目的探讨间歇性热量限制对辐射所致小鼠认知功能障碍的作用及可能机制。方法将36只7周龄c57BL/6J雄性小鼠按随机数表法分为假辐射+随意饮食组(sham-irradiation and ad libitum,Sham-AL)、辐射+随意饮食组(irradiation and ad libitum,I...目的探讨间歇性热量限制对辐射所致小鼠认知功能障碍的作用及可能机制。方法将36只7周龄c57BL/6J雄性小鼠按随机数表法分为假辐射+随意饮食组(sham-irradiation and ad libitum,Sham-AL)、辐射+随意饮食组(irradiation and ad libitum,IR-AL)、辐射+间歇性热量限制组(irradiation and intermittent fasting,IR-IF),每组12只。采用新旧事物识别实验检测各组小鼠认知功能;Western blot分别检测小鼠海马自噬相关基因5(ATG5)、微管相关蛋白1轻链3-II(LC3II)、自噬接头蛋白P62、线粒体阴离子通道蛋白1(VDAC1)、白介素1(IL-1β)、突触囊泡膜蛋白(SYP)、突触蛋白1(SYN-1)、突触后致密物-95(PSD95);免疫荧光法确定VDAC1在小鼠海马中定位。结果与Sham-AL组新事物识别指数(30.02±9.05)相比,IR-AL组小鼠新事物识别指数(-22.45±16.76)下降,两组比较差异有统计学意义(t=3.03,P<0.05)。与Sham-AL组相比,IR-AL组自噬标记蛋白ATG5和LC3II表达下降,抗自噬蛋白P62表达升高,VDAC1蛋白表达下降,IL-1β蛋白表达上升,SYP、SYN-1、PSD95蛋白表达降低(t=2.49、2.19、2.40、3.47、2.87、2.25、2.17、2.31,P<0.05)。与IR-AL组新事物识别指数(-22.45±16.76)相比,IR-IF组小鼠新事物识别指数(21.22±5.62)上升,两组比较差异有统计学意义(t=2.70,P<0.05)。与IR-AL组相比,IR-IF组自噬标记蛋白ATG5和LC3II表达上升,抗自噬蛋白P62表达降低,VDAC1蛋白表达升高,IL-1β蛋白表达下降,SYP、SYN-1、PSD95蛋白表达升高(t=2.88、2.71、3.18、3.18、3.11、3.30、3.35、2.53,P<0.05)。免疫荧光显示,VDAC1与离子钙接头蛋白分子1(IBA-1,小胶质细胞标记物)和神经胶质纤维酸性蛋白(GFAP,星形胶质细胞标记物)共表达,但与神经元核抗原蛋白(NEUN,神经元标记物)不共表达。结论间歇性热量限制可改善小鼠放射性认知功能障碍,其机制可能与上调海马区VDAC1蛋白表达、诱导自噬发生,最终抑制炎症因子释放和保护神经元突触可塑性�展开更多
Autophagy is a highly evolutionarily conserved pathway that depends on lysosome to degrade misfolded proteins and damaged organelles. Besides canonical autophagy, studies have shown some chemicals could bypass sever...Autophagy is a highly evolutionarily conserved pathway that depends on lysosome to degrade misfolded proteins and damaged organelles. Besides canonical autophagy, studies have shown some chemicals could bypass several core genes to induce autophagy, but the targets and regulatory mechanism is still unclear. In this work one novel chemical, G1, was screened out which could trigger both canonical autophagy and non-canonical autophagy by recruiting Atgl6L1 to pre-autophagosomal site and causing LC3 lipidation. The Gl-induced non-canonical auto- phagy was ULK1, and Beclinl-independent but ubiquitin-like conjugation system-dependent, indicating G1 might target the upstream of Atgl6L1. Moreover, inhibition of V-ATPase by specific V-ATPase inhibitiors could suppress the formation of Gl-indueed autophagosomes in FIP200-defieient MEF cells. While other classic lysosomal inhibi- tors could not block the puneta of Atgl2, Atgl6L1 and LC3, in different stages, suggesting V-ATPase activity in- stead of lysosome function is required for Gl-indueed non-canonical autophagy. These studies broaden the under- standing of different working pattern of autophagy and the crucial roles of V-ATPase in the regulation of different au- tophagy.展开更多
文摘目的探讨间歇性热量限制对辐射所致小鼠认知功能障碍的作用及可能机制。方法将36只7周龄c57BL/6J雄性小鼠按随机数表法分为假辐射+随意饮食组(sham-irradiation and ad libitum,Sham-AL)、辐射+随意饮食组(irradiation and ad libitum,IR-AL)、辐射+间歇性热量限制组(irradiation and intermittent fasting,IR-IF),每组12只。采用新旧事物识别实验检测各组小鼠认知功能;Western blot分别检测小鼠海马自噬相关基因5(ATG5)、微管相关蛋白1轻链3-II(LC3II)、自噬接头蛋白P62、线粒体阴离子通道蛋白1(VDAC1)、白介素1(IL-1β)、突触囊泡膜蛋白(SYP)、突触蛋白1(SYN-1)、突触后致密物-95(PSD95);免疫荧光法确定VDAC1在小鼠海马中定位。结果与Sham-AL组新事物识别指数(30.02±9.05)相比,IR-AL组小鼠新事物识别指数(-22.45±16.76)下降,两组比较差异有统计学意义(t=3.03,P<0.05)。与Sham-AL组相比,IR-AL组自噬标记蛋白ATG5和LC3II表达下降,抗自噬蛋白P62表达升高,VDAC1蛋白表达下降,IL-1β蛋白表达上升,SYP、SYN-1、PSD95蛋白表达降低(t=2.49、2.19、2.40、3.47、2.87、2.25、2.17、2.31,P<0.05)。与IR-AL组新事物识别指数(-22.45±16.76)相比,IR-IF组小鼠新事物识别指数(21.22±5.62)上升,两组比较差异有统计学意义(t=2.70,P<0.05)。与IR-AL组相比,IR-IF组自噬标记蛋白ATG5和LC3II表达上升,抗自噬蛋白P62表达降低,VDAC1蛋白表达升高,IL-1β蛋白表达下降,SYP、SYN-1、PSD95蛋白表达升高(t=2.88、2.71、3.18、3.18、3.11、3.30、3.35、2.53,P<0.05)。免疫荧光显示,VDAC1与离子钙接头蛋白分子1(IBA-1,小胶质细胞标记物)和神经胶质纤维酸性蛋白(GFAP,星形胶质细胞标记物)共表达,但与神经元核抗原蛋白(NEUN,神经元标记物)不共表达。结论间歇性热量限制可改善小鼠放射性认知功能障碍,其机制可能与上调海马区VDAC1蛋白表达、诱导自噬发生,最终抑制炎症因子释放和保护神经元突触可塑性�
文摘Autophagy is a highly evolutionarily conserved pathway that depends on lysosome to degrade misfolded proteins and damaged organelles. Besides canonical autophagy, studies have shown some chemicals could bypass several core genes to induce autophagy, but the targets and regulatory mechanism is still unclear. In this work one novel chemical, G1, was screened out which could trigger both canonical autophagy and non-canonical autophagy by recruiting Atgl6L1 to pre-autophagosomal site and causing LC3 lipidation. The Gl-induced non-canonical auto- phagy was ULK1, and Beclinl-independent but ubiquitin-like conjugation system-dependent, indicating G1 might target the upstream of Atgl6L1. Moreover, inhibition of V-ATPase by specific V-ATPase inhibitiors could suppress the formation of Gl-indueed autophagosomes in FIP200-defieient MEF cells. While other classic lysosomal inhibi- tors could not block the puneta of Atgl2, Atgl6L1 and LC3, in different stages, suggesting V-ATPase activity in- stead of lysosome function is required for Gl-indueed non-canonical autophagy. These studies broaden the under- standing of different working pattern of autophagy and the crucial roles of V-ATPase in the regulation of different au- tophagy.
