STAT3 mediates PDGF-BB-induced Pim-1 expression in rat vascular smooth muscle cells

AIM: To study the expression of Pim-1 in vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor BB (PDGF-BB). METHODS: VSMCs isolated from rats were treated with different concentrations of PDGF-BB for different time. The proliferation of VSMCs was detected by cell counting. The mRNA expression of Pim-1 was measured by real-time RT-PCR. The STAT3 activity was determined by Western blotting. Actinomycin D, AG490, and small interfering RNA (siRNA) for Pim-1 or STAT3 were used to investigate the underlying mechanisms. RESULTS: Pim-1 gene silencing attenuated the proliferation of VSMCs in response to PDGF-BB. The mRNA expression of Pim-1 was up-regulated by PDGF-BB at concentrations of 10 μg/L~50 μg/L for 1 h, and was maximally induced at the concentration of 20 μg/L. The time of Pim-1 mRNA expression maximally occurred 30 min after PDGF-BB exposure. Incubation of VSMCs with PDGF-BB resulted in a significant activation of STAT3. VSMCs pretreated with actinomycin D showed a significant decrease in the mRNA expression of Pim-1. Treatment with AG490 or knockdown of STAT3 in VSMCs resulted in inactivation of STAT3, and significantly suppressed the mRNA expression of Pim-1. CONCLUSION: PDGF-BB-induced VSMC proliferation is partly attributed to Pim-1. VSMCs strongly increase Pim-1 mRNA upon stimulation with PDGF-BB, and STAT3 signaling pathway appears to be efficient for regulation of Pim-1 expression. This process may play a critical role in development of vascular remodeling.     

JAK/STAT通路抑制剂AG490对单核细胞增生李斯特菌感染的影响

为检测Janus激酶/信号转导与转录激活子(JAK/STAT)信号通路抑制剂α-氰-3, 4-二羟基-N-苄基肉桂酰胺(AG490)对单核细胞增生李斯特菌(LM)感染小鼠脑微血管内皮细胞和小鼠的影响,将不同浓度抑制剂AG490与细胞共同孵育,然后将LM分别感染细胞,MTT法检测不同浓度抑制剂AG490对细胞活性的影响;利用菌落计数法计算LM的侵袭率和胞内细菌数量,检测AG490对LM介导的小鼠生存曲线的影响;利用试剂盒检测抑制剂AG490对LM介导的乳酸脱氢酶(LDH)、白细胞介素-1β(IL-1β)、干扰素-γ(IFN-γ)和白细胞介素-6(IL-6)分泌的影响;利用实时定量PCR检测AG490对LM介导的炎性小体NLRP3的mRNA水平影响。结果:抑制剂AG490在5、10或15μmol/L浓度时未见对细胞活性有明显影响,AG490显著抑制了LM的侵袭、胞内和脏器内细菌数量,提高小鼠成活率,显著提高LM介导的LDH、IFN-γ、IL-6、IL-1β和NLRP3的mRNA水平。本研究表明,JAK/STAT信号通路抑制剂AG490可调控细胞内LM的存活,增强LM介导的细胞炎性小体水平,为调查LM引起脑膜炎的致病机制提供科学依据。     

Brucine induces apoptosis in colon cancer SW480 cells via inhibiting IL-6/STAT3 signaling pathway

AIM: To observe the effects of brucine on the viability and apoptosis of colon cancer SW480 cells.METHODS: The SW480 cells were divided into control group, 1 μmol/L brucine treatment group, 100 μg/L IL-6 treatment group and IL-6＋brucine treatment group. The cell viability was detected by CCK-8 assay. The apoptotic rate was measured by flow cytometry using fluorescein-labeled Annexin V/PI. The changes of apoptosis-related proteins were determined by Western blot. The protein level of p-STAT3 was also detected by immunofluorescence staining. RESULTS: Brucine inhibited SW480 cell growth, and the viability inhibition rate of the SW480 cells treated with brucine alone was more efficient than using brucine combined with IL-6 (P < 0.05). The apoptotic SW480 cells increased significantly after 1 μmol/L brucine treatment as compared with brucine treatment alone (P < 0.05). The apoptotic SW480 cells were significantly reduced in brucine and IL-6 combination treatment group (P < 0.05). Brucine inhibited the protein level of p-STAT3 significantly. The protein level of p-STAT3 was significantly increased in 100 μg/L IL-6 treatment group. Compared with 1 μmol/L brucine treatment alone, the expression of Bcl-2 was increased and the protein levels of p-STAT3, Bax and cleaved PARP were reduced in brucine and IL-6 combination treatment group (P < 0.05).CONCLUSION: Brucine may inhibit the activation of STAT3 phosphorylation in IL-6/STAT3 pathway to exert an antitumor effect on SW480 cells in vitro.     

Inhibitory effects of paeonol on keratinocyte viability,cytokines secretion stimulated by IL-17A via STAT3 signaling pathway

AIM To observe the effect of paeonol on interleukin-17A （IL-17A）-induced human keratinocyte viability, cytokine secretion, and related signal transduction pathways. METHODS In vitro HaCaT cells stimulated by IL-17A （200 μg/L） were co-cultured with paeonol （200 mg/L and 100 mg/L） for 24 h. The cell viability was measured by CCK-8 assay. The Th1/Th2/Th17 cytokine （including IL-6, etc.） levels were measured by cytometric bead array assay. The IL-23 level was measured by ELISA. The mRNA expression of IL-23, IL-6, CXCL2, CXCL8, CCL20 and STAT3 was detected by real-time PCR, and Western blot was used to determine the protein levels of STAT3 and ERK1/2. RESULTS Paeonol significantly inhibited IL-17A-induced HaCaT cell viability （P<0.05）, as well as reduced IL-6 level. Meanwhile, paeonol decreased mRNA levels of IL-23, CXCL2, CXCL8, and CCL20. Paeonol also inhibited the expression of STAT3 at mRNA and protein levels. However, no significant effect of paeonol on ERK1/2 protein expression was observed. CONCLUSION Paeonol inhibits HaCaT cell viability and cytokine secretion induced by IL-17A, and its mechanism might be related to STAT3 singaling pathway.     

干扰素α对小鼠Tfh细胞分化的调控作用

通过体外试验观察干扰素-α(IFN-α)对滤泡辅助性T细胞(Tfh)分化及功能的影响。无菌分离小鼠脾细胞,磁珠分选CD4^+T细胞,加入抗CD3单克隆抗体、抗CD28单克隆抗体,重组IL-21、IL-6进行刺激,同时加入重组IFN-α(设不加IFN-α的阴性对照)进行培养,分别在培养后的1、3、5d,流式细胞术检测Tfh细胞(CD4^+CXCR5^+)和CD4^+CXCR5^+PD-1^+细胞的比例;ELISA检测细胞上清中Tfh细胞功能性细胞因子IL-4、IL-21的水平;real-time PCR检测培养3d后Tfh细胞相关转录因子STAT1、Bcl-6以及细胞因子IL-21mRNA的表达水平。结果表明,与阴性组相比,IFN-α能明显促进Tfh细胞的分化及Tfh细胞中PD-1的表达(P<0.05),能提高Tfh细胞功能性细胞因子IL-4、IL-21的分泌水平(P<0.05);IFN-α能正向调控Tfh细胞相关转录因子STAT1、Bcl-6以及细胞因子IL-21mRNA的表达水平(P<0.05)。说明IFN-α可通过正向调控STAT1、Bcl-6的表达,促进Tfh细胞的分化和细胞因子的分泌。     

IL-11对小鼠骨骼肌发育和能量代谢相关基因mRNA表达的影响

利用IL-11激活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,研究该通路对骨骼肌发育和能量代谢相关基因mRNA表达水平的影响。对雄性健康昆明小白鼠用IL-11连续皮下注射15 d,定期测量体质量和体温,小鼠处死后取腿肌提取总RNA,利用RT-PCR检测JAK2/STAT3信号通路关键因子JAK2和STAT3、骨骼肌发育相关基因成肌分化因子(MyoD)和生肌决定因子(Myf5)及能量代谢相关基因肝X受体(LXRα)和解偶联蛋白3(UCP3)mRNA表达。结果显示:处理组小鼠的体质量和腿部肌肉质量显著高于空白对照组(P0.05);两组小鼠体温均维持在正常浮动范围内。与空白对照组相比,处理组小鼠JAK2和STAT3 mRNA表达量上升(P0.05);骨骼肌发育相关基因MyoD和Myf5 mRNA表达量均上升(P0.05);能量代谢相关基因LXRαmRNA表达量不变(P0.05),UCP3 mRNA表达量上升(P0.05)。表明IL-11激活JAK2/STAT3信号通路后可通过提高骨骼肌发育和能量代谢相关基因的mR-NA表达而调节骨骼肌发育和能量代谢。