17-Hydroxysteroid Dehydrogenases

17 - 羟基类固醇脱氢酶
  • 文章类型: Journal Article
    人们对羟基类固醇脱氢酶12(HSD17B12)在脂质代谢中的功能知之甚少。为了进一步研究这一点,我们创建了肝细胞特异性敲除HSD17B12(LiB12cKO)的小鼠。从两个月开始,这些小鼠的肝脏显示出显著的脂肪积累。随着年龄的增长,他们的全身脂肪百分比也降低了。有趣的是,肝脏脂肪积累并没有导致典型的大脂滴(LD)的形成;相反,小液滴更普遍。因此,随着脂肪含量的增加,LiB12KO肝脏未显示大泡性脂肪变性增加,而微泡脂肪变性是肝脏的主要特征。这表示LD扩展失败。这与肝损伤有关,可能是由于脂毒性。值得注意的是,脂质组学数据不支持HSD17B12在脂肪酸(FA)延伸中的重要作用.然而,我们确实观察到含有碳链长度为18和20个原子的FAs的特定脂质种类的数量减少,包括油酸。其中,磷脂酰胆碱和磷脂酰乙醇胺已被证明在LD形成中起关键作用,这些脂质的数量有限可能是导致LD扩张功能障碍的机制的一部分。Cidec表达的增加进一步支持了LiB12cKO肝脏中LD扩增的缺陷。这种蛋白质对LD的融合和生长至关重要,随着主要尿蛋白家族蛋白质的几个成员的下调,最近被证明在内质网应激过程中发生了改变。
    The function of hydroxysteroid dehydrogenase 12 (HSD17B12) in lipid metabolism is poorly understood. To study this further, we created mice with hepatocyte-specific knockout of HSD17B12 (LiB12cKO). From 2 months on, these mice showed significant fat accumulation in their liver. As they aged, they also had a reduced whole-body fat percentage. Interestingly, the liver fat accumulation did not result in the typical formation of large lipid droplets (LD); instead, small droplets were more prevalent. Thus, LiB12KO liver did not show increased macrovesicular steatosis with the increasing fat content, while microvesicular steatosis was the predominant feature in the liver. This indicates a failure in the LD expansion. This was associated with liver damage, presumably due to lipotoxicity. Notably, the lipidomics data did not support an essential role of HSD17B12 in fatty acid (FA) elongation. However, we did observe a decrease in the quantity of specific lipid species that contain FAs with carbon chain lengths of 18 and 20 atoms, including oleic acid. Of these, phosphatidylcholine and phosphatidylethanolamine have been shown to play a key role in LD formation, and a limited amount of these lipids could be part of the mechanism leading to the dysfunction in LD expansion. The increase in the Cidec expression further supported the deficiency in LD expansion in the LiB12cKO liver. This protein is crucial for the fusion and growth of LDs, along with the downregulation of several members of the major urinary protein family of proteins, which have recently been shown to be altered during endoplasmic reticulum stress.
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  • 文章类型: Journal Article
    在哺乳动物中,17-β羟基类固醇脱氢酶2(Hsd17b2)酶特异性催化C17羟基的氧化,并有效调节雌激素和雄激素的活性,以预防由激素紊乱引起的疾病。然而,hsd17b2基因在动物性别分化中的功能尚不清楚。稻田鳗鱼(黄翅目),一种具有小基因组大小(2n=24)的雌雄同体的雌雄同体鱼,通常用作研究脊椎动物性别分化机制的理想模型。因此,在这项研究中,克隆了鳗鱼hsd17b2基因cDNA,并测定了其mRNA表达谱。克隆的hsd17b2cDNA片段为1230bp,包括一个1107bp的开放阅读框,编码368个氨基酸残基,具有保守的催化亚基。此外,实时定量逆转录聚合酶链反应(RT-qPCR)分析表明,hsd17b2mRNA在发育早期的卵巢中表达强烈,在肝脏和肠道中较弱,几乎不在睾丸和其他组织中。特别是,早期发现hsd17b2mRNA在幼鱼和卵睾丸的卵巢中表达达到峰值,并最终在性腺中从晚期卵睾丸下降到睾丸。同样,化学原位杂交结果表明,在I-II期卵细胞和卵母细胞的细胞质中主要检测到hsd17b2mRNA信号,随后在Ⅲ-Ⅳ期集中在卵母细胞周围的颗粒细胞中,但在成熟卵母细胞和雄性生殖细胞中检测不到。有趣的是,在稻田鳗鱼卵巢里,17β-雌二醇(E2)或他莫昔芬(17β-雌二醇抑制剂,E2I)在低浓度(10ng/mL)下诱导,而在高浓度(100ng/mL)下通过E2I诱导增加。另一方面,褪黑激素(MT)和氟他胺(雄激素抑制剂,AI)诱导可显著降低鳗鱼卵巢hsd17b2mRNA的表达。本研究为揭示鱼类性别分化的机制提供了线索。我们的研究结果表明,hsd17b2基因可能是稻田鳗鱼和其他雌雄同体鱼类的性分化和性别逆转的关键调节因子。
    In mammals, 17-beta hydroxysteroid dehydrogenase 2 (Hsd17b2) enzyme specifically catalyzes the oxidation of the C17 hydroxyl group and efficiently regulates the activities of estrogens and androgens to prevent diseases induced by hormone disorders. However, the functions of the hsd17b2 gene involved in animal sex differentiation are still largely unclear. The ricefield eel (Monopterus albus), a protogynous hermaphroditic fish with a small genome size (2n = 24), is usually used as an ideal model to study the mechanism of sex differentiation in vertebrates. Therefore, in this study, hsd17b2 gene cDNA was cloned and its mRNA expression profiles were determined in the ricefield eel. The cloned cDNA fragment of hsd17b2 was 1230 bp, including an open reading frame of 1107 bp, encoding 368 amino acid residues with conserved catalytic subunits. Moreover, real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis showed that hsd17b2 mRNA expressed strongly in the ovaries at early developmental stages, weakly in liver and intestine, and barely in testis and other tissues. In particular, hsd17b2 mRNA expression was found to peak in ovaries of young fish and ovotestis at the early stage, and eventually declined in gonads from the late ovotestis to testis. Likewise, chemical in situ hybridization results indicated that the hsd17b2 mRNA signals were primarily detected in the cytoplasm of oogonia and oocytes at stage I-II, subsequently concentrated in the granulosa cells around the oocytes at stage Ⅲ-Ⅳ, but undetectable in mature oocytes and male germ cells. Intriguingly, in ricefield eel ovaries, hsd17b2 mRNA expression could be significantly reduced by 17β-estradiol (E2) or tamoxifen (17β-estradiol inhibitor, E2I) induction at a low concentration (10 ng/mL) and increased by E2I induction at a high concentration (100 ng/mL). On the other hand, both the melatonin (MT) and flutamide (androgen inhibitor, AI) induction could significantly decrease hsd17b2 mRNA expression in the ovary of ricefield eel. This study provides a clue for demonstrating the mechanism of sexual differentiation in fish. The findings of our study imply that the hsd17b2 gene could be a key regulator in sexual differentiation and modulate sex reversal in the ricefield eel and other hermaphroditic fishes.
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  • 文章类型: Journal Article
    乳腺癌(BC)仍然是世界健康的主要问题之一,尤其是对女性来说,这就需要创新的治疗策略。在这项研究中,我们研究了维甲酸衍生物作为17β-羟基类固醇脱氢酶1型(17β-HSD1)抑制剂的功效,在雌激素的生物合成和代谢中起着至关重要的作用,从而影响BC的进展,这项调查的主要目的是通过计算药物设计方法,包括PASS预测,确定针对BC的可能候选药物。分子对接,ADMET分析,分子动力学模拟(MD)和密度泛函理论(DFT)计算。结果已经报道了115个衍生物中总共8个具有高结合亲和力和有希望的药代动力学性质。特别是,配体04和07表现出更高的结合亲和力,值为-9.9kcal/mol和-9.1kcal/mol,分别,比标准药物盐酸表柔比星,其具有-8.2kcal/mol的结合亲和力。通过MD模拟在100-ns的轨迹上进一步证实了配体-蛋白质复合物的稳定性,其中包括对氢键的评估,均方根偏差(RMSD),均方根波动(RMSF),动态互相关矩阵(DCCM)和主成分分析。该研究强调需要进行实验验证以确认这些化合物的治疗效用。这项研究增强了对新BC药物的计算搜索,并为后续的实验和临床研究奠定了坚实的基础。
    Breast cancer (BC) is still one of the major issues in world health, especially for women, which necessitates innovative therapeutic strategies. In this study, we investigated the efficacy of retinoic acid derivatives as inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), which plays a crucial role in the biosynthesis and metabolism of oestrogen and thereby influences the progression of BC and, the main objective of this investigation is to identify the possible drug candidate against BC through computational drug design approach including PASS prediction, molecular docking, ADMET profiling, molecular dynamics simulations (MD) and density functional theory (DFT) calculations. The result has reported that total eight derivatives with high binding affinity and promising pharmacokinetic properties among 115 derivatives. In particular, ligands 04 and 07 exhibited a higher binding affinity with values of -9.9 kcal/mol and -9.1 kcal/mol, respectively, than the standard drug epirubicin hydrochloride, which had a binding affinity of -8.2 kcal/mol. The stability of the ligand-protein complexes was further confirmed by MD simulations over a 100-ns trajectory, which included assessments of hydrogen bonds, root mean square deviation (RMSD), root mean square Fluctuation (RMSF), dynamic cross-correlation matric (DCCM) and principal component analysis. The study emphasizes the need for experimental validation to confirm the therapeutic utility of these compounds. This study enhances the computational search for new BC drugs and establishes a solid foundation for subsequent experimental and clinical research.
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  • 文章类型: Journal Article
    类固醇激素表现出强大的内分泌干扰活性,并已被证明会破坏水生生态系统的平衡,并通过其持续和致癌作用对公众健康构成威胁。中华芽孢杆菌HN14是一种中度嗜盐细菌,能够有效降解各种多环芳烃和其他有机污染物,以前是孤立的。此外,菌株HN14在各种环境胁迫条件下表现出较强的环境适应性。在这项研究中,首次研究了菌株HN14对类固醇的降解。我们证明了菌株HN14可以降解雌二醇(E2)以维持菌株的生长,并可以将E2转化为雌酮。此外,证明了在高盐度和高底物浓度条件下,中华黄连HN14的高效底物降解效率。此外,一种17β-羟基类固醇脱氢酶,17β-HSD(HN14),在菌株HN14中鉴定。比较分析表明,17β-HSD(HN14)与来自红球菌属的17β-HSDx具有约38%的序列同一性。P14.此外,100µg纯化的17β-HSD(HN14)可以在1小时内有效转化约40%的0.25mME2,酶活性为17.5U/mg,并在C-17位催化E2和睾酮的脱氢。纯化的酶特性的表征表明,即使在高达20%的高盐度条件下,17β-HSD(HN14)也表现出出色的结构稳健性和酶促效力。总的来说,这项研究增强了我们对菌株HN14中类固醇生物降解的理解,并为推进针对高盐环境中类固醇污染的生物修复技术提供了新的思路和理论基础。
    Steroid hormones exhibit potent endocrine disrupting activity and have been shown to disrupt the equilibrium of aquatic ecosystems and pose a threat to public health through their persistent and carcinogenic effects. Pontibacillus chungwhensis HN14, a moderately halophilic bacterium with the capacity to effectively degrade various polycyclic aromatic hydrocarbons and other organic pollutants, was previously isolated. Additionally, the strain HN14 showed strong environmental adaptability under various environmental stress conditions. In this study, the steroid degradation by strain HN14 was studied for the first time. We demonstrated that strain HN14 could degrade estradiol (E2) to maintain the growth of the strain and could convert E2 to estrone. Additionally, the efficient substrate degradation efficiency of P. chungwhensis HN14 under high salinity and high substrate concentration conditions was demonstrated. Furthermore, a 17β-hydroxysteroid dehydrogenase, 17β-HSD(HN14), was identified in strain HN14. Comparative analysis reveals that 17β-HSD(HN14) shares approximately 38% sequence identity with 17β-HSDx from Rhodococcus sp. P14. In addition, 100 µg of purified 17β-HSD(HN14) could effectively convert about 40% of 0.25 mM of E2 within 1 h period, with an enzyme activity of 17.5 U/mg, and catalyze the dehydrogenation of E2 and testosterone at the C-17 position. The characterization of purified enzyme properties reveals that 17β-HSD(HN14) exhibits exceptional structural robustness and enzymatic efficacy even under high salinity conditions of up to 20%. Overall, this study enhances our comprehension of steroid biodegradation in strain HN14 and contributes novel ideas and theoretical underpinnings for advancing bioremediation technologies targeting steroid pollution in high-saline environments.
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  • 文章类型: Journal Article
    对羟基苯甲酸酯是化妆品中常用的防腐剂,食物,和医药产品。这项研究的目的是检查九种对羟基苯甲酸酯对人和大鼠17β-羟基类固醇脱氢酶1(17β-HSD1)在人胎盘和大鼠卵巢细胞溶胶中的作用,以及BeWo细胞中雌二醇的合成。结果表明,这些化合物的IC50值从对人17β-HSD1的抑制作用最弱的对羟基苯甲酸甲酯(106.42μM)到抑制作用最强的对羟基苯甲酸己酯(2.05μM)不等。模式作用分析表明,这些化合物充当混合抑制剂。对老鼠来说,IC50值范围从对羟基苯甲酸甲酯的最弱抑制作用(100μM时无抑制作用)到对羟基苯甲酸己酯的最有效抑制作用(0.87μM),它们起混合抑制剂的作用。对接分析表明,对羟基苯甲酸酯结合到桥接人17β-HSD1的NADPH和类固醇结合位点以及大鼠17β-HSD1的NADPH结合位点的区域。双变量相关分析表明LogP之间呈负相关,分子量,重原子,和非极性去溶剂化能量,和这些化合物的IC50值。总之,这项研究确定了对羟基苯甲酸酯的抑制作用及其对人和大鼠17β-HSD1的结合机制,以及它们对激素合成的影响。
    Parabens are commonly used preservatives in cosmetics, food, and pharmaceutical products. The objective of this study was to examine the effect of nine parabens on human and rat 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1) in human placental and rat ovarian cytosols, as well as on estradiol synthesis in BeWo cells. The results showed that the IC50 values for these compounds varied from methylparaben with the weakest inhibition (106.42 μM) to hexylparaben with the strongest inhibition (2.05 μM) on human 17β-HSD1. Mode action analysis revealed that these compounds acted as mixed inhibitors. For rats, the IC50 values ranged from the weakest inhibition for methylparaben (no inhibition at 100 μM) to the most potent inhibition for hexylparaben (0.87 μM), and they functioned as mixed inhibitors. Docking analysis indicated that parabens bind to the region bridging the NADPH and steroid binding sites of human 17β-HSD1 and the NADPH binding site of rat 17β-HSD1. Bivariate correlation analysis demonstrated negative correlations between LogP, molecular weight, heavy atoms, and apolar desolvation energy, and the IC50 values of these compounds. In conclusion, this study identified the inhibitory effects of parabens and their binding mechanisms on human and rat 17β-HSD1, as well as their impact on hormone synthesis.
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  • 文章类型: Journal Article
    目的:慢性乙型肝炎(CHB)的病毒抑制限制了肝细胞癌(HCC)的进展;然而,一些患者尽管接受了抗病毒治疗,但仍有进展。单核苷酸多态性(SNPs)的存在,如PNPLA3rs738409和TM6SF2rs58542926与脂肪肝病的发展和进展为HCC相关。而HSD17B13rs72613567:TA中的剪接变体已被证明具有保护性。我们调查了这些SNP在肝癌的发展或预后在纯CHB病因的作用,在没有肝脏脂肪变性的情况下,仍然未知。
    方法:我们分析了由健康对照组成的前瞻性招募队列(n=323)中的PNPLA3rs738409、TM6SF2rs58542926和HSD17B13rs72613567SNP,CHB和CHB-HCC患者无肝脂肪变性。通过PCR分析确定SNP,并使用调整逻辑回归分析研究等位基因和基因型的关联。从CHB-HCC患者收集总生存(OS)数据进行生存分析。
    结果:PNPLA3rs738409,TM6SF2rs58542926和HSD17B13rs72613567的基因型和等位基因分布在健康对照组之间相似,CHB,和CHB-HCC组。没有基因型,等位基因或单倍型分析被发现与CHB-HCC风险增加相关。生存分析显示,在CHB-HCC患者中,没有基因型或等位基因与OS相关。
    结论:我们无法证明PNPLA3rs738409,TM6SF2rs58542926和HSD17B13rs72613567与CHB-HCC的发展或预后的任何关联,支持最初的假设,即它们应被视为以肝脏脂肪变性为特征的肝脏疾病的特定热点。
    OBJECTIVE: Progression to hepatocellular carcinoma (HCC) is restricted by viral suppression in chronic hepatitis B (CHB); however, some patients still progress despite antiviral therapy. Presence of single nucleotide polymorphisms (SNPs) such as PNPLA3 rs738409 and TM6SF2 rs58542926 are associated with the development and progression of steatotic liver disease to HCC, whereas a splice variant in HSD17B13 rs72613567:TA has been shown to be protective. We investigated the role of these SNPs in the development or prognosis of HCC in pure CHB etiology, in the absence of hepatic steatosis, remains unknown.
    METHODS: We analysed PNPLA3 rs738409, TM6SF2 rs58542926, and HSD17B13 rs72613567 SNPs in a prospectively recruited cohort (n=323) consisting of healthy controls, CHB and CHB-HCC patients without hepatic steatosis. SNPs were determined by PCR analysis and associations for the alleles and genotypes were investigated using adjusted-logistic regression analyses. The overall survival (OS) data were collected from CHB-HCC patients for survival analysis.
    RESULTS: The genotype and allelic distribution of PNPLA3 rs738409, TM6SF2 rs58542926, and HSD17B13 rs72613567 were similar between healthy controls, CHB, and CHB-HCC groups. No genotype, allele or haplotype analysis was found to be associated with increased risk for CHB-HCC. Survival analysis revealed no genotype or allele to be associated with OS in patients with CHB-HCC.
    CONCLUSIONS: We could not demonstrate any association of PNPLA3 rs738409, TM6SF2 rs58542926, and HSD17B13 rs72613567 with the development or prognosis of CHB-HCC, supporting the initial hypothesis that they should be considered specific hotspots for liver diseases characterized with hepatic steatosis.
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  • 文章类型: Journal Article
    L-岩藻糖(6-脱氧-L-半乳糖),哺乳动物细胞产生的糖脂和糖蛋白中丰富的单糖,已广泛研究了其在细胞内生物合成和GDP-L-岩藻糖用于岩藻糖基化的再循环中的作用。然而,在某些哺乳动物物种中,L-岩藻糖在鲜为人知的代谢途径中被有效地分解为丙酮酸和乳酸。在1970年代,L-岩藻糖脱氢酶,负责该途径初始步骤的酶,从猪和兔肝脏中部分纯化,并进行了生化表征。然而,直到最近,它的分子身份仍然难以捉摸。这项研究报告了纯化,identification,和哺乳动物L-岩藻糖脱氢酶的生化特征。该酶从兔肝脏中纯化约340倍。纯化的蛋白质制剂的质谱分析鉴定哺乳动物羟基类固醇17-β脱氢酶14(HSD17B14)为唯一的候选酶。兔和人HSD17B14在HEK293T和大肠杆菌中表达,分别,纯化并证明催化L-岩藻糖氧化为L-fucono-1,5-内酯,通过质谱和核磁共振分析证实。底物特异性研究揭示L-岩藻糖是两种酶的优选底物。人酶对L-岩藻糖的催化效率比其对雌二醇的效率高359倍。此外,重组大鼠HSD17B14对L-岩藻糖的活性可忽略不计,与该物种中缺乏L-岩藻糖代谢一致。编码哺乳动物L-岩藻糖脱氢酶的基因的鉴定提供了对属于17-β-羟基类固醇脱氢酶家族的酶的底物特异性的新见解。这一发现也为揭示L-岩藻糖降解途径的生理功能铺平了道路。这仍然是神秘的。
    L-Fucose (6-deoxy-L-galactose), a monosaccharide abundant in glycolipids and glycoproteins produced by mammalian cells, has been extensively studied for its role in intracellular biosynthesis and recycling of GDP-L-fucose for fucosylation. However, in certain mammalian species, L-fucose is efficiently broken down to pyruvate and lactate in a poorly understood metabolic pathway. In the 1970s, L-fucose dehydrogenase, an enzyme responsible for the initial step of this pathway, was partially purified from pig and rabbit livers and characterized biochemically. However, its molecular identity remained elusive until recently. This study reports the purification, identification, and biochemical characterization of the mammalian L-fucose dehydrogenase. The enzyme was purified from rabbit liver approximately 340-fold. Mass spectrometry analysis of the purified protein preparation identified mammalian hydroxysteroid 17-β dehydrogenase 14 (HSD17B14) as the sole candidate enzyme. Rabbit and human HSD17B14 were expressed in HEK293T and Escherichia coli, respectively, purified, and demonstrated to catalyze the oxidation of L-fucose to L-fucono-1,5-lactone, as confirmed by mass spectrometry and NMR analysis. Substrate specificity studies revealed that L-fucose is the preferred substrate for both enzymes. The human enzyme exhibited a catalytic efficiency for L-fucose that was 359-fold higher than its efficiency for estradiol. Additionally, recombinant rat HSD17B14 exhibited negligible activity towards L-fucose, consistent with the absence of L-fucose metabolism in this species. The identification of the gene-encoding mammalian L-fucose dehydrogenase provides novel insights into the substrate specificity of enzymes belonging to the 17-β-hydroxysteroid dehydrogenase family. This discovery also paves the way for unraveling the physiological functions of the L-fucose degradation pathway, which remains enigmatic.
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  • 文章类型: Journal Article
    肠道菌群可能影响代谢功能障碍相关的脂肪变性肝病(MASLD)的严重程度和进展。我们旨在通过磁共振弹性成像评估肠道菌群失调和纤维化分期的临床参数。这项研究包括156名MASLD患者,分为无/轻度纤维化(F0-F1)和中度/重度纤维化(F2-F4)。针对16SrRNA基因的V4区域对粪便标本进行测序,并使用生物信息学进行分析。PNPLA3,TM6SF2和HSD17B13的基因分型通过等位基因区分测定进行评估。我们的数据表明,组间的肠道微生物谱在β-多样性方面存在显着差异,但在α-多样性指数方面没有显着差异。富梭菌和大肠杆菌志贺氏菌,与F0-F1组相比,在F2-F4组中发现了耗尽的Lachnospira。与F0-F1相比,F2-F4组肠上皮通透性和细菌易位的血浆替代标志物升高。细菌属,PNPLA3多态性,老年,在多变量分析中,糖尿病与晚期纤维化独立相关.使用随机森林分类器,3个属的肠道微生物特征可以以很高的诊断准确率(AUC为0.93)区分各组.这些结果表明,富集病原菌和减少有益菌的失衡,与一些临床和遗传因素有关,是MASLD发病机制和进展的潜在贡献者。
    Gut microbiota might affect the severity and progression of metabolic dysfunction-associated steatotic liver disease (MASLD). We aimed to characterize gut dysbiosis and clinical parameters regarding fibrosis stages assessed by magnetic resonance elastography. This study included 156 patients with MASLD, stratified into no/mild fibrosis (F0-F1) and moderate/severe fibrosis (F2-F4). Fecal specimens were sequenced targeting the V4 region of the 16S rRNA gene and analyzed using bioinformatics. The genotyping of PNPLA3, TM6SF2, and HSD17B13 was assessed by allelic discrimination assays. Our data showed that gut microbial profiles between groups significantly differed in beta-diversity but not in alpha-diversity indices. Enriched Fusobacterium and Escherichia_Shigella, and depleted Lachnospira were found in the F2-F4 group versus the F0-F1 group. Compared to F0-F1, the F2-F4 group had elevated plasma surrogate markers of gut epithelial permeability and bacterial translocation. The bacterial genera, PNPLA3 polymorphisms, old age, and diabetes were independently associated with advanced fibrosis in multivariable analyses. Using the Random Forest classifier, the gut microbial signature of three genera could differentiate the groups with high diagnostic accuracy (AUC of 0.93). These results indicated that the imbalance of enriched pathogenic genera and decreased beneficial bacteria, in association with several clinical and genetic factors, were potential contributors to the pathogenesis and progression of MASLD.
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  • 文章类型: Journal Article
    乳腺癌细胞的生长通常依赖于类固醇激素的存在。17β-羟基类固醇脱氢酶1型同工型(17βHSD1)催化NADPH依赖性的雌酮转化为雌二醇,更有效的雌激素,并代表了乳腺癌治疗的潜在药物靶标。为抑制剂筛选提供活性酶,17βHSD1通常在昆虫或哺乳动物细胞中表达,或从人胎盘中分离出来。在本研究中,我们描述了从BL21(DE3)大肠杆菌细胞中表达和纯化活性人17βHSD1的简单方案。使用基于pET28a(+)的质粒表达可溶性人17βHSD1,编码与蛋白质N末端融合的六组氨酸标签,并通过镍亲和层析纯化。纯化的17βHSD1的酶活性通过三种方法进行验证:薄层色谱法,碱测定和光谱测定。这些非放射性酶检测只需要标准的实验室设备,并且可用于筛选调节17βHSD1活性的化合物。
    Breast cancer cell growth is often dependent on the presence of steroidal hormones. The 17β-hydroxysteroid dehydrogenase type 1 isoform (17βHSD1) catalyzes NADPH-dependent conversion of estrone to estradiol, a more potent estrogen, and represents potential drug target for breast cancer treatment.  To provide active enzyme for inhibitor screening, 17βHSD1 is usually expressed in insect or mammalian cells, or isolated from human placenta. In the present study we describe a simple protocol for expression and purification of active human 17βHSD1 from BL21(DE3) Escherichia coli cells. Soluble human 17βHSD1 was expressed using a pET28a(+)-based plasmid, which encodes a hexahistidine tag fused to the N-terminus of the protein, and purified by nickel affinity chromatography. The enzyme activity of purified 17βHSD1 was verified by three methods: thin-layer chromatography, an alkali assay and a spectroscopic assay. These non-radioactive enzyme assays require only standard laboratory equipment, and can be used for screening compounds that modulate 17βHSD1 activity.
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  • 文章类型: Journal Article
    背景:基因检测可用于评估疾病风险。我们评估了是否使用三个单核苷酸多态性(SNP),单独或合并为遗传风险评分(GRS),可以帮助识别患有代谢功能障碍相关脂肪变性肝病(MASLD)的受试者中的显著纤维化。
    方法:我们评估了三种已知的风险变异:PNPLA3rs738409、TM6SF2rs58542926和HSD17B13rs72613567。该研究包括414名来自丹麦人口的成年人,由于ALT升高和体重指数(BMI)>25kg/m2而被定义为MASLD的风险。对参与者进行临床评估,并通过纤维化-4(FIB-4)指数和Fibroscan进行评估。
    结果:总计,17名参与者(4.1%)患有酒精相关性肝病,79(19.1%)没有肝病的证据,4人(1.0%)被诊断出患有其他肝脏疾病,包括恶性疾病。其余314名参与者(75.8%)被诊断为MASLD。在27名因疑似纤维化而接受肝活检的患者中,15例具有显著纤维化(≥F2),12例无/轻度纤维化(F0/F1)。GRS与明显的纤维化无关(p=0.09),但PNPLA3的风险比为6.75(95%CI1.29-50.7;p=0.039)。PNPLA3联合增加的Fib-4(>1.3)的诊断准确性对于检测显着的纤维化是极好的,灵敏度为1.00(95%CI0.72-1.00),但特异性并不比单独的FIB-4好。
    结论:本研究没有证据支持使用GRS诊断MASLD中的显著纤维化。然而,PNPLA3和Fib-4的组合大大提高了灵敏度。此外,ALT仍然是筛选诊断其他肝脏疾病比MASLD有用的工具。
    BACKGROUND: Genetic testing can be used to evaluate disease risk. We evaluated if the use of three Single Nucleotide Polymorphisms (SNPs), alone or combined into a genetic risk score (GRS), can aid identify significant fibrosis in subjects with metabolic dysfunction-associated steatotic liver disease (MASLD).
    METHODS: We assessed three known risk variants: PNPLA3 rs738409, TM6SF2 rs58542926, and HSD17B13 rs72613567. The study included 414 adult individuals invited from the Danish population, who were defined as at-risk of MASLD due to elevated ALT and body mass index (BMI) >25 kg/m2. Participants were assessed clinically and by the Fibrosis-4 (FIB-4) index and Fibroscan.
    RESULTS: In total, 17 participants (4.1 %) had alcohol-related liver disease, 79 (19.1 %) had no evidence of liver disease, and four (1.0 %) were diagnosed with other liver diseases, including malignant disease. The remaining 314 participants (75.8 %) were diagnosed with MASLD. Of the 27 who underwent a liver biopsy for suspected fibrosis, 15 had significant fibrosis (≥F2) and 12 had no/mild fibrosis (F0/F1). The GRS was not associated with significant fibrosis (p = 0.09) but PNPLA3 was with an odds ratio of 6.75 (95 % CI 1.29 - 50.7; p = 0.039) risk allele CG/GG versus CC. The diagnostic accuracy of PNPLA3 combined with an increased Fib-4 (>1.3) was excellent for detecting significant fibrosis with a sensitivity of 1.00 (95 % CI 0.72-1.00), but the specificity was no better than for FIB-4 alone.
    CONCLUSIONS: This study found no evidence to support the use of GRS for diagnosing significant fibrosis in MASLD. However, the combination of PNPLA3 and Fib-4 increased sensitivity considerably. In addition, ALT remains a useful tool for screening diagnosing other liver diseases than MASLD.
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