Objective: The study aimed to explore the protective mechanism of Ganoderic acid A (GAA) in renal fibrosisand to verify that GAA can ameliorate renal fibrosis by regulating the Niemann-pick C1-like 1 (NPC1L1) gene. Meth...Objective: The study aimed to explore the protective mechanism of Ganoderic acid A (GAA) in renal fibrosisand to verify that GAA can ameliorate renal fibrosis by regulating the Niemann-pick C1-like 1 (NPC1L1) gene. Methods:Transforming growth factor beta1 (TGF-β1) was used to treat Human Kidney-2 (HK-2) cells to establish a renal fibrosismodel. The differentially expressed genes in the control (CTRL) group, TGF-β1 group, and TGF-β1 + GAA group werescreened via transcriptome sequencing technology and verified by qPCR and Western blot experiments. The NPC1L1gene overexpression plasmid was constructed. The expression levels of N-cad, E-cad, and Slug-related proteins inCTRL, TGF-β1, TGF-β1+GAA (25 μg/mL), and TGF-β1+GAA (25 μg/mL) + NPC1L1 Overexpression (OE) groupswere detected by qPCR and Western blot analysis. Western blot analysis was used to identify the extracellular matrixassociated proteins Tenascin-C, α-SMA, and fibrosis-related protein Collagen I. Fibrosis marker protein Fibronectinwas detected and quantified by immunofluorescence. Results: Transcriptomic sequencing revealed that TGF-β1stimulation led to 267 differentially regulated genes, with 118 up-regulated and 149 down-regulated, while furthermodulation of 213 genes, comprising 112 up-regulated and 101 down-regulated genes, was observed in the GAAintervention group. The target gene in these processes was found to be NPC1L1 by investigations using GeneOntology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). qPCR and Western blot resultsconfirmed that TGF-β1 increased NPC1L1 expression, which was attenuated by GAA. Additionally, TGF-β1upregulated N-cad and Slug. However, GAA reversed this effect and NPC1L1 overexpression partially rescued theGAA effect. TGF-β1 also decreased E-cad expression, reversed by GAA, and NPC1L1 overexpression antagonized thisreversal. Furthermore, TGF-β1 promoted Collagen I, α-SMA, and Tenascin-C expression, and GAA reduced theselevels, effects that were reversed by NPC1L1 overexpression. Immunofluorescence results showed that TGF-β1increased fibronectin expression, which was decreased by GAA, and increased by NPC1L1 overexpression.Conclusion: GAA ameliorates renal fibrosis by antagonizing NPC1L1 gene expression inhibiting epithelialmesenchymal transition and reducing extracellular matrix formation.展开更多
目的探究干酪乳酪杆菌Zhang(Lacticaseibacillus casei Zhang)在小鼠胆囊胆固醇结石形成过程中对肝脏SR-BI以及小肠NPC1L1、ABCG8基因表达的影响。方法购买8周龄,体重(18.70±0.89)g,SPF级雌性C57BL/6J小鼠40只,适应性喂养1周后,称...目的探究干酪乳酪杆菌Zhang(Lacticaseibacillus casei Zhang)在小鼠胆囊胆固醇结石形成过程中对肝脏SR-BI以及小肠NPC1L1、ABCG8基因表达的影响。方法购买8周龄,体重(18.70±0.89)g,SPF级雌性C57BL/6J小鼠40只,适应性喂养1周后,称重,采用随机数表法分为基础饲料盐水对照组(G-NS组,n=10)、致石饲料盐水对照组(L-NS组,n=10)、基础饲料L.casei Zhang干预组(G-L-casei组,n=10)及致石饲料L.casei Zhang干预组(L-L-casei组,n=10)。给予G-NS组、G-L-casei组小鼠喂饲基础饲料,L-NS组、L-L-casei组小鼠喂饲致石饲料,同时给予G-L-casei组、L-L-casei组小鼠L.casei Zhang菌液灌胃,对照组小鼠灌胃等体积的生理盐水,1次/d,持续8周。饲养8周后,经内眦静脉取血后处死小鼠,检测四组小鼠甘油三酯(TG)、血清总胆固醇(CHO)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)及载脂蛋白(Apo-AI)水平。肉眼与偏光显微镜下观察小鼠胆囊胆固醇结石形成情况,分别采集小鼠肝脏和小肠组织检测SR-BI、NPC1L1、ABCG8表达水平。结果成石率:G-NS组和G-L-casei组小鼠未见胆囊结石,L-NS组、L-L.casei组小鼠均出现胆囊结石,且L-L-casei组成石率较L-NS组低。血清CHO表达水平:L-L-casei组小鼠低于L-NS组,差异有统计学意义(t=3.873,P<0.05)。血清TG水平:L-L-casei组小鼠低于L-NS组,差异有统计学意义(t=3.481,P<0.01)。血清LDL-C水平:L-L-casei组小鼠低于L-NS组(t=3.815,P<0.01)。血清HDL-C水平:L-L-casei组小鼠高于L-NS组(t=3.235,P<0.01),G-L-casei组小鼠高于G-NS组(t=2.649,P<0.05),差异均有统计学意义。血清Apo-AI水平:L-L-casei组小鼠高于L-NS组,差异有统计学意义(t=3.938,P<0.05)。RT-qPCR小肠NPC1L1表达水平:L-L-casei组低于L-NS组,差异有统计学意义(t=3.147,P<0.01),G-NS组低于L-NS组,差异有统计学意义(t=2.537,P<0.05)。RT-qPCR小肠ABCG8表达水平:L-L-casei组高于L-NS组,差异有统计学意义(t=2.829,P<0.05)。RT-qPCR肝脏SR-BI的表达水平:L-L-casei组小鼠高于L-NS组,差异有统计学意义(t=2.446,P<0.05)。结论L.casei Zhang可以有效改善小鼠血清的高脂状态,降低血清CHO、TG、LDL-C水平,升高血清HDL-C和ApoAI水平;同时可以使小肠NPC1L1表达水平降低,小肠ABCG8和肝脏SR-BI表达水平增加,降低小鼠胆囊结石形成概率。展开更多
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
本刊讯 7月17日,清华大学生命科学学院隋森芳院士课题组在Science Advances上在线发表了题为"人NPC1L1介导胆固醇吸收机制的结构研究"(Structural insights into the mechanism of human NPC1L1-mediated cholesterol uptake...本刊讯 7月17日,清华大学生命科学学院隋森芳院士课题组在Science Advances上在线发表了题为"人NPC1L1介导胆固醇吸收机制的结构研究"(Structural insights into the mechanism of human NPC1L1-mediated cholesterol uptake)的研究论文。通过结构生物学、细胞生物学以及生物化学等手段,阐明了NPC1L1感应胆固醇的机制和Ezetimibe的抑制机理,为进一步降脂药物的开发提供了重要的结构基础。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
基金sponsored by KeyResearch and Development Project of Science andTechnology Department of Tibet (No. XZ202201ZY0033G).
文摘Objective: The study aimed to explore the protective mechanism of Ganoderic acid A (GAA) in renal fibrosisand to verify that GAA can ameliorate renal fibrosis by regulating the Niemann-pick C1-like 1 (NPC1L1) gene. Methods:Transforming growth factor beta1 (TGF-β1) was used to treat Human Kidney-2 (HK-2) cells to establish a renal fibrosismodel. The differentially expressed genes in the control (CTRL) group, TGF-β1 group, and TGF-β1 + GAA group werescreened via transcriptome sequencing technology and verified by qPCR and Western blot experiments. The NPC1L1gene overexpression plasmid was constructed. The expression levels of N-cad, E-cad, and Slug-related proteins inCTRL, TGF-β1, TGF-β1+GAA (25 μg/mL), and TGF-β1+GAA (25 μg/mL) + NPC1L1 Overexpression (OE) groupswere detected by qPCR and Western blot analysis. Western blot analysis was used to identify the extracellular matrixassociated proteins Tenascin-C, α-SMA, and fibrosis-related protein Collagen I. Fibrosis marker protein Fibronectinwas detected and quantified by immunofluorescence. Results: Transcriptomic sequencing revealed that TGF-β1stimulation led to 267 differentially regulated genes, with 118 up-regulated and 149 down-regulated, while furthermodulation of 213 genes, comprising 112 up-regulated and 101 down-regulated genes, was observed in the GAAintervention group. The target gene in these processes was found to be NPC1L1 by investigations using GeneOntology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). qPCR and Western blot resultsconfirmed that TGF-β1 increased NPC1L1 expression, which was attenuated by GAA. Additionally, TGF-β1upregulated N-cad and Slug. However, GAA reversed this effect and NPC1L1 overexpression partially rescued theGAA effect. TGF-β1 also decreased E-cad expression, reversed by GAA, and NPC1L1 overexpression antagonized thisreversal. Furthermore, TGF-β1 promoted Collagen I, α-SMA, and Tenascin-C expression, and GAA reduced theselevels, effects that were reversed by NPC1L1 overexpression. Immunofluorescence results showed that TGF-β1increased fibronectin expression, which was decreased by GAA, and increased by NPC1L1 overexpression.Conclusion: GAA ameliorates renal fibrosis by antagonizing NPC1L1 gene expression inhibiting epithelialmesenchymal transition and reducing extracellular matrix formation.
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.
文摘本刊讯 7月17日,清华大学生命科学学院隋森芳院士课题组在Science Advances上在线发表了题为"人NPC1L1介导胆固醇吸收机制的结构研究"(Structural insights into the mechanism of human NPC1L1-mediated cholesterol uptake)的研究论文。通过结构生物学、细胞生物学以及生物化学等手段,阐明了NPC1L1感应胆固醇的机制和Ezetimibe的抑制机理,为进一步降脂药物的开发提供了重要的结构基础。
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.
