JAK/STAT 信号通路在肺纤维化中作用的研究进展

蛋白酪氨酸激酶(JAK)/信号转导转录激活因子(STAT)信号通路是介导炎症反应的重要信号通路之一,广泛参与细胞的增生、分化、凋亡、免疫调节等病理生理过程。JAK/STAT 信号通路通过介导肺巨噬细胞、成纤维细胞、肺泡上皮细胞的病理性活化及损伤,对肺纤维化进程具有重要调控作用。此文就 JAK/STAT 信号通路在肺纤维化中的作用机制及研究进展作一综述。     

JAK/STAT信号转导通路在肝纤维化形成中的作用

JAK/STAT信号转导通路是一条由多种细胞因子和生长因子介导的信号转导通路,能参与细胞的增殖、分化、凋亡及炎症等病理生理过程。研究表明,JAK/STAT通路在肝纤维化的形成进程中具有重要的调控作用。本文就近年JAK/STAT信号转导通路在肝纤维化形成过程中的作用作一综述。     

JAK2/STAT3信号通路介导小鼠骨性关节炎中软骨细胞代谢和抗氧化应激的研究

目的 在小鼠骨性关节炎（OA）模型中观察Janus酪氨酸蛋白激酶2/信号转导子与转录激活子蛋白3（JAK2/STAT3）信号通路对软骨细胞代谢的影响以及线粒体抗氧化应激能力的改变,探讨JAK2/STAT3信号通路在此过程中的作用。方法 将10只C57BL/6小鼠随机分为两组,选择其中一组小鼠建立OA模型,3周后取材,培养软骨细胞作为实验组,其余小鼠正常培养细胞作为对照组。在对照组和实验组中分别加入JAK2/STAT3信号通路激动剂SC-39100,运用蛋白印迹法（Western blotting）检测各组细胞p-JAK2、p-STAT3、B淋巴细胞瘤?2（Bcl-2）蛋白和Bax蛋白的表达,同时检测各组线粒体氧化应激指标琥珀酸脱氢酶（SDH）、细胞色素c氧化酶（COX）、丙二醛（MDA）改变。结果 与对照组相比,OA模型组软骨细胞p-JAK2、p-STAT3、Bcl-2蛋白的表达偏低（P＜0.05）、Bax蛋白的表达水平偏高（P＜0.05）,且OA模型组软骨细胞SDH和COX的表达水平均偏低（P＜0.05）、MDA的含量偏高（P＜0.05）;当OA模型组加入SC-39100后,p-JAK2、p-STAT3、Bcl-2表达均较OA模型组升高（P＜0.05）、Bax蛋白表达下降（P＜0.05）,SDH和COX的表达水平均较OA模型组升高（P＜0.05）,MDA的含量较OA模型组降低（P＜0.05）;对照组中加入SC-39100后的各指标与加入SC-39100前比较,差异均无统计学意义（P＞0.05）;OA模型加入SC-39100组后的各指标与对照组加入SC-39100比较,差异均有统计学意义（P＜0.05）。结论 JAK2/STAT3信号通路和OA中软骨细胞变化密切相关,JAK2/STAT3信号通路激活后可抑制软骨细胞的凋亡;当激活的JAK2/STAT3信号通路活化时会增加软骨细胞线粒体抗氧化应激能力。     

IL-27调控JAK/STAT通路在博来霉素诱导肺纤维化中的作用

目的探讨白细胞介素(IL)-27是否通过酪氨酸激酶(JAK)/信号转导与转录激活子(STAT)信号通路抑制肺纤维化。方法将40只雄性C57/BL6小鼠随机分为空白对照组(A组)、博来霉素(BLM)组(B组)、BLM+IL-27组(C组)和BLM+IL-27抗体组(D组),每组10只。于造模后第7、28天每组各处死5只小鼠,取右肺组织用Western印迹方法检测JAK2、STAT1、STAT3、STAT5及SOCS3蛋白的表达。结果 BLM诱导肺纤维化模型中,7、28 d肺组织均有p-JAK2、p-STAT1、p-STAT3、p-STAT5表达量升高,IL-27治疗后降低SOCS3的表达;D组p-JAK2、p-STAT1、p-STAT3、p-STAT5表达量在造模后7、28 d均最高,C组在造模后7 d表达量少于B组。结论外源性IL-27可能通过抑制JAK/STAT通路相关蛋白的磷酸化减轻肺纤维化。     

Regulation of YKL‐40 production by human articular chondrocytes

Objective

YKL‐40 (human cartilage glycoprotein 39) is one of the most abundant proteins secreted by cultured chondrocytes. The objectives of the present study were to identify regulators of YKL‐40 production in cartilage and chondrocytes and to map the localization of YKL‐40 in chondrocytes.

Methods

Human articular chondrocytes and cartilage explants (obtained from subjects at autopsy, from a tissue bank, and from osteoarthritis [OA] patients undergoing total joint replacement surgery) were stimulated with cytokines, growth factors, and other agents. YKL‐40 expression was analyzed by Northern blot and polymerase chain reaction. YKL‐40 secretion into the media was determined by enzyme‐linked immunosorbent assay.

Results

YKL‐40 production increased to very high levels during the early phase of chondrocyte monolayer culture and in normal cartilage explant cultures as a response to tissue injury. Spontaneous YKL‐40 release was higher in OA than in normal cartilage explant cultures. In chondrocyte monolayer cultures, interleukin‐1β (IL‐1β) and transforming growth factor β (TGFβ) decreased the levels of secreted YKL‐40, and this was associated with a reduction in YKL‐40 messenger RNA levels. IL‐1β, but not TGFβ, reduced YKL‐40 production in cartilage explant cultures. Media from explants treated with cycloheximide had no detectable YKL‐40, suggesting that the released protein was newly synthesized. Immunofluorescence microscopy showed YKL‐40 staining in the Golgi system of the chondrocytes, but YKL‐40 could not be detected in the extracellular matrix.

Conclusion

The spontaneous increase in the production of YKL‐40 in the early phase of culture appears to represent a cellular response to changes in the extracellular matrix environment. This, coupled with the profound suppressive effects of IL‐1β and TGFβ on YKL‐40 production, identifies a novel regulatory pattern for this major chondrocyte‐derived protein.

     

Histamine stimulates the proliferation of human articular chondrocytes in vitro and is expressed by chondrocytes in osteoarthritic cartilage

OBJECTIVES: To determine the effects of histamine on the proliferative rate of human articular chondrocytes (HAC) in vitro, and to demonstrate whether HAC in osteoarthritic (OA) cartilage express histamine and histidine decarboxylase (HDC). METHODS: HAC in vitro were incubated with and without histamine in 96 well culture plates and the extent of cell proliferation was determined using the naphthol blue-black method. Histamine effects were analysed with the histamine H(1) and H(2) receptor antagonists, mepyramine and ranitidine, respectively. Rabbit polyclonal antibodies and alkaline phosphatase conjugated secondary antibodies were used, and histamine and HDC were demonstrated by immunohistochemistry in OA cartilage tissues. RESULTS: Histamine stimulated the proliferation of HAC in culture. This stimulation was blocked by the addition of mepyramine, but not ranitidine, suggesting that the effect is mediated through H(1) histamine receptors. The addition of alpha-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase (the enzyme responsible for histamine production), reduced the rate of proliferation of HAC. Both histamine and histidine decarboxylase were demonstrated in chondrocytes of OA cartilage by immunohistochemistry. CONCLUSION: Changes induced by histamine in the proliferative rate of HAC may contribute to the formation of chondrocyte clusters associated with OA cartilage; an observation supported by the demonstration of histamine and HDC expression by chondrocytes of OA cartilage in situ.     

Cytokine signaling through the JAK/STAT pathway is required for long-term memory in Drosophila

Cytokine signaling through the JAK/STAT pathway regulates multiple cellular responses, including cell survival, differentiation, and motility. Although significant attention has been focused on the role of cytokines during inflammation and immunity, it has become clear that they are also implicated in normal brain function. However, because of the large number of different genes encoding cytokines and their receptors in mammals, the precise role of cytokines in brain physiology has been difficult to decipher. Here, we took advantage of Drosophila's being a genetically simpler model system to address the function of cytokines in memory formation. Expression analysis showed that the cytokine Upd is enriched in the Drosophila memory center, the mushroom bodies. Using tissue- and adult-specific expression of RNAi and dominant-negative proteins, we show that not only is Upd specifically required in the mushroom bodies for olfactory aversive long-term memory but the Upd receptor Dome, as well as the Drosophila JAK and STAT homologs Hop and Stat92E, are also required, while being dispensable for less stable memory forms.     

Janus激酶2/信号转导和转录激活子3信号通路在心肌细胞缺氧损伤中的作用

目的探讨Janus激酶2/信号转导和转录激活子3(JAK2/STAT3)信号通路在心肌细胞缺氧损伤中的作用。方法体外培养的新生大鼠心肌细胞,构建缺氧模型,按随机数字表法分为正常对照组、缺氧组、JAK2抑制剂AG490处理组和STAT3抑制剂Statti c处理组。采用细胞计数试剂盒CCK-8检测心肌细胞活力,采用比色法检测细胞上清液中的乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)含量,并采用缺口末端标记法(TUNEL)检测各组细胞的凋亡率。采用蛋白质印迹(Western blot)检测JAK2及STAT3蛋白表达及磷酸化情况。结果与正常对照组相比,缺氧组心肌细胞成活率明显降低,为对照组的30.14%±6.23%(P<0.01),细胞上清液中LDH、MDA含量升高明显,分别为(50.11±2.58)U/L和(19.55±1.81)mol/L(均为P<0.01),SOD活力则显著降低,为(10.21±0.57)U/ml(P<0.01),缺氧组凋亡率明显升高,为24.24%±4.37%(P<0.01),JAK2、STAT3磷酸化水平上调。AG490及Stattic预处理后,JAK2及STAT3磷酸化水平降低,心肌细胞成活率明显升高(P<0.01),LDH、MDA含量显著低于缺氧组(均为P<0.01),SOD活力则高于缺氧组(P<0.01)。结论 JAK2/STAT3信号通路参与了缺氧所致心肌细胞损伤,抑制JAK/STAT通路有助于减轻缺氧所致心肌损伤。     

茵陈蒿汤抑制JAK2/STAT3信号通路调控肝癌细胞增殖与凋亡

目的 探索茵陈蒿汤的抗肝癌作用及其机制.方法 体外培养HepG2肝癌细胞,分为对照组和茵陈蒿汤低、中、高浓度(12.5、25、50μg/mL)组.通过CCK-8检测细胞活力、EdU染色观察茵陈蒿汤对HepG2肝癌细胞增殖的影响,钙黄绿素/PI细胞染色观察茵陈蒿汤对肝癌细胞存活能力的影响.通过Western blot检测...     

姜黄素对大鼠脑缺血后Janus蛋白酪氨酸激酶2信号转导和转录激活子3信号通路的影响

目的探讨姜黄素对大鼠脑缺血损伤后Janus蛋白酪氨酸激酶2(JAK2)/信号转导和转录激活子3(STAT3)信号通路的影响。方法将68只SD大鼠随机分为假手术组、脑缺血组、姜黄素组(尾静脉注射姜黄素40mg/kg)和对照组(注射等量生理盐水),每组17只,后3组建立大鼠局灶性脑缺血模型,观察各组大鼠神经行为学变化,ELISA检测大鼠脑组织TNF-α、白细胞介素(IL)-1β、IL-6表达,Western blot检测大鼠脑组织JAK2、磷酸化JAK2(p-JAK2)、STAT3、磷酸化STAT3(p-STAT3)、高迁移率族蛋白1(HMGB1)表达。结果与脑缺血组比较,姜黄素组大鼠神经行为学评分减低[(1.53±0.62)分vs(2.94±0.87)分,P0.05];与脑缺血组比较,对照组TNF-α、IL-1β和IL-6无明显变化(P0.05),姜黄素组大鼠TNF-α[(57.63±10.27)ng/L vs(99.35±8.97)ng/L]、IL-1β[(33.67±9.10)ng/L vs(58.43±7.22)ng/L]和IL-6[(31.97±6.91)ng/L vs(49.23±6.28)ng/L]表达降低(P0.01),p-JAK2/JAK2、p-STAT3/STAT3相对含量及HMGB1表达降低(P0.05,P0.01)。结论姜黄素可保护大鼠缺血后脑损伤,其机制可能是通过抑制JAK2/STAT3信号通路,减少HMGB1表达,减轻炎性反应。     

Integrin‐mediated adhesion of human articular chondrocytes to cartilage

Objective

To determine 1) the kinetics and strength of adhesion of human articular chondrocytes to a cut cartilage surface, and 2) the role of specific integrins in mediating such adhesion, using an in vitro model.

Methods

Human articular chondrocytes isolated from cadaveric donors (mean ± SD age 38 ± 13 years) were cultured in high‐density or low‐density monolayer. Following release from culture with trypsin and a 2–2.5‐hour recovery period, chondrocytes were analyzed either for adhesion to cartilage or for integrin expression by flow cytometry.

Results

Following culture in monolayer, adhesion of chondrocytes to cartilage increased with time, from 6–16% at 10 minutes to a maximum of 59–82% at 80–320 minutes. After 80 minutes of adhesion, the resistance of cells to flow‐induced shear stress (50% detachment) was ∼21 Pa. Chondrocyte adhesion to cartilage decreased with pretreatment of cells with monoclonal antibodies that bound to and blocked certain integrins. After an 80‐minute incubation time, adhesion of chondrocytes cultured in high‐density monolayer decreased from the value of IgG1‐treated controls (55%) with blocking of the β1 integrin subunit (to 23%) or with blocking of α5β1 (to 36%). Following expansion of chondrocytes in low‐density monolayer, the mechanisms of adhesion to cartilage were generally similar. After an 80‐minute incubation time, adhesion of chondrocytes cultured in low‐density monolayer decreased from the value of IgG1‐treated controls (62%) with blocking of the β1 integrin subunit (to 30%) or with blocking of α5β1 (to 44%). Additionally, adhesion of these cells decreased to 46% by blocking of αvβ5, with a similar trend in effect for chondrocytes cultured in high‐density monolayer. Blocking of the α1 or α3 integrin subunits or αvβ3 had no detectable effect on adhesion, even though these receptors were detected by flow cytometry.

Conclusion

Under the culture and seeding conditions studied, β1, α5β1, and αvβ5 integrins mediate human chondrocyte adhesion to cartilage. These chondrocyte integrins have a potential role in the initial adhesion and retention of chondrocytes at a cartilage defect site following clinical procedures of chondrocyte transplantation.

     

N-乙酰半胱氨酸通过抑制JAK2/STAT3信号通路对高糖诱导的内皮细胞损伤的保护作用

目的 探讨N-乙酰半胱氨酸(NAC)能否通过抑制JAK2/STAT3信号通路对高糖诱导的内皮细胞损伤起保护作用。方法 体外分别应用不同浓度NAC对人脐静脉内皮细胞(HUVEC)进行预处理,筛选出NAC减轻高糖诱导的HUVEC细胞毒性的最佳浓度。使用最佳浓度的NAC预处理高糖诱导的HUVEC,通过CCK-8检测细胞存活率,Hoechst33258核染色荧光显微镜照相法检测内皮细胞凋亡的形态学改变,Western blot检测JAK2/STAT3信号通路的蛋白表达水平,DCFH-DA检测细胞内活性氧水平,ELISA检测相关炎症因子细胞间黏附分子1(ICAM-1)、核因子κB(NF-κB)、肿瘤坏死因子α(TNF-α)及白细胞介素1β(IL-1β)、IL-6和IL-8的含量;荧光探针JC-1检测线粒体膜电位的变化。结果 应用7 mmol/L NAC预处理HUVEC 30 min可明显减轻高糖诱导的HUVEC损伤,使细胞存活率升高,细胞凋亡数量减少,细胞凋亡蛋白cleaved Caspase-3表达减少,胞内活性氧堆积及线粒体膜电位丢失减少(P＜0.01)。NAC能抑制高糖对p-JAK2、p-STAT3表达的上调作用(P＜0.01),同时可抑制高糖诱导的炎症反应,使炎症因子ICAM-1、NF-κB、TNF-α及白细胞介素1β(IL-1β)、IL-6和IL-8的水平下降(P＜0.01)。结论NAC能够通过抑制JAK2/STAT3信号通路对高糖诱导的HUVEC损伤起到保护作用。     

Histamine stimulates matrix metalloproteinase-3 and -13 production by human articular chondrocytes in vitro

OBJECTIVES: To determine the effects of histamine on matrix metalloproteinase (MMP) production by human articular chondrocytes (HAC) in vitro. METHODS: Conditioned culture medium from HAC cultures incubated with and without 20 microM histamine was assayed by enzymne linked immunosorbent assay (ELISA) for MMP-1, MMP-8, MMP-13 (collagenases 1, 2, and 3, respectively) and MMP-3 (stromelysin). Monolayer cultures of HAC were also immunostained for MMP-13 and MMP-3. RESULTS: The HAC cultures showed a significant increase in MMP-13 and MMP-3 production (2.2- and 1.9-fold, respectively) after treatment with 20 microM histamine for 24 hours, but MMP-1 and MMP-8 were unaffected. All cultures showed MMP-13 and MMP-3 detectable by immunolocalisation. MMP-3 was the more prominent enzyme as shown by both ELISA and immunolocalisation techniques. CONCLUSIONS: Histamine exposure increased both MMP-13 and MMP-3 production by HAC in vitro, thereby suggesting a pathophysiological role in the chondrocytic phenotype associated with degenerative changes in osteoarthritis.     

Histamine stimulates prostaglandin E production by rheumatoid synovial cells and human articular chondrocytes in culture

Histamine stimulates prostaglandin E (PGE) production by cultures of adherent rheumatoid synovial cells and human articular chondrocytes. When subcultured synovial fibroblasts or human articular chondrocytes were "primed" by preincubation with conditioned media from primary adherent rheumatoid synovial cell cultures (synovial factor), each produced even higher PGE levels upon histamine exposure. This histamine stimulation was prevented by histamine H1, but not H2, antagonists and was more marked if serum was absent from the culture media. Thus, histamine-induced PGE production by these cells is mediated via H1 receptor activation and subsequent arachidonic acid liberation.     

人生长激素慢性刺激对小鼠肝细胞JAK/STAT5信号通路的影响

目的研究正常幼年小鼠长期注射人生长激素（hGH）对其肝细胞生长激素JAK/STAT5信号通路的影响。方法实验组小鼠连续2wk按1μg/g体重每天皮下注射人生长激素（hGH）,对照组小鼠连续2wk每天皮下注射磷酸盐缓冲液（PBS）,浓度为0.1M。在注射完最后一次hGH和PBS16h之后,每组处死1/2小鼠。两组中另外1/2小鼠处死前30min皮下按1μg/g体重注射单剂量hGH。蛋白质印迹法（WesternBlot）检测小鼠肝组织细胞核和细胞总蛋白中人信号传导子及转录激活子5（STAT5）、STAT5的磷酸化形式（p-STAT5）和生长激素受体（GHR）的表达,凝胶迁移实验分析（EMSA）肝细胞核STAT5与DNA粘附能力,酶标记免疫吸附测定（ELISA）法测定血清GH浓度。结果 hGH慢性刺激组小鼠与PBS对照组小鼠相比体重明显增加;与PBS对照组小鼠相比,hGH慢性刺激组小鼠的GH信号通路的人信号传导子及转录激活子5（STAT5）磷酸化水平显著减低;处死前30min按1μg/g体重单剂量注射hGH的小鼠中,hGH慢性刺激组小鼠的STAT5b磷酸化水平与PBS对照组相比也同样显著减低;细胞膜GHR水平没有变化。结论 hGH慢性刺激可抑制小鼠肝细胞的JAK/STAT5信号转导通路。     

JAK/STAT信号通路相关基因在人胃腺癌5-氟尿嘧啶耐药细胞株中的表达变化

目的 用基因芯片技术研究人胃腺癌5-氟尿嘧啶(5-Fu)耐药细胞株SGC7901／R及其亲本细胞株SGC7901 JAK／STAT信号传导通路相关基因表达谱的差异。方法 分别抽提两种细胞mRNA，经逆转录反应，用生物素标记的16-duTP将两种细胞的mRNA分别标记成两种CDNA探针，并与载有-组靶基因的人JAlL／STAT信号通路芯片进行杂交，通过软件对扫描图像进行数字化处理和分析，筛选2种细胞在该信号通路中有表达差异的基因。通过RT-PCR法分别对耐药细胞及其亲本细胞中的Stat3、核因子(NF)-KB1和bcl-x mRNA的表达水平进行检测。结果通过两种细胞株JAKIC／STAT信号通路的基因谱平行比较，筛选出人胃腺癌5-Fu耐药细胞株及其亲本细胞株中有表达差异2倍以上的基因共70个，其中表达上调＞2倍的40个，表达下调＞2倍的30个。RT-PCR的验证结果与芯片的筛查结果基本相符。结论 人胃腺癌5-Fu耐药细胞株中JAK／STAT信号通路基因表达谱的变化研究是对胃癌多药耐药分子机制研究的有力补充。