Microwave assisted one-pot synthesis of novel molecular clefts with only one chiral arm based on deoxycholic acid

A rapid, safe, and efficient method for the synthesis of novel molecular clefts based on deoxycholic acid was reported. Seven new molecular clefts have been synthesized in good yields （89-98%）. This method proved to be extremely simple and highly efficient. The structures of these receptors were confirmed by 1H NMR, IR, MS spectra and elemental analysis. 2007 Zhi Gang Zhao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Ursodeoxycholic acid improves gastrointestinal motility defects in gallstone patients

AIM: To simultaneously evaluate the presence of defects in gallbladder and gastric emptying, as well as in intestinal transit in gallstone patients (GS) and the effect of chronic ursodeoxycholic acid (UDCA) administration on these parameters and on serum bile acids and clinical outcome in GS and controls (CTR). METHODS: After a standard liquid test meal, gallbla-dder and gastric emptying (by ultrasound), oroileal transit time (OITT) (by an immunoenzymatic technique) and serum bile acids (by HPLC) were evaluated before and after 3 mo of UDCA (12 mg/kg bw/d) or placebo administration in 10 symptomatic GS and 10 matched healthy CTR. RESULTS: OITT was longer in GS than in CTR (P < 0.0001); UDCA significantly reduced OITT in GS (P < 0.0001), but not in CTR. GS had longer gastric half-emptying time (t1/2) than CTR (P < 0.0044) at baseline; after UDCA, t1/2 significantly decreased (P < 0.006) in GS but not in CTR. Placebo administration had no effect on gastric emptying and intestinal transit in both GS and CTR. CONCLUSION: The gallstone patient has simultaneous multiple impairments of gallbladder and gastric emptying, as well as of intestinal transit. UDCA administration restores these defects in GS, without any effect in CTR. These results confirm the pathogenetic role of gastrointestinal motility in gallstone disease and suggest an additional mechanism of action for UDCA in reducing bile cholesterol supersaturation.

Design and synthesis of novel tweezer anion receptors based on deoxycholic acid

A novel type of molecular tweezer receptors based on deoxycholic acid has been designed and synthesized and their binding properties were examined by UV-vis spectral titration. These molecular tweezers showed a high selectivity toward F^- over Cl^-, Br^-, I^-, AcO^-, H2PO4^-.

High level of deoxycholic acid in human bile does not promote cholesterol gallstone formation

AIM:To study whether patients with excess deoxycholic acid (DCA) differ from those with normal percentage of DCA with respect to biliary lipid composition and cholesterol saturation of gallbladder bile. METHODS:Bile was collected during operation through puncturing into the gallbladder from 122 cholesterol gallstone patients and 46 gallstone-free subjects undergoing cholecystectomy.Clinical data,biliary lipids,bile add composition, presence of crystals and nucleation time were analyzed. RESULTS:A subgroup of gallstone patients displayed a higher proportion of DCA in bile than gallstone free subjects. By choosing a cut-off level of the 90th percentile,a group of 13 gallstone patients with high DCA levels (mean 50 percent of total bile acids) and a large group of 109 patients with normal DCA levels (mean 21 percent of total bile acids) were obtained.The mean age of the patients with high DCA levels was higher than that of the group with normal levels (mean age:62 years vs45 years) and so was the mean BMI (28.3 vs.24.7).Plasma levels of cholesterol and triglycerides were slightly higher in the DCA excess groups compared with those in the normal DCA group.There was no difference in biliary lipid composition,cholesterol saturation,nucleation time or occurrence of cholesterol crystals in bile between patients with high and normal levels of DCA. CONCLUSION:Gallstone patients with excess DCA were of older age and had higher BMI than patients with normal DCA.The two groups of patients did not differ with respect to biliary lipid composition,cholesterol saturation,nucleation time or occurrence of cholesterol crystals.It is concluded that DCA in bile does not seem to contribute to gallstone formation in cholesterol gallstone patients.

Design and Synthesis of Chiral Molecular Tweezers Based on Deoxycholic Acid

A series of new chiral molecular tweezers have been designed and synthesized by using deoxycholic acid as spacer and aromatic amines as arms. Instead of using toxic phosgene, the triphosgene was employed in synthesis of the molecular tweezers receptors. These chiral molecular tweezers showed good enantioselectivity for D-amino acid methyl esters.

Design and synthesis of novel chiral molecular tweezers based on deoxycholic acid

A novel type of chiral molecular tweezers has been designed and synthesized by using deoxycholic acid as backbone and ethanoyl and the chiral unsymmetrical urea unit as arms. Their structures were characterized by 1H NMR, IR, MS spectra and elemental analysis. These molecular tweezers showed good binding ability for neutral molecules and chiral molecules.

Dichloroacetic Acid (DCA)-Induced Cytotoxicity in Human Breast Cancer Cells Accompanies Changes in Mitochondrial Membrane Permeability and Production of Reactive Oxygen Species

Cancer cells utilize cytosolic glycolysis for their energy production even in the presence of adequate levels of oxygen (Warbug effect) due to mitochondrial defects. Dichloroacetic acid (DCA) shifts cytosolic glucose metabolism to aerobic oxidation by inhibiting mitochondrial pyruvate dehydrogenase kinase (PDK) and increasing pyruvate uptake. Therefore, DCA has potential in reversing the glycolytic metabolism defect in cancerous cells. DCA is also known to induce apoptosis in a number of cancer cell lines, the mechanism of which is not well understood. In this study, an attempt has been made to investigate the effects of DCA on aggressive human breast cancer (MCF-7) cells as compared with less aggressive mouse osteoblastic (MC3T3) cells. Cell cytotoxicity was determined by MTT, crystal violet and Trypan blue exclusion assays. Western blot was used to detect any changes in the expression of apoptotic markers. Flow cytometry was used to measure apoptotic and necrotic effects of DCA. Mitochondrial integrity was determined by change in mitochondrial membrane potential (Δψm), whereas oxidative damage was determined by production of reactive oxygen species (ROS). DCA caused a concentration-dependent cytotoxicity both in MCF-7 and MC3T3 cell lines. MCF-7 cells were most affected. Flow cytometry results showed a significantly higher apoptosis in MCF-7 even at lower concentrations of DCA. However, higher concentrations of DCA were necrotic. Western blotting showed an increased expression of Mn-SOD-1 upon DCA treatment. Further, DCA decreased Δψm and increased ROS production. The effects of DCA were more pronounced on MCF-7 cells as compared to MC3T3 cells. Our results suggest that DCA-induced cytotoxicity in cancerous cells is mediated via changes in Δψm and production of ROS.

MNNG、CDCA构建胃黏膜上皮细胞GES-1肠化生模型的可行性

目的探讨N-甲基-N'-硝基-N-亚硝基胍(MNNG)、鹅脱氧胆酸(CDCA)构建胃黏膜上皮细胞GES-1肠化生模型的可行性。方法体外培养GES-1细胞,分别给予不同浓度CDCA、MNNG处理24 h,采用CCK-8法检测450 nm波长处各孔光密度(OD)值,确定CDCA、MNNG对GES-1细胞具有生长抑制作用,二者的10%抑制浓度(IC10)分别为0.14、51.89μmol/L。另取GES-1细胞随机分为CDCA组、MNNG组,分别加入IC10的CDCA、MNNG,同时设未加药物处理的细胞为对照组,培养24 h时采用实时荧光定量PCR法检测黏蛋白1(MUC1)mRNA、黏蛋白2(MUC2)mRNA及尾型同源框转录因子2(CDX2)mRNA相对表达量。结果与对照组比较,CDCA组、MNNG组MUC1 mRNA相对表达量均降低,MNNG组MUC2 mRNA相对表达量降低,组间比较P均<0.05。三组间CDX2mRNA相对表达量比较P>0.05。结论 CDCA、MNNG可抑制胃黏膜上皮细胞生长,可用于构建胃黏膜上皮细胞的肠化生模型;下调胃特异性基因MUC1表达、上调肠特异性基因CDX2表达可能是二者的作用机制。

线性电位滴定法测定CS-DCA的接枝率

[目的]建立一种壳聚糖去氧胆酸聚合物(deoxycholic acid grafted chitosan,CS-DCA)接枝率测定的新方法。[方法]分别建立并采用线性电位滴定法(linear potentiometric titration,LPT)和2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenesulfonicacid,TNBS)法测定CS-DCA的接枝率,以氢核磁共振波谱(1H-nuclear magnetic resonance,1H-NMR)法为参比,比较分析可替代的接枝率测定方法。[结果]比较CS-DCA的接枝率测定数据,经典方法1H-NMR法与LPT无统计学差异(P>0.05),而与TNBS法具有显著性差异(P<0.05),表明LPT与经典方法的准确度相当;LPT的最大相对误差为3.8%(n=15),而TNBS法最大相对误差为12.8%(n=15),提示LPT有更高的精密度。[结论]LPT可作为一种新的经济、快速且准确的CS-DCA接枝率测定方法。

A secondary bile acid from microbiota metabolism attenuates ileitis and bile acid reduction in subclinical necrotic enteritis in chickens

Background:Clostridium perfringens-induced chicken necrotic enteritis(NE)is responsible for substantial economic losses worldwide annually.Recently,as a result of antibiotic growth promoter prohibition,the prevalence of NE in chickens has reemerged.This study was aimed to reduce NE through titrating dietary deoxycholic acid(DCA)as an effective antimicrobial alternative.Materials and methods:Day-old broiler chicks were assigned to six groups and fed diets supplemented with 0(basal diet),0.8,1.0 and 1.5 g/kg(on top of basal diet)DCA.The birds were challenged with Eimeria maxima(20,000 oocysts/bird)at d 18 and C.perfringens(109 CFU/bird per day)at d 23,24,and 25 to induce NE.The birds were sacrificed at d 26 when ileal tissue and digesta were collected for analyzing histopathology,mRNA accumulation and C.perfringens colonization by real-time PCR,targeted metabolomics of bile acids,fluorescence in situ hybridization(FISH),or terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay.Results:At the cellular level,birds infected with E.maxima and C.perfringens developed subclinical NE and showed shortening villi,crypt hyperplasia and immune cell infiltration in ileum.Dietary DCA alleviated the NE-induced ileal inflammation in a dose-dependent manner compared to NE control birds.Consistent with the increased histopathological scores,subclinical NE birds suffered body weight gain reduction compared to the uninfected birds,an effect attenuated with increased doses of dietary DCA.At the molecular level,the highest dose of DCA at 1.5 g/kg reduced C.perfringens luminal colonization compared to NE birds using PCR and FISH.Furthermore,the dietary DCA reduced subclinical NE-induced intestinal inflammatory gene expression and cell apoptosis using PCR and TUNEL assays.Upon further examining ileal bile acid pool through targeted metabolomics,subclinical NE reduced the total bile acid level in ileal digesta compared to uninfected birds.Notably,dietary DCA increased total bile acid and DCA levels in a dose-dependent manner compared to NE birds.Conclusion:These results indicate that DCA attenuates NE-induced intestinal inflammation and bile acid reduction and could be an effective antimicrobial alternative against the intestinal disease.

Bile acids in serum and bile of patients with cholesterol gallstone

IM To analyze serum bile acids and biliary lipids of patients with cholesterol gallstone(CS) and explore the relationship between deoxycholic acid (DCA) and CS disease.METHODS Analysis of bile acids in serum was done with gaschromatography in two groups: CS group (n=151) and control group (n=256). Serum bile acids and biliary lipids were also studied in 90 matched samples..RESULTS The serum DCA was 0955μmol/L±0078μmol/L in CS group, which was more than that of control group (0696μmol/L±0047μmol/L), P<001. The ratio of DCA/chenodeoxycholic acids (CDCA) was 176±030 in CS group, about two times that in control group (092±014). The mole percent of DCA in bile was positively related to cholesterol saturation index (CSI) (P<001) and the mole percent of CDCA in bile negatively to CSI (P=001). There was correlation between the mole percent of DCA, CDCA and cholic acid in bile and in serum.CONCLUSION It is suggested that DCA is lithogenic and the increased amount of DCA or the ratio of DCA/CDCA in serum may be one of the features of cholesterol gallstone patients.

脱氧胆酸通过ROS/NF-κB通路对Barrett食管细胞氧化应激的影响

目的 探讨脱氧胆酸(DCA)对人BAR-T细胞氧化应激的影响及机制。方法 体外培养人Barrett上皮细胞株BAR-T,采用不同浓度的DCA(100、200、300μmol/L)和不同作用时间(30 min、60 min、3 h、6 h)干预BAR-T细胞。采用Real-time PCR法和Western blotting法检测环氧酶-2(COX-2)的mRNA和蛋白表达;采用显微镜观察和流式细胞仪检测细胞内活性氧(reactive oxygen species, ROS)含量,并与200μmol/L DCA+5 mmol/L ROS清除剂N-乙酰半胱胺酸(NAC)组进行比较;采用细胞免疫荧光法检测细胞p65蛋白入核情况,并与200μmol/L DCA+100μmol/L NF-κB通路抑制剂吡咯烷二硫代甲酸铵(PDTC)组进行比较。结果 与对照组相比,DCA可以显著升高BAR-T细胞中ROS的含量,呈剂量依赖性,5 mmol/L NAC明显抑制DCA诱导的ROS释放。与对照组相比,相同干预时间下,DCA(200、300μmol/L组)均可以显著升高COX-2 mRNA表达。与1 h组相比,200、300μmol/L DCA 6 h组均可以显著升高COX-2 mRNA表达。与对照组比较,200μmol/L DCA可以显著升高COX-2蛋白表达。同时,200μmol/L DCA可以促进p65蛋白的入核,PDTC可以抑制DCA的作用。结论 DCA可能通过升高细胞内ROS水平,促进p65蛋白入核以激活NF-κB信号通路,进而上调COX-2的表达。

胆酸和去氧胆酸抗惊厥作用的血清代谢组学研究

目的:探究胆汁酸单体化合物胆酸(cholic acid,CA)和去氧胆酸(deoxycholic acid,DCA)的抗惊厥作用机制。方法:将3周龄雄性SD大鼠随机分为对照组、模型组、丙戊酸钠组(sodium valproate/valproic acid,VPA,189 mg/kg)、CA组(60 mg/kg)和DCA组(60 mg/kg),每组9只,对照组及模型组为假给药,各给药组于造模前1 h预给药,连续给药16 d。采用(45.0±0.5)℃水浴建立惊厥大鼠模型,每隔1 d水浴1次,共计8次,观察记录模型组及各给药组大鼠惊厥发作时间、惊厥结束时间,对大鼠惊厥发作行为的严重程度进行评分。检测大鼠血清及海马组织中白介素1β(interleukin 1β,IL-1β)、肿瘤坏死因子α(tumor necrosis factor α,TNF-α)和IL-6含量及海马组织中谷氨酸(glutamic acid,Glu)和γ-氨基丁酸(γ-aminobutyric acid,GABA)含量,苏木精-伊红(hematoxylin-eosin,HE)染色观察海马神经元病理损伤。超高效液相色谱仪串联高分辨质谱技术对大鼠血清进行代谢组学分析。结果:与模型组相比,各给药组大鼠惊厥潜伏期均显著延长,惊厥持续时间显著缩短(P均<0.001);VPA组和DCA组惊厥发作等级显著降低(P <0.001,P <0.01),CA组差异无统计学意义。与对照组相比,模型组血清和海马组织中TNF-α,IL-6和IL-1β含量均显著升高(P <0.001),海马组织中Glu和GABA含量均显著升高(P <0.001);与模型组相比,DCA组和VPA组各项生化指标水平均显著降低(P <0.001),而与模型组相比,除血清中IL-1β和海马组织IL-6的水平外,CA组其他各项指标均显著降低(P <0.01)。海马组织HE染色结果显示,与对照组相比,模型组大鼠海马组织中锥体细胞胞体紧缩,体积变小,染色加深,嗜碱性增强,胞质胞核分界不清;与模型组相比,各给药组大鼠海马神经元细胞形态得到明显改善。其中,DCA组大鼠海马神经元细胞形态与VPA组相近。血清代谢组学分析结果经过数据处理、文献及数据库比对,共鉴定出312个差异化合物。通过主成分分析(principal component analysis,PCA)及正交偏最小二乘法判别分析,共筛选出9个差异化合物;代谢通路富集结果显示,CA和DCA的抗惊厥作用主要涉及柠檬酸循环、氨基酸代谢和丁酸代谢通路。结论:CA和DCA对热性惊厥大鼠的行为学和生化指标等均具有一定的改善作用,其作用机制可能与惊厥过程中的能量代谢、氨基酸代谢和丁酸等短链脂肪酸代谢有关。

动物双歧杆菌乳亚种对便秘的改善研究

便秘是常见胃肠道疾病,部分动物双歧杆菌乳亚种(Bifidobacterium animalis subsp.lactis,B.lactis)已被证明对便秘有改善作用,但效果存在差异。为研究不同动物双歧杆菌乳亚种菌株对便秘的缓解效果,建立便秘动物模型,通过3株动物双歧杆菌乳亚种菌株的干预试验,测定便秘相关指标、肠道菌群及其代谢产物水平,探究各菌株缓解便秘的作用及机制。结果表明,CCFM1278和BB12干预显著改善便秘小鼠排便质量、排便粒数、粪便含水质量分数、首粒黑便时间和小肠推进率,而FJSSZ6M5的干预效果不显著。CCFM1278干预调节便秘小鼠肠道菌群中Akkermansia、Ruminiclostridium 6、Ruminococcus1和Alloprevotella相对丰度,改善短链脂肪酸和脱氧胆酸水平,促进胃泌素、生长抑素分泌,抑制水通道蛋白3和水通道蛋白8的基因转录水平的表达。综上,动物双歧杆菌乳亚种CCFM1278具有缓解便秘的潜力,可将为便秘患者的治疗提供新方向。

新型酯键型鹅去氧胆酸分子钳的设计合成

以鹅去氧胆酸为隔离基、芳香族化合物为手臂设计合成了一类新型的分子钳,其结构经1HNMR,IR,MS和元素分析确证,并且考察了其对D/L-氨基酸甲酯的对映选择性识别性能.初步的结果表明,这类手性分子钳对D-氨基酸甲酯有较好的识别性能.

Ca^(2+)、 La^(3+)及Eu^(3+)对NaDC胶团的作用

在水溶液中将脱氧胆酸钠（ NaDC）分别与 CaCl2、 EuCl3及 LaCl3反应，改变反应物浓度和配比，合成了系列脱氧胆酸络合物 .利用红外光谱（ FTIR）、元素分析、 ICP分析及 X射线粉末衍射谱，对它们的组成和结构进行了研究 .结果表明：水溶液中 CaCl2与 NaDC的反应不是简单离子间的反应，改变其反应物浓度和配比，生成组成和结构不同的络合物 ;而 LnCl3与 NaDC反应时，反应物浓度和配比的改变不影响产物的组成和结构 .说明虽然 Ln3＋与 Ca2＋有相似之处，但它们与 NaDC的作用却有很大差别 .

氨基甲酸酯型脱氧胆酸分子钳对氨基酸甲酯的手性识别研究

以脱氧胆酸为spacer,通过三光气桥连各种芳胺 ,合成了新的氨基甲酸酯型分子钳受体 1～ 4.这些化合物的结构经IR ,1HNMR和元素分析所证实 .利用差光谱滴定法考察了其与D/L氨基酸甲酯的对映选择性识别性能 .结果表明 ,分子钳 1～ 4对所考察的氨基酸甲酯均具有识别能力 ,其对D 氨基酸甲酯的识别优于对L 氨基酸甲酯的识别 .从主客体间的大小形状匹配及几何互补关系等方面对这些受体的识别能力及对映选择性进行了讨论 .

内质网应激介导滋养细胞凋亡在妊娠期肝内胆汁淤积症中的作用及机制

目的探讨内质网应激介导滋养细胞凋亡在妊娠期肝内胆汁淤积症(ICP)中的作用及机制。方法收集2015年12月~2016年12月在南方医科大学南方医院住院分娩的ICP孕妇20例为病例组,另以同期因社会因素行择期剖宫产术的20例无合并症的正常孕妇为正常对照组。利用HE染色计量观察正常对照孕妇及ICP孕妇胎盘合体细胞结节数量;RT-PCR法检测正常孕妇及ICP孕妇胎盘组织中GRP78、CHOP、caspase-3及caspase-7的m RNA表达;建立不同浓度胆酸(0、10、50、100μmol/L)体外刺激HTR-8/SVneo人早孕滋养细胞株模型,利用RT-PCR法检测HTR-8/SVneo细胞GRP78、CHOP、caspase-3及caspase-7的m RNA表达及利用蛋白印迹法检测GRP78、CHOP蛋白的表达;caspase-3、caspase-7活性检测试剂盒分别检测其活化程度;电镜观察细胞凋亡形态。结果 ICP组较对照组孕妇胎盘组织中合体细胞结节数明显增多(P<0.01);GRP78、CHOP、caspase-3及caspase-7的m RNA表达明显上调(P<0.05);不同浓度(0、10、50、100μmol/L)去氧胆酸作用细胞24 h后,GRP78、CHOP、caspase-3及caspase-7 m RNA的表达水平较对照组显著增高(P<0.05),GRP78、CHOP蛋白的表达水平较对照组也显著增高,且均存在浓度依赖效应;50μmol/L去氧胆酸作用细胞24 h后检测caspase-3及caspase-7活性增高(P<0.05);50μmol/L去氧胆酸作用细胞12 h后电镜观察HTR-8/SVneo细胞呈现凋亡小体。结论 ICP孕妇胎盘组织中存在滋养细胞过度凋亡及内质网应激激活的现象。去氧胆酸可以呈浓度依赖性地促进滋养细胞内质网应激相关基因m RNA和蛋白的表达,引起细胞凋亡,提示内质网应激介导的滋养细胞凋亡在ICP的发病机制中可能起着重要的作用。

内质网应激对膝骨关节炎大鼠关节软骨细胞的影响

背景:研究认为,内质网应激与糖尿病、扩张性心肌病、神经退行性疾病等许多慢性疾病的发生相关,而它与骨关节炎的发生也密切相关。目的:分析内质网应激对大鼠关节软骨细胞生存状态的影响,以及内质网应激在体内对大鼠骨关节炎发生发展的影响。方法:在体外分离培养大鼠关节软骨细胞,应用10 mg/L衣霉素建立内质网应激模型。Western Blot检测内质网应激相关蛋白葡萄糖调节蛋白78和C/EBP同源蛋白的表达,并分别应用CCK-8和Annexin V-FITC(流式法)检测软骨细胞增殖活性及细胞凋亡情况。体内实验选取SD大鼠15只,行前交叉韧带切断、内侧半月板切除术制作膝关节骨关节炎模型,于关节腔分别注射内质网应激激动剂衣霉素、抑制剂牛磺熊去氧胆酸和空白对照PBS,4周后应用苏木精-伊红染色评估各组关节退变情况。结果与结论:(1)应用衣霉素刺激大鼠关节软骨细胞后,葡萄糖调节蛋白78和C/EBP同源蛋白的表达均显著升高;同时,软骨细胞的细胞增殖活性在衣霉素刺激后逐渐下降,而细胞凋亡率则明显升高;(2)在大鼠骨关节炎模型中,大体标本及苏木精-伊红染色结果均显示,关节腔注射衣霉素组骨关节炎进展加速,而关节腔注射牛磺熊去氧胆酸组骨关节炎进展减慢;(3)结论:内质网应激的过度激活导致体外培养的大鼠关节软骨细胞增殖活性降低及细胞凋亡增加,这可能是导致骨关节炎发生发展的重要机制之一;应用内质网应激抑制剂牛磺熊去氧胆酸可以有效地减少内质网应激导致的骨关节炎的进展。