BACKGROUND Intestinal ischemia reperfusion(I/R)occurs in various diseases,such as trauma and intestinal transplantation.Excessive reactive oxygen species(ROS)accumulation and subsequent apoptotic cell death in intesti...BACKGROUND Intestinal ischemia reperfusion(I/R)occurs in various diseases,such as trauma and intestinal transplantation.Excessive reactive oxygen species(ROS)accumulation and subsequent apoptotic cell death in intestinal epithelia are important causes of I/R injury.PTEN-induced putative kinase 1(PINK1)and phosphorylation of dynamin-related protein 1(DRP1)are critical regulators of ROS and apoptosis.However,the correlation of PINK1 and DRP1 and their function in intestinal I/R injury have not been investigated.Thus,examining the PINK1/DRP1 pathway may help to identify a protective strategy and improve the patient prognosis.AIM To clarify the mechanism of the PINK1/DRP1 pathway in intestinal I/R injury.METHODS Male C57BL/6 mice were used to generate an intestinal I/R model via superior mesenteric artery occlusion followed by reperfusion.Chiu’s score was used to evaluate intestinal mucosa damage.The mitochondrial fission inhibitor mdivi-1 was administered by intraperitoneal injection.Caco-2 cells were incubated in vitro in hypoxia/reoxygenation conditions.Small interfering RNAs and overexpression plasmids were transfected to regulate PINK1 expression.The protein expression levels of PINK1,DRP1,p-DRP1 and cleaved caspase 3 were measured by Western blotting.Cell viability was evaluated using a Cell Counting Kit-8 assay and cell apoptosis was analyzed by TUNEL staining.Mitochondrial fission and ROS were tested by MitoTracker and MitoSOX respectively.RESULTS Intestinal I/R and Caco-2 cell hypoxia/reoxygenation decreased the expression of PINK1 and p-DRP1 Ser637.Pretreatment with mdivi-1 inhibited mitochondrial fission,ROS generation,and apoptosis and ameliorated cell injury in intestinal I/R.Upon PINK1 knockdown or overexpression in vitro,we found that p-DRP1 Ser637 expression and DRP1 recruitment to the mitochondria were associated with PINK1.Furthermore,we verified the physical combination of PINK1 and p-DRP1 Ser637.CONCLUSION PINK1 is correlated with mitochondrial fission and apoptosis by regulating DRP1 phosphorylation in intestinal I/R.These results suggest that the PINK1/DRP1 pathway is involved in intestinal I/R injury,and provide a new approach for prevention and treatment.展开更多
Tau, a primary component of microtubule-associated protein, promotes microtubule assembly and/or disassembly and maintains the stability of the microtubule structure. Although the importance of tau in neurodegenerativ...Tau, a primary component of microtubule-associated protein, promotes microtubule assembly and/or disassembly and maintains the stability of the microtubule structure. Although the importance of tau in neurodegenerative diseases has been well demonstrated, wheth- er tau is involved in peripheral nerve regeneration remains unknown. In the current study, we obtained sciatic nerve tissue from adult rats 0, 1, 4, 7, and 14 days after sciatic nerve crush and examined tau mRNA and protein expression levels and the location of tau in the sciatic nerve following peripheral nerve injury. The results from our quantitative reverse transcription polymerase chain reaction analysis showed that compared with the uninjured control sciatic nerve, mRNA expression levels for both tau and tau tubulin kinase 1, a serine/ threonine kinase that regulates tau phosphorylation, were decreased following peripheral nerve injury. Our western blot assay results suggested that the protein expression levels of tau and phosphorylated tau initially decreased 1 day post nerve injury but then gradually increased. The results of our immunohistochemical labeling showed that the location of tau protein was not altered by nerve injury. Thus, these results showed that the expression of tau was changed following sciatic nerve crush, suggesting that tau may be involved in periph- eral nerve repair and regeneration.展开更多
[目的]探究shRNA靶向敲低Cx43基因表达对人多发性骨髓瘤细胞株MM.1S生存的影响及其机制。[方法]针对Cx43 mRNA不同位点构建sh/Cx43-1、sh/Cx43-2、sh/Con重组质粒,感染体外培养的MM.1S细胞,RT-PCR或Western Blotting检测Cx43 mRNA或蛋...[目的]探究shRNA靶向敲低Cx43基因表达对人多发性骨髓瘤细胞株MM.1S生存的影响及其机制。[方法]针对Cx43 mRNA不同位点构建sh/Cx43-1、sh/Cx43-2、sh/Con重组质粒,感染体外培养的MM.1S细胞,RT-PCR或Western Blotting检测Cx43 mRNA或蛋白表达,CCK-8、流式细胞术检测细胞增殖、凋亡情况,Western Blotting检测磷脂酸肌醇-3-激酶(PI3K)、丝苏氨酸蛋白激酶(AKT)通路蛋白表达。[结果]sh/Cx43-1组、sh/Cx43-2组Cx43 mRNA或蛋白均低于sh/Con组,且sh/Cx43-1组低于sh/Cx43-2组(0.18±0.05 vs 0.75±0.07,0.21±0.03 vs 0.81±0.06,P<0.05);培养24h时各组细胞增殖水平无明显差异(P>0.05),培养48 h、72 h时sh/Cx43-1组、sh/Cx43-2组细胞增殖水平均低于sh/Con组(0.42±0.05 vs 0.58±0.09,1.02±0.10 vs 1.08±0.13,P<0.05);sh/Cx43-1组、sh/Cx43-2组细胞总凋亡率高于sh/Con组,PI3K、AKT蛋白表达均低于sh/Con组,且sh/Cx43-1组变化较sh/Cx43-2组更明显(P<0.05)。[结论]通过shRNA靶向敲低Cx43基因表达能显著抑制MM.1S细胞增殖,促进其凋亡,这可能与敲低Cx43后PI3K/AKT通路受到抑制有关。展开更多
前期研究发现烟草中一个壳寡糖诱导的Ser/Thr蛋白激酶(Oligochitosan induced protein kinase,OIPK)在壳寡糖诱导的烟草抗性反应中起着重要作用.目前在烟草中发现OIPK基因的两个选择性剪接体:OIPKL和OIPKS,其中OIPKL是3个选择性剪接体...前期研究发现烟草中一个壳寡糖诱导的Ser/Thr蛋白激酶(Oligochitosan induced protein kinase,OIPK)在壳寡糖诱导的烟草抗性反应中起着重要作用.目前在烟草中发现OIPK基因的两个选择性剪接体:OIPKL和OIPKS,其中OIPKL是3个选择性剪接体中序列最长的.为进一步研究OIPK在壳寡糖诱导植物抗性中起的作用,本研究采用酵母双杂交方法,以pGBKT7-OIPKL为诱饵质粒,筛选构建在pACT2载体上的烟草cDNA文库,寻找OIPKL的相互作用蛋白.结果表明OIPKL蛋白在酵母中可以和烟草的S-腺苷甲硫氨酸合成酶、泛素激活酶E1、丙酮酸激酶、过氧化物酶相互作用.实验结果为研究壳寡糖诱导抗性机理提供了一定依据.展开更多
基金the National Natural Science Foundation of China,No.81679154,No.81871547.
文摘BACKGROUND Intestinal ischemia reperfusion(I/R)occurs in various diseases,such as trauma and intestinal transplantation.Excessive reactive oxygen species(ROS)accumulation and subsequent apoptotic cell death in intestinal epithelia are important causes of I/R injury.PTEN-induced putative kinase 1(PINK1)and phosphorylation of dynamin-related protein 1(DRP1)are critical regulators of ROS and apoptosis.However,the correlation of PINK1 and DRP1 and their function in intestinal I/R injury have not been investigated.Thus,examining the PINK1/DRP1 pathway may help to identify a protective strategy and improve the patient prognosis.AIM To clarify the mechanism of the PINK1/DRP1 pathway in intestinal I/R injury.METHODS Male C57BL/6 mice were used to generate an intestinal I/R model via superior mesenteric artery occlusion followed by reperfusion.Chiu’s score was used to evaluate intestinal mucosa damage.The mitochondrial fission inhibitor mdivi-1 was administered by intraperitoneal injection.Caco-2 cells were incubated in vitro in hypoxia/reoxygenation conditions.Small interfering RNAs and overexpression plasmids were transfected to regulate PINK1 expression.The protein expression levels of PINK1,DRP1,p-DRP1 and cleaved caspase 3 were measured by Western blotting.Cell viability was evaluated using a Cell Counting Kit-8 assay and cell apoptosis was analyzed by TUNEL staining.Mitochondrial fission and ROS were tested by MitoTracker and MitoSOX respectively.RESULTS Intestinal I/R and Caco-2 cell hypoxia/reoxygenation decreased the expression of PINK1 and p-DRP1 Ser637.Pretreatment with mdivi-1 inhibited mitochondrial fission,ROS generation,and apoptosis and ameliorated cell injury in intestinal I/R.Upon PINK1 knockdown or overexpression in vitro,we found that p-DRP1 Ser637 expression and DRP1 recruitment to the mitochondria were associated with PINK1.Furthermore,we verified the physical combination of PINK1 and p-DRP1 Ser637.CONCLUSION PINK1 is correlated with mitochondrial fission and apoptosis by regulating DRP1 phosphorylation in intestinal I/R.These results suggest that the PINK1/DRP1 pathway is involved in intestinal I/R injury,and provide a new approach for prevention and treatment.
基金supported by the National Natural Science Foundation of China,No.81130080,31300942the National Key Basic Research Program of China(973 Program)+5 种基金No.2014CB542202the Natural Science Foundation of Jiangsu Province,China,No.BK20150409the Natural Science Foundation of Jiangsu Higher Education Institutions of China,No.15KJB180013the Scientific Research Foundation of Nantong University of China,No.14R29the Natural Science Foundation of Nantong City in China,No.MS12015043the Priority Academic Program Development of Jiangsu Higher Education Institutions of China
文摘Tau, a primary component of microtubule-associated protein, promotes microtubule assembly and/or disassembly and maintains the stability of the microtubule structure. Although the importance of tau in neurodegenerative diseases has been well demonstrated, wheth- er tau is involved in peripheral nerve regeneration remains unknown. In the current study, we obtained sciatic nerve tissue from adult rats 0, 1, 4, 7, and 14 days after sciatic nerve crush and examined tau mRNA and protein expression levels and the location of tau in the sciatic nerve following peripheral nerve injury. The results from our quantitative reverse transcription polymerase chain reaction analysis showed that compared with the uninjured control sciatic nerve, mRNA expression levels for both tau and tau tubulin kinase 1, a serine/ threonine kinase that regulates tau phosphorylation, were decreased following peripheral nerve injury. Our western blot assay results suggested that the protein expression levels of tau and phosphorylated tau initially decreased 1 day post nerve injury but then gradually increased. The results of our immunohistochemical labeling showed that the location of tau protein was not altered by nerve injury. Thus, these results showed that the expression of tau was changed following sciatic nerve crush, suggesting that tau may be involved in periph- eral nerve repair and regeneration.
文摘[目的]探究shRNA靶向敲低Cx43基因表达对人多发性骨髓瘤细胞株MM.1S生存的影响及其机制。[方法]针对Cx43 mRNA不同位点构建sh/Cx43-1、sh/Cx43-2、sh/Con重组质粒,感染体外培养的MM.1S细胞,RT-PCR或Western Blotting检测Cx43 mRNA或蛋白表达,CCK-8、流式细胞术检测细胞增殖、凋亡情况,Western Blotting检测磷脂酸肌醇-3-激酶(PI3K)、丝苏氨酸蛋白激酶(AKT)通路蛋白表达。[结果]sh/Cx43-1组、sh/Cx43-2组Cx43 mRNA或蛋白均低于sh/Con组,且sh/Cx43-1组低于sh/Cx43-2组(0.18±0.05 vs 0.75±0.07,0.21±0.03 vs 0.81±0.06,P<0.05);培养24h时各组细胞增殖水平无明显差异(P>0.05),培养48 h、72 h时sh/Cx43-1组、sh/Cx43-2组细胞增殖水平均低于sh/Con组(0.42±0.05 vs 0.58±0.09,1.02±0.10 vs 1.08±0.13,P<0.05);sh/Cx43-1组、sh/Cx43-2组细胞总凋亡率高于sh/Con组,PI3K、AKT蛋白表达均低于sh/Con组,且sh/Cx43-1组变化较sh/Cx43-2组更明显(P<0.05)。[结论]通过shRNA靶向敲低Cx43基因表达能显著抑制MM.1S细胞增殖,促进其凋亡,这可能与敲低Cx43后PI3K/AKT通路受到抑制有关。
文摘前期研究发现烟草中一个壳寡糖诱导的Ser/Thr蛋白激酶(Oligochitosan induced protein kinase,OIPK)在壳寡糖诱导的烟草抗性反应中起着重要作用.目前在烟草中发现OIPK基因的两个选择性剪接体:OIPKL和OIPKS,其中OIPKL是3个选择性剪接体中序列最长的.为进一步研究OIPK在壳寡糖诱导植物抗性中起的作用,本研究采用酵母双杂交方法,以pGBKT7-OIPKL为诱饵质粒,筛选构建在pACT2载体上的烟草cDNA文库,寻找OIPKL的相互作用蛋白.结果表明OIPKL蛋白在酵母中可以和烟草的S-腺苷甲硫氨酸合成酶、泛素激活酶E1、丙酮酸激酶、过氧化物酶相互作用.实验结果为研究壳寡糖诱导抗性机理提供了一定依据.
