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
β-Sitosterol is a type of phytosterol that occurs naturally in plants.Previous studies have shown that it has anti-oxidant,anti-hyperlipidemic,anti-inflammatory,immunomodulatory,and anti-tumor effects,but it is unkno...β-Sitosterol is a type of phytosterol that occurs naturally in plants.Previous studies have shown that it has anti-oxidant,anti-hyperlipidemic,anti-inflammatory,immunomodulatory,and anti-tumor effects,but it is unknown whetherβ-sitosterol treatment reduces the effects of ischemic stroke.Here we found that,in a mouse model of ischemic stroke induced by middle cerebral artery occlusion,β-sitosterol reduced the volume of cerebral infarction and brain edema,reduced neuronal apoptosis in brain tissue,and alleviated neurological dysfunction;moreover,β-sitosterol increased the activity of oxygen-and glucose-deprived cerebral cortex neurons and reduced apoptosis.Further investigation showed that the neuroprotective effects ofβ-sitosterol may be related to inhibition of endoplasmic reticulum stress caused by intracellular cholesterol accumulation after ischemic stroke.In addition,β-sitosterol showed high affinity for NPC1L1,a key transporter of cholesterol,and antagonized its activity.In conclusion,β-sitosterol may help treat ischemic stroke by inhibiting neuronal intracellular cholesterol overload/endoplasmic reticulum stress/apoptosis signaling pathways.展开更多
A new isochromanone,cladosporinisochromanone(1),accompanied by 15 known compounds(2–16)were obtained from secondary metabolites produced by marine-derived fungus Cladosporium sp.DLT-5.NMR and HRESIMS spectra elucidat...A new isochromanone,cladosporinisochromanone(1),accompanied by 15 known compounds(2–16)were obtained from secondary metabolites produced by marine-derived fungus Cladosporium sp.DLT-5.NMR and HRESIMS spectra elucidation determined the planar structure of 1.Subsequent electronic circular dichroism(ECD)experiment assigned the absolute configuration of 1.Compounds 1,2,4–6,and 10 displayed different degrees of neuroprotective activities on human neuroblastoma cells SH-SY5Y.Five compounds(1,3–5,and 13)emerged resistance to protein tyrosine phosphatase 1B(PTP1B),further kinetic analysis and molecular docking study indicated that the most potent compound 13(IC50value of 10.74±0.61μmol/L)was found as a noncompetitive inhibitor for PTP1B.Surface plasmon resonance(SPR)and molecular docking studies also demonstrated the interaction between compound 12 and Niemann-Pick C1 Like 1(NPC1L1),which has been identified as significant therapeutic target for hypercholesteremia.In addition,compounds 3,6,and 14 showed attractive inhibitory activity against the phytopathogenic fungi:Colletotrichum capsici.Therefore,library of Cladosporium metabolites is enriched and new active uses of known compounds are explored.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.
基金supported by the National Natural Science Foundation of China,Nos.82104158(to XT),31800887(to LY),31972902(to LY),82001422(to YL)China Postdoctoral Science Foundation,No.2020M683750(to LY)partially by Young Talent Fund of University Association for Science and Technology in Shaanxi Province of China,No.20200307(to LY).
文摘β-Sitosterol is a type of phytosterol that occurs naturally in plants.Previous studies have shown that it has anti-oxidant,anti-hyperlipidemic,anti-inflammatory,immunomodulatory,and anti-tumor effects,but it is unknown whetherβ-sitosterol treatment reduces the effects of ischemic stroke.Here we found that,in a mouse model of ischemic stroke induced by middle cerebral artery occlusion,β-sitosterol reduced the volume of cerebral infarction and brain edema,reduced neuronal apoptosis in brain tissue,and alleviated neurological dysfunction;moreover,β-sitosterol increased the activity of oxygen-and glucose-deprived cerebral cortex neurons and reduced apoptosis.Further investigation showed that the neuroprotective effects ofβ-sitosterol may be related to inhibition of endoplasmic reticulum stress caused by intracellular cholesterol accumulation after ischemic stroke.In addition,β-sitosterol showed high affinity for NPC1L1,a key transporter of cholesterol,and antagonized its activity.In conclusion,β-sitosterol may help treat ischemic stroke by inhibiting neuronal intracellular cholesterol overload/endoplasmic reticulum stress/apoptosis signaling pathways.
基金Supported by the China Agriculture Research System of MOF and MARA(CARS-21)the Financial Fund of the Ministry of Agriculture and Rural Affairs,China(No.NFZX2021)the National Natural Science Foundation of China(No.81973568)。
文摘A new isochromanone,cladosporinisochromanone(1),accompanied by 15 known compounds(2–16)were obtained from secondary metabolites produced by marine-derived fungus Cladosporium sp.DLT-5.NMR and HRESIMS spectra elucidation determined the planar structure of 1.Subsequent electronic circular dichroism(ECD)experiment assigned the absolute configuration of 1.Compounds 1,2,4–6,and 10 displayed different degrees of neuroprotective activities on human neuroblastoma cells SH-SY5Y.Five compounds(1,3–5,and 13)emerged resistance to protein tyrosine phosphatase 1B(PTP1B),further kinetic analysis and molecular docking study indicated that the most potent compound 13(IC50value of 10.74±0.61μmol/L)was found as a noncompetitive inhibitor for PTP1B.Surface plasmon resonance(SPR)and molecular docking studies also demonstrated the interaction between compound 12 and Niemann-Pick C1 Like 1(NPC1L1),which has been identified as significant therapeutic target for hypercholesteremia.In addition,compounds 3,6,and 14 showed attractive inhibitory activity against the phytopathogenic fungi:Colletotrichum capsici.Therefore,library of Cladosporium metabolites is enriched and new active uses of known compounds are explored.
