目的基于实时细胞分析(real time cell analysis,RTCA)技术对中药挥发性组分的细胞毒性及抗人腺病毒3型(human adenovirus 3,HAdV-3)的作用进行毒/效整合分析,构建抗病毒药物高通量筛选的新策略。方法采用RTCA技术动态监测不同接种密度...目的基于实时细胞分析(real time cell analysis,RTCA)技术对中药挥发性组分的细胞毒性及抗人腺病毒3型(human adenovirus 3,HAdV-3)的作用进行毒/效整合分析,构建抗病毒药物高通量筛选的新策略。方法采用RTCA技术动态监测不同接种密度的A549细胞48 h生长曲线及10倍梯度稀释的HAdV-3感染A549细胞生长曲线,获得A549细胞最佳接种密度及HAdV-3最佳稀释浓度用于后续实验。以利巴韦林为阳性对照,基于RTCA技术采用曲线下面积(Area Under the Curve,AUC)方法对5种中药挥发性组分的细胞毒性及抗HAdV-3的作用进行毒/效整合分析,并与传统终点法数据处理进行对比。结果终点法中艾叶、柴胡、薄荷、荆芥、牛蒡子、酸枣仁和利巴韦林对细胞的保护率分别为0.73%、12.50%、20.99%、44.50%、27.99%、51.50%和82.70%;AUC法中艾叶、柴胡、薄荷的选择性指数(Selective Index,SI)为负数,分别为-0.57、-0.21和-0.08。荆芥、牛蒡子、酸枣仁和利巴韦林的SI值分别为0.14、0.40、0.72和5.33。以利巴韦林的抗病毒活性为参照,终点法中酸枣仁、牛蒡子和荆芥的抗病毒活性为利巴韦林抗病毒活性的62.27%、33.85%和53.81%;AUC法中酸枣仁、牛蒡子和荆芥的抗病毒活性为利巴韦林抗病毒活性的13.51%、7.50%和2.63%。结论传统终点法与研究采用的AUC法计算结果有较大的差异。传统的抗病毒活性筛选研究多采用终点法检测受试药物对病毒感染细胞的保护作用,无法反映整个感染周期的变化,缺乏细胞毒的数据也会对结果的判断产生偏差。研究提示基于RTCA技术对中药挥发性组分开展抗HAdV-3的毒/效整合评价,采用AUC方法计算药物的选择性指数作为高通量筛选新策略,能快速、精准地判断受试药物的抗病毒活性,具有准确性高、重复性好的优势。展开更多
Progressive beta cell apoptosis is a major cause leading to a decline in beta cell mass in type 2 diabetes. While it has been discovered that mitochondrial protein Sirtuin 5 （ SIRT5 ） functions as the primary site o...Progressive beta cell apoptosis is a major cause leading to a decline in beta cell mass in type 2 diabetes. While it has been discovered that mitochondrial protein Sirtuin 5 （ SIRT5 ） functions as the primary site of oxidative metabolism and plays crucial roles in apoptosis and intracellular signaling, the contribution of SIRT5 in beta cell re- mains to be fully defined. Given the potential benefit of SIRT5 activation in cell metabolism, this study aimed to examine whether overexpression of SIRT5 in beta cells is sufficient to prevent saturated fatty acid induced impair- ment in insulin secretion and apoptosis, thus elucidating the role of SIRT5 in beta cell protection. In line with our previous study, two kinds of pancreatic beta cell lines were selected for the detection. Mouse-derived NIT-1 cells as well as human-derived PANC-1 cells were transfected with GFP-SIRT5-Ad plasmid and cell apoptosis was induced by palmitic acid （0.5 mM） , validated with TUNEL-DAPI double staining and RTCA iCelligence cell growth moni- tor system. Compared with control group, it was shown that SIRT5 overexpression could significantly reduce the quantity of apoptotic beta cells under chronic exposure to palmitic acid, accompanied with decreased Caspase 3 and Caspase 9 activities. Accordingly, cytochrome C oxidase activity in cells was also suppressed. Meanwhile, palmitic acid suppressed glucose-stimulated insulin secretion, but SIRT5 overexpression could recover the beta cell insulin secretion capacity against glucose fluctuation. Moreover, it is discovered that novel binding relationship exists be- tween SIRT5 and Bcl-XL, providing a reliable explanation for the anti-apoptosis role of SIRT5. Together, these re- sults reveal a potential role of SIRT5 in improvement of saturated fatty acid -induced beta cell dysfunction and apop- tosis. Considering the role of beta cell apoptosis in T2DM, overexpression or activation of SIRT5 may provide an e-rumpent approach as a potential target for beta cell protection. This approach might actually reverse the disease to a degree rather than just palliate glycemia.展开更多
文摘目的基于实时细胞分析(real time cell analysis,RTCA)技术对中药挥发性组分的细胞毒性及抗人腺病毒3型(human adenovirus 3,HAdV-3)的作用进行毒/效整合分析,构建抗病毒药物高通量筛选的新策略。方法采用RTCA技术动态监测不同接种密度的A549细胞48 h生长曲线及10倍梯度稀释的HAdV-3感染A549细胞生长曲线,获得A549细胞最佳接种密度及HAdV-3最佳稀释浓度用于后续实验。以利巴韦林为阳性对照,基于RTCA技术采用曲线下面积(Area Under the Curve,AUC)方法对5种中药挥发性组分的细胞毒性及抗HAdV-3的作用进行毒/效整合分析,并与传统终点法数据处理进行对比。结果终点法中艾叶、柴胡、薄荷、荆芥、牛蒡子、酸枣仁和利巴韦林对细胞的保护率分别为0.73%、12.50%、20.99%、44.50%、27.99%、51.50%和82.70%;AUC法中艾叶、柴胡、薄荷的选择性指数(Selective Index,SI)为负数,分别为-0.57、-0.21和-0.08。荆芥、牛蒡子、酸枣仁和利巴韦林的SI值分别为0.14、0.40、0.72和5.33。以利巴韦林的抗病毒活性为参照,终点法中酸枣仁、牛蒡子和荆芥的抗病毒活性为利巴韦林抗病毒活性的62.27%、33.85%和53.81%;AUC法中酸枣仁、牛蒡子和荆芥的抗病毒活性为利巴韦林抗病毒活性的13.51%、7.50%和2.63%。结论传统终点法与研究采用的AUC法计算结果有较大的差异。传统的抗病毒活性筛选研究多采用终点法检测受试药物对病毒感染细胞的保护作用,无法反映整个感染周期的变化,缺乏细胞毒的数据也会对结果的判断产生偏差。研究提示基于RTCA技术对中药挥发性组分开展抗HAdV-3的毒/效整合评价,采用AUC方法计算药物的选择性指数作为高通量筛选新策略,能快速、精准地判断受试药物的抗病毒活性,具有准确性高、重复性好的优势。
文摘Progressive beta cell apoptosis is a major cause leading to a decline in beta cell mass in type 2 diabetes. While it has been discovered that mitochondrial protein Sirtuin 5 （ SIRT5 ） functions as the primary site of oxidative metabolism and plays crucial roles in apoptosis and intracellular signaling, the contribution of SIRT5 in beta cell re- mains to be fully defined. Given the potential benefit of SIRT5 activation in cell metabolism, this study aimed to examine whether overexpression of SIRT5 in beta cells is sufficient to prevent saturated fatty acid induced impair- ment in insulin secretion and apoptosis, thus elucidating the role of SIRT5 in beta cell protection. In line with our previous study, two kinds of pancreatic beta cell lines were selected for the detection. Mouse-derived NIT-1 cells as well as human-derived PANC-1 cells were transfected with GFP-SIRT5-Ad plasmid and cell apoptosis was induced by palmitic acid （0.5 mM） , validated with TUNEL-DAPI double staining and RTCA iCelligence cell growth moni- tor system. Compared with control group, it was shown that SIRT5 overexpression could significantly reduce the quantity of apoptotic beta cells under chronic exposure to palmitic acid, accompanied with decreased Caspase 3 and Caspase 9 activities. Accordingly, cytochrome C oxidase activity in cells was also suppressed. Meanwhile, palmitic acid suppressed glucose-stimulated insulin secretion, but SIRT5 overexpression could recover the beta cell insulin secretion capacity against glucose fluctuation. Moreover, it is discovered that novel binding relationship exists be- tween SIRT5 and Bcl-XL, providing a reliable explanation for the anti-apoptosis role of SIRT5. Together, these re- sults reveal a potential role of SIRT5 in improvement of saturated fatty acid -induced beta cell dysfunction and apop- tosis. Considering the role of beta cell apoptosis in T2DM, overexpression or activation of SIRT5 may provide an e-rumpent approach as a potential target for beta cell protection. This approach might actually reverse the disease to a degree rather than just palliate glycemia.
