Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient...Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided into 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003.58±1 626.57) and the expression of IL-5 mRNA (0.8300±0.0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888.47±1103.56 and 0.3050±0.0208, respectively, P< 0.01), while the indexes (23 219.83±1 024.86 and 0.3425±0.0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P< 0.01). The indexes (87 107.41±1 342.92 and 0.8225±0.0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P>0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P< 0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.展开更多
The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs ...The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.展开更多
AIM:Coptidis Rhizoma(CR),the dried rhizomes of Asian herbs(including Coptis chinensis Franch),has been used to treat diabetes mellitus for thousands of years.We explored the possibility that CR acts directly on skelet...AIM:Coptidis Rhizoma(CR),the dried rhizomes of Asian herbs(including Coptis chinensis Franch),has been used to treat diabetes mellitus for thousands of years.We explored the possibility that CR acts directly on skeletal muscle,the major organ responsible for glucose homeostasis,and activates 5'-AMP-activated protein kinase(AMPK),a signaling intermediary leading to metabolic enhancement of skeletal muscle.METHODS:Isolated rat epitrochlearis and soleus muscles were incubated in a buffer containing a CR water extract(CE),and activation of AMPK and related events were examined.RESULTS:In response to CE treat-ment,phosphorylation of Thr172 at the catalyticαsubunit of AMPK,an essential step for full kinase activation,increased in both mus-cles.Phosphorylation of Ser79 of acetyl CoA carboxylase(ACC),an endogenous substrate of AMPK,increased concomitantly.Analysis of isoform-specific AMPK activity revealed that CE activated both the α1 and α2 isoforms of the catalytic subunit.Importantly,the maximal effect of CE on AMPK phosphorylation was significantly greater than that of berberine(BBR),indicating that the action of CE is not totally ascribed to BBR.CONCLUSION:We propose that CE is an acute activator of AMPK in both fast-and slow-twitch skeletal muscles.展开更多
Objective：To investigate the effect of Dan-gua Fang（丹瓜方） on adenosine 5’-monophosphate（AMP） activated protein kinase（AMPK） α expression in liver and subsequent improvement of glucose and lipid metabolism.M...Objective：To investigate the effect of Dan-gua Fang（丹瓜方） on adenosine 5’-monophosphate（AMP） activated protein kinase（AMPK） α expression in liver and subsequent improvement of glucose and lipid metabolism.Methods：Forty 13-week-old diabetic Goto-Kakizaki（GK） rats were randomly divided into model,Dan-gua Fang,metformin and simvastatin groups（n=10 for each）,and fed high-fat diet ad libitum.Ten Wistar rats were used as normal group and fed normal diet.After 24 weeks,liver expression of AMPK α mRNA was assessed by real-time PCR.AMPK α and phospho-AMPK α protein expression in liver was evaluated by Western blot.Liver histomorphology was carried out after hematoxylin-eosin staining,and blood glucose（BG）,glycosylated hemoglobin A1c（HbA1c）,food intake and body weight recorded.Results：Similar AMPK α mRNA levels were found in the Dan-gua Fang group and normal group,slightly higher than the values obtained for the remaining groups（P〈0.05）.AMPK α protein expression in the Dan-gua Fang group animals was similar to other diabetic rats,whereas phospho-AMPK α（Thr-172） protein levels were markedly higher than in the metformin group and simvastatin group（P〈0.05）,respectively.However,phosphor-AMPKa/AMPK α ratios were similar in all groups.Dan-gua Fang reduced fasting blood glucose with similar strength to metformin,and was superior in reducing cholesterol,triglycerides,high-density lipoprotein cholesterol as well as improving low-density lipoprotein cholesterol in comparison with simvastatin and metformin.Dan-gua Fang decreases plasma alanine aminotransferase（ALT） significantly.Conclusion：Dan-gua Fang,while treating phlegm-stasis,could decrease BG and lipid in type 2 diabetic GK rats fed with high-fat diet,and effectively protect liver histomorphology and function.This may be partly explained by increased AMPK expression in liver.Therefore,Dan-gua Fang might be an ideal drug for comprehensive Intervention for glucose and lipid metabolism disorders in type 2 diabetes mellitus.展开更多
AIM: To investigate the functional significance of insulin-like growth factor binding protein-5 (IGFBP-5) overexpression in pancreatic cancer (PaC).METHODS: The effects of IGFBP-5 on cell growth were assessed by stabl...AIM: To investigate the functional significance of insulin-like growth factor binding protein-5 (IGFBP-5) overexpression in pancreatic cancer (PaC).METHODS: The effects of IGFBP-5 on cell growth were assessed by stable transfection of BxPC-3 and PANC-1 cell lines and measuring cell number and DNA synthesis. Alterations in the cell cycle were assessed by flow cytometry and immunoblot analyses. Changes in cell survival and signal transduction were evaluated after mitogen activated protein kinase and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.RESULTS: After serum deprivation, IGFBP-5 expression increased both cell number and DNA synthesis in BxPC-3 cells, but reduced cell number in PANC-1 cells. Consistent with this observation, cell cycle analysis of IGFBP-5-expressing cells revealed accelerated cell cycle progression in BxPC-3 and G2/M arrest of PANC-1 cells. Signal transduction analysis revealed that Akt activation was increased in BxPC-3, but reduced in PANC-1 cells that express IGFBP-5. Inhibition of PI3K with LY294002 suppressed extracellular signal-regulated kinase-1 and -2 (ERK1/2) activation in BxPC-3, but enhanced ERK1/2 activation in PANC-1 cells that express IGFBP-5. When MEK1/2 was blocked, Akt activation remained elevated in IGFBP-5 expressing PaC cells; however, inhibition of PI3K or MEK1/2 abrogated IGFBP-5-mediated cell survival.CONCLUSION: These results indicate that IGFBP-5 expression affects the cell cycle and survival signal pathways and thus it may be an important mediator of PaC cell growth.展开更多
Objective: To investigate whether ginsenoside-Rb1(Gs-Rb1) improves the CoCl2-induced autophagy of cardiomyocytes via upregulation of adenosine 5’-monophosphate-activated protein kinase(AMPK) pathway. Methods: Ventric...Objective: To investigate whether ginsenoside-Rb1(Gs-Rb1) improves the CoCl2-induced autophagy of cardiomyocytes via upregulation of adenosine 5’-monophosphate-activated protein kinase(AMPK) pathway. Methods: Ventricles from 1-to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group(normal level oxygen), hypoxia group(500 μmol/L CoCl2), Gs-Rb1 group(200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), Ara A group(500 μmol/L Ara A + 500 μmol/L CoCl2), Ara A+ Gs-Rb1 group(500 μmol/L Ara A + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide(AICAR)+ 500 μmol/L CoCl2], and AICAR+Gs-Rb1 group(1 mmol/L AICAR + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2). Cel s were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay and cardiac troponin I(cTnI) levels were detected by enzyme-linked immunosorbent assay(ELISA). AMPK activity was assessed by 2’,7’-dichlorofluorescein diacetate(DCFH-DA) ELISA assay. The protein expressions of Atg4 B, Atg5, Atg6, Atg7, microtubule-associated protein 1 A/1 B-light chain 3(LC3), P62, and active-cathepsin B were measured by Western blot. Results: Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes(P<0.01). However, the viability of hypoxia-treated cardiomyocytes was significantly inhibited by Ara A(P<0.01). Gs-Rb1 increased the AMPK activity of hypoxia-treated cardiomyocytes. The AMPK activity of hypoxia-treated cadiomyocytes was inhibited by Ara A(P<0.01) and was not affected by AICAR(P=0.983). Gs-Rb1 up-regulated Atg4B, Atg5, Beclin-1, Atg7, LC3B Ⅱ, the LC3BⅡ/Ⅰ ratio and cathepsin B activity of hypoxia cardiomyocytes(P<0.05), each of these protein levels was significantly enhanced by Ara A(all P<0.01), but was not affected by AICAR(all P>0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes(P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A(P<0.05) and were not affected by AICAR(P=0.871). Conclusion: Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.展开更多
OBJECTIVE: To assess the effect of a mixture of five herbal extracts(FT-5) on insulin resistance, glucose/lipid metabolism, hepatic steatosis, and to investigate whether the combination of FT-5 and pioglitazone would ...OBJECTIVE: To assess the effect of a mixture of five herbal extracts(FT-5) on insulin resistance, glucose/lipid metabolism, hepatic steatosis, and to investigate whether the combination of FT-5 and pioglitazone would provide a robust effect on diabetes treatment, while may minimize undesirable side-effects of pioglitazone in diabetic Ay gene(KKAy)mice.METHODS: Seven-week-old KKAy mice were randomly divided into five groups: control(CON)group, FT-5(2.0 g/kg) group, pioglitazone(20 mg/kg)(PIO) group, pioglitazone(20 mg/kg) + FT-5(2.0 g/kg)(P + F) group. Age-matched C57 BL/6 J micewere used as the control group. After seven weeks of continuous intragastric administration of medication, the glucose metabolism, insulin sensitivity and lipid metabolism of KKAy mice were evaluated by assessing the fasting blood glucose(FBG), oral glucose tolerance test(OGTT), fasting serum insulin(FINS), insulin tolerance test(ITT), homeostasis model of assessment-insulin resistance index(HOMA-IR), total cholesterol(TC), total triglycerides(TG), and free fatty acids(FFA) in plasma and liver.Plasma and hepatic adiponectin were measured via enzyme-linked immunosorbent assays. Genes related to adipogenesis and lipolysis in white adipose tissues(WAT) and liver were examined by real-time polymerase chain reaction. Lipid metabolism-related protein expression in the liver of KKAy mice were detected by Western blotting.RESULTS: PIO treatment remarkably improved insulin resistance. However, it also showed substantial side effects. FT-5 group exhibited no significant decrease in serum glucose. However, it reduced fasting plasma TG levels and improved hepatic steatosis of KKAy mice. P + F group showed improved insulin resistance and similar body weight gain, as compared with control group. The m RNA expression of genes related to fatty acid oxidation was markedly up-regulated in the liver of P + F group.Pioglitazone administration markedly decreased the phosphorylation levels of AMPK, as compared with all other groups. Besides, even though plasma adiponectin increased in PIO, FT-5, P + F group, adipo R2 gene expression significantly decreased in the liver of PIO group.CONCLUSION: FT-5 decreased plasma TG and alleviated aggravating hepatic steatosis induced by pioglitazone in KKAy mice. FT-5's mechanism might be associated with its ability to activate the Adipo R2/AMPK pathway.展开更多
信号转导及转录活化蛋白5(signal transducer and activator of transcription 5,STAT5)在乳腺发育、免疫应答、细胞代谢、造血及肿瘤的发生发展中发挥重要作用,但对胰岛细胞功能影响研究甚少。本研究旨在探讨STAT5对小鼠胰岛肿瘤MIN6...信号转导及转录活化蛋白5(signal transducer and activator of transcription 5,STAT5)在乳腺发育、免疫应答、细胞代谢、造血及肿瘤的发生发展中发挥重要作用,但对胰岛细胞功能影响研究甚少。本研究旨在探讨STAT5对小鼠胰岛肿瘤MIN6细胞胰岛素分泌的影响及作用机制。电穿孔法转染小干扰RNA(siRNA)敲减STAT5基因表达后,实时定量PCR和蛋白质印迹法显示,与对照干扰RNA转染的细胞比较,Stat5 siRNA转染细胞的mRNA及蛋白质表达水平分别约70%和67%(P<0.01),提示成功构建了Stat5基因沉默模型。酶联免疫吸附法结合蛋白质印迹法显示,与对照组细胞比较,Stat5 siRNA转染细胞在不同浓度葡萄糖刺激下,胰岛素分泌能力提高大约1倍(P<0.05);同时,钙调蛋白依赖的蛋白激酶Ⅱ(calmodulin-dependent protein kinaseⅡ,Ca MKⅡ)的磷酸化水平亦随之加强。加入Ca MKⅡ抑制剂AIPⅡ(auto camtide-2 related inhibitory peptideⅡ)可明显抑制Stat5 siRNA转染导致的细胞胰岛素分泌水平增加(P<0.01)。上述结果表明,沉默Stat5基因后可通过增强Ca MKⅡ的磷酸化促进MIN6细胞胰岛素的分泌。展开更多
运动导致骨骼肌细胞内的能量平衡状态被破坏,因此,机体恢复与维持能量状态的平衡对运动能力有重要影响,5′-一磷酸腺苷激活的蛋白激酶(5′-AMP activated protein kinase,AMPK)作为细胞内的能量监控器具有这种作用。AMPK在一次性运动中...运动导致骨骼肌细胞内的能量平衡状态被破坏,因此,机体恢复与维持能量状态的平衡对运动能力有重要影响,5′-一磷酸腺苷激活的蛋白激酶(5′-AMP activated protein kinase,AMPK)作为细胞内的能量监控器具有这种作用。AMPK在一次性运动中能以强度和时间依赖性方式激活,其机制主要与运动中AMP/ATP比值改变有关,AMP通过3条途径激活AMPK:直接别构激活;使AMPK成为上游激酶的更适底物;阻遏蛋白磷酸酶对AMPK的抑制,ATP与AMP的作用相拮抗。其他能量状态的变化如磷酸肌酸、肌酸、葡萄糖和肌糖原也对AMPK的激活产生影响。阐明运动激活AMPK的分子机制对理解骨骼肌在运动中的能量代谢具有一定意义。展开更多
目的研究二十二碳六烯酸(DHA)联合5氟尿嘧啶(5-FU)对人肝癌细胞HepG2耐药相关蛋白ERK、JNK及p38的表达。方法 Western blot检测5-FU5μg/mL,或5-FU5μg/mL联合DHA30μg/mL作用HepG2细胞48h后ERK、JNK及p38蛋白表达。结果与单用5-FU组比...目的研究二十二碳六烯酸(DHA)联合5氟尿嘧啶(5-FU)对人肝癌细胞HepG2耐药相关蛋白ERK、JNK及p38的表达。方法 Western blot检测5-FU5μg/mL,或5-FU5μg/mL联合DHA30μg/mL作用HepG2细胞48h后ERK、JNK及p38蛋白表达。结果与单用5-FU组比较,5-FU联合DHA组对耐药相关蛋白的影响,p-ERK明显受到抑制,而p-JNK表达显著增加,p-p38表达无差别。结论 DHA对肝癌细胞耐药相关蛋白的调控可能通过抑制ERK、激活JNK信号通路来增强5-FU的细胞毒活动,发挥增敏化疗药物的作用。展开更多
文摘Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided into 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003.58±1 626.57) and the expression of IL-5 mRNA (0.8300±0.0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888.47±1103.56 and 0.3050±0.0208, respectively, P< 0.01), while the indexes (23 219.83±1 024.86 and 0.3425±0.0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P< 0.01). The indexes (87 107.41±1 342.92 and 0.8225±0.0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P>0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P< 0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.
基金supported by the National Natural Science Foundation of China,No.81202620the Domestic Visiting Scholar Program for Young Talent Teachers in University of Shandong Province
文摘The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression.
基金supported by the research grants from the Japan Society for the Promotion of Science (No.20500576)Japan Vascular Disease Research Foundation
文摘AIM:Coptidis Rhizoma(CR),the dried rhizomes of Asian herbs(including Coptis chinensis Franch),has been used to treat diabetes mellitus for thousands of years.We explored the possibility that CR acts directly on skeletal muscle,the major organ responsible for glucose homeostasis,and activates 5'-AMP-activated protein kinase(AMPK),a signaling intermediary leading to metabolic enhancement of skeletal muscle.METHODS:Isolated rat epitrochlearis and soleus muscles were incubated in a buffer containing a CR water extract(CE),and activation of AMPK and related events were examined.RESULTS:In response to CE treat-ment,phosphorylation of Thr172 at the catalyticαsubunit of AMPK,an essential step for full kinase activation,increased in both mus-cles.Phosphorylation of Ser79 of acetyl CoA carboxylase(ACC),an endogenous substrate of AMPK,increased concomitantly.Analysis of isoform-specific AMPK activity revealed that CE activated both the α1 and α2 isoforms of the catalytic subunit.Importantly,the maximal effect of CE on AMPK phosphorylation was significantly greater than that of berberine(BBR),indicating that the action of CE is not totally ascribed to BBR.CONCLUSION:We propose that CE is an acute activator of AMPK in both fast-and slow-twitch skeletal muscles.
基金Supported by the National Natural Science Foundation of China(No.81173179)the Natural Science Foundation of Fujian Province(No.2011J01198)+5 种基金the Fujian Medical Innovation Project(No.2009-CX-19)the Research Foundation of Fujian Health Department(No.Zlcnfm02)the Fujian Provincial Department of Education Category A Projects(No.JA09131)the Fujian Health Department of Traditional Chinese Medicine Research(No.WZY0920)the CHEN Ke-ji Integrative Medicine Development Fund(No.CKJ2008047,CKJ2009004)the Integrative Medicine of Fujian Key Laboratory of Age-related Diseases Funded Projects(No.2008J1004-10)
文摘Objective：To investigate the effect of Dan-gua Fang（丹瓜方） on adenosine 5’-monophosphate（AMP） activated protein kinase（AMPK） α expression in liver and subsequent improvement of glucose and lipid metabolism.Methods：Forty 13-week-old diabetic Goto-Kakizaki（GK） rats were randomly divided into model,Dan-gua Fang,metformin and simvastatin groups（n=10 for each）,and fed high-fat diet ad libitum.Ten Wistar rats were used as normal group and fed normal diet.After 24 weeks,liver expression of AMPK α mRNA was assessed by real-time PCR.AMPK α and phospho-AMPK α protein expression in liver was evaluated by Western blot.Liver histomorphology was carried out after hematoxylin-eosin staining,and blood glucose（BG）,glycosylated hemoglobin A1c（HbA1c）,food intake and body weight recorded.Results：Similar AMPK α mRNA levels were found in the Dan-gua Fang group and normal group,slightly higher than the values obtained for the remaining groups（P〈0.05）.AMPK α protein expression in the Dan-gua Fang group animals was similar to other diabetic rats,whereas phospho-AMPK α（Thr-172） protein levels were markedly higher than in the metformin group and simvastatin group（P〈0.05）,respectively.However,phosphor-AMPKa/AMPK α ratios were similar in all groups.Dan-gua Fang reduced fasting blood glucose with similar strength to metformin,and was superior in reducing cholesterol,triglycerides,high-density lipoprotein cholesterol as well as improving low-density lipoprotein cholesterol in comparison with simvastatin and metformin.Dan-gua Fang decreases plasma alanine aminotransferase（ALT） significantly.Conclusion：Dan-gua Fang,while treating phlegm-stasis,could decrease BG and lipid in type 2 diabetic GK rats fed with high-fat diet,and effectively protect liver histomorphology and function.This may be partly explained by increased AMPK expression in liver.Therefore,Dan-gua Fang might be an ideal drug for comprehensive Intervention for glucose and lipid metabolism disorders in type 2 diabetes mellitus.
基金Supported by A grant from the Arkansas Master Tobacco Settlement and Arkansas Biosciences Institute
文摘AIM: To investigate the functional significance of insulin-like growth factor binding protein-5 (IGFBP-5) overexpression in pancreatic cancer (PaC).METHODS: The effects of IGFBP-5 on cell growth were assessed by stable transfection of BxPC-3 and PANC-1 cell lines and measuring cell number and DNA synthesis. Alterations in the cell cycle were assessed by flow cytometry and immunoblot analyses. Changes in cell survival and signal transduction were evaluated after mitogen activated protein kinase and phosphatidylinositol 3-kinase (PI3K) inhibitor treatment.RESULTS: After serum deprivation, IGFBP-5 expression increased both cell number and DNA synthesis in BxPC-3 cells, but reduced cell number in PANC-1 cells. Consistent with this observation, cell cycle analysis of IGFBP-5-expressing cells revealed accelerated cell cycle progression in BxPC-3 and G2/M arrest of PANC-1 cells. Signal transduction analysis revealed that Akt activation was increased in BxPC-3, but reduced in PANC-1 cells that express IGFBP-5. Inhibition of PI3K with LY294002 suppressed extracellular signal-regulated kinase-1 and -2 (ERK1/2) activation in BxPC-3, but enhanced ERK1/2 activation in PANC-1 cells that express IGFBP-5. When MEK1/2 was blocked, Akt activation remained elevated in IGFBP-5 expressing PaC cells; however, inhibition of PI3K or MEK1/2 abrogated IGFBP-5-mediated cell survival.CONCLUSION: These results indicate that IGFBP-5 expression affects the cell cycle and survival signal pathways and thus it may be an important mediator of PaC cell growth.
基金Supported by Shenyang Innovation Foundation of Science and Technology–the Application Projects of Basic Research(No.F15-199-1-06)Liaoning Province Science and Technique Foundation of China(No.2015020282)
文摘Objective: To investigate whether ginsenoside-Rb1(Gs-Rb1) improves the CoCl2-induced autophagy of cardiomyocytes via upregulation of adenosine 5’-monophosphate-activated protein kinase(AMPK) pathway. Methods: Ventricles from 1-to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group(normal level oxygen), hypoxia group(500 μmol/L CoCl2), Gs-Rb1 group(200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), Ara A group(500 μmol/L Ara A + 500 μmol/L CoCl2), Ara A+ Gs-Rb1 group(500 μmol/L Ara A + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide(AICAR)+ 500 μmol/L CoCl2], and AICAR+Gs-Rb1 group(1 mmol/L AICAR + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2). Cel s were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay and cardiac troponin I(cTnI) levels were detected by enzyme-linked immunosorbent assay(ELISA). AMPK activity was assessed by 2’,7’-dichlorofluorescein diacetate(DCFH-DA) ELISA assay. The protein expressions of Atg4 B, Atg5, Atg6, Atg7, microtubule-associated protein 1 A/1 B-light chain 3(LC3), P62, and active-cathepsin B were measured by Western blot. Results: Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes(P<0.01). However, the viability of hypoxia-treated cardiomyocytes was significantly inhibited by Ara A(P<0.01). Gs-Rb1 increased the AMPK activity of hypoxia-treated cardiomyocytes. The AMPK activity of hypoxia-treated cadiomyocytes was inhibited by Ara A(P<0.01) and was not affected by AICAR(P=0.983). Gs-Rb1 up-regulated Atg4B, Atg5, Beclin-1, Atg7, LC3B Ⅱ, the LC3BⅡ/Ⅰ ratio and cathepsin B activity of hypoxia cardiomyocytes(P<0.05), each of these protein levels was significantly enhanced by Ara A(all P<0.01), but was not affected by AICAR(all P>0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes(P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A(P<0.05) and were not affected by AICAR(P=0.871). Conclusion: Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.
基金Supported by the Science and Technology Support Program of China(No.2014BAI10B04)
文摘OBJECTIVE: To assess the effect of a mixture of five herbal extracts(FT-5) on insulin resistance, glucose/lipid metabolism, hepatic steatosis, and to investigate whether the combination of FT-5 and pioglitazone would provide a robust effect on diabetes treatment, while may minimize undesirable side-effects of pioglitazone in diabetic Ay gene(KKAy)mice.METHODS: Seven-week-old KKAy mice were randomly divided into five groups: control(CON)group, FT-5(2.0 g/kg) group, pioglitazone(20 mg/kg)(PIO) group, pioglitazone(20 mg/kg) + FT-5(2.0 g/kg)(P + F) group. Age-matched C57 BL/6 J micewere used as the control group. After seven weeks of continuous intragastric administration of medication, the glucose metabolism, insulin sensitivity and lipid metabolism of KKAy mice were evaluated by assessing the fasting blood glucose(FBG), oral glucose tolerance test(OGTT), fasting serum insulin(FINS), insulin tolerance test(ITT), homeostasis model of assessment-insulin resistance index(HOMA-IR), total cholesterol(TC), total triglycerides(TG), and free fatty acids(FFA) in plasma and liver.Plasma and hepatic adiponectin were measured via enzyme-linked immunosorbent assays. Genes related to adipogenesis and lipolysis in white adipose tissues(WAT) and liver were examined by real-time polymerase chain reaction. Lipid metabolism-related protein expression in the liver of KKAy mice were detected by Western blotting.RESULTS: PIO treatment remarkably improved insulin resistance. However, it also showed substantial side effects. FT-5 group exhibited no significant decrease in serum glucose. However, it reduced fasting plasma TG levels and improved hepatic steatosis of KKAy mice. P + F group showed improved insulin resistance and similar body weight gain, as compared with control group. The m RNA expression of genes related to fatty acid oxidation was markedly up-regulated in the liver of P + F group.Pioglitazone administration markedly decreased the phosphorylation levels of AMPK, as compared with all other groups. Besides, even though plasma adiponectin increased in PIO, FT-5, P + F group, adipo R2 gene expression significantly decreased in the liver of PIO group.CONCLUSION: FT-5 decreased plasma TG and alleviated aggravating hepatic steatosis induced by pioglitazone in KKAy mice. FT-5's mechanism might be associated with its ability to activate the Adipo R2/AMPK pathway.
文摘信号转导及转录活化蛋白5(signal transducer and activator of transcription 5,STAT5)在乳腺发育、免疫应答、细胞代谢、造血及肿瘤的发生发展中发挥重要作用,但对胰岛细胞功能影响研究甚少。本研究旨在探讨STAT5对小鼠胰岛肿瘤MIN6细胞胰岛素分泌的影响及作用机制。电穿孔法转染小干扰RNA(siRNA)敲减STAT5基因表达后,实时定量PCR和蛋白质印迹法显示,与对照干扰RNA转染的细胞比较,Stat5 siRNA转染细胞的mRNA及蛋白质表达水平分别约70%和67%(P<0.01),提示成功构建了Stat5基因沉默模型。酶联免疫吸附法结合蛋白质印迹法显示,与对照组细胞比较,Stat5 siRNA转染细胞在不同浓度葡萄糖刺激下,胰岛素分泌能力提高大约1倍(P<0.05);同时,钙调蛋白依赖的蛋白激酶Ⅱ(calmodulin-dependent protein kinaseⅡ,Ca MKⅡ)的磷酸化水平亦随之加强。加入Ca MKⅡ抑制剂AIPⅡ(auto camtide-2 related inhibitory peptideⅡ)可明显抑制Stat5 siRNA转染导致的细胞胰岛素分泌水平增加(P<0.01)。上述结果表明,沉默Stat5基因后可通过增强Ca MKⅡ的磷酸化促进MIN6细胞胰岛素的分泌。
文摘运动导致骨骼肌细胞内的能量平衡状态被破坏,因此,机体恢复与维持能量状态的平衡对运动能力有重要影响,5′-一磷酸腺苷激活的蛋白激酶(5′-AMP activated protein kinase,AMPK)作为细胞内的能量监控器具有这种作用。AMPK在一次性运动中能以强度和时间依赖性方式激活,其机制主要与运动中AMP/ATP比值改变有关,AMP通过3条途径激活AMPK:直接别构激活;使AMPK成为上游激酶的更适底物;阻遏蛋白磷酸酶对AMPK的抑制,ATP与AMP的作用相拮抗。其他能量状态的变化如磷酸肌酸、肌酸、葡萄糖和肌糖原也对AMPK的激活产生影响。阐明运动激活AMPK的分子机制对理解骨骼肌在运动中的能量代谢具有一定意义。
文摘目的研究二十二碳六烯酸(DHA)联合5氟尿嘧啶(5-FU)对人肝癌细胞HepG2耐药相关蛋白ERK、JNK及p38的表达。方法 Western blot检测5-FU5μg/mL,或5-FU5μg/mL联合DHA30μg/mL作用HepG2细胞48h后ERK、JNK及p38蛋白表达。结果与单用5-FU组比较,5-FU联合DHA组对耐药相关蛋白的影响,p-ERK明显受到抑制,而p-JNK表达显著增加,p-p38表达无差别。结论 DHA对肝癌细胞耐药相关蛋白的调控可能通过抑制ERK、激活JNK信号通路来增强5-FU的细胞毒活动,发挥增敏化疗药物的作用。
