JAK/STAT途径调节瘦素诱导的肝星状细胞Ⅰ型胶原基因的表达

目的研究瘦素对肝星状细胞(HSC)α1(Ⅰ)型胶原mRNA表达和蛋白合成的影响,探讨Janus激酶/信号传导及活化转录因子(JAK/STAT)信号传导通路在瘦素诱导的HSC胶原基因表达过程中的作用。方法采用RT-PCR法、ELISA法和Western blot法分别检测不同浓度的瘦素对人肝星状细胞株LX-2α1(Ⅰ)型胶原mRNA表达、蛋白合成以及JAK1、STAT3磷酸化状态的影响;采用Western blot法和RT-PCR法分别观察JAK1抑制剂AG490对瘦素诱导的JAK1磷酸化和α1(Ⅰ)型胶原mRNA表达的影响;采用Western blot法分别观察AG490、LX-2转染STAT3反义寡核苷酸(STAT3-ASON)对瘦素诱导的STAT3磷酸化状态的影响;应用RT-PCR法检测LX-2转染STAT3-ASON对瘦素诱导的α1(Ⅰ)型胶原mRNA表达的影响。结果瘦素增加LX-2α1(Ⅰ)型胶原mRNA表达和蛋白合成,呈剂量效应关系,当瘦素浓度为80μg·L-1时达到最大值;瘦素促进JAK1、STAT3磷酸化,呈时间效应关系;AG490完全阻断瘦素诱导的JAK1、STAT3磷酸化和α1(Ⅰ)型胶原mRNA的表达;LX-2转染STAT3-ASON阻断瘦素诱导的STAT3磷酸化和α1(Ⅰ)型胶原mRNA的表达。结论瘦素通过增加HSCα1(Ⅰ)型胶原mRNA表达和蛋白合成而在肝纤维化发生发展过程中发挥重要的作用,JAK/STAT信号传导通路参与并调节该过程,AG490和LX-2转染STAT3-ASON可有效阻滞此传导途径。在人HSC中,活化的JAK1和STAT3信号可作为肝纤维化治疗新的分子靶点。     

JAK2/STAT3信号转导通路在子宫内膜异位症发病过程中的作用

目的 研究JAK2/STAT3信号转导通路在子宫内膜异位症 （EMS） 发展过程中的作用。方法 将60只大鼠随机分为假手术组、 模型组、 JAK2/STAT3通路抑制剂组 （AG组）, 每组20只。模型组、 AG组大鼠通过自身子宫内膜移植建立EMS模型。AG组造模前给予AG490 1 mg/kg, 手术后给予AG490 4 mg/kg, 每周2次, 连续4周; 模型组给予生理盐水。处理结束后记录各组大鼠异位内膜体积, 计算异位内膜生长抑制率, HE染色观察病理改变; 蛋白质印迹法 （Western blot） 检测内膜组织中p-JAK2、 JAK2、 p-STAT3、 STAT3蛋白表达水平。结果 治疗后AG组异位内膜组织生长发育不良, 异位内膜体积明显小于模型组, 异位内膜生长的抑制率为65.66%。Western blot结果显示, EMS模型大鼠异位内膜病灶中JAK2、 STAT3总蛋白及其磷酸化水平 （p-JAK2/JAK2、 p-STAT3/STAT3） 明显高于假手术组; 而 AG组大鼠异位内膜病灶中JAK2、 STAT3总蛋白及其磷酸化水平明显低于模型组。结论 JAK2/STAT3通路在EMS 发病过程中可能发挥了重要作用, 而抑制JAK2/STAT3信号通路则能够对EMS的治疗产生积极作用。     

Janus激酶-信号转导子与转录激活子信号通路在硫化氢后处理离体缺血再灌注大鼠心肌中的作用

目的 探讨Janus激酶-信号转导子与转录激活子(JAK2/STAT3)信号通路在硫化氢后处理(H2S)减轻离体大鼠心脏缺血/再灌注(I/R)损伤的作用.方法 应用Langendorff离体心脏灌流装置、通过停灌30 min/复灌60 min的方法建立SD大鼠I/R模型.按照处理及再灌注成分分为持续灌注对照组,I/R组...     

Inhibition of Jak2 phosphorylation attenuates pressure overload cardiac hypertrophy

RationaleWe examined the role of Jak2 kinase phosphorylation in the development of pressure overload hypertrophy in mice subjected to transverse aortic constriction (TAC) and treated with tyrphostin AG490, a pharmacological inhibitor of Jak2.MethodsControl mice (sham), subjected to TAC for 15 days (TAC) or to TAC and treated with 48 μg/kg/day i.p. of tyrphostin AG490 (TAC + AG490) were evaluated for morphological, physiological, and molecular changes associated with pressure overload hypertrophy.ResultsMice subjected to TAC alone developed concentric hypertrophy that accompanied activation of the components of the Jak/STAT signaling pathway manifested by an increase in phosphorylation of Jak2 and STAT3. We also observed increased phosphorylation of MAPK p44/p42, p38 MAPK and JNK in the TAC group, as well as, an increase in expression of MKP-1 phosphatase which negatively regulates MAPK kinases. Treatment of aortic constricted mice with tyrphostin AG490 failed to develop hypertrophy and showed a marked reduction in phosphorylation of Jak2 and STAT3. There was, however, in TAC and AG490 treated mice, a notable increase in the phosphorylation state of the MAPK p44/42, whereas MKP-1 phosphatase was downregulated.ConclusionThese findings suggest that Jak2 kinase plays an important role in left ventricular remodeling during pressure overload hypertrophy. Pharmacological inhibition of Jak2 kinase during pressure overload blocks the development of concentric hypertrophy.     

褪黑素对PDGF诱导的肝星状细胞中JAK2/STAT3信号通路的影响

目的探讨褪黑素对血小板衍生生长因子(PDGF)诱导的肝星状细胞-T6(HSC-T6)JAK2/STAT3信号通路的影响。方法将HSC-T6细胞分为6组:对照组、模型组、实验组(褪黑素1 nmol·L^-1、1μmol·L^-1、0.1 mmol·L^-1)、抑制剂组(AG490为JAK2/STAT3通路的抑制剂),MTT实验检测褪黑素对PDGF激活的HSC-T6细胞增殖的影响;免疫组化和Western blot检测褪黑素HSC-T6细胞中p-JAK2、p-STAT3蛋白的表达。结果与对照组比较,PDGF能明显激活HSC-T6细胞增殖,明显上调HSC-T6细胞中p-JAK2、p-STAT3的表达;与模型组比较,褪黑素和抑制剂均可抑制PDGF激活的HSC-T6细胞增殖,显著下调p-JAK2、p-STAT3的表达。结论褪黑素可抑制PDGF诱导的HSC-T6的活化与增殖,其机制可能与抑制JAK2/STAT3信号通路有关。     

Inhibition of Jak2 phosphorylation attenuates pressure overload cardiac hypertrophy

RATIONALE: We examined the role of Jak2 kinase phosphorylation in the development of pressure overload hypertrophy in mice subjected to transverse aortic constriction (TAC) and treated with tyrphostin AG490, a pharmacological inhibitor of Jak2. METHODS: Control mice (sham), subjected to TAC for 15 days (TAC) or to TAC and treated with 48 microg/kg/day i.p. of tyrphostin AG490 (TAC+AG490) were evaluated for morphological, physiological, and molecular changes associated with pressure overload hypertrophy. RESULTS: Mice subjected to TAC alone developed concentric hypertrophy that accompanied activation of the components of the Jak/STAT signaling pathway manifested by an increase in phosphorylation of Jak2 and STAT3. We also observed increased phosphorylation of MAPK p44/p42, p38 MAPK and JNK in the TAC group, as well as, an increase in expression of MKP-1 phosphatase which negatively regulates MAPK kinases. Treatment of aortic constricted mice with tyrphostin AG490 failed to develop hypertrophy and showed a marked reduction in phosphorylation of Jak2 and STAT3. There was, however, in TAC and AG490 treated mice, a notable increase in the phosphorylation state of the MAPK p44/42, whereas MKP-1 phosphatase was downregulated. CONCLUSION: These findings suggest that Jak2 kinase plays an important role in left ventricular remodeling during pressure overload hypertrophy. Pharmacological inhibition of Jak2 kinase during pressure overload blocks the development of concentric hypertrophy.     

Inhibition of cytokine-induced JAK-STAT signalling pathways by an endonuclease inhibitor aurintricarboxylic acid

1. Inducible nitric oxide (iNOS) is thought to involve in host defence and tissue damage in inflammatory loci. In previous study, we have found that the endonuclease inhibitor aurintricarboxylic acid (ATA) can protect macrophages from cell death induced by bacterial lipopolysaccharide. This action is through the interruption with signalling pathways for NF-kappa B and AP-1 activation, and thus iNOS expression. In this study we have addressed the effects of ATA on JAK-STAT signalling pathways. 2. In murine RAW 264.7 macrophages, IFN-gamma-mediated NO production and iNOS expression were concentration-dependently reduced by the presence of 3-100 micro M ATA. 3. IFN-gamma-induced STAT1 activation, as assessed from its tyrosine phosphorylation, nuclear translocation, binding to specific DNA response element and evoked IRF-1 reporter gene assay, were concomitantly inhibited by ATA. However, ATA did not alter IFN-gamma binding to RAW 264.7 cells. 4. The activities of JAK1 and JAK2, the upstream kinases essential for STAT1 signalling in response to IFN-gamma, were also reduced by ATA. 5. Moreover, IL-4, IL-10, GM-CSF and M-CSF elicited tyrosine phosphorylation of STAT3, STAT5 and/or STAT6 in macrophages were diminished by the presence of ATA. 6. Taken together, we conclude that ATA can interfere JAK-STAT signalling pathways in response to cytokines. This action contributes to the inhibition of IFN-gamma-induced iNOS expression.     

Down-regulation of the JAK2/PI3K-mediated signaling activation is involved in Taiwan cobra cardiotoxin III-induced apoptosis of human breast MDA-MB-231 cancer cells

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. Exposure of MDA-MB-231 cells with 0.03, 0.09, and 0.15 μM of CTX III for 18 h, CTX III-induced cell apoptosis, as evidenced by accumulation of sub-G1 population, externalization of phosphatidylserine, loss of mitochondrial membrane potential (ΔΨm) with subsequent release of cytochrome c, and activation of both capases-9 and caspase-3. This correlated with up-regulation in Bax and Bad, and down-regulation of various anti-apoptotic proteins, including Bcl-2, Bcl-X_L, and survivin in CTX III-treated cells. Mechanistic studies showed that CTX III suppressed the phosphorylation of JAK2, STAT3, Akt, and activation of PI3K. Moreover, the PI3K inhibitor wortmannin blocked activation of STAT3 and Akt without affecting the JAK2 activation, whereas JAK2 inhibitor AG490 suppressed the levels of phospho-STAT3, phospho-Akt, and PI3K, suggesting that PI3K activation occurs after JAK2 phosphorylation, and both PI3K and JAK2 kinases cooperate to mediate STAT3 and Akt phosphorylation. Both AG490 and wortmannin also led to up-regulation in Bax and Bad, and down-regulation of Bcl-2, Bcl-X_L, and survivin in MDA-MB-231 cells. Taken together, these results indicate that CTX III induces apoptosis in MDA-MB-231 cells via concomitant inactivation of the JAK2, STAT3, PI3K, and Akt signaling pathways.     

miRNA-21与JAK2-STAT3通路在心肌缺血后处理保护心肌中的相互影响*

【摘要】目的 观察miRNA-21在缺血后处理心肌保护机制中的作用,并探讨其与JAK2-STAT3通路在心肌缺血后处理保护心肌机制中的相互影响。方法 将健康雄性成年Wistar大鼠随机分为假手术组（Sham组）、缺血再灌注损伤组(I/R组)、缺血后处理组（PostC组）、缺血后处理+miRNA-21抑制物处理组（PostC+antagomiR-21组）和缺血后处理+AG490组（PostC+AG490组）。原位末端脱氧核苷酸转移酶标记（TUNEL）法检测心肌细胞凋亡指数;Western blot法检测总STAT3（t-STAT3）及磷酸化STAT3（p-STAT3）蛋白水平;荧光实时定量（qRT）-PCR检测心肌组织miRNA-21表达水平。结果 与I/R组相比,PostC组再灌注后心肌细胞凋亡指数显著减少,miRNA-21表达水平及STAT3磷酸化水平显著上调。使用antagomiR-21可显著削弱PostC心肌保护作用。PostC+antagomiR-21组与PostC组相比,STAT3的磷酸化水平显著降低。PostC+AG490组与PostC组比较,STAT3的磷酸化水平及miRNA-21表达均显著降低。结论miRNA-21在PostC心肌保护机制中发挥调控作用。在后处理适应性机制中STAT3与miRNA-21可能存在某种回路调控机制。     

Naphtho[1,2-b]furan-4,5-dione disrupts Janus kinase-2 and induces apoptosis in breast cancer MDA-MB-231 cells

Naphtho[1,2-b]furan-4,5-dione (NFD), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. NFD-induced apoptosis in MDA-MB-231 cells, as indicated by the accumulation of sub-G1 population, externalization of phosphatidylserine, loss of mitochondrial membrane potential (ΔΨm) with subsequent release of cytochrome c, and activation of both capase-9 and caspase-3. This correlated with up-regulation in Bax and Bad, and down-regulation of various anti-apoptotic proteins, including Bcl-2, Bcl-X_L, Mcl-1, and survivin in NFD-treated cells. In the analysis of signal transduction pathway, NFD suppressed the phosphorylation of JAK2 in MDA-MB-231 cells without altering the expression of JAK2 protein. Activation of STAT3, Src, and PI3K/Akt were also inhibited by NFD. Moreover, the JAK2 inhibitor AG490 blocked JAK2, STAT3, Src, PI3K, and Akt activation, whereas both Src inhibitor PP2 and PI3K inhibitor wortmannin did not affect JAK2 activation. This suggests that STAT3, Src, and PI3K/Akt are downstream molecules of the JAK2 signaling pathway. AG490 treatment also mimics the cytotoxic effects of NFD. Taken together, these results indicate that NFD disrupts JAK2 pathway and induces apoptosis in MDA-MB-231 cells.