姜黄素抑制Caco-2细胞胆固醇吸收的作用及机制研究

目的探讨姜黄素对Caco-2细胞胆固醇吸收的影响以及可能机制。方法建立Caco-2细胞单层模型,分别用25、50、100μmol/L姜黄素或胆固醇吸收转运蛋白尼曼-匹克C1型类似蛋白1(NPC1L1)的抑制剂依折麦布孵育细胞24h或2h后,再用[14C]-胆固醇微胶溶液孵育细胞2h。用液闪计数仪检测细胞胆固醇吸收量,荧光定量PCR和Western blot检测NPC1L1的基因和蛋白表达量。结果 Caco-2细胞[14C]-胆固醇吸收可被依折麦布呈浓度依赖性地抑制,表明本研究建立的单层Caco-2细胞模型的胆固醇吸收是由NPC1L1所介导的。姜黄素能有效的下调NPC1L1的基因及蛋白表达水平,降低Caco-2细胞胆固醇吸收,100μmol/L姜黄素可引起40%胆固醇吸收下降。结论姜黄素能够降低Caco-2细胞胆固醇吸收,这可能与其抑制NPC1L1表达有关。     

燕麦膳食纤维对肠道胆固醇吸收的影响及机制研究

目的研究燕麦膳食纤维(dietary fiber,DF)对高脂高胆固醇饮食小鼠肠道胆固醇吸收的影响及其机制。方法 36只雄性C57BL/6J小鼠,按体质量随机分为:阴性对照组、模型对照组、燕麦DF组,预防性干预24 w。实验结束后测定各组小鼠血清总胆固醇(TC)、甘油三酯(TG)及高密度脂蛋白胆固醇(HDL-C),计算低密度脂蛋白(LDL-c)。western blot方法测定肠道中与胆固醇吸收相关的GTP结合蛋白Sar1B、尼曼-匹克C1型类似蛋白1(NPC1L1)、清道夫受体B1(SR-B1)蛋白的表达水平。结果实验结束时,与模型对照组相比,燕麦DF组的体重、血清TC、LDL-C水平均显著下降(P0.05),HDL-C水平显著升高(P0.05);与胆固醇吸收相关的NPC1L1水平显著降低(P0.05),SR-B1无显著变化,调控NPC1L1和SR-B1的Sar1B蛋白表达水平显著升高(P0.05)。结论燕麦DF可以通过上调Sar1B降低肠道NPC1L1表达水平,从而减少肠道胆固醇吸收,降低血脂水平,改善高脂高胆固醇饮食引起的高脂血症。     

小麦纤维通过调控小鼠肠道胆固醇代谢抗动脉粥样硬化研究

目的探讨小麦纤维对ApoE-/-小鼠肠道胆固醇代谢的影响及其发挥抗动脉粥样硬化(AS)的机制。方法将ApoE-/-雄性小鼠按血清总胆固醇水平随机分为AS模型(AS)组和小麦纤维(WF)组,分别用不添加或添加小麦纤维的高脂高胆固醇饲料喂养18w。测定两组小鼠的血脂四项、血糖和游离脂肪酸水平;取小鼠整体主动脉和血管横截面进行油红O染色;采用Westernblot法检测小鼠小肠组织中参与胆固醇吸收和逆转运相关蛋白的表达,包括过氧化物酶体增殖物激活受体α(PPARα),肝X受体α(LXRα),ATP结合盒转运子A1(ABCA1)和G1(ABCG1),分泌及ras相关的GTP结合蛋白1B(Sar1B),尼曼-匹克C1型类似蛋白1(NPC1L1)和清道夫受体B1(SR-B1)。结果干预18w后,与AS组相比,WF组低密度脂蛋白胆固醇(LDL-C)水平显著下降;小麦纤维降低了ApoE-/-小鼠血管内壁动脉粥样硬化斑块面积;小麦纤维激活了核转录因子PPARα和LXRα,上调了ABCA1和ABCG1的蛋白表达;小麦纤维增加了Sar1B的蛋白表达,降低了NPC1L1的蛋白表达。结论小麦纤维可通过促进肠道胆固醇逆转运,增加胆固醇的外流和减少胆固醇的吸收,改善血清胆固醇水平,发挥抗AS的作用。[营养学报,2019,41(5):465-469]     

乳清蛋白对高脂饮食小鼠肠道胆固醇代谢的影响

目的探讨浓缩乳清蛋白粉对高脂饮食诱导肥胖小鼠肠道胆固醇代谢的影响。方法40只C57BL/6J小鼠随机分为对照、高脂饲料(HFD)、12.5%乳清蛋白(WP)和25%WP组,干预组在HFD中添加相应含量WP,喂养12w后测定各组小鼠的血脂水平、游离脂肪酸、空腹血糖和胰岛素,并计算胰岛素抵抗指数(HOMA-IR)。取小肠组织,Western blot法测定胆固醇吸收相关的Niemann-Pick C1-Like 1(NPC1L1)、清道夫受体B1(SR-B1)、以及胆固醇外流相关的肝X受体α(LXRα)及ATP结合盒转运子G1(ABCG1)蛋白的表达水平。结果与HFD组比,25%WP组小鼠血清总胆固醇、空腹胰岛素和HOMA-IR值显著下降;12.5%和25%WP组小鼠的血清低密度脂蛋白胆固醇水平亦显著下降。与HFD组比,25%WP组小鼠的小肠组织中NPC1L1、SR-B1蛋白表达显著下调,而LXRα和ABCG1蛋白表达显著上调。结论乳清蛋白可能通过减少肠道胆固醇吸收,增加肠道胆固醇外流,改善了高脂饮食引起的糖脂代谢紊乱。     

小知识（9）

血清总胆固醇（serum total cholesterol） 胆固醇以游离胆固醇及胆固醇酯（软脂酸、硬脂酸和油酸酯）的形式，存在于一切动物组织中，植物组织中没有胆固醇。我国人血清总胆固醇约为2．9～6．0mmol／L。胆固醇酯约占总胆固醇的2／3。血清胆固醇含量高于正常范围是动脉粥样硬化的危险因素。     

高危和极高危动脉粥样硬化的调脂治疗

大量循证研究证明,低密度脂蛋白胆固醇是导致动脉粥样硬化和心血管病的元凶,而它每降低1mmol/L,可令主要心血管事件减少20%~25%。对于血脂异常的管理,当前国内外指南强调,尽可能地降低低密度脂蛋白胆固醇LDL-C水平,以预防动脉粥样硬化性心脑血管疾病。     

1 220名学生血清脂蛋白及低密度脂蛋白胆固醇含量

目的为了解青少年血清脂蛋白[Lp(a)]及低密度脂蛋白胆固醇(LDL-C)含量的变化.尽早发现青少年中血清高[Lp(a)]和高LDL-C潜在危险因素,以期有效地预防早发性心脑血管疾病.方法 Lp(a)采用酶免疫比浊法、LDL-C采用抗原抗体直接测定法.用HITACH 7150全自动生化分析仪进行检测.计量资料以x±s表示,t检验.结果在1 220名被检学生中Lp(a)和LDL-C均在合适水平的占82%(1 027/1 220),11%的学生(134/1 220)Lp(a)>200 mg/L,5%(61/1 220)Lp(a)>300 mg/L,10%(124/1 220)LDL-C>2.85 mmol/L,5%(68/1 220)的学生LDL-C>3.4 mmol/L,4%(49/1 220)的学生Lp(a)>300 mg/L同时LDL-C>3.4 mmol/L.结论 Lp(a)和LDL-C被认为是引起心脑血管疾病的两个独立危险因素,若高Lp(a)和高LDL-C同时发生,其相对危险性可增加6倍.实验测得约17%的学生不同程度伴有血清Lp(a)或LDL-C含量升高.显示潜在的早发动脉粥样硬化及血管性疾病的危险性,应引起高度的重视.     

权威专家告诉你保健误区在哪里

误区之1 鸡蛋内含有大量胆固醇,中老年人不宜食用 血液内胆固醇含量过高,是动脉粥样硬化的危险因素。群众对这一观点了解较多,但对胆固醇是维持人体生存的必需物质又缺乏了解,所以出现了这样那样的行为误区。因此,对胆固醇应有全面正确的认识。 新生红细胞的细胞膜,需要一定量的胆固醇;胆固醇是生物膜(细胞膜、神经鞘膜等)的重要组成部分;是合成肾上腺素、性激素的主要原料并参与维生素D的合成,因此,胆固醇具有十分重要的生理作用。正常成人每日约需胆固醇1.1克,才能满足维持上述功能的需要。 胆固醇的来源分为内源、外源两种。血液内的胆固醇水平主要取决于肝脏的合成。外源胆固醇的吸收和内源的合成具有互相制约作用,这是机体的自身调节机制。同位素标记实验表明,摄入胆固醇     

载脂蛋白E基因敲除小鼠主动脉壁胆固醇含量的测定及意义

目的　探讨载脂蛋白E(apoE)基因敲除小鼠主动脉胆固醇含量的检测方法 ,并分析其对apoE基因敲除小鼠动脉粥样硬化斑块面积的评估价值。 方法　分离apoE基因敲除小鼠和正常小鼠主动脉 ,用酶法检测主动脉胆固醇的含量 ;用油红O染色法和图像分析法测量小鼠动脉粥样斑块面积。结果　apoE基因敲除小鼠主动脉胆固醇的含量显著高于正常小鼠 (P <0 .0 1) ,apoE基因敲除小鼠的动脉粥样硬化斑块面积也显著高于正常小鼠 ,且二者之间呈显著性相关 (相关系数r =0 .982 ,P <0 .0 1)。结论　apoE基因敲除小鼠主动脉胆固醇含量与其动脉粥样斑块面积的大小密切相关 ,是apoE基因敲除小鼠动脉粥样硬化斑块形成的一个重要指标。     

高脂血症的非药物治疗

高脂血症是各种原因导致的血浆中胆固醇和／或甘油三脂水平升高的一种疾病，其诊断标准为：近期2次(相隔2周以上)空腹血清总胆固醇(Tc)≥6．45mmol／L，甘油三酯(TG)≥1．53mmol／L或高密度脂蛋白胆固醇(HDL-ch)男性≤1．04mmol／L，女性≤1．17mmol／L。高脂血症是人体脂质代谢障碍，是引发和加重动脉粥样硬化的病理基础及导致动脉硬化、心绞痛、心肌梗死、脑梗塞、肾损害等动脉栓塞性疾病的独立危害因素。近年来其发病率呈上升及发病年龄提前趋势。     

Sitosterol reduces messenger RNA and protein expression levels of Niemann-Pick C1-like 1 in FHs 74 Int cells

Intake of plant sterols has long been shown to reduce cholesterol absorption and subsequently plasma cholesterol concentrations. Despite competition between plant sterols and cholesterol for incorporation into mixed micelles as a suggested major mechanism for the inhibition of cholesterol absorption by plant sterols, studies exist to support an alternative mechanism. For example, another mechanism may be the action of plant sterols to reduce cholesterol absorption at the cellular level. This study was undertaken to test the hypothesis that plant sterols can modulate the expression of transporters such as Niemann-Pick C1-like 1 (NPC1L1) and scavenger receptor class B, type I (SR-BI) to lower intestinal cholesterol absorption. FHs 74 Int cells, a human small intestine epithelial cell line, were used as a model of enterocytes. The cells were treated with 25α-hydroxycholesterol (25 μmol/L) or 250 μmol/L of sitosterol, stigmasterol, and cholesterol for 24 hours to measure genes involved in cholesterol absorption and metabolism by quantitative real-time polymerase chain reaction. 25α-Hydroxycholesterol, cholesterol, and sitosterol significantly reduced the messenger RNA (mRNA) expression of NPC1L1 and hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, whereas SR-BI mRNA was not altered by the sterols. Western blot analysis confirmed the reduction in NPC1L1 by sterols. Depletion of cellular cholesterol by mevinolin, a cholesterol synthesis inhibitor, increased NPC1L1 and HMG-CoA reductase mRNA; and repletion of cholesterol abolished the increase. Sitosterol, but not stigmasterol, reduced the mRNA levels of NPC1L1 and HMG-CoA reductase to a similar extent of cholesterol. In conclusion, sitosterol can inhibit the expression of NPC1L1 in the enterocytes, which could be an alternate mechanism for plant sterols to reduce intestinal cholesterol uptake.     

NPC1L1 haplotype is associated with inter-individual variation in plasma low-density lipoprotein response to ezetimibe

Background  

NPC1L1 encodes a putative intestinal sterol transporter which is the likely target for ezetimibe, a new type of lipid-lowering medication. We previously reported rare non-synonymous mutations in NPC1L1 in an individual who had no plasma lipoprotein response to ezetimibe. We next hypothesized that common variants in NPC1L1 would underlie less extreme inter-individual variations in the plasma LDL cholesterol response to ezetimibe.     

Reduction in cholesterol absorption in Caco-2 cells through the down-regulation of Niemann-Pick C1-like 1 by the putative probiotic strains Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74 from fermented foods

Hypercholesterolaemia is a major risk factor related to atherosclerosis, and it may be influenced by our diet. This study addresses the impact of Lactobacillus rhamnosus BFE5264 (isolated from Maasai fermented milk) and Lactobacillus plantarum NR74 (from Korean kimchi) on the control of cholesterol absorption through down-regulation of Niemann-Pick C1-like 1 (NPC1L1) expression. Caco-2 enterocytes were treated with the live, heat-killed (HK) bacteria, bacterial cell wall extracts and metabolites; mRNA level and protein expression were measured. Caco-2 cells showed lower NPC1L1 expression in the presence of the live test strains than the control, elucidating down-regulation of cholesterol uptake, and were compared well with the positive control, L. rhamnosus GG. This effect was also observed with HK bacteria and cell wall fractions but not with their metabolites. The potential of some Lactobacillus strains associated with traditional fermented foods to suppress cholesterol uptake and promote its efflux in enterocytes has been suggested from these data.     

Lycopene absorption in human intestinal cells and in mice involves scavenger receptor class B type I but not Niemann-Pick C1-like 1

Cholesterol membrane transporters scavenger receptor class B type I (SR-BI) and (cluster determinant 36) are involved in intestinal uptake of lutein and beta-carotene, 2 of the 3 main carotenoids of the human diet. The aim of this work was therefore to determine whether SR-BI and NPC1L1 (Niemann-Pick C1-like 1), another cholesterol transporter, are implicated in absorption of lycopene, the 3rd main carotenoid of the human diet. Anti-human SR-BI antibody and block lipid transport 1 (BLT1) (a chemical inhibitor of lipid transport by SR-BI) impaired up to 60% (all-E) and (5Z)-lycopene uptake (P < 0.05) by Caco-2 cell monolayers, which were used as a model of human intestinal epithelium. The involvement of SR-BI in lycopene absorption in vivo was then verified by comparing plasma lycopene concentrations in wild-type and SR-BI transgenic mice that were fed a diet enriched with 0.25 g/kg (all-E)-lycopene for 1 mo. Plasma lycopene concentrations were approximately 10-fold higher (P < 0.001) in mice overexpressing SR-BI in the intestine than in wild-type mice, confirming the involvement of SR-BI in lycopene absorption. Further experiments showed that (all-E)-lycopene did not affect SR-BI mRNA levels in Caco-2 cells or mouse intestine. In contrast to SR-BI, neither anti-human NPC1L1 antibody nor ezetimibe, used as inhibitors of lycopene uptake via NPC1L1, significantly impaired (all-E) or (5Z)-lycopene uptake by Caco-2 monolayers. Thus, the present data show that lycopene absorption is, at least in part, mediated by SR-BI but not by NPC1L1.     

Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression

Background  

Curcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1) protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells.     

Carotenoid transport is decreased and expression of the lipid transporters SR-BI, NPC1L1, and ABCA1 is downregulated in Caco-2 cells treated with ezetimibe

Data suggest that intestinal carotenoid absorption is a facilitated process. The present study was conducted to determine whether carotenoids and cholesterol share common pathways (transporters) for their intestinal absorption. Differentiated Caco-2 cells on membranes were incubated (16 h) with a carotenoid (1 micromol/L) with or without ezetimibe (EZ; Zetia, an inhibitor of cholesterol transport), and with or without antibodies against the receptors, cluster determinant 36 (CD36) and scavenger receptor class B, type I (SR-BI). Carotenoid transport in Caco-2 cells (cellular uptake + secretion) was decreased by EZ (10 mg/L) as follows: beta-carotene approximately alpha-carotene (50% inhibition) > beta-cryptoxanthin approximately lycopene (20%) > lutein:zeaxanthin (1:1) (7%). EZ reduced cholesterol transport by 31%, but not retinol transport. beta-Carotene transport was also inhibited by anti-SR-BI, but not by anti-CD36. The inhibitory effects of EZ and anti-SR-BI on beta-carotene transport were additive, indicating that they may have different targets. Finally, differentiated Caco-2 cells treated with EZ showed a significant decrease in mRNA expression for the surface receptors SR-BI, Niemann-Pick type C1 Like 1 protein (NPC1L1), and ATP-binding cassette transporter, subfamily A (ABCA1) and for the nuclear receptors retinoid acid receptor (RAR)gamma, sterol-regulatory element binding proteins (SREBP)-1 and -2, and liver X receptor (LXR)beta as assessed by real-time PCR analysis. The data indicate that 1) EZ is an inhibitor of carotenoid transport, an effect that decreases with increasing polarity of the carotenoid molecule, 2) SR-BI is involved in carotenoid transport, and 3) EZ may act, not only by interacting physically with cholesterol transporters as previously suggested, but also by downregulating expression of these proteins. The cellular uptake and efflux of carotenoids, like that of cholesterol, likely involve more than one transporter.     

In silico assessment of phosphorylation and O-β-GlcNAcylation sites in human NPC1 protein critical for Ebola virus entry

Ebola is a highly pathogenic enveloped virus responsible for deadly outbreaks of severe hemorrhagic fever. It enters human cells by binding a multifunctional cholesterol transporter Niemann–Pick C1 (NPC1) protein. Post translational modification (PTM) information for NPC1 is crucial to understand Ebola virus (EBOV) entry and action due to changes in phosphorylation or glycosylation at the binding site. It is difficult and costly to experimentally assess this type of interaction, so in silico strategy was employed. Identification of phosphorylation sites, including conserved residues that could be possible targets for 21 predicted kinases was followed by interplay study between phosphorylation and O-β-GlcNAc modification of NPC1. Results revealed that only 4 out of 48 predicted phosphosites exhibited O-β-GlcNAc activity. Predicted outcomes were integrated with residue conservation and 3D structural information. Three Yin Yang sites were located in the α-helix regions and were conserved in studied vertebrate and mammalian species. Only one modification site S425 was found in β-turn region located near the N-terminus of NPC1 and was found to differ in pig, mouse, cobra and humans. The predictions suggest that Yin Yang sites may not be important for virus attachment to NPC1, whereas phosphosite 473 may be important for binding and hence entry of Ebola virus. This information could be useful in addressing further experimental studies and therapeutic strategies targeting PTM events in EBOV entry.     

Modulation of paraoxonases during infectious diseases and its potential impact on atherosclerosis

Background

Niemann-pick C1-like 1 (NPC1L1) is a key protein for intestinal cholesterol transportation. Common single nucleotide polymorphisms (SNPs) in the NPC1L1 gene have been associated with cholesterol absorption and serum lipid levels. The present study was undertaken to explore the possible association of NPC1L1 rs2072183 1735?C?>?G SNP and several environmental factors with serum lipid levels in the Mulao and Han populations.

Methods

Genotyping of the rs2072183 SNP was performed in 688 subjects of Mulao and 738 participants of Han Chinese. The interactions between NPC1L1 1735?C?>?G polymorphism and several environmental factors on serum lipid phenotypes were tested using the factorial design covariance analysis after controlling for potential confounders.

Results

The frequency of G allele was lower in Mulao than in Han (29.72% vs. 37.26%, P?<?0.001). The frequency of CC, CG and GG genotypes was 49.85%, 40.84% and 9.31% in Mulao, and 39.30%, 46.88% and 13.82% in Han (P?<?0.001); respectively. The levels of low-density lipoprotein cholesterol (LDL-C), apolipoprotein (Apo) B and the ratio of ApoAI/ApoB in Han but not in Mulao were different among the three genotypes (P?<?0.05 for all), the subjects with GG and CG genotypes had higher LDL-C, ApoB levels and lower ApoAI/ApoB ratio than the subjects with CC genotype. Subgroup analysis showed that the G allele carriers in Han had higher total cholesterol (TC), LDL-C and ApoB levels in males (P?<?0.05) and lower ApoAI/ApoB ratio in both sexes (P?<?0.05) than the G allele noncarriers. The G allele carriers in Mulao had higher TC and LDL-C levels in males (P?<?0.05) and lower high-density lipoprotein cholesterol (HDL-C) levels in both sexes (P?<?0.05) than the G allele noncarriers. Serum TC, LDL-C, ApoB levels and ApoAI/ApoB ratio were correlated with genotypes in Han males (P?<?0.05) but not in females. Serum lipid parameters were also correlated with several environmental factors. The genotypes of rs2072183 SNP were interacted with gender or cigarette smoking to influence serum TC and HDL-C levels in Mulao, whereas the genotypes of rs2072183 SNP were interacted with several environmental factors to influence all seven lipid traits in Han (P?<?0.05-0.01).

Conclusions

The present study suggests that the rs2072183 SNP in NPC1L1 gene and its association with serum lipid profiles are different between the Mulao and Han populations. The difference in serum lipid profiles between the two ethnic groups might partly result from different rs2072183 SNP or NPC1L1 gene-environmental interactions.