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。     

钙激活的氯离子通道 A4通过抑制 JAK激酶 2/信号转导及转录激活蛋白 3信号通路对食管癌细胞增殖、迁移及侵袭的影响

目的 探讨钙激活的氯离子通道A4(CLCA4)对食管癌细胞增殖、迁移、侵袭及JAK激酶2/信号转导及转录激活蛋白3(JAK2/STAT3)信号通路的影响.方法 实时荧光定量逆转录聚合酶链反应(qRT-PCR)与蛋白质印迹法(Western blotting)分别检测正常人食管鳞状上皮细胞与人食管癌细胞中CLCA4的表达;将合成的CLCA4过表达载体及其对照分别转染至食管癌细胞Eca109,分别记作CLCA4过表达(pcDNA-CLCA4)组、CLCA4阴性对照(pcDNA-NC)组,并将未转染的细胞作为阴性对照(NC)组.四甲基偶氮唑盐微量酶反应比色法(MTT法)检测细胞增殖能力;细胞迁移实验(Transwell)检测细胞迁移及侵袭能力.JAK2/STAT3信号通路激活剂p-JAK2多肽对细胞增殖、迁移及侵袭的影响.Western blotting检测细胞周期蛋白D1(Cy-clinD1)、依赖性激酶抑制因子(P21)、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)、JAK2、STAT3、磷酸化JAK激酶2(p-JAK2)、磷酸化信号转导及转录激活蛋白3(p-STAT3)的表达水平.结果 与Het-1A相比,人食管癌细胞KYSE170、Eca109、TE10中CLCA4 mRNA及蛋白表达水平降低(P<0.05);与pcDNA-NC组比较,pcDNA-CLCA4组细胞存活率显著降低[(52.16±11.41)％比(99.57±13.49)％,P<0.05],迁移细胞数[(56.47±10.03)％比(112.49±13.52)％]与侵袭细胞数[(63.43±9.87)％比(123.47±16.58)％]减少(P<0.05),CyclinD1、MMP-2、MMP-9、p-JAK2、p-STAT3的表达水平降低(P<0.05),P21的表达水平升高(P<0.05);激活JAK2/STAT3信号通路可逆转CLCA4过表达对Eca109细胞增殖、迁移及侵袭的抑制作用.结论 CL-CA4过表达可抑制食管癌细胞增殖、迁移及侵袭,其作用机制可能与抑制JAK2/STAT3信号通路活化有关.     

木犀草素抑制JAK2/STAT3信号通路减轻大鼠脑缺血 再灌注损伤作用的研究

目的 观察木犀草素减轻大鼠脑缺血再灌注损伤的作用并探讨其潜在机制。方法 SD大鼠按随机数字 表法分为假手术组、大脑中动脉栓塞（MCAO）组、木犀草素低剂量组（50 mg/kg）、木犀草素高剂量组（100 mg/kg）及尼 莫地平组（15 mg/kg）,灌胃给药,共7 d。采用线栓法建立MCAO大鼠模型,比较神经功能损伤评分、脑组织含水量、 脑梗死体积,测定各组肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-6、IL-1β、B细胞淋巴瘤因子2（Bcl-2）、Bcl-2相关 X蛋白（Bax）、半胱氨酸蛋白酶3（Caspase-3）含量及磷酸化Janus蛋白酪氨酸激酶2（p-JAK2）、磷酸化信号转导及转 录激活蛋白3（p-STAT3）蛋白表达等变化情况。结果 与假手术组比较,MCAO组大鼠神经功能损伤评分、脑组织含 水量、脑梗死体积、脑组织TNF-α、IL-6、IL-1β、Bax、Caspase-3含量及JAK2、STAT3蛋白磷酸化水平均明显增加（P＜ 0.05）,而脑组织Bcl-2含量明显降低（P＜0.05）;与MCAO组比较,木犀草素低、高剂量组及尼莫地平组大鼠神经功能 损伤评分、脑组织含水量、脑梗死体积、脑组织TNF-α、IL-6、IL-1β、Bax、Caspase-3含量及JAK2、STAT3蛋白磷酸化 水平均明显降低（P＜0.05）,而脑组织Bcl-2含量明显增加（P＜0.05）。结论 木犀草素具有减轻大鼠脑缺血再灌注 损伤的作用,该作用与抑制JAK2/STAT3信号通路活化,进而减弱炎症反应及减少细胞凋亡有关。     

Inhibition of STAT3 expression by siRNA suppresses growth and induces apoptosis in laryngeal cancer cells

Aim: To determine the inhibitory effect of the synthetic STAT3 siRNA on the expression of STAT3 gene in human laryngeal cancer cell lines Hep2 and to investigate the effect of STAT3 siRNA on growth and apoptosis in Hep2 cells. Methods: A pair of DNA templates coding siRNA against STAT3-mRNA was synthesized to reconstruct plasmid of pSilencer1.0-U6 siRNA-STAT3. Hep2 cells were transfected with RPMI-1640 media (untreated), plasmid (empty), and STAT3 siRNA, respectively. Northern blot and Western blot analysis of STAT3 and pTyr-STAT3 expression in Hep2 cells and Western blot analysis of Bcl-2 expression in the Hep2 cell was performed 72 h after transfection. MTT, flow cytometry, and AO/EB assay were used for determination of cells proliferation and apoptosis in Hep2 cells. Results: pTyr-STAT3 was markedly expressed in untreated Hep2 cells and the vector-treated Hep2 cells, whereas pTyr-STAT3 expression was significantly reduced in STAT3 siRNA-transfected Hep2 cells, indicating that STAT3 siRNA inhibited the activity of STAT3. Transfection of Hep2 cells with STAT3 siRNA significantly inhibited STAT3 expression at both mRNA and protein level in Hep2 cells and the inhibition was characterized by time-dependent transfection. Treatment of Hep2 cells with STAT3 siRNA resulted in dose-dependent growth inhibition of Hep2, this significantly increased apoptotic cell rate, and decreased Bcl-2 expression level in Hep2 cells. STAT3 siRNA had an effect on induction of either early or late stage apoptosis. Conclusion: This study demonstrates that STAT3 siRNA effectively inhibits STAT3 gene expression in Hep2 cells leading to growth suppression and induction of apoptosis in Hep2 cells. The use of siRNA technique may provide a novel therapeutic approach to treat laryngeal cancer and other malignant tumors expressing constitutively activated STAT3.     

白藜芦醇通过阻断 JAK激酶 2/信号转导及转录活化因子 3信号通路抑制食管癌细胞增殖和迁移并诱导其凋亡的作用研究

目的探究白藜芦醇对人食管癌细胞增殖、凋亡、迁移及 JAK激酶 2/信号转导及转录活化因子 3(JAK2/STAT3)信号通路的调控作用。方法 2021年 7月至 2022年 7月,体外培养人食管癌 OE19细胞,将细胞分为对照组(不做干预)和实验组( 15、 30、60、90和 120 μmol/L白藜芦醇干预 24 h)进行预实验,根据细胞计数试剂盒( CCK-8)预实验结果,筛选有显著作用且细胞活力高于 50%的 30、60、90 μmol/L白藜芦醇进行后续的实验,后续实验又分为对照组(不做干预)、 30、60、90 μmol/L白藜芦醇组(30、60和 90 μmol/L白藜芦醇)和 30 μmol/L白藜芦醇 +抑制剂组( 30 μmol/L白藜芦醇 +10 μmol/L JAK2/STAT3通路抑制剂 AG490),干预 24 h。用 5-乙炔基 -2''脱氧尿嘧啶核苷( EdU)、 Hoechst 33258染色、 Transwell、实时荧光定量 PCR(RT-qPCR)及蛋白质印迹法对细胞增殖率、凋亡情况、迁移数及细胞周期蛋白 D1(cyclin D1)、胱天蛋白酶 -3(caspase-3)和 JAK2/STAT3相关因子表达水平进行分析。结果不同浓度实验组的细胞活力与对照组相比逐渐降低,其中 30、60、90和 120 μmol/L白藜芦醇差异有统计学意义( P<0.05)但 120 μmol/L白藜芦醇组细胞活力低于 50%,所以本研究选择 30、60和 90 μmol/L白藜芦醇继续后续实验; 60 μmol/L白藜芦醇组,细胞增殖率(41.49±5.06)%、迁移细胞数( 36.67±2.52)个显著低于对照组( 53.34±1.99)%、(58.00±     

YD383和YD439对IL-4诱导的BJAB细胞CD23表达的影响

目的:初步探讨2种化合物YD383、YD439对JAK/STAT6信号传导通路的抑制机制。方法:将不同剂量的化合物YD383和YD439加入对数生长期的BJAB细胞作用30min,终浓度为20μg/L的白细胞介素(IL)-4共同孵育3h后,收集细胞并加入PE标记的鼠抗人CD23抗体,流式细胞仪检测各细胞群体CD23分子的表达情况。结果:化合物YD383和YD439均能降低由IL-4诱导的BJAB细胞CD23分子的表达,且随着化合物剂量的增大,CD23分子的表达水平逐渐降低。2种化合物各自不同的剂量组间CD23分子表达的差异均有统计学意义(P<0.05)。结论:化合物YD383和YD439对IL-4诱导的CD23分子的表达均有明显的抑制作用,并且具有剂量依赖性。     

Emodin inhibits growth and induces apoptosis in an orthotopic hepatocellular carcinoma model by blocking activation of STAT3

BACKGROUND AND PURPOSE

Aberrant activation of STAT3 is frequently encountered and promotes proliferation, survival, metastasis and angiogenesis in hepatocellular carcinoma (HCC). Here, we have investigated whether emodin mediates its effect through interference with the STAT3 activation pathway in HCC.

EXPERIMENTAL APPROACH

The effect of emodin on STAT3 activation, associated protein kinases and apoptosis was investigated using various HCC cell lines. Additionally, we also used a predictive tumour technology to analyse the effects of emodin. The in vivo effects of emodin were assessed in an orthotopic mouse model of HCC.

KEY RESULTS

Emodin suppressed STAT3 activation in a dose- and time-dependent manner in HCC cells, mediated by the modulation of activation of upstream kinases c-Src, JAK1 and JAK2. Vanadate treatment reversed emodin-induced down-regulation of STAT3, suggesting the involvement of a tyrosine phosphatase and emodin induced the expression of the tyrosine phosphatase SHP-1 that correlated with the down-regulation of constitutive STAT3 activation. Interestingly, silencing of the SHP-1 gene by siRNA abolished the ability of emodin to inhibit STAT3 activation. Finally, when administered i.p., emodin inhibited the growth of human HCC orthotopic tumours in male athymic nu/nu mice and STAT3 activation in tumour tissues.

CONCLUSIONS AND IMPLICATIONS

Emodin mediated its effects predominantly through inhibition of the STAT3 signalling cascade and thus has a particular potential for the treatment of cancers expressing constitutively activated STAT3.     

Arecoline inhibits myogenic differentiation of C2C12 myoblasts by reducing STAT3 phosphorylation

Areca nut (Areca catechu) is chewed regularly as a medical and psychoactive food by about 10% of the world population, in countries including India, Taiwan and parts of Southern Asia. Areca nut chewing during pregnancy has been associated with both lower birth weight and premature birth. Animals of low birth weights showed retardation of muscle development. Our previous study showed that arecoline, the major areca alkaloid, decreased the number of implanted embryos. Here we sought to determine the effects of arecoline in myogenic differentiation by in vitro assays using C2C12 myoblast cells. The results showed that arecoline higher than 0.4 mM significantly increased apoptosis and decreased viability of C2C12 cells. Morphometric measurements of myotube formation and analyses of myogenic markers, myosin heavy chain and myogenin, revealed that myogenic differentiation was inhibited by 0.04–0.08 mM arecoline. Moreover, phosphorylated but not total STAT3 was significantly inhibited by arecoline during myotube formation. These results indicate that arecoline inhibits the myogenic differentiation of C2C12 cells by reducing the activation of STAT3, an upstream regulator of myogenesis. Improved understanding of the effects of arecoline during myogenic differentiation may help to establish public health policies and to develop potential treatments for such patients.     

没食子乙醇提取物对结肠癌细胞JAK2/STAT3信号通路的调控作用研究

目的 基于蛋白酪氨酸激酶2(JAK2)/信号传导及转录活化因子3(STAT3)信号通路研究没食子乙醇提取物对结肠癌细胞HCT-116、Caco-2增殖、迁移的调控机制.方法 采用CCK-8法检测0.05、0.1、0.2、0.3、0.4、0.5 mg/mL没食子乙醇提取物作用12、24、48、72 h后对HCT-116、...     

Allitridi induces apoptosis by affecting Bcl-2 expression and caspase-3 activity in human gastric cancer cells

INTRODUCTION Gastric cancer is the second leading cause of can-cer mortality in the world and is the leading cause ofcancer mortality in China[1]. Numerous epidemiologicalinvestigations have demonstrated that an inverse corre-lation between gastric cancer incidence and garlic con-sumption[2-8]. For example, Linqu County has gastriccancer rate that was 15 times higher than that of Cang-shan County in Shangdong Province[10], and the deathrate of gastric cancer in Linqu County was 7…     

慢病毒介导的PRL-3 shRNA对结肠癌细胞增殖、侵袭、凋亡的影响

目的 观察慢病毒介导的 shRNA 沉默肝再生磷酸酶-3（PRL-3）基因对结肠癌 SW480 细胞增殖、侵袭、凋亡的影响。方法 实验分组为空白对照组、阴性对照组、转染组。将携带 PRL-3 shRNA 的慢病毒载体转染结肠癌SW480 细胞,建立稳定沉默 PRL-3 的细胞株,real-time PCR 检测转染后 PRL-3 mRNA 的相对表达水平。采用 MTT法、平板克隆形成实验检测转染后细胞增殖能力;采用 Transwell 侵袭实验、侵袭小室法检测转染后细胞迁移及侵袭能力;采用流式细胞术检测转染后细胞凋亡率变化。结果 稳定沉默 PRL-3 的细胞株构建成功,转染组 PRL-3mRNA 的相对表达水平低于空白对照组、阴性对照组（P＜0.05）,空白对照组、阴性对照组比较差异无统计学意义。PRL-3 shRNA 转染 SW480 细胞 72 h 后,转染组与空白对照组、阴性对照组比较,细胞增殖能力受到抑制,转染 120 h时最明显（P＜0.05）。转染组克隆形成能力较空白对照组、阴性对照组下降（P＜0.05）。转染组与空白对照组、阴性对照组比较,细胞迁移、侵袭能力下降,凋亡率增加（P＜0.05）。结论 结肠癌 SW480 细胞转染 PRL-3 shRNA 可减少 PRL-3 的表达,有效抑制 SW480 细胞增殖,促进其凋亡,PRL-3 可能成为治疗结肠癌的靶基因。     

STAT3 down‐regulation induces mitochondria‐dependent G2/M cell cycle arrest and apoptosis in oesophageal carcinoma cells

STAT3 is persistently activated in a wide variety of human tumours, and aberrant STAT3 activity promotes tumour growth, invasion and metastasis. To explore STAT3 down‐regulation in human oesophageal cancer cells, cell proliferation, apoptosis and mitochondrial mechanisms were explored in oesophageal carcinoma TE1 cell cultures. We demonstrate for the first time that STAT3 down‐regulation by RNAi is sufficient to inhibit oesophageal cancer cell proliferation inducing cell apoptosis. Further, we demonstrate that mitochondrial transmembrane potential is impaired thereby leading to collapsed mitochondrial membrane potential, abnormal mitochondrial membrane depolarization, nuclear DNA fragmentation and cell cycle G2/M arrest under the conditions of STAT3 down‐regulation. Thus, our results suggest that STAT3 inhibition is a valid approach to induce oesophageal carcinoma cell mitochondrial‐dependent apoptosis in therapeutic strategies against oesophageal cancers.     

Interleukin-13-induced activation of signal transducer and activator of transcription 6 is mediated by an activation of Janus kinase 1 in cultured human bronchial smooth muscle cells

BackgroundThe current study was carried out to identify the JAK molecule(s) that is involved in the IL-13-induced activation of STAT6 in cultured human bronchial smooth muscle cells (hBSMCs).MethodsCultured hBSMCs were stimulated with IL-13 in the absence and presence of JAK inhibitor-I (a nonspecific JAKs inhibitor), tyrphostin-AG490 (a specific JAK2 inhibitor), WHI-P131 (a specific JAK3 inhibitor), or tyrphostin-AG9 (a specific Tyk2 inhibitor), and levels of phosphorylated STAT6 were measured by immunoblot analyses.ResultsThe IL-13-induced phosphorylation of STAT6 was abolished by JAK inhibitor-I, whereas the other inhibitors had no significant effect.ConclusionThese findings indicate that the STAT6 phosphorylation/activation induced by IL-13 is mediated by an activation of JAK1 in cultured hBSMCs.     

MiR-599 regulates LPS-mediated apoptosis and inflammatory responses through the JAK2/STAT3 signalling pathway via targeting ROCK1 in human umbilical vein endothelial cells

MicroRNA plays an integral role in the development of atherosclerosis. Our study aimed to investigate the roles of miR-599 in lipopolysaccharide (LPS)-induced endothelial damage in human umbilical vein endothelial cells (HUVECs). HUVECs were transfected with a miR-599 mimic and negative control, and then exposed to LPS. The expression of miR-599 was detected by quantitative real time-polymerase chain reaction (RT-qPCR). Cell viability was analyzed by CCK-8 assay and trypan blue exclusion assay; the formation of DNA fragments was tested by Cell Death Detection ELISA Plus kit; the incidence of apoptosis was detected by flow cytometry; the expression of p53 and cleaved-caspase 3 (c-caspase 3) was evaluated by western blot. Moreover, the mRNA levels and concentrations of tumour necrosis factor (TNF)-α, interleukin (IL)-6, ICAM-1 and VCAM-1 were assayed by RT-qPCR and ELISA. The results showed that overexpression of miR-599 increased cell viability, reduced DNA fragments, the incidence of apoptosis, as well as the protein levels of p53 and c-caspase 3 in the presence of LPS. TNF-α, IL-6, ICAM-1 and VCAM-1 mRNA levels and concentrations were also decreased upon miR-599 upregulation. In addition, the dual luciferase reporter assay demonstrated that ROCK1 is a direct target of miR-599. MiR-599 overexpression inhibited ROCK1 expression. Induced expression of ROCK1 reversed the roles of miR-599 in apoptosis and inflammation. The gain function of miR-599 function inhibited activation of the JAK2/STAT3 signalling pathway, which was abrogated by overexpression of ROCK1. Taken together, our results indicate that miR-599 attenuates LPS-caused cell apoptosis and inflammatory responses through the JAK2/STAT3 signalling pathway via targeting ROCK1.     

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.