinating with VDAC1regulate the apoptosis process. Therefore, mitochondrial dysfunction may play an important role in synovial apoptosis.更多还原