Proline-Rich Tyrosine Kinase 2 （PYK2）在系统性红斑狼疮患者发病机制和治疗中的作用研究
摘要
研究背景和目的
富含脯氨酸的非受体酪氨酸激酶(Proline-Rich Tyrosine Kinase 2,PYK2)和粘着斑激酶(FAK)同属于粘着斑激酶家族成员,二者具有相同的结构域和很高的序列相似性。PYK2的多肽链包含三个结构域,即N末端结构域、中央酪氨酸激酶结构域和C末端结构域。N末端结构域包含一个受体结合域,该结构域可与酪氨酸激酶受体如EGF受体(EGFR)及细胞骨架蛋白如埃兹蛋白(Ezrin)等结合。C末端结构域包含两个富含脯氨酸的区域：(aa712-713)和(aa876-877),其中PXXPK、PXXLG基序是SH3结构域的结合位点,分别结合含有SH3结构域的蛋白,如p130cas, Graf, PI3K, Hic-5,paxillin等。中央酪氨酸激酶结构域包含三个酪氨酸磷酸化位点402,579,580(Tyr402,Tyr579,Tyr580),是PYK2的活性部位,其中,Tyr402可以发生自动磷酸化,磷酸化的Tyr402可与含有SH2结构域的蛋白质,如Src,Abl等结合,Src与Tyr402结合并被磷酸化而活化,活化后的Src反过来磷酸化PYK2的其它磷酸化位点,使PYK2活性进一步增强。故PYK2蛋白活化的标志是其402位点的磷酸化。
PYK2分布在神经系统和造血来源的细胞,可以感受包括细胞外基质在内的多种物理和化学的细胞外信号而激活,活化后的PYK2作为细胞内信号转导途径的上游调节分子,通过调节细胞内多条信号转导途径(PI3K、MAPK、JAK/STAT途径)参与胞内信号传递,从而在细胞粘附、迁移、生存、增殖、凋亡、分化等生理过程中发挥重要作用。由于PYK2主要在造血系统来源的细胞表达,因此PYK2对淋巴细胞功能的影响更为重要。活化后的PYK2参与免疫细胞形态的维持和细胞迁移、调节淋巴细胞的活性和增值、扩增淋巴细胞表面多种受体信号。由此可见,PYK2途径的活化是多种细胞外信号促进淋巴细胞活化的一条重要通路。
系统性红斑狼疮是最具代表性的多系统受累的自身免疫性炎性疾病,其主要特征是自身反应性T、B淋巴细胞的异常活化和多种自身抗体的出现。SLE发病机理仍然不清楚,但多种细胞因子、趋化因子、生长因子、整合素以及外界物理化学刺激(如紫外线、某些药物等)的调节异常导致的T、B淋巴细胞异常活化,是SLE发病机制的一个必然环节,并且已经成为治疗SLE的靶目标。
目前研究已经证实,和PYK2同一家族的成员FAK介导的整合素β1信号促进了SLE患者T淋巴细胞共刺激分子CD40L的表达和T淋巴细胞的活化。既往研究也表明,PYK2的异常活化及对细胞内多条信号途径的失调控反应是造成细胞异常生长、分化或迁移的重要因素,参与了多种肿瘤、血管疾病和急慢性炎症,特别是有免疫因素参与的炎症的病理生理过程。鉴于PYK2信号分子在调节淋巴细胞活化中的重要作用以及淋巴细胞的异常活化在SLE发病机制中的重要地位,我们推测PYK2可能通过干扰淋巴细胞的异常活化而参与了SLE的发病。但是PYK2信号途径的活化在SLE的研究国内外相关报道极少,本研究将通过测定SLE患者外周血单个核细胞(PBMCs)PYK2蛋白和磷酸化水平探讨PYK2的活化；通过测定促进或阻断PYK2活化后对SLE患者PBMCs共刺激分子CD40L、CTLA4蛋白表达以及PBMCs增值的影响,探讨PYK2活化后的功能,从而探讨PYK2活化在SLE发病机制中的作用。
自从半个多世纪以前人们开始应用皮质激素治疗系统性红斑狼疮以来,激素治疗己成为狼疮肾炎治疗的主流。然而,重症狼疮肾炎(主要是Ⅳ型及部分Ⅲ型)的高死亡率并未因此得到解决。上个世纪80年代美国国立卫生研究院(NIH)开始倡导环磷酞胺(cyclophosphamide, CTX)大剂量静脉注射疗法,从此糖皮质激素结合免疫抑制剂的药物治疗方案成为SLE治疗的首选。糖皮质激素和免疫抑制剂对免疫反应的许多环节都有抑制作用,对合并有肾脏等重要脏器损害患者有重要价值,是现有治疗SLE最重要的药物。然而,在糖皮质激素结合免疫抑制剂的治疗过程中,由于用药量大、时间长,容易出现副作用和并发症,60%以上患者最后死于它们的毒副作用,严重影响患者的生存期和生活质量。近年来发展起来的血液净化、生物制剂和造血干细胞移植等治疗方法虽然能在不同程度上缓解病情,但均不能达到根治的目的。SLE的治疗仍然是当今的难点问题,人们一直在探索一种疗效确切又无副作用的药物以改善患者的长期预后和生存质量。
姜黄素(Curcumin)是古老亚洲的一种药用植物姜黄的有效成分,自从1937年Lancet刊登了一篇姜黄素治疗人类疾病的文献后,至今已有2700多篇关于研究姜黄素的文献发表。姜黄素可以在基因、细胞信号传导、阻滞细胞周期、免疫调节等多途径上发挥抗肿瘤、抗炎、抗调亡、抗氧化作用。从目前动物实验及临床应用的效果看,姜黄素是一种疗效好、无毒、无副作用的新型抗肿瘤和免疫调节药物,姜黄素与免疫抑制剂联合应用均有良好的协同作用。目前姜黄素已在治疗肿瘤、粥样硬化、感染]、多发性硬化和类风湿关节炎发挥了重要作用。但对于姜黄素治疗SLE的研究目前尚无报道。鉴于姜黄素在自身免疫性疾病治疗中具有良好的应用前景,并且本研究第一部分已经探讨了PYK2信号蛋白的活化可能在SLE发病机理和治疗中具有重要意义,因此阐明姜黄素的抗自身免疫作用是否与PYK2途径相关显得尤为重要。本研究将测定姜黄素对SLE患者PBMCs PYK2表达和活性的影响,探讨姜黄素对SLE的治疗作用是否与姜黄素对PYK2的活化的影响有关。
研究对象与方法
第一部分研究PYK2信号蛋白在SLE患者PBMCs的表达和活化及活化后的功能。选用48例SLE患者、32例类风湿关节炎患者(RA)和24例健康自愿者作为研究对象。晨起空腹抽取静脉血20ml,分离PBMCs,一部分直接用于PYK2表达和活化的测定,另一部分PBMCs用RPMI 1640培养液稀释成1 x 109／ml,取细胞悬液置6孔板中,每孔各1ml,设3个组：①单纯培养组；②PMA刺激组；③PMA刺激组+TyrA9阻断组。Western Blotting测定各组患者PBMCs PYK2总蛋白和其活化蛋白(p-PYK2)的表达,细胞免疫组化测定p-PYK2表达的细胞类型,流式细胞术测定PYK2活化对PBMCs表达共刺激分子CD40L和CTLA4的影响,3H-thymidine渗入法测定PYK2活化对PBMCs增值的影响。第二部分研究姜黄素对SLE患者PBMCsPYK2表达和活化的影响。选用20例活动期SLE患者和20例健康志愿者作为研究对象。晨起空腹抽取静脉血20ml,分离PBMCs,用RPMI 1640培养液稀释成1 X 106／ml,取细胞悬液置6孔板中,每孔各1ml,设4个组：①单纯培养组；②PMA刺激组；③PMA刺激组+TyrA9阻断组；④PMA刺激组+姜黄素组。培养结束收集各孔细胞,Western Blotting测定各组患者PBMC PYK2总蛋白和其活化蛋白(p-PYK2)的表达；细胞免疫组化测定p-PYK2表达的细胞类型；SYBR GreenⅠreal-time定量PCR研究姜黄素对PBMCs表达共刺激分子CD40L和CTLA4 mRNA的影响；流式细胞术测定姜黄素对PBMCs表达共刺激分子CD40L和CTLA4蛋白表达的影响,3H-thymidine渗入法测定姜黄素对PBMCs增值的影响。
结果
1、与健康自愿者比较,非活动期SLE患者和活动期SLE患者的PBMCPYK2表达和活化均明显增高,而RA患者的PBMCs PYK2表达和活化均未见明显升高。
2、WHOⅣ型狼疮肾炎的患者PBMCs p-PYK2的表达明显高于健康自愿者、其他类型狼疮肾炎和伴有中枢神经合并症者。p-PYK2的活化和SLE患者血清总补体水平呈明显负相关(P〈0.01,r=-0.668),与抗核小体抗体呈正相关(P〈0.05,r=0.535),与其他抗体的滴度均无相关性。p-PYK2的表达水平和SLEDAI积分亦无相关性。
3、PMA刺激后,SLE组、RA组和健康对照组PBMCs PYK2活化均明显增高。
4、应用PMA促进PYK2的活化后,SLE患者、RA患者和健康志愿者PBMCs CD40L和CTLA4的表达明显增高,TyrA9阻断PYK2的活化,则PMA的刺激不能诱导SLE患者PBMCs CD40L和CTLA4的表达,但仍能诱导RA患者和健康志愿者PBMCs CD40L和CTLA4的表达。这些结果提示,p-PYK2是PMA诱导SLE患者PBMC共刺激分子CD40L和CTLA4上调表达的一个调节因子。
5、单纯体外培养SLE患者、RA患者和健康志愿者PBMCs, RA患者和健康志愿者PBMCs增值弱于SLE患者,PMA刺激促进PBMCs PYK2活化后,SLE患者、RA患者和健康志愿者PBMCs的增殖均明显增高。TyrA9阻断PYK2的活化,则SLE患者PBMCs增殖被抑制,RA患者和健康志愿者PBMCs增值未见明显抑制。
6、健康对照组PBMCs PYK2的表达和活化明显弱于SLE患者组,PMA刺激后,两组PBMCs PYK2的表达和活化均明显增高,应用姜黄素或TyrA9预处理,两组PBMCs PYK2的表达和活化均明显减弱。姜黄素可以发挥酪氨酸激酶抑制剂样作用。
7、PMA刺激后,SLE患者和正常人外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达均明显升高。姜黄素或TyrA9预处理,姜黄素能抑制两组PBMCs CD40L、CTLA4 mRNA和蛋白的表达,TyrA9仅能抑制SLE患者外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达,不能抑制正常人外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达。
8、体外细胞培养,正常人PBMCs的增值弱于SLE患者PBMCs的增值。PMA刺激后,正常人和SLE组PBMCs的增值均明显增高。姜黄素预处理,可抑制正常人和SLE患者PBMCs的增值。TyrA9预处理仅能抑制SLE患者PBMCs的增值,不能抑制正常人PBMCs的增值。
9、姜黄素对SLE患者外周血单个核细胞PYK2活化的抑制率与与SLE患者血清补体水平C3(r=-0.52)、C4(r=-0.42)呈负相关(P<0.05)；与24小时尿蛋白定量成正相关(P<0.01),相关系数r=0.63；而与SLEDAI积分无相关性。
结论及意义
1、PYK2信号蛋白的表达和活化在SLE患者PBMCs明显升高,推测PYK2信号途径的异常可能参与和促进了SLE体内淋巴细胞的高度活化。
2、SLE患者PBMCs PYK2的表达和活化明显升高而类风湿关节炎患者PBMCs PYK2的表达和活化无明显变化,提示PYK2的异常活化可能对SLE具有特异性。
3、活动期SLE患者和非活动期SLE患者外周血单个核细胞PYK2的表达和活化均明显升高,并且PYK2的活化与SLEDAI积分无相关性,提示PYK2的表达和活化可能参与非活动期SLE多系统损伤的过程,具体作用机制尚需进一步研究。
4、在Ⅳ型狼疮肾炎患者中,p-PYK2表达明显增高,并且与血清补体水平呈负相关,与血清核小体抗体呈正相关,提示PYK2信号途径可能和狼疮肾炎的发生、发展有关。
5、PMA刺激后,SLE患者PBMCs共刺激分子CD40L、CTLA4表达增高,应用PYK2特异性抑制剂TyrA9阻断PYK2的活化,PMA刺激则不能诱导SLE患者PBMCs CD40L、CTLA4的表达,提示PYK2信号蛋白可能是PMA诱导的SLE患者PBMCs共刺激分子CD40L、CTLA4表达的一个关键信号通路蛋白。
6、PMA刺激可诱导SLE患者PBMCs的增值,应用PYK2特异性抑制剂阻断PYK2的活化,则PMA刺激不能诱导SLE患者PBMCs的增值,提示SLE患者PYK2活化可促进PBMCs增值。
7、PMA刺激后,健康组和RA组PBMCs的增值及共刺激分子CD40L和CTLA4表达均明显升高,但不能被PYK2特异性抑制剂TyrA9抑制,这表明在RA患者,除PYK2信号途径外,PMA还可通过其他途径促进PBMCs的增值和共刺激分子的表达,进一步提示PYK2的异常活化可能对SLE具有特异性。
8、健康对照组PBMCs PYK2的表达和活化明显弱于SLE患者组,PMA刺激后,两组PBMCs PYK2的表达和活化均明显增高,应用姜黄素或TyrA9预处理,两组PBMCs PYK2的表达和活化均明显减弱。证实姜黄素可以发挥酪氨酸激酶抑制剂样作用。
9、PMA刺激后,SLE患者和正常人外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达均明显升高。姜黄素或TyrA9预处理,姜黄素能抑制两组PBMCs CD40L、CTLA4 mRNA和蛋白的表达,TyrA9仅能抑制SLE患者外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达,不能抑制正常人外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达。提示姜黄素除可通过抑制PYK2信号蛋白的活化进而抑制SLE患者PBMCs共刺激分子CD40L、CTLA4 mRNA和蛋白的表达外,还可能通过抑制其他途径的信号蛋白进而抑制正常人PBMCs共刺激分子CD40L、CTLA4 mRNA和蛋白的表达。由此推测,姜黄素可能为一种多靶点抑制剂。
10、.姜黄素可通过抑制SLE患者外周血单个核细胞PYK2的活化进而抑制PBMCs的增殖,由此推测,姜黄素可能成为治疗系统性红斑狼疮的有效药物。
11、姜黄素对SLE患者外周血单个核细胞PYK2活化的抑制率与SLE患者血清补体水平C3(r=-0.52)、C4(r=-0.42)呈负相关(P<0.05)；与24小时尿蛋白定量成正相关(P<0.01),相关系数r=0.63；提示姜黄素的抑制作用与SLE患者的病情相关,特别是有肾脏损伤的SLE患者。
Backgroud and objective
Proline-rich tyrosine kinase 2 (PYK2)is a non-receptor protein tyrosine kinase that belongs to the focal adhesion kinase(FAK) subfamily. Similar to the prototype FAK,PYK2 contains three structural domains:a centrally located kinase domain, C-terminal domain and N-terminal domain.N-terminal domain contains a receptor combine domain and cytoskeletal protein combine domain.In the C-terminal tail,there are two proline-rich regions that are aa712-713 and aa876-877,PXXPKand PXXLG are domains binding of SH3 domain-containing proteins,including p130cas,Graf, PI3K,Hic-5,paxillin.The centrally located kinase domain contains tree sites of tyrosine phosphorylation402,579,580(Tyr402,Tyr579,Tyr580),which are the active sites of PYK2.Tyr 402 has been shown recently to be the PYK2 autophosphorylation site and was first shown to be bound by the SH2 domain of Src and later to other Src-family protein.Binding of Src is thought to allow for the subsequent phosphorylation of other tyrosine residues on PYK2 leading to increased activity. Phosphorylation of Tyr402 means the activation of PYK2 molecules.
PYK2 is primarily expressed in the central nervous system and the hematopoietic system.PYK2 is activated in response to a myriad of stimuli in many different cell types.In response to these stimuli,PYK2 is thought to mediate activation of mitogen-activated protein kinases(MAPK),phosphatidylinositol 3'-kinase(PI3),the Janus kinase/signal transducer and activator of transcription(JAK/STAT) pathway. PYK2 is a signaling molecule that regulates fundamental cellular processes,including adhesion,survival,proliferation,apoptosis differentiation.Because PYK2 is expressed in hematopoietic system,we focus here only on the role of PYK2 in lymphocyte. PYK2 phosphorylation participates to maintain the immunocyte morphological, regulates lymphocytic activity and proliferation,amplifies signals transmitted through various receptors on lymphocyte.Thus,the activation of PYK2 is an important pathway by which a variety of extracellular signals promote lymphocyte activation.
Systemic lupus erythematosus is the most representative multi-system involvement in autoimmune disease,it's main feature is the abnormal activation of self-reactive T,B lymphocytes and a variety of the emergence of autoantibodies.The pathogenesis of SLE is still not clear,However,the abnormal activation of lymphocytes resulted by the abnormal regulation of T,B caused by a variety of cytokines,chemokines,growth factors,integrins as well as the outside of physical or chemical irritants (such as ultraviolet light,certain drugs, etc)is an inevitable part of the pathogenesis of SLE,and has become the target goal of treatment of SLE.
The present study has confirmed that FAK,members of the same family with PYK2,mediated integrinβ1 signaling promotes SLE patients with T-lymphocyte costimulatory molecules CD40L expression and T lymphocyte activation.
Many researches have shown that the abnormal activation of PYK2 and the loss of regulatory response to a number of signaling pathways are the important factors resulted to abnormal cell growth,differentiation, or migration,involved in a variety of tumors,vascular diseases and acute or chronic inflammation,especially those with immune factors involved in the pathophysiological process of inflammation.In view of the important role of PYK2 signaling molecule in the regulation of lymphocyte activation,as well as the important role of the abnormal activation of lymphocytes in the pathogenesis of SLE,We speculate that PYK2 may be involved in the pathogenesis of SLE by interfering with the abnormal activation of lymphocytes. However,the related research about the activation of PYK2 signaling pathway in SLE reported rarely at home and abroad.In this study,we will reseach the activation of PYK2 in SLE by measuring the levels of PYK2 protein and phosphorylation in the peripheral blood mononuclear cells from SLE patients,to explore the function of activated PYK2 by measuring the expression of costimulatory molecules CD40L, CTLA4 protein in SLE patients'PBMCs cultured with promoting or blocking the PYK2 activation.And to explore the role of PYK2 activation in the pathogenesis of SLE.
Over half a century ago people began to use corticosteroid to treat systemic lupus erythematosus patients,the hormone therapy has become the mainstream treatment of lupus nephritis. However,high mortality rates of severe lupus nephritis (mainly typeⅣand someⅢ-type) has not been resolved.80s of last century,the U.S. National Institutes of Health (NIH) began to advocate high-dose intravenous therapy of CTX (cyclophosphamide,CTX),and since then,glucocorticoid hormone-binding immunosuppressant drug treatment programs has been a first treatment of choice for SLE.
Glucocorticoid and immunosuppressive drugs can inhibite immune response in many aspects,and have important value in SLE patients invovled in important organ damage including lupus nephritis.In the current treatment of SLE,Glucocorticoid and immunosuppressive are the most important drugs.However,in the process of treatment with glucocorticoid and immunosuppressive agents,because of a large quantity of drugs,long duration and prone to side effects or complications, more than 60% of patients died of their toxic side-effects,which seriously affects the patient's survival and quality of life.
In recent years,the development of blood purification,biological agents and hematopoietic stem cell transplantation therapy can alleviate the disease to some degrees.But all of these threatments can not achieve the purpose of cure.The treatment of SLE is still a difficult problems to date.Exploring curative effect drugs without side effects to improve the patient's long-term prognosis and quality of life is still clinicians' goal.
Curcumin is a component of turmeric,the yellow spice derived from the roots of the plant Curcuma longa used in ancient Asian. Since the first article referring to the use of curcumin to treat human disease was published in The Lancet in 1937,>2,600 research studies using curcumin or turmeric have been published. The mechanisms implicated in the inhibition of tumorigenesis by curcumin are diverse and appear to involve a combination of antiinflammatory,antioxidant,immunomodulatory,pro-apoptotic,and antiangiogenic properties via pleiotropic effects on genes and cell-signaling pathways at multiple levels.Judging from the current animal experiments and clinical application of the effect,curcumin is a new anti-tumor and immuno-modulatory drugs with good curative effect,neither toxic nor side-effects.Curcumin in combination with immunosuppressive agents have good synergy.Curcumin has played an important role in the treatment of cancer,atherosclerosis,infections,multiple sclerosis and rheumatoid arthritis.But for the study about the treatment of SLE with curcumin is currently no reports.In view of a good application prospect of curcumin in the treatment of autoimmune diseases,and the first part of this study has explored that the activation of PYK2 signaling protein may be importantly significant in the pathogenesis and treatment of SLE,to clarify the role of curcumin's anti-autoimmunity is associated with PYK2 or not show a particular importantane.This study will explore the therapeutic effect of curcumin on SLE by determining the effect of curcumin on SLE patients PBMC PYK2 expression and activition.
Objects and methods
The first part of the research studyed the expression and activation and functions of PYK2 signaling protein in SLE PBMCs.48 cases of SLE patients,32 cases of patients with rheumatoid arthritis (RA) and 24 healthy volunteers were selected in the present study.Peripheral blood mononuclear cells(PBMCs) from healthy volunteers, RA patients,and SLE patients were isolated from 20ml heparinized peripheral blood by Ficoll-Paque gradient centrifugation,The isolated PBMCs were divided into two groups:one group was used for Western blotting and Immunocytochemistry;in the other group,the PBMCs were resuspended at 1×109 PBMC/ml in RPMI-1640 medium cultured with PMA or TyrA9.Control cultures without stimulants were included in each experiment.The cultures were incubated at 37℃in a humidified atmosphere containing 5% CO2 for 24h.Conditioned cells were then collected and analyzed for the expression of CD40L and CTLA4 by flow cytometric analysis.PBMCs proliferation was determined with [3H]-thymidine incorporation.The second part of the reseach studyed the impact of curcumin on SLE PBMCs' PYK2 expression and activation.20 cases of SLE patients and 20 healthy volunteers were selected in the present study.PBMCs from healthy volunteers and SLE patients were isolated from 20ml heparinized peripheral blood by Ficoll-Paque gradient centrifugation,The isolated PBMCs were resuspended at 1×106 PBMCs/ml in RPMI-1640 medium cultured with PMA or TyrA9 and PMA or curcumin and PMA.Control cultures without stimulants were included in each experiment.The cultures were incubated at 37℃in a humidified atmosphere containing 5% CO2 for 24h.Conditioned cells were then collected and analyzed for the expression and activation of PYK2 in PBMCs by Western blotting and Immunocytochemistry,the expression of CD40L and CTLA4 mRNA was measured by SYBR green dye I real-time PCR,the expression of CD40L and CTLA4 protein was measured by flow cytometric analysis.PBMCs proliferation was determined with [3H]-thymidine incorporation.
Result
1、PYK2 is increased and activated in PBMCs from patients with SLE: Quantitative analysis shows PYK2 in PBMCs from SLE,but not RA patients,was significantly up-regulated in inactive and active SLE patients,respectively,compared with that from healthy donors.
2、The correlation between the levels of p-PYK2 and clinical manifestation of SLE:the expression of p-PYK2 was markedly up-regulated in PBMCs from SLE patients with class IV lupus nephritis,whereas this up-regulation was not seen in either healthy donors or SLE patients with CNS disease or nephritis other than classⅣ.PYK2 activation showed a significant negative correlation with serum complement level(CH50)(p (0.01,r=-0.668),and a positive correlation with the level of AnuA.PYK2 activation did not show a correlation with the other autoantibodies and the SLEDAI score.
3、The effect of PMA on phosphorylation of PYK2 in SLE:Quantitative analysis shows that p-PYK2 in PBMCs stimulated by PMA,but not by medium,was significantly up-regulated in healthy control,RA and SLE patients.
4、The impact of phosphorylation of PYK2 in the expression of costimulatory molecules in SLE PBMCs:Using PMA to stimulate PBMCs from active SLE resulted in a significant upregulation of CD40L and CTLA4,whereas this upregulation is not observed in PBMCs pretreated with chemical inhibitor of PYK2 kinase activity (TyrA9).In PBMCs from normal individuals and RA patients,CD40L and CTLA4 expression were also significantly upregulated by stimulation with PMA.This effect, however,cannot be suppressed by administration of TyrA9.
5、The phosphorylation of PYK2 promotes the proliferation of SLE PBMCs:The proliferation of PBMCs from all sources were enhanced by PMA.However,in the presence of TyrA9,only PBMCs from SLE patients showed a repressed proliferation when stimulated with PMA.
6、Effect of curcumin on the expression and activation of PYK2 in cultured PBMCs from patients with SLE.The expression and activation of PYK2 in normal individuals PBMCs were significantly weaker than those of SLE patient group.Using PMA to stimulate PBMCs,The expression and activation of PYK2 in PBMCs from two groups were upregulated.However, pretreated with curcumin or TyrA9 before PMA stimulus,the expression and activation of PYK2 in PBMs from two groups were decreased. So curcumin can play a role like as tyrosine kinase inhibitor.
7、The effect of curcumin on the expression of CD40L,CTLA4 mRNA and protein:PMA induced the upregulation of CD40L,CTLA4 mRNA and protein in PBMCs from SLE as well as normal individuals.Pretreated with curcumin,the expression of CD40L,CTLA mRNA and protein in PBMCs from SLE and normal groups was inhibited.however,TyrA9,PYK2 inhibitor,can only inhibit the expression of CD40L,CTLA4 mRNA and protein in PBMCs from SLE patients,but not from normal groups.
8、The effect of curcumin on the proliferation of SLE PBMCs:In vitro, proliferation of PBMCs from normal individuals was weaker than that from SLE patients.With stimulation by PMA,proliferation of PBMCs from SLE was signi-ficantly increased as well as normal idividuals.Pretreated with curcumin,the proliferation of PBMCs from SLE and normal idividuals was inhibited.But,TyrA9 can only inhibit the proliferation of PBMCs from SLE,not from normal individuals.
9、The correlation between the effect of curcumin on the activation of PYK2 and the clinical indices in SLE patients:In SLE group,the inhibition rate of PYK2 activation caused by curcumin showed a negative correlation with the level of serum complements (P<0.05) and a positive correlation with quantity of 24 hours' urinary protein(P<0.01,r=0.63).How-ever,we can see that the inhibition rate of PYK2 activation caused by curcumin in SLE patients displayed no correlation with the SLEDAI score.
Conclusions and significances
1、The expression and activation of PYK2 signaling protein were both elevated in PBMCs from active SLE and inactive SLE patients.which implys that disregulated activation of PYK2 signaling protein may be of pathogenic significance in the abnormal activation of lymphocyte in SLE.
2、The expression and activation of PYK2 in SLE PBMCs was significantly higher than that in rheumatoid arthritis,suggesting that the abnormal activation of PYK2 maybe specific to SLE.
3、The expression and activation of PYK2 in PBMCs from active SLE and inactive SLE patients were significantly higher,and did not show a correlation with the SLEDAI score,suggesting that disregulated activation of PYK2 signaling protein may be of pathogenic significance in the organ involverrient and progression of SLE in inactive phase.But the mechanism needs further study.
4、The expression of p-PYK2 was markedly up-regulated in PBMCs from SLE patients with class IV lupus nephritis,and showed negative correlation with the level of serum complements,positive correlation with the level of AnuA,which imply that PYK2 signaling protein may be associated with the development of Lupus nephritis.
5、Using PMA to stimulate PBMCs from active SLE resulted in a significant upregulation of CD40L and CTLA4,whereas this upregulation is not observed in PBMCs pretreated with TyrA9,suggesting PYK2 signaling protein may be a key signaling pathway protein through which PMA-induced the expression of costimulatory molecules CD40L,CTLA4 in SLE PBMCs.
6、The proliferation of PBMCs from all sources were enhanced by PMA. However,in the presence of TyrA9,only PBMCs from SLE patients showed a repressed proliferation when stimulated with PMA.Which suggests that the phosphorylation of PYK2 promotes the proliferation of SLE PBMCs.
7、The proliferation of PBMCs and expression of costimulatory molecules CD40L and CTLA4 were significantly increased in normal individuals as well as RA patients,but this upregulation was not inhibited by TyrA9,suggesting that besides of PYK2 signaling protein,PMA can also promote the proliferation of PBMCs and the expression of costimulatory molecules CD40L and CTLA4.
8、The expression and activation of PYK2 in normal individuals PBMCs were significantly weaker than those of SLE patient group.Using PMA to stimulate PBMCs,The expression and activation of PYK2 in PBMCs from two groups were upregulated.However,pretreated with curcumin or TyrA9 before PMA stimulus,the expression and activation of PYK2 in PBMs from two groups were decreased.So curcumin can play a role like as tyrosine kinase inhibitor.
9、PMA induced the upregulation of CD40L,CTLA4 mRNA and protein in PBMCs from SLE as well as normal individuals.Pretreated with curcumin,the expression of CD40L,CTLA mRNA and protein in PBMCs from SLE and normal groups was inhibited.however,TyrA9,PYK2 inhibitor,can only inhibit the expression of CD40L,CTLA4 mRNA and protein in PBMCs from SLE patients,but not from normal groups.Which implys that curcumin may be multi-target inhibitors.
10、Curcumin supressed the proliferation of PBMCs through inhibiting the activation of PYK2,So we are promising curcumin may be therapeutic drugs for future interventions in lupus nephritis inflammation.
11、In SLE group,the inhibition rate of PYK2 activation caused by curcumin showed a negative correlation with the level of serum complements (P<0.05) and a positive correlation with quantity of 24 hours' urinary protein(P<0.01,r=0.63). Those results suggests that the inhibition of curcumin in patients with SLE was related with patients'condition,especially SLE patients with renal injury.
富含脯氨酸的非受体酪氨酸激酶(Proline-Rich Tyrosine Kinase 2,PYK2)和粘着斑激酶(FAK)同属于粘着斑激酶家族成员,二者具有相同的结构域和很高的序列相似性。PYK2的多肽链包含三个结构域,即N末端结构域、中央酪氨酸激酶结构域和C末端结构域。N末端结构域包含一个受体结合域,该结构域可与酪氨酸激酶受体如EGF受体(EGFR)及细胞骨架蛋白如埃兹蛋白(Ezrin)等结合。C末端结构域包含两个富含脯氨酸的区域：(aa712-713)和(aa876-877),其中PXXPK、PXXLG基序是SH3结构域的结合位点,分别结合含有SH3结构域的蛋白,如p130cas, Graf, PI3K, Hic-5,paxillin等。中央酪氨酸激酶结构域包含三个酪氨酸磷酸化位点402,579,580(Tyr402,Tyr579,Tyr580),是PYK2的活性部位,其中,Tyr402可以发生自动磷酸化,磷酸化的Tyr402可与含有SH2结构域的蛋白质,如Src,Abl等结合,Src与Tyr402结合并被磷酸化而活化,活化后的Src反过来磷酸化PYK2的其它磷酸化位点,使PYK2活性进一步增强。故PYK2蛋白活化的标志是其402位点的磷酸化。
PYK2分布在神经系统和造血来源的细胞,可以感受包括细胞外基质在内的多种物理和化学的细胞外信号而激活,活化后的PYK2作为细胞内信号转导途径的上游调节分子,通过调节细胞内多条信号转导途径(PI3K、MAPK、JAK/STAT途径)参与胞内信号传递,从而在细胞粘附、迁移、生存、增殖、凋亡、分化等生理过程中发挥重要作用。由于PYK2主要在造血系统来源的细胞表达,因此PYK2对淋巴细胞功能的影响更为重要。活化后的PYK2参与免疫细胞形态的维持和细胞迁移、调节淋巴细胞的活性和增值、扩增淋巴细胞表面多种受体信号。由此可见,PYK2途径的活化是多种细胞外信号促进淋巴细胞活化的一条重要通路。
系统性红斑狼疮是最具代表性的多系统受累的自身免疫性炎性疾病,其主要特征是自身反应性T、B淋巴细胞的异常活化和多种自身抗体的出现。SLE发病机理仍然不清楚,但多种细胞因子、趋化因子、生长因子、整合素以及外界物理化学刺激(如紫外线、某些药物等)的调节异常导致的T、B淋巴细胞异常活化,是SLE发病机制的一个必然环节,并且已经成为治疗SLE的靶目标。
目前研究已经证实,和PYK2同一家族的成员FAK介导的整合素β1信号促进了SLE患者T淋巴细胞共刺激分子CD40L的表达和T淋巴细胞的活化。既往研究也表明,PYK2的异常活化及对细胞内多条信号途径的失调控反应是造成细胞异常生长、分化或迁移的重要因素,参与了多种肿瘤、血管疾病和急慢性炎症,特别是有免疫因素参与的炎症的病理生理过程。鉴于PYK2信号分子在调节淋巴细胞活化中的重要作用以及淋巴细胞的异常活化在SLE发病机制中的重要地位,我们推测PYK2可能通过干扰淋巴细胞的异常活化而参与了SLE的发病。但是PYK2信号途径的活化在SLE的研究国内外相关报道极少,本研究将通过测定SLE患者外周血单个核细胞(PBMCs)PYK2蛋白和磷酸化水平探讨PYK2的活化；通过测定促进或阻断PYK2活化后对SLE患者PBMCs共刺激分子CD40L、CTLA4蛋白表达以及PBMCs增值的影响,探讨PYK2活化后的功能,从而探讨PYK2活化在SLE发病机制中的作用。
自从半个多世纪以前人们开始应用皮质激素治疗系统性红斑狼疮以来,激素治疗己成为狼疮肾炎治疗的主流。然而,重症狼疮肾炎(主要是Ⅳ型及部分Ⅲ型)的高死亡率并未因此得到解决。上个世纪80年代美国国立卫生研究院(NIH)开始倡导环磷酞胺(cyclophosphamide, CTX)大剂量静脉注射疗法,从此糖皮质激素结合免疫抑制剂的药物治疗方案成为SLE治疗的首选。糖皮质激素和免疫抑制剂对免疫反应的许多环节都有抑制作用,对合并有肾脏等重要脏器损害患者有重要价值,是现有治疗SLE最重要的药物。然而,在糖皮质激素结合免疫抑制剂的治疗过程中,由于用药量大、时间长,容易出现副作用和并发症,60%以上患者最后死于它们的毒副作用,严重影响患者的生存期和生活质量。近年来发展起来的血液净化、生物制剂和造血干细胞移植等治疗方法虽然能在不同程度上缓解病情,但均不能达到根治的目的。SLE的治疗仍然是当今的难点问题,人们一直在探索一种疗效确切又无副作用的药物以改善患者的长期预后和生存质量。
姜黄素(Curcumin)是古老亚洲的一种药用植物姜黄的有效成分,自从1937年Lancet刊登了一篇姜黄素治疗人类疾病的文献后,至今已有2700多篇关于研究姜黄素的文献发表。姜黄素可以在基因、细胞信号传导、阻滞细胞周期、免疫调节等多途径上发挥抗肿瘤、抗炎、抗调亡、抗氧化作用。从目前动物实验及临床应用的效果看,姜黄素是一种疗效好、无毒、无副作用的新型抗肿瘤和免疫调节药物,姜黄素与免疫抑制剂联合应用均有良好的协同作用。目前姜黄素已在治疗肿瘤、粥样硬化、感染]、多发性硬化和类风湿关节炎发挥了重要作用。但对于姜黄素治疗SLE的研究目前尚无报道。鉴于姜黄素在自身免疫性疾病治疗中具有良好的应用前景,并且本研究第一部分已经探讨了PYK2信号蛋白的活化可能在SLE发病机理和治疗中具有重要意义,因此阐明姜黄素的抗自身免疫作用是否与PYK2途径相关显得尤为重要。本研究将测定姜黄素对SLE患者PBMCs PYK2表达和活性的影响,探讨姜黄素对SLE的治疗作用是否与姜黄素对PYK2的活化的影响有关。
研究对象与方法
第一部分研究PYK2信号蛋白在SLE患者PBMCs的表达和活化及活化后的功能。选用48例SLE患者、32例类风湿关节炎患者(RA)和24例健康自愿者作为研究对象。晨起空腹抽取静脉血20ml,分离PBMCs,一部分直接用于PYK2表达和活化的测定,另一部分PBMCs用RPMI 1640培养液稀释成1 x 109／ml,取细胞悬液置6孔板中,每孔各1ml,设3个组：①单纯培养组；②PMA刺激组；③PMA刺激组+TyrA9阻断组。Western Blotting测定各组患者PBMCs PYK2总蛋白和其活化蛋白(p-PYK2)的表达,细胞免疫组化测定p-PYK2表达的细胞类型,流式细胞术测定PYK2活化对PBMCs表达共刺激分子CD40L和CTLA4的影响,3H-thymidine渗入法测定PYK2活化对PBMCs增值的影响。第二部分研究姜黄素对SLE患者PBMCsPYK2表达和活化的影响。选用20例活动期SLE患者和20例健康志愿者作为研究对象。晨起空腹抽取静脉血20ml,分离PBMCs,用RPMI 1640培养液稀释成1 X 106／ml,取细胞悬液置6孔板中,每孔各1ml,设4个组：①单纯培养组；②PMA刺激组；③PMA刺激组+TyrA9阻断组；④PMA刺激组+姜黄素组。培养结束收集各孔细胞,Western Blotting测定各组患者PBMC PYK2总蛋白和其活化蛋白(p-PYK2)的表达；细胞免疫组化测定p-PYK2表达的细胞类型；SYBR GreenⅠreal-time定量PCR研究姜黄素对PBMCs表达共刺激分子CD40L和CTLA4 mRNA的影响；流式细胞术测定姜黄素对PBMCs表达共刺激分子CD40L和CTLA4蛋白表达的影响,3H-thymidine渗入法测定姜黄素对PBMCs增值的影响。
结果
1、与健康自愿者比较,非活动期SLE患者和活动期SLE患者的PBMCPYK2表达和活化均明显增高,而RA患者的PBMCs PYK2表达和活化均未见明显升高。
2、WHOⅣ型狼疮肾炎的患者PBMCs p-PYK2的表达明显高于健康自愿者、其他类型狼疮肾炎和伴有中枢神经合并症者。p-PYK2的活化和SLE患者血清总补体水平呈明显负相关(P〈0.01,r=-0.668),与抗核小体抗体呈正相关(P〈0.05,r=0.535),与其他抗体的滴度均无相关性。p-PYK2的表达水平和SLEDAI积分亦无相关性。
3、PMA刺激后,SLE组、RA组和健康对照组PBMCs PYK2活化均明显增高。
4、应用PMA促进PYK2的活化后,SLE患者、RA患者和健康志愿者PBMCs CD40L和CTLA4的表达明显增高,TyrA9阻断PYK2的活化,则PMA的刺激不能诱导SLE患者PBMCs CD40L和CTLA4的表达,但仍能诱导RA患者和健康志愿者PBMCs CD40L和CTLA4的表达。这些结果提示,p-PYK2是PMA诱导SLE患者PBMC共刺激分子CD40L和CTLA4上调表达的一个调节因子。
5、单纯体外培养SLE患者、RA患者和健康志愿者PBMCs, RA患者和健康志愿者PBMCs增值弱于SLE患者,PMA刺激促进PBMCs PYK2活化后,SLE患者、RA患者和健康志愿者PBMCs的增殖均明显增高。TyrA9阻断PYK2的活化,则SLE患者PBMCs增殖被抑制,RA患者和健康志愿者PBMCs增值未见明显抑制。
6、健康对照组PBMCs PYK2的表达和活化明显弱于SLE患者组,PMA刺激后,两组PBMCs PYK2的表达和活化均明显增高,应用姜黄素或TyrA9预处理,两组PBMCs PYK2的表达和活化均明显减弱。姜黄素可以发挥酪氨酸激酶抑制剂样作用。
7、PMA刺激后,SLE患者和正常人外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达均明显升高。姜黄素或TyrA9预处理,姜黄素能抑制两组PBMCs CD40L、CTLA4 mRNA和蛋白的表达,TyrA9仅能抑制SLE患者外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达,不能抑制正常人外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达。
8、体外细胞培养,正常人PBMCs的增值弱于SLE患者PBMCs的增值。PMA刺激后,正常人和SLE组PBMCs的增值均明显增高。姜黄素预处理,可抑制正常人和SLE患者PBMCs的增值。TyrA9预处理仅能抑制SLE患者PBMCs的增值,不能抑制正常人PBMCs的增值。
9、姜黄素对SLE患者外周血单个核细胞PYK2活化的抑制率与与SLE患者血清补体水平C3(r=-0.52)、C4(r=-0.42)呈负相关(P<0.05)；与24小时尿蛋白定量成正相关(P<0.01),相关系数r=0.63；而与SLEDAI积分无相关性。
结论及意义
1、PYK2信号蛋白的表达和活化在SLE患者PBMCs明显升高,推测PYK2信号途径的异常可能参与和促进了SLE体内淋巴细胞的高度活化。
2、SLE患者PBMCs PYK2的表达和活化明显升高而类风湿关节炎患者PBMCs PYK2的表达和活化无明显变化,提示PYK2的异常活化可能对SLE具有特异性。
3、活动期SLE患者和非活动期SLE患者外周血单个核细胞PYK2的表达和活化均明显升高,并且PYK2的活化与SLEDAI积分无相关性,提示PYK2的表达和活化可能参与非活动期SLE多系统损伤的过程,具体作用机制尚需进一步研究。
4、在Ⅳ型狼疮肾炎患者中,p-PYK2表达明显增高,并且与血清补体水平呈负相关,与血清核小体抗体呈正相关,提示PYK2信号途径可能和狼疮肾炎的发生、发展有关。
5、PMA刺激后,SLE患者PBMCs共刺激分子CD40L、CTLA4表达增高,应用PYK2特异性抑制剂TyrA9阻断PYK2的活化,PMA刺激则不能诱导SLE患者PBMCs CD40L、CTLA4的表达,提示PYK2信号蛋白可能是PMA诱导的SLE患者PBMCs共刺激分子CD40L、CTLA4表达的一个关键信号通路蛋白。
6、PMA刺激可诱导SLE患者PBMCs的增值,应用PYK2特异性抑制剂阻断PYK2的活化,则PMA刺激不能诱导SLE患者PBMCs的增值,提示SLE患者PYK2活化可促进PBMCs增值。
7、PMA刺激后,健康组和RA组PBMCs的增值及共刺激分子CD40L和CTLA4表达均明显升高,但不能被PYK2特异性抑制剂TyrA9抑制,这表明在RA患者,除PYK2信号途径外,PMA还可通过其他途径促进PBMCs的增值和共刺激分子的表达,进一步提示PYK2的异常活化可能对SLE具有特异性。
8、健康对照组PBMCs PYK2的表达和活化明显弱于SLE患者组,PMA刺激后,两组PBMCs PYK2的表达和活化均明显增高,应用姜黄素或TyrA9预处理,两组PBMCs PYK2的表达和活化均明显减弱。证实姜黄素可以发挥酪氨酸激酶抑制剂样作用。
9、PMA刺激后,SLE患者和正常人外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达均明显升高。姜黄素或TyrA9预处理,姜黄素能抑制两组PBMCs CD40L、CTLA4 mRNA和蛋白的表达,TyrA9仅能抑制SLE患者外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达,不能抑制正常人外周血单个核细胞共刺激分子CD40L、CTLA4 mRNA和蛋白的表达。提示姜黄素除可通过抑制PYK2信号蛋白的活化进而抑制SLE患者PBMCs共刺激分子CD40L、CTLA4 mRNA和蛋白的表达外,还可能通过抑制其他途径的信号蛋白进而抑制正常人PBMCs共刺激分子CD40L、CTLA4 mRNA和蛋白的表达。由此推测,姜黄素可能为一种多靶点抑制剂。
10、.姜黄素可通过抑制SLE患者外周血单个核细胞PYK2的活化进而抑制PBMCs的增殖,由此推测,姜黄素可能成为治疗系统性红斑狼疮的有效药物。
11、姜黄素对SLE患者外周血单个核细胞PYK2活化的抑制率与SLE患者血清补体水平C3(r=-0.52)、C4(r=-0.42)呈负相关(P<0.05)；与24小时尿蛋白定量成正相关(P<0.01),相关系数r=0.63；提示姜黄素的抑制作用与SLE患者的病情相关,特别是有肾脏损伤的SLE患者。
Backgroud and objective
Proline-rich tyrosine kinase 2 (PYK2)is a non-receptor protein tyrosine kinase that belongs to the focal adhesion kinase(FAK) subfamily. Similar to the prototype FAK,PYK2 contains three structural domains:a centrally located kinase domain, C-terminal domain and N-terminal domain.N-terminal domain contains a receptor combine domain and cytoskeletal protein combine domain.In the C-terminal tail,there are two proline-rich regions that are aa712-713 and aa876-877,PXXPKand PXXLG are domains binding of SH3 domain-containing proteins,including p130cas,Graf, PI3K,Hic-5,paxillin.The centrally located kinase domain contains tree sites of tyrosine phosphorylation402,579,580(Tyr402,Tyr579,Tyr580),which are the active sites of PYK2.Tyr 402 has been shown recently to be the PYK2 autophosphorylation site and was first shown to be bound by the SH2 domain of Src and later to other Src-family protein.Binding of Src is thought to allow for the subsequent phosphorylation of other tyrosine residues on PYK2 leading to increased activity. Phosphorylation of Tyr402 means the activation of PYK2 molecules.
PYK2 is primarily expressed in the central nervous system and the hematopoietic system.PYK2 is activated in response to a myriad of stimuli in many different cell types.In response to these stimuli,PYK2 is thought to mediate activation of mitogen-activated protein kinases(MAPK),phosphatidylinositol 3'-kinase(PI3),the Janus kinase/signal transducer and activator of transcription(JAK/STAT) pathway. PYK2 is a signaling molecule that regulates fundamental cellular processes,including adhesion,survival,proliferation,apoptosis differentiation.Because PYK2 is expressed in hematopoietic system,we focus here only on the role of PYK2 in lymphocyte. PYK2 phosphorylation participates to maintain the immunocyte morphological, regulates lymphocytic activity and proliferation,amplifies signals transmitted through various receptors on lymphocyte.Thus,the activation of PYK2 is an important pathway by which a variety of extracellular signals promote lymphocyte activation.
Systemic lupus erythematosus is the most representative multi-system involvement in autoimmune disease,it's main feature is the abnormal activation of self-reactive T,B lymphocytes and a variety of the emergence of autoantibodies.The pathogenesis of SLE is still not clear,However,the abnormal activation of lymphocytes resulted by the abnormal regulation of T,B caused by a variety of cytokines,chemokines,growth factors,integrins as well as the outside of physical or chemical irritants (such as ultraviolet light,certain drugs, etc)is an inevitable part of the pathogenesis of SLE,and has become the target goal of treatment of SLE.
The present study has confirmed that FAK,members of the same family with PYK2,mediated integrinβ1 signaling promotes SLE patients with T-lymphocyte costimulatory molecules CD40L expression and T lymphocyte activation.
Many researches have shown that the abnormal activation of PYK2 and the loss of regulatory response to a number of signaling pathways are the important factors resulted to abnormal cell growth,differentiation, or migration,involved in a variety of tumors,vascular diseases and acute or chronic inflammation,especially those with immune factors involved in the pathophysiological process of inflammation.In view of the important role of PYK2 signaling molecule in the regulation of lymphocyte activation,as well as the important role of the abnormal activation of lymphocytes in the pathogenesis of SLE,We speculate that PYK2 may be involved in the pathogenesis of SLE by interfering with the abnormal activation of lymphocytes. However,the related research about the activation of PYK2 signaling pathway in SLE reported rarely at home and abroad.In this study,we will reseach the activation of PYK2 in SLE by measuring the levels of PYK2 protein and phosphorylation in the peripheral blood mononuclear cells from SLE patients,to explore the function of activated PYK2 by measuring the expression of costimulatory molecules CD40L, CTLA4 protein in SLE patients'PBMCs cultured with promoting or blocking the PYK2 activation.And to explore the role of PYK2 activation in the pathogenesis of SLE.
Over half a century ago people began to use corticosteroid to treat systemic lupus erythematosus patients,the hormone therapy has become the mainstream treatment of lupus nephritis. However,high mortality rates of severe lupus nephritis (mainly typeⅣand someⅢ-type) has not been resolved.80s of last century,the U.S. National Institutes of Health (NIH) began to advocate high-dose intravenous therapy of CTX (cyclophosphamide,CTX),and since then,glucocorticoid hormone-binding immunosuppressant drug treatment programs has been a first treatment of choice for SLE.
Glucocorticoid and immunosuppressive drugs can inhibite immune response in many aspects,and have important value in SLE patients invovled in important organ damage including lupus nephritis.In the current treatment of SLE,Glucocorticoid and immunosuppressive are the most important drugs.However,in the process of treatment with glucocorticoid and immunosuppressive agents,because of a large quantity of drugs,long duration and prone to side effects or complications, more than 60% of patients died of their toxic side-effects,which seriously affects the patient's survival and quality of life.
In recent years,the development of blood purification,biological agents and hematopoietic stem cell transplantation therapy can alleviate the disease to some degrees.But all of these threatments can not achieve the purpose of cure.The treatment of SLE is still a difficult problems to date.Exploring curative effect drugs without side effects to improve the patient's long-term prognosis and quality of life is still clinicians' goal.
Curcumin is a component of turmeric,the yellow spice derived from the roots of the plant Curcuma longa used in ancient Asian. Since the first article referring to the use of curcumin to treat human disease was published in The Lancet in 1937,>2,600 research studies using curcumin or turmeric have been published. The mechanisms implicated in the inhibition of tumorigenesis by curcumin are diverse and appear to involve a combination of antiinflammatory,antioxidant,immunomodulatory,pro-apoptotic,and antiangiogenic properties via pleiotropic effects on genes and cell-signaling pathways at multiple levels.Judging from the current animal experiments and clinical application of the effect,curcumin is a new anti-tumor and immuno-modulatory drugs with good curative effect,neither toxic nor side-effects.Curcumin in combination with immunosuppressive agents have good synergy.Curcumin has played an important role in the treatment of cancer,atherosclerosis,infections,multiple sclerosis and rheumatoid arthritis.But for the study about the treatment of SLE with curcumin is currently no reports.In view of a good application prospect of curcumin in the treatment of autoimmune diseases,and the first part of this study has explored that the activation of PYK2 signaling protein may be importantly significant in the pathogenesis and treatment of SLE,to clarify the role of curcumin's anti-autoimmunity is associated with PYK2 or not show a particular importantane.This study will explore the therapeutic effect of curcumin on SLE by determining the effect of curcumin on SLE patients PBMC PYK2 expression and activition.
Objects and methods
The first part of the research studyed the expression and activation and functions of PYK2 signaling protein in SLE PBMCs.48 cases of SLE patients,32 cases of patients with rheumatoid arthritis (RA) and 24 healthy volunteers were selected in the present study.Peripheral blood mononuclear cells(PBMCs) from healthy volunteers, RA patients,and SLE patients were isolated from 20ml heparinized peripheral blood by Ficoll-Paque gradient centrifugation,The isolated PBMCs were divided into two groups:one group was used for Western blotting and Immunocytochemistry;in the other group,the PBMCs were resuspended at 1×109 PBMC/ml in RPMI-1640 medium cultured with PMA or TyrA9.Control cultures without stimulants were included in each experiment.The cultures were incubated at 37℃in a humidified atmosphere containing 5% CO2 for 24h.Conditioned cells were then collected and analyzed for the expression of CD40L and CTLA4 by flow cytometric analysis.PBMCs proliferation was determined with [3H]-thymidine incorporation.The second part of the reseach studyed the impact of curcumin on SLE PBMCs' PYK2 expression and activation.20 cases of SLE patients and 20 healthy volunteers were selected in the present study.PBMCs from healthy volunteers and SLE patients were isolated from 20ml heparinized peripheral blood by Ficoll-Paque gradient centrifugation,The isolated PBMCs were resuspended at 1×106 PBMCs/ml in RPMI-1640 medium cultured with PMA or TyrA9 and PMA or curcumin and PMA.Control cultures without stimulants were included in each experiment.The cultures were incubated at 37℃in a humidified atmosphere containing 5% CO2 for 24h.Conditioned cells were then collected and analyzed for the expression and activation of PYK2 in PBMCs by Western blotting and Immunocytochemistry,the expression of CD40L and CTLA4 mRNA was measured by SYBR green dye I real-time PCR,the expression of CD40L and CTLA4 protein was measured by flow cytometric analysis.PBMCs proliferation was determined with [3H]-thymidine incorporation.
Result
1、PYK2 is increased and activated in PBMCs from patients with SLE: Quantitative analysis shows PYK2 in PBMCs from SLE,but not RA patients,was significantly up-regulated in inactive and active SLE patients,respectively,compared with that from healthy donors.
2、The correlation between the levels of p-PYK2 and clinical manifestation of SLE:the expression of p-PYK2 was markedly up-regulated in PBMCs from SLE patients with class IV lupus nephritis,whereas this up-regulation was not seen in either healthy donors or SLE patients with CNS disease or nephritis other than classⅣ.PYK2 activation showed a significant negative correlation with serum complement level(CH50)(p (0.01,r=-0.668),and a positive correlation with the level of AnuA.PYK2 activation did not show a correlation with the other autoantibodies and the SLEDAI score.
3、The effect of PMA on phosphorylation of PYK2 in SLE:Quantitative analysis shows that p-PYK2 in PBMCs stimulated by PMA,but not by medium,was significantly up-regulated in healthy control,RA and SLE patients.
4、The impact of phosphorylation of PYK2 in the expression of costimulatory molecules in SLE PBMCs:Using PMA to stimulate PBMCs from active SLE resulted in a significant upregulation of CD40L and CTLA4,whereas this upregulation is not observed in PBMCs pretreated with chemical inhibitor of PYK2 kinase activity (TyrA9).In PBMCs from normal individuals and RA patients,CD40L and CTLA4 expression were also significantly upregulated by stimulation with PMA.This effect, however,cannot be suppressed by administration of TyrA9.
5、The phosphorylation of PYK2 promotes the proliferation of SLE PBMCs:The proliferation of PBMCs from all sources were enhanced by PMA.However,in the presence of TyrA9,only PBMCs from SLE patients showed a repressed proliferation when stimulated with PMA.
6、Effect of curcumin on the expression and activation of PYK2 in cultured PBMCs from patients with SLE.The expression and activation of PYK2 in normal individuals PBMCs were significantly weaker than those of SLE patient group.Using PMA to stimulate PBMCs,The expression and activation of PYK2 in PBMCs from two groups were upregulated.However, pretreated with curcumin or TyrA9 before PMA stimulus,the expression and activation of PYK2 in PBMs from two groups were decreased. So curcumin can play a role like as tyrosine kinase inhibitor.
7、The effect of curcumin on the expression of CD40L,CTLA4 mRNA and protein:PMA induced the upregulation of CD40L,CTLA4 mRNA and protein in PBMCs from SLE as well as normal individuals.Pretreated with curcumin,the expression of CD40L,CTLA mRNA and protein in PBMCs from SLE and normal groups was inhibited.however,TyrA9,PYK2 inhibitor,can only inhibit the expression of CD40L,CTLA4 mRNA and protein in PBMCs from SLE patients,but not from normal groups.
8、The effect of curcumin on the proliferation of SLE PBMCs:In vitro, proliferation of PBMCs from normal individuals was weaker than that from SLE patients.With stimulation by PMA,proliferation of PBMCs from SLE was signi-ficantly increased as well as normal idividuals.Pretreated with curcumin,the proliferation of PBMCs from SLE and normal idividuals was inhibited.But,TyrA9 can only inhibit the proliferation of PBMCs from SLE,not from normal individuals.
9、The correlation between the effect of curcumin on the activation of PYK2 and the clinical indices in SLE patients:In SLE group,the inhibition rate of PYK2 activation caused by curcumin showed a negative correlation with the level of serum complements (P<0.05) and a positive correlation with quantity of 24 hours' urinary protein(P<0.01,r=0.63).How-ever,we can see that the inhibition rate of PYK2 activation caused by curcumin in SLE patients displayed no correlation with the SLEDAI score.
Conclusions and significances
1、The expression and activation of PYK2 signaling protein were both elevated in PBMCs from active SLE and inactive SLE patients.which implys that disregulated activation of PYK2 signaling protein may be of pathogenic significance in the abnormal activation of lymphocyte in SLE.
2、The expression and activation of PYK2 in SLE PBMCs was significantly higher than that in rheumatoid arthritis,suggesting that the abnormal activation of PYK2 maybe specific to SLE.
3、The expression and activation of PYK2 in PBMCs from active SLE and inactive SLE patients were significantly higher,and did not show a correlation with the SLEDAI score,suggesting that disregulated activation of PYK2 signaling protein may be of pathogenic significance in the organ involverrient and progression of SLE in inactive phase.But the mechanism needs further study.
4、The expression of p-PYK2 was markedly up-regulated in PBMCs from SLE patients with class IV lupus nephritis,and showed negative correlation with the level of serum complements,positive correlation with the level of AnuA,which imply that PYK2 signaling protein may be associated with the development of Lupus nephritis.
5、Using PMA to stimulate PBMCs from active SLE resulted in a significant upregulation of CD40L and CTLA4,whereas this upregulation is not observed in PBMCs pretreated with TyrA9,suggesting PYK2 signaling protein may be a key signaling pathway protein through which PMA-induced the expression of costimulatory molecules CD40L,CTLA4 in SLE PBMCs.
6、The proliferation of PBMCs from all sources were enhanced by PMA. However,in the presence of TyrA9,only PBMCs from SLE patients showed a repressed proliferation when stimulated with PMA.Which suggests that the phosphorylation of PYK2 promotes the proliferation of SLE PBMCs.
7、The proliferation of PBMCs and expression of costimulatory molecules CD40L and CTLA4 were significantly increased in normal individuals as well as RA patients,but this upregulation was not inhibited by TyrA9,suggesting that besides of PYK2 signaling protein,PMA can also promote the proliferation of PBMCs and the expression of costimulatory molecules CD40L and CTLA4.
8、The expression and activation of PYK2 in normal individuals PBMCs were significantly weaker than those of SLE patient group.Using PMA to stimulate PBMCs,The expression and activation of PYK2 in PBMCs from two groups were upregulated.However,pretreated with curcumin or TyrA9 before PMA stimulus,the expression and activation of PYK2 in PBMs from two groups were decreased.So curcumin can play a role like as tyrosine kinase inhibitor.
9、PMA induced the upregulation of CD40L,CTLA4 mRNA and protein in PBMCs from SLE as well as normal individuals.Pretreated with curcumin,the expression of CD40L,CTLA mRNA and protein in PBMCs from SLE and normal groups was inhibited.however,TyrA9,PYK2 inhibitor,can only inhibit the expression of CD40L,CTLA4 mRNA and protein in PBMCs from SLE patients,but not from normal groups.Which implys that curcumin may be multi-target inhibitors.
10、Curcumin supressed the proliferation of PBMCs through inhibiting the activation of PYK2,So we are promising curcumin may be therapeutic drugs for future interventions in lupus nephritis inflammation.
11、In SLE group,the inhibition rate of PYK2 activation caused by curcumin showed a negative correlation with the level of serum complements (P<0.05) and a positive correlation with quantity of 24 hours' urinary protein(P<0.01,r=0.63). Those results suggests that the inhibition of curcumin in patients with SLE was related with patients'condition,especially SLE patients with renal injury.
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