IL-12调节蛋白酶激活受体在肥大细胞的表达

目的:探讨IL-12对肥大细胞蛋白酶激活受体(PARs)表达的影响.方法:不同浓度IL-12刺激肥大细胞后, 在不同时间点, 用实时定量逆转录聚合酶链式反应(q-RT-PCR)和流式细胞术(FCM), 在mRNA水平和蛋白水平检测PAR-1、 PAR-2、 PAR-3和PAR-4在肥大细胞P815表面和胞内的表达情况.结果:IL-12下调肥大细胞膜表面PAR-2蛋白的表达, 上调肥大细胞膜表面和胞质内PAR-4蛋白的表达, IL-12抗体的应用可阻断IL-12对肥大细胞PARs蛋白表达的影响;IL-12上调肥大细胞PAR-1、 3、 4 mRNA的表达, 下调PAR-2 mRNA的表达.结论:IL-12在过敏性炎症反应中的调节作用可能与IL-12调节肥大细胞PARs表达有关.     

蛋白酶激活受体2介导肥大细胞IL-4分泌

目的:探讨蛋白酶激活受体2(Proteinase activated receptor-2,PAR-2)的激活对肥大细胞P815介质分泌的影响.方法:肥大细胞培养后用PAR-2激动肽,胰蛋白酶、类胰蛋白酶、弹性蛋白酶结合PAR-2拮抗肽激发肥大细胞,收集上清并用ELISA方法检测组胺、IL-4和IL-6的水平.结果:PAR-2激动肽、胰蛋白酶、类胰蛋白酶以浓度依赖的方式促进肥大细胞P815分泌IL-4.PAR-2拮抗肽能阻断胰蛋白酶和类胰蛋白酶引起的肥大细胞IL-4分泌(P<0.05).胰蛋白酶、类胰蛋白酶以及PAR-2激动肽对肥大细胞IL-6的分泌和组胺释放无明显影响.弹性蛋白酶对肥大细胞IL-4、IL-6分泌及组胺释放功能无影响.结论:PAR-2的激活介导肥大细胞P815分泌IL-4;胰蛋白酶和类胰蛋白酶刺激肥大细胞IL-4分泌的发现为肥大细胞激活的自我放大机制提供了新的理论依据.     

蛋白酶激活受体-2与呼吸系统疾病

蛋白酶激活受体（protease-activated receptors,PARs）属于G蛋白偶联的受体家族,目前发现四个亚型,分别命名为：PAR-1、PAR-2、PAR-3和PAR-4。凝血酶可激活PAR-1、PAR-3及PAR-4;胰蛋白酶、纤维蛋白溶酶及TF/VIIa（组织因子/活化的凝血因子Ⅶ）复合物、活化的凝血因子X（Xa）等可以激活PAR-2。PAR-3和PAR-4主要表达于血小板膜表面,介导凝血酶反应,引起血小板激活、血液凝固等反应;     

TNF刺激肥大细胞IL-6分泌的信号转导通路

目的:检测TNF对肥大细胞IL-6、IL-10分泌和组胺释放的影响并探讨其可能的信号转导途径。方法:用不同浓度TNF激发肥大细胞系P815后收集细胞和上清,细胞用细胞激发信号ELISA(CASE)方法检测信号转导通路蛋白ERK、p38、和STAT3磷酸化水平,上清用ELISA检测组胺、IL-6和IL-10的水平。结果:ELISA结果显示TNF促进P815肥大细胞IL-6分泌(P<0.05),但对IL-10分泌和组胺释放无明显影响。ERK信号转导通路抑制剂PD98059和U0126抑制TNF引起的P815肥大细胞IL-6分泌(P<0.05),而p38信号转导通路抑制剂SB203580和STAT3信号转导通路抑制剂AG490不能抑制TNF引起的P815肥大细胞IL-6分泌。CASE结果显示ERK信号转导通路抑制剂PD98059,U0126抑制TNF引起的P815肥大细胞内ERK蛋白磷酸化(P<0.05)而p38信号转导通路抑制剂SB203580和STAT3信号转导通路抑制剂AG490不能抑制TNF引起的P815肥大细胞内p38和STAT3蛋白磷酸化。结论:TNF刺激小鼠肥大细胞P815分泌IL-6可能与ERK信号转导通路的激活有关的。     

类胰蛋白酶通过激活PAR-2促进肠黏膜上皮IEC-6细胞损伤

目的:观察类胰蛋白酶(tryptase)对小肠黏膜上皮细胞IEC-6的作用及机制。方法:体外培养IEC-6细胞,随机分为对照(control)组、tryptase处理组、蛋白酶激活受体2(PAR-2)抑制剂(FSLLRY-NH2,FS)处理组以及tryptase+FS处理组。采用MTT法检测细胞生存率;测定分泌的乳酸脱氢酶(LDH)活性;Western blotting测定PAR-2和cleaved-caspase 3的表达。结果:与对照组比,100μg/L和1 000μg/L类胰蛋白酶导致细胞生存率明显降低(P0.05)。与对照组相比,10μg/L到1 000μg/L类胰蛋白酶导致LDH的活性明显增加(P0.05)。与对照组相比,100μg/L和1 000μg/L类胰蛋白酶导致细胞的PAR-2及cleaved-caspase 3表达明显升高(P0.05)。与1 000μg/L tryptase处理组比较,FS能够明显增加细胞生存率、降低LDH活性及cleaved-caspase 3表达(P0.05)。结论:类胰蛋白酶通过激活PAR-2促进IEC-6细胞损伤,且这种损伤呈浓度依赖性。     

脑血康片对急性脑出血大鼠PAR-1表达及动态演变的影响(英)

目的：探讨脑出血后蛋白酶激活的受体-1（PAR-1）的动态表达及脑血康片(NXKT)的干预作用。 方法： 将72只Wistar大鼠随机分为正常组、脑出血模型6 h、24 h、3 d、7 d组和脑血康片6 h、24 h、3 d、7 d组。用Ⅶ-S型胶原酶诱导大鼠脑出血模型。免疫组化方法测定各时间点大鼠脑出血后血肿周围水肿组织PAR-1蛋白的表达；RT-PCR检测PAR-1 mRNA的表达。 结果： 正常组大鼠大脑PAR-1蛋白和PAR-1 mRNA表达轻度阳性, 模型组6 h时PAR-1表达强度开始增强，24 h PAR-1表达进一步增加，于3 d达到高峰，然后开始下降，7 d时明显下降。在6 h、24 h、3 d及7 d各时点，模型组、NXKT组PAR-1 mRNA吸光度比值及PAR-1阳性细胞数升高与正常组比较均有显著差异(P<0.05或P<0.01)，NXKT组PAR-1阳性细胞数、PAR-1 mRNA吸光度比值降低与模型组比较各时点均有显著差异(P＜0.05或P＜0.01)。 结论： 脑出血后PAR-1受到凝血酶的持续活化，脑出血后凝血酶的作用可能是通过PAR-1介导的; NXKT可抑制PAR-1活化，从而使行为学得到改善,这可能是NXKT促进神经功能修复的主要机制之一。     

蛋白酶激活受体2/P38MAPK信号通路在缺血后处理对抗老年糖尿病大鼠心肌缺血再灌注损伤中的作用

目的:研究蛋白酶激活受体2/P38MAPK(PAR-2/P38MAPK)信号通路在缺血后处理(IPost)对抗老年糖尿病大鼠心肌缺血再灌注(I/R)损伤中的作用。方法:22-24月龄雄性Wistar大鼠32只,随机选取24只采用链脲佐菌素(STZ)腹腔注射制备老年糖尿病模型。成模大鼠随机分成3组(每组8只):I/R组、IPost组、PAR-2抑制剂组(PAR-2组),PAR-2组大鼠于缺血处理前1h尾静脉注射PAR-2抑制剂FSLLRY-NH2,其余处理同IPost组;未造模的8只大鼠作为对照组(SC组)。采用黄嘌呤氧化酶法和硫代巴比妥比色法分别测定血清超氧化物歧化酶(SOD)活性及丙二醛(MDA)浓度,TUNEL法检测心肌细胞凋亡率,Western Blotting测定PAR-2及P38MAPK、P-P38MAPK蛋白表达水平。结果:I/R组MDA浓度、心肌细胞凋亡率及P-P38MAPK水平均较SC组显著增加(P0.05或P0.01),SOD活性、PAR-2蛋白表达水平明显降低(P0.01)。IPost组SOD活性及PAR-2水平较I/R组显著增加(P0.05),P-P38MAPK水平、MDA浓度及心肌细胞凋亡率均下降(P0.01)。PAR-2抑制剂则可抑制IPost作用,使P-P38MAPK蛋白表达水平上调、心肌细胞凋亡率增加、MDA浓度升高、SOD活性降低(P0.05或P0.01)。结论:IPost对老年糖尿病大鼠心肌I/R损伤有保护作用,其机制与PAR-2/P38MAPK信号通路的调节有关。     

大鼠脑缺血再灌注后脑组织PAR-1表达与MDA含量的相关性研究

目的：观察脑缺血再灌注（cerebral ischemia reperfusion,CIR）后脑组织中PAR-1的表达与MDA含量变化之间的关系。方法：40只SD大鼠随机分缺血再灌注（CIR）后3h、6h、12h、1d、2d、3d、7d及假手术组8个组每组5只大鼠。于脑缺血2h后再灌注相应时间断头取脑,免疫组化法和脑组织匀浆法观察蛋白酶激活受体-1（protease—activated receptor-1,PAR-1）和丙二醛（malondialdehyde,MDA）变化情况。结果：随着CIR时间延长,PAR-1表达和MDA含量增多,呈正相关关系（r=0．844,P〈0．05）。结论：CIR后PAR-1表达增多可加重脑损伤。     

慢性血小板减少性紫癜患者血小板Gα蛋白及多种G蛋白偶联受体的表达

目的 研究慢性特发性血小板减少性紫癜(chronic idiopathic thrombocytopenic purpura,CITP)患者血小板G蛋白信号相关分子的表达变化.方法 纳入2015年7月~2016年1月我院收治的CITP患者92例为观察组、同期健康体检志愿者92例为对照组,取空腹静脉血10 ml,检测对象G蛋白偶联的跨膜受体,包括二磷酸腺苷受体P2Y1、P2Y12,α2A-肾上腺素能受体(α2A-AR),血栓烷A2受体(thromboxane A2 receptor,TXA2-R),蛋白酶激活受体(PAR)-1,PAR-4,同时检测血小板Gα蛋白的表达.结果 观察组P2Y1、P2Y12、α2A-AR、TXA2-R、PAR-1、PAR-4及血小板Gα蛋白的表达均明显高于对照组,差异有统计学意义(P<0.05).P2Y1、P2Y12、α2A-AR、TXA2-R、PAR-1、PAR-4及血小板Gα蛋白的表达均与血小板计数呈负相关(P<0.05).结论 CITP患者可检出血小板Gα蛋白及多种G蛋白偶联受体的高表达,且上述信号分子的表达与血小板计数呈负相关,提示G蛋白偶联受体的信号转导途径可能参与了CITP的免疫病理机制.     

γ干扰素(IFN-γ)刺激小鼠肥大细胞释放组织蛋白酶S

目的探讨γ干扰素(IFN-γ)对P815小鼠肥大细胞以及小鼠髓源性肥大细胞(BMMC)表达组织蛋白酶S(CTSS)的影响。方法用(10、25、50)ng/m L IFN-γ分别刺激P815细胞及原代培养的小鼠BMMC,利用实时定量PCR、ELISA分别检测P815细胞及BMMC CTSS的mRNA和蛋白水平。用25 ng/m L IFN-γ处理P815细胞及小鼠BMMC,P815细胞分别处理6、12、18、24、48、72 h,BMMC分别处理12、24、48 h,重复上述检测步骤。结果 IFN-γ可增加P815细胞及BMMC CTSS的mRNA和蛋白水平,并具有一定的时间、剂量依赖关系。结论 IFN-γ可促进小鼠肥大细胞表达CTSS。     

Induction of T-helper type 2 cytokine release and up-regulated expression of protease-activated receptors on mast cells by recombinant American cockroach allergen Per a 7

Background The cockroach is a major source of indoor allergens, which cause perennial rhinitis and asthma. Per a 7 is one of the major allergens from the American cockroach, eliciting IgE reaction in over 50% of sera from allergic individuals. However, the actions of Per a 7 in the pathogenesis of allergic diseases remain poorly understood.
Objective The aim of the study was to investigate the effects of Per a 7 on the expression of protease-activated receptors (PARs) on murine mast cells and the release of T-helper type 2 (Th2) cytokines from these cells.
Methods Per a 7 gene was cloned and expressed in Escherichia coli . The purified recombinant Per a 7 (rPer a 7) was used to challenge murine mast cells P815 and the expression of PARs was determined by using real-time quantitative RT-PCR and flow cytometry. The release of IL-4 and IL-13 was detected with ELISA.
Results rPer a 7 was solubly expressed in E. coli and the purified rPer a 7 reacted with 75% (six out of eight) of the sera from cockroach-allergic patients. Following stimulation of rPer a 7, the expression of PAR-1, PAR-2, PAR-3 and PAR-4 mRNAs on P815 cells increased by 8.7-, 6.8-, 14.4- and 8.8-fold, respectively, following 2- and 6-h incubation periods, and the expression of PAR-1, PAR-2 and PAR-4 proteins was enhanced following 16-h incubation. rPer a 7 provoked approximately up to a 4.8- and 2.3-fold increase in IL-4 and IL-13 release from P815 cells following 6- and 16-h incubation periods.
Conclusion rPer a 7 induced up-regulation of PAR expression on P815 cells and provoked Th2 cytokines IL-4 and IL-13 secretion from these murine mast cells, which implicated that this major cockroach allergen is likely to contribute to the development of cockroach-related allergic disease.     

Analysis of properties and proinflammatory functions of cockroach allergens Per a 1.01s

Cockroaches have been identified as one of the major indoor allergens inducing perennial rhinitis and asthma. Per a 1s are a group of the major allergens from American cockroach. Although Per a 1s are major allergens from American cockroach, factors contributing to the allergenicity of Per a 1s are still poorly defined. To investigate the effects of Per a 1s on the expression of PARs and the release of proinflammatory cytokines from mast cells. Per a 1.0101 and Per a 1.0104 were cloned from American cockroach and then expressed in Eschericia coli. The purified allergens were used to stimulate P815 mast cells, and the expression of protease-activated receptors (PARs) was determined by real-time RT-PCR and flow cytometry. The levels of IL-4 and IL-13 in culture media were detected with ELISA. Sera from 80 and 77.3% of cockroach allergy patients reacted to recombinant Per a (rPer a) 1.0101 and rPer a 1.0104, confirming they are major allergens. Both rPer a 1.0101 and rPer a 1.0104 had no enzymatic activity, but rPer a 1.0101 upregulated the expression of PAR-1 and PAR-2, and rPer a 1.0104 enhanced the expression of PAR-1 and PAR-4 proteins. Both recombinant allergens were able to increase the release of IL-4 and IL-13 from P815 mast cells. This is the first study aiming to investigate functions of group 1 allergens of American cockroach. rPer a 1.0101 and rPer a 1.0104 have the capacity to upregulate the expression of PARs and to enhance Th2 cytokine production in mast cells.     

Expression of protease-activated receptor-1, -2, -3, and -4 in control and experimentally inflamed mouse bladder

Inflammation underlines all major bladder pathologies and represents a defense reaction to injury involving a mandatory participation of mast cells and sensory nerves. Mast cells are particularly frequent in close proximity to epithelial surfaces where they are strategically located in the bladder and release their mediators in response to inflammation. Tryptase is specifically produced by mast cells and modulates inflammation by activating protease-activated receptors (PARs). We recently found that PAR-4 mRNA is up-regulated in experimental bladder inflammation regardless of the initiating stimulus. Because it has been reported that PAR-1, PAR-2, and PAR-3 may also be involved in the processes of inflammation, we used immunohistochemistry to characterize the expression of all known PARs in normal, acute, and chronic inflamed mouse bladder. We found that all four PARs are present in the control mouse bladder, and follow a unique distribution. All four PARs are co-expressed in the urothelium, whereas PAR-1 and PAR-2 are predominant in the detrusor muscle, and PAR-4 is expressed in peripheral nerves and plexus cell bodies. The strong expression of PARs in the detrusor muscle indicates the need for studies on the role of these receptors in motility whereas the presence of PAR-4 in nerves may indicate its participation in neurogenic inflammation. In addition, PARs are differentially modulated during inflammation. PAR-1 and PAR-2 are down-regulated in acute inflammation whereas PAR-3 and PAR-4 are up-regulated. Bladder fibroblasts were found to present a clear demarcation in PAR expression secondary to acute and chronic inflammation. Our findings provide evidence of participation of PARs in the urinary system, provide a working model for mast cell tryptase signaling in the mouse bladder, and evoke testable hypotheses regarding the roles of PARs in bladder inflammation. It is timely to understand the role of tryptase signaling and PARs in the context of bladder biology.     

Mast cell tryptase activates peripheral blood eosinophils to release granule-associated enzymes

BACKGROUND: Mast cells and eosinophils are important effector cells in asthma. Understanding their interactions is essential for studying asthma pathophysiology. Inflammatory mediators released from mast cells, such as arachidonic acid metabolites, TNF and IL-5, are important in eosinophil biology. However, little is known about the effects of mast cell-specific mediators, such as tryptase, on eosinophils. Our objective was to investigate the effects of mast cell tryptase on human peripheral blood eosinophils. METHODS: Peripheral blood eosinophils isolated from asthmatic individuals were activated using various concentrations of tryptase- and protease-activated receptor-2 (PAR-2)-activating peptides (PAR-2 AP). Eosinophil activation was evaluated by the release of granule mediators, superoxide release, estimation of eosinophil survival, changes in intracellular Ca2+ concentration and mitogen-activated protein kinase activation. RESULTS: Tryptase induced the release of eosinophil peroxidase and beta-hexosaminidase from peripheral blood eosinophils but had no effect on RANTES release. Eosinophils isolated from two thirds of our donors responded to tryptase, while the remainder appeared not to respond. Release of granule mediators was dependent on tryptase enzymatic activity. To identify the mechanism of eosinophil activation by tryptase, we studied the expression of PAR-2 by eosinophils and its function. Using RT-PCR, we amplified PAR-2 from eosinophils. However, flow cytometry failed to detect significant PAR-2 expression on the surface of eosinophils. The PAR-2 AP SLIGRL-NH2 did not induce eosinophil activation by any of the methods we employed. CONCLUSION: Our data indicate that mast cell tryptase may affect eosinophil activation status independently of PAR-2.     

Inhibition by Triton X-100 of histamine liberation from mast cells induced by substance 48/80

Triton X-100, in concentrations below those liberating histamine, produced dose-dependent inhibition of histamine liberation from rat mast cells induced by substance 48/80. Triton X-100 (0.02 ml/liter) exhausted the ATP reserves in the mast cells and completely inhibited histamine liberation induced by substance 48/80, and exhaustion of the ATP content in the mast cells was abolished by glucose (10 mM). It was concluded that inhibition by Triton X-100 of histamine liberation induced by substance 48/80 depends on the inhibition of energy production.Allergologic Research Laboratory, Academy of Medical Sciences of the USSR, Moscow, Department of Pharmacology, Kuban Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 2, pp. 185–187, February, 1979.     

Effect of acute and chronic inflammatory stimuli on expression of protease-activated receptors 1 and 2 in alveolar macrophages

BACKGROUND: Protease-activated receptors 1 and 2 (PAR-1 and PAR-2) are 7-transmembrane G protein-coupled receptors activated by serine proteases in many cell types, including monocytes-macrophages, leading to the production of pro-inflammatory mediators, cytokines, and growth factors. OBJECTIVE: We determined the influence of chronic smoking and asthma on the expression of PAR-1 and PAR-2 receptors on alveolar macrophages (AMs). METHODS: We used RT-PCR and immunocytochemistry with confocal microscopy to determine mRNA and protein expression of PAR-1 and PAR-2 in AMs obtained from healthy smokers, asthmatic patients, and healthy subjects. In addition, we examined the effect of IL-1beta and LPS. RESULTS: PAR1 mRNA was decreased, whereas PAR2 mRNA was increased in 24-hour cultured AMs from smokers when compared with values in AMs from healthy subjects. Paradoxically, there was a higher degree of PAR-1 protein staining in AMs from smokers, whereas PAR-2 staining was similar in smokers and healthy subjects. PAR-1 and PAR-2 mRNA and protein expression were similar in asthmatic patients and control subjects. IL-1beta and LPS had no effect on PAR1 and PAR2 gene expression by AMs. CONCLUSIONS: There is a dissociation between gene and protein expression of PAR-1 and PAR-2. PAR-1 protein overexpression in AMs from smokers might be important in the pathophysiology of chronic airways disease.