酶法合成阿托伐他汀侧链中间体（S）-4-氯-3-羟基丁酸乙酯的研究进展

第三代羟甲戊二酰辅酶A （HMG-CoA）还原酶抑制剂——阿托伐他汀是年销售额超过百亿的重磅降血脂药物,因其疗效显著而广受医生患者好评.阿托伐他汀主要通过疏水性母核及手性侧链进行合成,而手性侧链的制备是合成关键.目前,生物催化技术在手性药物合成中的应用备受关注,而阿托伐他汀手性侧链中间体（S）-4-氯-3-羟基丁酸乙酯的酶法合成也成为研究热点.本文主要介绍近年酶法合成在（S）-4-氯-3-羟基丁酸乙酯中的研究进展.     

瑞舒伐他汀侧链(3R,5S)-6-R-取代基-3,5-二羟基己酸叔丁酯合成研究进展

瑞舒伐他汀是一种高效的羟甲戊二酰辅酶A(HMG-CoA)还原酶抑制剂,能有效降低低密度脂蛋白(IDL)胆固醇,且无副作用,被称为“超级他汀”.瑞舒伐他汀是由疏水性母核和开环侧链构成,其手性侧链是合成中的难点.目前,化学法和生物酶法合成瑞舒伐他汀关键手性侧链(3R,5S)-6-R-取代基-3,5-二羟基己酸叔丁酯受到研究者广泛的关注.主要介绍了不对称还原合成(3R,5S)-6-R-取代基-3,5-二羟基己酸叔丁酯的研究进展.     

甜菊醇、异甜菊醇抑制α-葡萄糖苷酶和HMG-CoA还原酶的分子机制研究

本文研究了甜菊醇、异甜菊醇抑制高血糖、高血脂代谢关键酶α-葡萄糖苷酶、HMG-CoA还原酶活性的分子机制。采用分光光度法测定甜菊醇、异甜菊醇对α-葡萄糖苷酶、HMG-CoA还原酶抑制率,用双倒数作图法研究甜菊醇、异甜菊醇的酶抑制动力学,利用AutoDock软件对甜菊醇、异甜菊醇与α-葡萄糖苷酶、HMG-CoA还原酶的结合模式进行分析。结果表明:甜菊醇、异甜菊醇对α-葡萄糖苷酶的IC₅₀分别为70.75、49.65 mg/L,采用竞争性与非竞争性相混合的方式抑制α-葡萄糖苷酶;甜菊醇、异甜菊醇对HMG-CoA还原酶的IC₅₀分别为46.29、36.66 mg/L,竞争性抑制HMG-CoA还原酶。分子对接的分析结果表明甜菊醇、异甜菊醇分别位于α-葡萄糖苷酶、HMG-CoA还原酶的疏水口袋中,与多个氨基酸残基结合,并存在疏水作用。本研究对于开发新型的食源性α-葡萄糖苷酶和HMG-CoA还原酶抑制剂,推动甜菊醇、异甜菊醇在食品和医药领域的应用具有一定的参考意义。     

石榴皮多酚对脂变L-02肝细胞胆固醇合成的影响及机制探究

研究了石榴皮多酚纯化物、安石榴苷和石榴鞣花酸对脂变肝细胞胆固醇合成的影响及机制;采用MTT法筛选石榴皮多酚适宜浓度,体积分数50%胎牛血清的RPMI-1640培养基与L-02肝细胞孵育48 h建立脂肪变性肝细胞模型;实验分为正常组、模型组和治疗组,治疗组加入不同浓度的受试物,继续培养48 h,油红O染色定性观察细胞形态和脂滴堆积,高效液相色谱法定量检测细胞内胆固醇含量;紫外分光-速率法检测HMG-CoA还原酶活性变化;结果显示石榴皮多酚均能成剂量依赖性地减少细胞内脂滴的积累、减少细胞内总胆固醇的含量,同时抑制HMG-CoA还原酶的活性,其中以100μg/mL的安石榴苷标品效果最佳。表明石榴皮多酚具有降低肝细胞内总胆固醇的作用,可能是通过抑制HMG-CoA还原酶活性实现的,安石榴苷是石榴皮多酚中降血脂的主要活性形式。     

HMG-CoA还原酶抑制剂研究进展

HMG-CoA还原酶抑制剂与底物HMG-CoA具有类似的结构片段,能够竞争性抑制HMG-CoA还原为甲羟戊酸,有效降低胆固醇水平,为降脂药物中的首选药物,文章对这类药物的分类、结构特征、降脂作用机制、多效性研究和不良反应等几个方面进行综述。     

液态发酵法产Lovastatin的研究进展

Lovastatin是Aspergillus和Monascus等丝状真菌的次生代谢产物,在人体内通过竞争性地抑制胆固醇合成的限速酶3-羟-3-甲基戊二酰辅酶A还原酶而阻断内源性胆固醇的合成,强烈降低血胆固醇浓度,是预防和治疗高血脂症和高胆固醇症及相关病症的有效药物。近年来,研究人员对液态发酵产Lovastatin开展了广泛深入的研究,取得了一系列重要研究进展,促进了工业化生产Lovastatin的发展。文中对Lovastatin高产菌种及选育、碳源和氮源及发酵培养条件对不同菌种产Lovastatin的影响等方面的研究进展进行了综述,为相关的研究与应用提供参考。     

降胆固醇乳酸菌的体外筛选及其降胆固醇机理探讨

目的：从开菲尔粒和陈年泡菜水中分离出具有降胆固醇能力的乳酸杆菌,探讨其降解小鼠血清胆固醇的机理。方法：以耐酸性、胆盐耐受性、疏水性、胆盐水解酶活性以及降胆固醇特性筛选出1株性状优良的植物乳杆菌（Lactobacillus?plantarum?DMDL?9010）。将50?只8?周龄的Sprague?Dawley大鼠随机分为正常组、模型组、阳性组、9010高组、9010低组,分别饲喂28?d和70?d采血,测定总胆固醇（total cholesterol,TC）、甘油三酯（triglyceride,TG）、高密度脂蛋白胆固醇（high density lipoprotein-cholesterol,HDL-C）、低密度脂蛋白胆固醇（low density lipoprotein-cholesterol,LDL-C）的含量,70 d取肝组织细胞实时荧光逆转录聚合酶链式反应检测胆固醇合成限速酶3-羟基-3-甲基戊二酸单酰辅酶A还原酶（3-hydroxy-3-methylglutaryl-CoA reducase,HMGCR）的相对表达量。结果：筛选得到1株具有降胆固醇能力的L.?plantarum DMDL 9010,胆固醇去除率为37.58%,胆盐耐受性为35.48%,疏水性高达40%,具有较好的耐酸性。饲喂第28天,成功建出高脂模型大鼠,饲喂第70天,9010高组能显著降低高脂大鼠血清TC（23.03%）和LDL-C（28.00%）,9010低组和阳性组无明显差异。阳性组和9010高组分别下调肝脏中HMG-CoA基因mRNA的表达（79.92%和62.86%）（P＜0.05）。结论：实验获得了1?株在体内外均具有高效降胆固醇能力的L.?plantarum?DMDL?9010,可进一步开发为功能性微生态制剂。     

无斑鹞鲼蛋白质对大鼠胆固醇代谢的影响

目的：探讨无斑鹞鲼蛋白（Aetobatus flagelum protein,AFP）对高胆固醇模型大鼠胆固醇代谢的影响,并对其可能的作用机制加以研究。方法：5 周龄雄性Sprague-Dawley（SD）大鼠,适应性饲养1 周后,按体质量随机分为酪蛋白对照组、5% AFP组、10% AFP组,分别给予高胆固醇饲料及分别添加5%和10% AFP的高胆固醇饲料。28 d后测定大鼠血清总胆固醇（total cholesterol,TC）、高密度脂蛋白胆固醇（high density lipoprotein cholesterol,HDL-C）水平,肝脏TC、游离胆固醇水平,粪便中胆汁酸和中性固醇含量以及肝脏3-羟基-3-甲基戊二酸单酰辅酶A（3-hydroxy-3-methyl glutaryl coenzyme A reductase,HMG-CoA）、胆固醇酰基转移酶2（acyl coenzyme Acholesterolacyltransferase 2,ACAT2）、胆固醇7α-羟化酶1（cholesterol 7α-hydroxylase 1,CYP7A1）的mRNA表达量。结果：与酪蛋白对照组相比,无斑鹞鲼蛋白可显著降低大鼠血清和肝脏中的TC含量（P＜0.05）,明显增加血清HDL-C含量（P＜0.05）,极显著降低大鼠动脉粥样硬化指数（P＜0.01）。此外,无斑鹞鲼蛋白可明显增加大鼠粪便中胆汁酸和中性固醇的排出量（P＜0.05）,并可降低大鼠肝脏中ACAT2 mRNA的表达量（P＜0.05）,增加CYP7A1 mRNA的表达量（P＜0.05）,而对HMG-CoA mRNA的作用不显著。结论：无斑鹞鲼蛋白可明显降低大鼠血清和肝脏中的TC含量,降低大鼠血清动脉粥样硬化指数,其作用机制主要与增加肝脏内胆固醇的分解代谢以及促进粪便中性固醇和胆汁酸的排出有关。     

石榴皮多酚对脂变L-02肝细胞HMG-CoA还原酶mRNA表达的影响

研究了石榴皮多酚纯化物、安石榴苷和石榴鞣花酸对脂变L-02肝细胞胆固醇合成限速酶HMG-CoA还原酶mRNA表达的影响;采用MTT法筛选石榴皮多酚适宜浓度;体积分数50%胎牛血清的RPMI-1640培养基与L-02肝细胞孵育48 h建立脂肪变性肝细胞模型;实验分为正常组、模型组和治疗组,治疗组加入不同浓度的受施物,继续培养48 h,采用RT-PCR法检测不同受施物对脂变L-02肝细胞HMG-CoA还原酶mRNA表达的影响;结果显示:石榴皮多酚均能呈剂量依赖性地减弱脂变L-02肝细胞mRNA表达,且以100μg/mL的安石榴苷标品抑制作用最强;表明石榴皮多酚降肝细胞总胆固醇作用是通过降低HMG-CoA还原酶mRNA表达实现的,安石榴苷是石榴皮多酚中降血脂的主要活性形式。     

尿石素A对巨噬细胞极化及巨噬-泡沫细胞形成的作用

旨在探究尿石素A对巨噬细胞极化及对巨噬-泡沫细胞形成的影响及相关的分子机制。结果发现,尿石素A通过调控不同标志基因的表达,不仅能够抑制RAW264.7小鼠巨噬细胞向促炎的M1型巨噬细胞极化,还能促进自然状态巨噬细胞向M2型巨噬细胞极化;油红O染色发现尿石素A能够显著抑制巨噬-泡沫细胞形成,实时荧光定量聚合酶链式反应检测说明尿石素A能够抑制胆固醇合成基因羟甲基戊二酸单酰辅酶A还原酶和脂肪酸合成酶基因的转录;此外,尿石素A显著上调三磷酸腺苷结合盒转运蛋白A1和G1基因的表达,该两个基因表达与促进了胆固醇的排出相关。本研究证明了尿石素A对巨噬细胞极化具有调控作用,同时尿石素A能够抑制巨噬-泡沫细胞形成和胆固醇合成。这些结果揭示了尿石素A具有潜在的抗动脉粥样硬化的作用,为之后深入研究提供了理论参考。     

Dietary factors and the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for breast cancer and development

A role for mevalonate in cancer development has long been suggested by findings that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity is elevated in malignant cells. Increased synthesis mevalonate and mevalonate-derived nonsterol isoprenoids supports increased cell proliferation through the activation of growth-regulatory proteins and oncoproteins, and by promoting DNA synthesis. We have recently shown that mevalonate promotes the growth of human breast cancer cells both in culture and as tumors grown in nude mice. Inhibition mevalonate synthesis, therefore, may be an effective strategy to impair the growth of malignant breast cells. Several dietary compounds with known anti-cancer effects are also reported to inhibit HMG-CoA reductase activity. Here, we review evidence suggesting that inhibition of mevalonate synthesis may mediate the protective effects of cholesterol, plant isoprenoids, genistein, and long-chain n-3 polyunsaturated fatty acids (PUFAs) on experimental breast cancer.     

Proso millet醇溶蛋白调节小鼠血清胆固醇代谢的作用及其机理

通过比较分别饲喂不同蛋白源(酪蛋白、大豆分离蛋白、Proso m illet醇溶蛋白)饲料的小鼠血清总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-c)、低密度脂蛋白胆固醇(LDL-c)、甘油三酯(TG)浓度,旨在评价Proso m illet醇溶蛋白对小鼠胆固醇代谢的特定作用,并通过体外实验探讨其作用机理。结果显示,以Prosom illet醇溶蛋白(添加1.83%Lys和0.23%Trp)为唯一氮源的小鼠日粮对TC、TG无明显作用,却能显著提高小鼠血清HDL-c浓度(P<0.05),降低小鼠动脉硬化指数(AI)。但在体外其并不能抑制羟甲基戊二酸单酰辅酶A(HMG-CoA)还原酶的活性,即不能直接抑制胆固醇的合成,所以,其对胆固醇代谢的调节作用另有其他途径。     

Molecular,functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast,Schizosaccharomyces pombe

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase, hmg1+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmg1+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmg1+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMG1p, the hmg1+ protein induced membrane proliferations known as karmellae. A previously undescribed ‘feed-forward’ regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmg1+ protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.     

Deglycosylation of stilbene glucoside compounds improves inhibition of 3-hydroxy-3-methylglutaryl coenzyme a reductase and squalene synthase activities

The glycosylated stilbenes mulberroside A and rhapontin were converted to their aglycones (oxyresveratrol and rhapontigenin) by enzymatic transformation. Rhapontin, rhapontigenin, mulberroside A, oxyresveratrol-3-O-glucoside, and oxyresveratrol showed inhibitory activities against 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and squalene synthase, which are key enzymes in the cholesterol biosynthesis pathway. Aglycones showed stronger inhibitory activities than their glycosylated counterparts, and methoxylated stilbenes were stronger inhibitors of both enzymes than non-methoxylated stilbenes. The inhibitory activities of the stilbene compounds were significantly different from that of a negative control group (p<0.05).     

Herbicidal effects of statin pharmaceuticals in Lemna gibba

Statin pharmaceuticals, heavily prescribed in the treatment of hypercholesterolemia, are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR). In plants, these compounds also inhibit HMGR, which regulates cytosolic isoprenoid biosynthesis in the mevalonic acid (MVA) pathway. Phytotoxicity was evaluated in the higher aquatic plant Lemna gibba exposed to atorvastatin and lovastatin for 7-days by measuring the concentrations of sterols and ubiquinone; products downstream in the MVA pathway. The efficiency of the parallel and unaffected methylerythritol phosphate pathway (MEP) was also evaluated by measuring the end product, plastoquinone. Statin treatment caused an accumulation of plastoquinone, and unexpectedly, ubiquinone, an artifact likely due to metabolite sharing from the plastidial MEP pathway. Statins were, however, highly phytotoxic to L. gibba and HPLC-UV analysis of plant extracts showed significantly decreased concentrations of both stigmasterol and beta-sitosterol, which are critical components of plant membranes and regulate morphogenesis and development. EC10 values for atorvastatin and lovastatin were as small as 26.1 and 32.8 microg/L, respectively. However, hazard quotients indicated that statins present little risk to the model higher aquatic plant Lemna gibba at environmentally relevant concentrations, even though pathway-specific endpoints were 2-3 times more sensitive than traditional gross morphological endpoints typically used in risk assessment.     

Comparative effects of safflower oil and perilla oil on serum and hepatic lipid levels, fatty acid compositions of serum and hepatic phospholipids, and hepatic mRNA expressions of 3-hydroxy-3-methylglutaryl CoA reductase, LDL receptor, and cholesterol 7alpha-hydroxylase in young and adult rats

Male Wistar rats, 4 and 33 weeks of age, were fed the diets containing safflower oil (SO-diet, 77.3% linoleic acid) or perilla oil (PO-diet, 58.4% -linolenic acid) for 7 days. Serum total cholesterol was lower on the PO-diet in both ages. On the other hand, hepatic cholesterol and phospholipids were significantly higher in the PO group than in the SO group of the adult rats. The PO group showed significantly lower 20:4 n-6 but higher 18:2 n-6 in hepatic phosphatidylcholine compared with the SO group in both ages. Hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase mRNA was significantly lower on the PO-diet than on the SO-diet irrespective of age. The present results show that -linolenic acid has a higher hypocholesterolemic ability than linoleic acid in rats irrespective of age and these fatty acids behaved differently in affecting hepatic mRNA expressions of HMG-CoA reductase, LDL receptor, and cholesterol 7-hydroxylase.     

Hypocholesterolemic activity of onion is mediated by enhancing excretion of fecal sterols in hamsters

Onion has been shown to favorably modify the lipoprotein profile. However, research on its underlying mechanism is lacking. The present study investigated the interaction of dietary onion powder with the protein expression of key receptors and enzymes involved in cholesterol metabolism. Thirty-six male hamsters were randomly divided into three groups and fed a high-cholesterol control diet or the two experimental diets supplemented with 1% onion powder (OP-1) or 5% onion powder (OP-5), for a period of 8 weeks. It was found that onion dose-dependently decreased plasma total cholesterol (TC) level. The change in plasma lipoprotein profile was accompanied by a greater excretion of both fecal neutral and acidic sterols. Western blot analysis revealed that onion up-regulated sterol regulatory element binding protein 2 (SREBP-2), liver X receptor alpha (LXRα) and cholesterol-7α-hydroxylase (CYP7A1) with no effect on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and LDL receptor (LDL-R). It was concluded that the hypocholesterolemic activity of onion powder was mediated by enhancement of fecal sterol excretion and up-regulation of LXRα and CYP7A1.     

双蛋白对大鼠血脂的影响研究

目的 探讨双蛋白对高脂模型大鼠血脂影响及作用机制。 方法 饲喂高脂饲料制备高脂模型,造模后根据血液生化指标分为空白对照组、高血脂模型组、双蛋白低剂量组(0.83g/kg)、双蛋白中剂量组(1.67g/kg)、双蛋白高剂量组(5.00g/kg)五组。连续灌胃6周后处死,测定大鼠血清中总胆固醇(total cholesterol,TC)、总甘油三酯(total glyceride,TG)、低密度脂蛋白胆固醇(low Density Lipoprotein,LDL)、高密度脂蛋白胆固醇(high density liptein cholesterol,HDL-C)。ELISA测定乙酰辅酶A羧化酶(acetyl-CoA carboxylase,ACC)、胰高血糖素(glucagon,GC)、胰岛素(insulin,INS)、3-羟基-3-甲酰基-CoA还原酶(HMG-CoA)含量。 结果 与空白组比较模型组除HMG-CoA外其他指标均有显著差异;与模型组相比,高剂量组LDL-C、TG、TC含量显著下降(P<0.05),GC、HDL-C、INS含量显著提升(P<0.05),各组HMG-CoA水平无显著差异。 结论 双蛋白可降低模型大鼠血脂水平,具有改善血脂异常的功能。其作用机制可能与调节GC、ACC等水平有关。     

Cholesterol oxides and atherosclerosis: A review

Cholesterol is an important constituent of animal food products and has often been implicated in the etiology of atherosclerosis and coronary heart diseases. Recent reports have, however, shown the possible role of cholesterol oxidation products (COP), rather than cholesterol, in the initiation of atherosclerotic plaque formation. Cholestan-3β,5α,6β-triol and 25-hydroxycholesterol have been reported as the most potent atherogenic agents. Inhibition of HMG-CoA reductase (EC 1.1.1.34) enzyme activity and cholesterol biosynthesis by cholesterol oxidation products has also been thoroughly investigated in cultured cells. Various animal food products, viz meat products, egg products and dairy products (especially butter, butter oil, ghee, cheese etc) have been reported to contain various COP developed during certain processing treatments. The literature on the presence of COP in food products and their cytotoxic and atherogenic effects is reviewed.