Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia,which is an independent risk factor for atherosclerotic cardiovascular disease.Niemann-Pick C1-like 1(NPC1...Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia,which is an independent risk factor for atherosclerotic cardiovascular disease.Niemann-Pick C1-like 1(NPC1L1)is a major membrane protein responsible for cholesterol absorption,in which the physiological role of vesicular endocytosis is still controversial,and it lacks a feasible tool to visualize and evaluate the endocytosis of NPC1L1 vesicles in vivo.Here,we genetically labeled endogenous NPC1L1 protein with EGFP in a knock-in mouse model,and demonstrated fluorescent visualization and evaluation of the endocytic vesicles of NPC1L1-cago during intestinal cholesterol absorption.The homozygous NPC1L1-EGFP mice have normal NPC1L1 expression pattern as well as cholesterol homeostasis on chow or high-cholesterol diets.The fluorescence of NPC1L1-EGFP fusion protein localizes at the brush border membrane of small intestine,and EGFP-positive vesicles is visualized beneath the membrane as early as 5 min post oral gavage of cholesterol.Of note,the vesicles colocalize with the early endosomal marker early endosome antigen 1(EEA1)and the filipin-stained free cholesterol.Pretreatment with NPC1L1 inhibitor ezetimibe inhibits the formation of these cholesterol-induced endocytic vesicles.Our data support the notion that NPC1L1-mediated cholesterol absorption is a vesicular endocytic process.NPC1L1-EGFP mice are a useful model for visualizing cellular NPC1L1-cargo vesicle itineraries and for evaluating NPC1L1 activity in vivo in response to diverse pharmacological agents and nutrients.展开更多
目的观察三七总皂甙(PNS)对高脂饮食糖尿病SD大鼠血脂、血糖、炎症因子和主动脉脂质代谢基因的影响。方法 46只SD大鼠普通饲料饲养2周后,其中36只经腹腔内注射链脲佐霉素(STZ)建成糖尿病动物模型,成功30只。随后分为3组(每组10只):高脂...目的观察三七总皂甙(PNS)对高脂饮食糖尿病SD大鼠血脂、血糖、炎症因子和主动脉脂质代谢基因的影响。方法 46只SD大鼠普通饲料饲养2周后,其中36只经腹腔内注射链脲佐霉素(STZ)建成糖尿病动物模型,成功30只。随后分为3组(每组10只):高脂饮食合并糖尿病组(糖尿病组),高脂饮食合并糖尿病PNS干预组(PNS组),高脂饮食合并糖尿病阿托伐他汀钙干预组(他汀组),另10只大鼠设为单纯高脂饮食非糖尿病组(对照组)。4组大鼠均予高脂饲养12周,他汀组和PNS组第5周起分别在高脂饲养基础上给予阿托伐他汀钙[(20mg/(kg·d)]和PNS[(80mg/(kg·d)]连续灌胃8周,最后处死大鼠。测定4组大鼠体重、血糖、血脂和血清炎症因子水平,q RT-PCR法检测主动脉组织TLR4、SR-BI、ABCA1和β-actin基因表达,Western blot检测主动脉组织TLR-4、ABCA1和SR-BI蛋白表达。结果糖尿病组、PNS组和他汀组体重比对照组明显降低(均P<0.01),空腹血糖较对照组明显升高(均P<0.01)。与糖尿病组比较,PNS组和他汀组血清TG、TC、TNF-α、IL-1β和LDL-C水平均降低(均P<0.05),与PNS组比较,他汀组LDL-C降低更为明显(P<0.01)。糖尿病组血管内膜及外膜脂质含量明显增加;PNS组和他汀组主动脉内膜及外膜的脂质含量均明显改善,他汀组主动脉脂质含量降低更为明显。糖尿病组大鼠主动脉组织的TLR-4 m RNA表达明显较对照组升高(P<0.01)。与糖尿病组比较,他汀组TLR-4 m RNA表达明显降低(P<0.01),SB-RI m RNA和ABCA1 m RNA表达明显上调(均P<0.01),PNS组TLR-4 m RNA表达降低,SB-RI m RNA和ABCA1 m RNA水平升高,但未达统计学差异(均P>0.05)。与对照组比较,糖尿病组主动脉组织TLR-4蛋白明显升高(P<0.01),SB-RI蛋白和ABCA1蛋白表达明显降低(均P<0.01)。与糖尿病组比较,PNS组和他汀组TLR-4蛋白表达均明显降低(均P<0.01),SR-BI蛋白和ABCA1蛋白表达均明显升高(均P<0.01),而PNS组SR-BI蛋白和ABCA1蛋白表达水展开更多
高密度脂蛋白是一种高度异质型脂蛋白(high density lipoprotein,HDL),因其密度、颗粒大小、电荷和组成成分不同,可分为不同的亚类,不同的亚类对动脉粥样硬化(atherosclerosis,AS)的影响和在胆固醇的逆向转运、抗炎、抗氧化过程所发挥...高密度脂蛋白是一种高度异质型脂蛋白(high density lipoprotein,HDL),因其密度、颗粒大小、电荷和组成成分不同,可分为不同的亚类,不同的亚类对动脉粥样硬化(atherosclerosis,AS)的影响和在胆固醇的逆向转运、抗炎、抗氧化过程所发挥的作用均不相同。综述高密度脂蛋白亚类分布与动脉粥样硬化的相关性研究进展,为进一步阐明动脉粥样硬化的发生机制及寻找抗动脉粥样硬化治疗的新的靶点提供参考。展开更多
基金This work was supported by grants from the National Key R&D Program and the National Natural Science Foundation of China(2019YFA0802503,91857203,2018YFA0800602)Collaborative Innovation Program of Shanghai Municipal Health Commission(2020CXJQ01).
文摘Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia,which is an independent risk factor for atherosclerotic cardiovascular disease.Niemann-Pick C1-like 1(NPC1L1)is a major membrane protein responsible for cholesterol absorption,in which the physiological role of vesicular endocytosis is still controversial,and it lacks a feasible tool to visualize and evaluate the endocytosis of NPC1L1 vesicles in vivo.Here,we genetically labeled endogenous NPC1L1 protein with EGFP in a knock-in mouse model,and demonstrated fluorescent visualization and evaluation of the endocytic vesicles of NPC1L1-cago during intestinal cholesterol absorption.The homozygous NPC1L1-EGFP mice have normal NPC1L1 expression pattern as well as cholesterol homeostasis on chow or high-cholesterol diets.The fluorescence of NPC1L1-EGFP fusion protein localizes at the brush border membrane of small intestine,and EGFP-positive vesicles is visualized beneath the membrane as early as 5 min post oral gavage of cholesterol.Of note,the vesicles colocalize with the early endosomal marker early endosome antigen 1(EEA1)and the filipin-stained free cholesterol.Pretreatment with NPC1L1 inhibitor ezetimibe inhibits the formation of these cholesterol-induced endocytic vesicles.Our data support the notion that NPC1L1-mediated cholesterol absorption is a vesicular endocytic process.NPC1L1-EGFP mice are a useful model for visualizing cellular NPC1L1-cargo vesicle itineraries and for evaluating NPC1L1 activity in vivo in response to diverse pharmacological agents and nutrients.
文摘目的观察三七总皂甙(PNS)对高脂饮食糖尿病SD大鼠血脂、血糖、炎症因子和主动脉脂质代谢基因的影响。方法 46只SD大鼠普通饲料饲养2周后,其中36只经腹腔内注射链脲佐霉素(STZ)建成糖尿病动物模型,成功30只。随后分为3组(每组10只):高脂饮食合并糖尿病组(糖尿病组),高脂饮食合并糖尿病PNS干预组(PNS组),高脂饮食合并糖尿病阿托伐他汀钙干预组(他汀组),另10只大鼠设为单纯高脂饮食非糖尿病组(对照组)。4组大鼠均予高脂饲养12周,他汀组和PNS组第5周起分别在高脂饲养基础上给予阿托伐他汀钙[(20mg/(kg·d)]和PNS[(80mg/(kg·d)]连续灌胃8周,最后处死大鼠。测定4组大鼠体重、血糖、血脂和血清炎症因子水平,q RT-PCR法检测主动脉组织TLR4、SR-BI、ABCA1和β-actin基因表达,Western blot检测主动脉组织TLR-4、ABCA1和SR-BI蛋白表达。结果糖尿病组、PNS组和他汀组体重比对照组明显降低(均P<0.01),空腹血糖较对照组明显升高(均P<0.01)。与糖尿病组比较,PNS组和他汀组血清TG、TC、TNF-α、IL-1β和LDL-C水平均降低(均P<0.05),与PNS组比较,他汀组LDL-C降低更为明显(P<0.01)。糖尿病组血管内膜及外膜脂质含量明显增加;PNS组和他汀组主动脉内膜及外膜的脂质含量均明显改善,他汀组主动脉脂质含量降低更为明显。糖尿病组大鼠主动脉组织的TLR-4 m RNA表达明显较对照组升高(P<0.01)。与糖尿病组比较,他汀组TLR-4 m RNA表达明显降低(P<0.01),SB-RI m RNA和ABCA1 m RNA表达明显上调(均P<0.01),PNS组TLR-4 m RNA表达降低,SB-RI m RNA和ABCA1 m RNA水平升高,但未达统计学差异(均P>0.05)。与对照组比较,糖尿病组主动脉组织TLR-4蛋白明显升高(P<0.01),SB-RI蛋白和ABCA1蛋白表达明显降低(均P<0.01)。与糖尿病组比较,PNS组和他汀组TLR-4蛋白表达均明显降低(均P<0.01),SR-BI蛋白和ABCA1蛋白表达均明显升高(均P<0.01),而PNS组SR-BI蛋白和ABCA1蛋白表达水
文摘高密度脂蛋白是一种高度异质型脂蛋白(high density lipoprotein,HDL),因其密度、颗粒大小、电荷和组成成分不同,可分为不同的亚类,不同的亚类对动脉粥样硬化(atherosclerosis,AS)的影响和在胆固醇的逆向转运、抗炎、抗氧化过程所发挥的作用均不相同。综述高密度脂蛋白亚类分布与动脉粥样硬化的相关性研究进展,为进一步阐明动脉粥样硬化的发生机制及寻找抗动脉粥样硬化治疗的新的靶点提供参考。
