京尼平苷酸抗佐剂性关节炎大鼠滑膜细胞炎症及其MAPK信号转导通路的机制研究

发布时间：2018-07-08 18:03

本文选题：类风湿性关节炎 + 京尼平苷酸　；参考：《河北医科大学》2015年硕士论文

【摘要】：目的:类风湿关节炎(rheumatoid arthritis,RA)是一种以累及周围关节为主的多系统慢性自身免疫性疾病,以滑膜组织炎性增生、关节软骨进行性破坏为特征。该病遍布世界各地,各个种族均有发病。RA的传统治疗药物主要包括非甾体类抗炎药、抗风湿药、糖皮质激素、植物药、肿瘤坏死因子α拮抗剂等几大类。目前临床上应用的药物主要用于控制和缓解RA症状,且存在较多的不良反应,所以寻找高效低毒治疗RA的新药是今后研究的重要方向。我们前期研究发现,中药栀子具有显著抗炎镇痛作用,栀子浸膏及其单体京尼平苷酸明显抑制RA大鼠足肿胀,提示该药物对治疗RA有较好的作用前景。其中京尼平苷酸(geniposidic acid,GA),又名栀子苷酸,是一种环烯醚萜类化合物。MAPK信号转导通路包括ERK1/2、JNK1/2,P38共5条途径,是真核生物信号传递网络中的重要途径之一,在基因表达调控和细胞质功能活动中发挥关键作用,而信号转导途径的活化是类风湿性关节炎慢性滑膜炎的典型特征。因此我们猜测,京尼平苷酸治疗类风湿性关节炎可能与此途径有关。但京尼平苷酸如何作用于RA滑膜细胞、并进一步影响细胞内的MAPK信号转导通路,从而调节炎症介质分泌和功能蛋白的表达,目前并不清楚,尚需揭示其作用机制,为进一步将其筛选为有效药物提供理论依据。方法:本实验主要分为四个部分:第一部分实验:用弗氏完全佐剂建立大鼠佐剂型关节炎(AA)模型并分离培养成纤维样滑膜细胞。据文献报道FLS在体外培养后能够稳定增生。我们取第3代成纤维样滑膜细胞进行免疫细胞化学鉴定;第二部分实验分为5组:对照组(1640培养基,不含药物),京尼平苷酸高(终浓度为1×10-5 molL-1)、中(终浓度为1×10-6 molL-1)、低(终浓度为1×10-7 molL-1)给药组和甲氨蝶呤(methotrexate,MTX)阳性对照药组(终浓度为1×10-6 molL-1)。给予药物干预后,检测相关指标。分别在加入药物24小时、48小时和72小时用MTT法检测细胞增殖情况,用Hoechst33342和PI双染荧光显微镜观察给予不同浓度GA后凋亡和坏死的成纤维样滑膜细胞的细胞形态学改变。第三部分试验:研究京尼平苷酸对细胞上清液细胞因子的影响。分别用ELISA法测定给药后细胞上清液中IL-1β、TNF-α、IL-10的含量。通过检测各类细胞因子含量的变化探讨京尼平苷酸对类风湿性关节炎的治疗作用机制;第四部分实验:研究京尼平苷酸对成纤维样滑膜细胞的Ras-MAPKS途径的影响。用RT-PCR法检测JNK、ERK、P38基因m RNA表达,用western blotting检测磷酸化的JNK、ERK、P38蛋白表达的影响,从细胞膜信号传导途径探讨京尼平苷酸在RA中的作用机制。结果:第一部分实验结果发现AA模型建立成功:大鼠在用弗氏完全佐剂注射后第14天开始出现足部红肿。炎症出现的高峰期是在免疫后的第25天。大鼠逐渐开始出现活动障碍,并可见足部、尾根部“关节结节”。将关节炎指数3分以上的大鼠脱臼处死,取大鼠病变部位关节滑膜组织进行分离与培养。培养后的第3代成纤维样滑膜细胞进行免疫细胞化学染色,阳性细胞数量达到95%以上,符合实验要求,原代细胞培养理想。第二部分实验结果发现,京尼平苷酸高浓度组(10-5 molL-1)能够抑制滑膜细胞增殖,较之模型对照组比较明显降低(P0.05);京尼平苷酸高浓度组(10-5 molL-1)、京尼平苷酸中浓度组(10-6 molL-1)能够促进滑膜细胞凋亡,较之模型对照组比较明显降低(P0.05)。第三部分试验细胞因子的检测发现:加京尼平苷酸药物后,治疗组细胞上清液中京尼平苷酸高浓度组(10-5 molL-1),京尼平苷酸中浓度组(10-6 molL-1)中的IL-1β、TNF-α的含量较之模型对照组比较明显降低(P0.05);京尼平苷酸高浓度组(10-5 molL-1)中的IL-10的含量明显上调。第四部分试验由RT-PCR结果可以看出:京尼平苷酸高浓度组(10-5 molL-1),京尼平苷酸中浓度组(10-6 molL-1)能够抑制JNK、ERK、P38基因m RNA表达。由western blotting结果可以看出:京尼平苷酸高浓度组(10-5 molL-1),京尼平苷酸中浓度组(10-6 molL-1)能够抑制磷酸化JNK、ERK、P38蛋白的表达。结论:1足够量浓度的京尼平苷酸能够抑制成纤维样滑膜细胞的增生,促进成纤维样滑膜细胞的凋亡。2足够量浓度的京尼平苷酸对于成纤维样滑膜细胞的上清液中IL-1β、TNF-α的含量起到抑制分泌作用,对成纤维样滑膜细胞的上清液中IL-10的含量起到刺激分泌的作用。3足够量浓度的京尼平苷酸能抑制成纤维样滑膜细胞中磷酸化的JNK、ERK、P38-MAPKs蛋白及m RNA的表达。综合以上四部分实验结果,我们发现京尼平苷酸对于AA有很好的治疗作用,可以明显抑制成纤维样滑膜细胞的增殖,促进成纤维样滑膜细胞的凋亡。抑制成纤维样滑膜细胞释放炎性因子IL-1β、TNF-α,促进保护性因子IL-10的释放。同时还可以抑制AA大鼠成纤维样滑膜细胞中的JNK、ERK、P38-MAPKs信号通路的激活。
[Abstract]:Objective: rheumatoid arthritis (RA) is a multi system chronic autoimmune disease involving the surrounding joints, characterized by inflammatory hyperplasia of the synovial tissue and the progressive destruction of articular cartilage. The disease is all over the world, and the traditional therapeutic drugs for the pathogenesis of.RA, including non steroidal anti-inflammatory drugs, are all around the world. Antirheumatic drugs, glucocorticoids, plant drugs, tumor necrosis factor alpha antagonists, and other major categories. The current clinical drugs are mainly used to control and alleviate RA symptoms, and there are many adverse reactions. So it is an important direction for future research to find new drugs with high efficiency and low toxicity for the treatment of RA. Anti inflammatory and analgesic effects, gardenia extract and mono geniposide acid obviously inhibit foot swelling in RA rats, suggesting that the drug has a good role in the treatment of RA. Among them, geniposide acid (geniposidic acid, GA), also known as geniposide, is a.MAPK signal transduction pathway including ERK1/2, JNK1/2 and P38, which is a kind of enidoterpenoids. One of the important pathways in the eukaryotic signal transmission network plays a key role in the regulation of gene expression and cytoplasmic function, and the activation of signal transduction pathway is a typical characteristic of chronic synovitis in rheumatoid arthritis. Therefore, we suspect that geniposide may be related to this pathway in the treatment of rheumatoid arthritis. How niping glycoside acts on RA synovial cells and further affects the MAPK signal transduction pathway in the cells, thus regulating the secretion of inflammatory mediators and the expression of functional proteins, is not clear at present. It is still necessary to reveal its mechanism of action and provide a theoretical basis for further screening of its effective drugs. Methods: this experiment is divided into four parts: the first part: A part of the experiment: the model of rat adjuvant arthritis (AA) was established with Freund's complete adjuvant and fibrous synovial cells were isolated and cultured. It was reported that FLS could be stable after culture in vitro. We took third generations of fibroblast like synovial cells for immunocytochemical identification; the second part of the experiment was divided into 5 groups: the control group (1640 medium, no) Drugs), geniposide acid high (final concentration is 1 x 10-5 mol? L-1), medium (final concentration is 1 * 10-6 mol? L-1), low (final concentration is 1 * 10-7 mol? L-1) administration group and methotrexate (methotrexate, MTX) positive control group (terminal concentration is 1 * 10-6 mol? L-1). Give drug drying prognosis, detection of related indicators, 24 hours, 48 hours and 72, respectively. The cell proliferation was detected by MTT method. The morphological changes of fibroid synovial cells with apoptosis and necrosis after different concentrations of GA were observed by Hoechst33342 and PI double staining fluorescence microscope. The third part experiment: To study the effect of geniposide on cytokine of cell supernatant. The cells were measured by ELISA method respectively. The content of IL-1 beta, TNF- a, IL-10 in the clear liquid. The therapeutic mechanism of Geniposide in the treatment of rheumatoid arthritis was explored by detecting the changes in the content of various cytokines. The fourth part of the experiment was to study the effect of geniposide on the Ras-MAPKS pathway of fibroid synovial cells. JNK, ERK, P38 gene m RNA expression was detected by RT-PCR, and West was used. Ern blotting detected the effects of phosphorylated JNK, ERK, P38 protein expression, and explored the mechanism of Geniposide acid in RA from cell membrane signal transduction pathway. Results: the first part of the experiment found that the AA model was established successfully: the rats began to appear foot swelling at fourteenth days after the injection of the Freund complete adjuvant. The peak period of the inflammation appeared at the peak period. After twenty-fifth days of immunization, the rats gradually began to appear activity disorder, and the foot and tail nodules were seen. The rats were dislocated and killed in the rats with the arthritis index more than 3 points. The synovial tissue of the lesion parts of the rats was isolated and cultured. The cultured third generation fibroblast like cells were stained with immunocytochemical staining and positive cells. The number reached more than 95%, which was in line with the experimental requirements and the primary cell culture was ideal. The second part of the experiment found that the high concentration group (10-5 mol? L-1) could inhibit the proliferation of synovial cells (P0.05) compared with the model control group (10-5 mol? L-1), and the concentration group of Geniposide (10-6 mol? L-1). The apoptosis of synovial cells was significantly reduced (P0.05) compared with the model control group (P0.05). After the test of cytokine, the content of IL-1 beta in the concentration group (10-6 mol? L-1) in the concentration group (10-6 mol? L-1) in the cell supernatant of the treatment group was compared with the model of IL-1 beta, and the content of TNF- a was more than that of the model. The control group was significantly lower (P0.05); the content of IL-10 in the high concentration group of Geniposide (10-5 mol? L-1) was obviously up-regulated. The fourth part of the test showed that the high concentration group of Geniposide acid (10-5 mol? L-1) and the concentration group of Geniposide (10-6 mol? L-1) could inhibit JNK, ERK, P38 gene expression. Ng results showed that the high concentration group of Geniposide (10-5 mol? L-1), the concentration group of Geniposide (10-6 mol? L-1) could inhibit the expression of phosphorylated JNK, ERK, P38 protein. Conclusion: 1 concentration of Geniposide can inhibit the proliferation of fibroid synovial cells and promote the concentration of apoptotic.2 in fibroid synovial cells. Geniposide acid inhibits the secretion of IL-1 beta, TNF- a in the supernatant of fibroid synovial cells and stimulates the secretion of IL-10 in the supernatant of fibroid synovial cells..3 sufficient concentration of Geniposide can inhibit the phosphorylation of JNK, ERK, P38-MAPKs protein in fibroblast like synovial cells. M RNA expression. Combined with the four parts of the experimental results, we found that geniposide has a good therapeutic effect on AA, which can obviously inhibit the proliferation of fibroid synovial cells and promote the apoptosis of fibroid synovial cells. Inhibition of fibroid synovial cells release inflammatory factor IL-1 beta, TNF- alpha, and promote the release of protective factor IL-10. It can also inhibit the activation of JNK, ERK and P38-MAPKs signaling pathways in fibroblast like synovial cells of AA rats.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22

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