Effect of bile salts and bile acids on human gastric mucosal epithelial cells

Objective：To explore the effect of bile salt and bile acid on cultured eternalized human gastric mucosa epithelium GES-1 cells. Methods：Cultured eternalized human gastric mucosa epithelium GES-1 cells were treated with media containing 6 different kinds of bile salts and 3 different kinds of bile acids and their mixture with different concentrations： GCDC（glycochenodeoxychoμte）, GDC （glycodeoxychoμte）, GC（glycochoμte）, TCDC（taurochenodeoxychoμte）, TDC（taurodeoxychoμte）, TC （taurochoμte）, LCA （lithocholicacid）, CA（cholic acid）, DCA（deoxycholic acid）（50 μ mol/L,250 μ mol/L,500 μ mol/L,1000 μ mol/L）, DY（mixture of bile salts） and DS（mixture of bile acids）（250 μ mol/L,500 μ mol/L,1000 μ mol/L,1500 μ mol/L, 2000 μ mol/L）, in comparison with the control group（in normal media without bile salts and bile acids）. Cell proliferation was assessed by MTT（3-[4,5-Dimethylthiaolyl]-2,5- diphenyl-tetrazolium bromide） assay for 72 hours with different concentrations and the apoptotic cells were assayed by flow cytometry （FCM） with Annex V-FITC conjugated with propidium iodide（PI） staining for 24 hours with different concentrations（1500,2000 μt mol/L）. Results：There was no significant difference in morphology and cell proliferation in GC group after 24-72 h. Low concentration（50 μ mol/L） of GCDC, GDC, TCDC, TDC and TC accelerated gastric epithelial cell growth in a dosage-time dependent manner. At middle concentration （250-500 μ mol/L）, it showed positive effect after 24-48 h, while negative effect after 72 h. At high concentration（1000 μ mol/L）, it accelerated gastric epithelial cell growth after 24h and show consistent inhibition even leading to necrosis after 48-72 h. LCA and CA showed a positive effect on the concentration of 50 μ mol/L after 24-72 h, while 250-1000 μ mol/L showed a trend towards apoptosis after 24-72 h. At 50-500 μmol/L, DCA showed proliferation after 24 h and apoptosis after 48-72 h, but showed necrosis after 24-72 h at 1000 μmol/L. DY and DS could facilitate normal gastric mucosa epithelial cell growth at low concentration （250-500 μ mol/L）, however at 1000-2000 μ mol/L the trend shifted from apoptosis to necrosis. FCM with Annexin-V conjugated with PI staining revealed that GCDC, GDC, GC, TCDC, TDC, TC, LCA, CA, DCA, DY and DS induced apoptosis of human gastric mucosal epithelial cells. They were all significantly higher than that of the control（P 〈 0.05）, but there was no significant difference in GC group （P 〉 0.05）. The bile salts induced apoptosis in a time-dose-dependent manner. Conclusion：Our results suggested that bile acid and bile salt is the trigger of injury in human gastric mucosal epithelial cells.

High-fat-induced intestinal permeability dysfunction associated with altered fecal bile acids

AIM： To investigate whether high-fat-feeding is associ- ated with increased intestinal permeability via altera- tions in bile acid metabolism.METHODS： Male C57BI/6J mice were fed on a high-fat （n = 26） or low-fat diet （n = 24） for 15 wk. Intestinal permeability was measured from duodenum, jejunum, ileum and colon in an Ussing chamber system using 4 kDa FITC-labeled dextran as an indicator. Fecal bile ac- ids were analyzed with gas chromatography. Segments of jejunum and colon were analyzed for the expression of farnesoid X receptor （FXR） and tumor necrosis factor

Experimental study on the relationship of bile acids and myocardium damage in obstructive jaundice

AIM To investigate the morphologic changes of the myocardium and its relationship to serum bile acids in obstructive jaundice. METHODS Part Ⅰ: 35 rats were randomly assigned to three groups: Group Ⅰ (BDL1, n =11), the common bile duct (CBD) was ligated and severed and killed after one week. Group Ⅱ (BDL2, n =11), the CBD was ligated and severed and killed after two weeks. Group Ⅲ (SO, n =13), the CBD was simply isolated. The hearts were taken for morphologic studies and blood was taken to determine the total serum bile acids (TAB). Part Ⅱ: 13 rats received gastric intubation of 10% 4ml/kg of sodium cholate, and their serum TBA and the morphologic changes of the heart were examined. RESULTS One to two weeks after the CBD was ligated and severed, the mitochondrium of the myocardium was damaged and the serum TBA obviously increased. When the rats were administered sodium cholate to make their peak blood concentration close to the average blood concentration in BDL2, their myocardium was damaged in a similar degree. CONCLUSION The myocardium was damaged in obstructive jaundice and the endogenous bile acids was one of the factors.

Cloning of Bile Salt Hydrolase Gene and Its Expression in Lactic Acid Bacteria

According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.

Changes in plasma total bile acid level of patients with essential hyper tension and of spontaneous hypertension rats and the correlation with systolic and diastolic blood pressures

To examine the hypothesis that plasma bile acid (BA) level is correlated with essential hypertension (EH). Methods: The level of plasma total bile acid (TBA) in 88 patients with EH and in 11 spontaneous hypertension rats (SHR) were measured, and regression analysis of systolic and diastolic blood pressures with plasma TBA was performed. Results: Plasma TBA level in EH patients was significantly higher than that in normotensive subjects (7. 35±3. 38μmol/L vs 4. 94±3. 25 μmol/L, PRO. of ); Plasma TBA level in SHR was significantly higher than that in Wistar--Kyoto (WKY) rats (13. 16±3. 58 μmol/L vs 10. 42±2. 24 μmol/L,P<0. 05); Plasma TBA level in patients with EH was the highest in stage Ⅲ (9. 54±4. 12 μmol/L, n ＝25), the lowest in stage Ⅰ (5. 76±3. 33 μmol/L, n＝33), and middle in stage Ⅱ (7. 32±4. 52 μmol/L, n＝30); Plasma TBA level in patients with EH was positively correlated with both systolic (r= 0. 33, P<0. 01 ) and diastolic blood pressure (r＝0. 46, P＜0．01 ); Plasma TBA level in SHR was positively correlated with both systolic (r＝0. 82, P<0. 01 ) and diastolic blood pressures (r＝0. 69, P<0. 01). Conclusion: elevated level of plasma TBA in patients with EH and in SHR may participate in the pathogenesis of hypertension.

妊娠期肝内胆汁淤积症与妊娠期糖尿病的相关性研究

目的:探讨妊娠期肝内胆汁淤积症(ICP)与妊娠期糖尿病(GDM)的相关性。方法:选取2013年2月至2021年2月在北京大学深圳医院产科分娩的GDM孕妇5517例(GDM组)和非GDM孕妇5517例(非GDM组),采用单因素、多因素和分层分析法分析ICP与GDM、ICP与血糖水平、总胆汁酸与GDM的相关性。结果:GDM组中ICP比例显著大于非GDM组,差异有统计学意义(P<0.05)。GDM合并ICP患者的OGTT-1h血糖显著高于GDM未合并ICP患者(P<0.05),两组患者OGTT-0h或2h血糖比较,差异无统计学意义(P>0.05)。与非GDM组比较,GDM组中重度ICP占ICP总体的比例显著增高(34.88%vs 9.68%,P<0.001);ICP病例中,与未合并GDM患者比较,合并GDM的患者血清总胆汁酸显著升高(中位数29.10μmol/L vs 20.65μmol/L,P<0.01)。多因素分析提示,ICP与GDM显著相关(OR=1.426,95%CI为1.011~2.011,P=0.043),这与非高龄妊娠、单胎妊娠、经产妇中的分层分析结果一致。结论:ICP与GDM存在显著相关性,并且这种相关性在重度ICP中更明显,ICP可能是GDM的风险因素之一。

血浆胆汁酸在肝肿瘤中的差异分析及应用价值

目的分析原发性肝癌与转移性肝癌患者血浆胆汁酸水平及其与临床指标的相关性,并评估其联合AFP对于原发性肝癌的诊断价值。方法纳入2020年8月—2021年9月于上海中医药大学附属曙光医院就诊且具有组织病理学及影像学明确的75例原发性肝癌患者及79例转移性肝癌患者,采集外周血并分别分离血清及血浆,比色法及色谱法检测生化指标,电化学发光免疫分析法检测肿瘤标志物水平,高效液相色谱-串联质谱法检测胆汁酸含量。符合正态分布的计量资料两组间比较采用成组t检验,不符合正态分布的计量资料两组间比较采用Mann-Whitney U检验。相关性检验采用Spearman相关分析。临床诊断效能采用受试者工作特征曲线(ROC曲线)评估。结果原发性肝癌组患者TC、TG、LDL-C及载脂蛋白(Apo)B显著低于转移性肝癌组患者,差异均具有统计学意义(U值分别为1598、1255、909、889,P值均<0.05)。原发性肝癌组患者AFP显著高于转移性肝癌组患者,癌胚抗原(CEA)显著低于转移性肝癌组患者,差异均具有统计学意义(U值分别为1873、926,P值均<0.05)。原发性肝癌组患者总胆汁酸(TBA)、胆酸(CA)、鹅脱氧胆酸(CDCA)、熊脱氧胆酸(UDCA)、牛磺胆酸(TCA)、牛磺鹅脱氧胆酸(TCDCA)、甘氨胆酸(GCA)、甘氨鹅脱氧胆酸(GCDCA)、牛磺熊脱氧胆酸(TUDCA)、甘氨熊脱氧胆酸(GUDCA)均显著高于转移性肝癌患者,脱氧胆酸(DCA)显著低于转移性肝癌患者,差异均具有统计学意义(P值均<0.05)。总人群中TBA、CDCA、GCA、GCDCA、GUDCA、TCA、TCDCA及TUDCA含量与AFP水平呈显著正相关(P值均<0.05)。原发性肝癌患者中GCA、TCA、TCDCA及TUDCA含量与AFP水平呈显著正相关(P值均<0.05)。AFP+TCA+GCA+TCDCA联合检测的AUC为0.822(95%CI:0.746~0.898,P<0.0001),效能最高。结论原发性肝癌与转移性肝癌患者血浆胆汁酸含量具有显著差异,差异性胆汁酸与肝损伤及AFP升高密切相关,联合AFP对原发性肝癌具有更优的临床诊断价值。

细胞焦亡:连接肠道菌群与肝脏疾病的新桥梁

自肠-肝轴的概念被提出以来,已有大量研究着眼于探索肠道菌群和肝病之间的联系,但以细胞焦亡为枢纽,探究肠-肝串扰的内在机制的观点仍处于萌芽阶段。本文主要通过叙述了肠道菌群失调通过影响黏膜屏障的完整性和胆汁酸的代谢,诱导细胞焦亡,进而影响肝脏相关疾病的发生和进展的过程,并总结出肠道菌群失调通过诱导NLRP3/AIM2/Caspase-1依赖型、Caspase-4/11/GSDMD依赖型和Caspase-3/GSDME依赖型细胞焦亡以影响肝脏相关疾病的观点。希望通过建立细胞焦亡与肠-肝免疫串扰之间的联系,为未来肝病的诊治提供新的思路和靶点。

冷冻干燥对益生菌传代稳定性的影响

益生菌随着传代的进行,菌株的产酸能力、耐胆盐能力、耐酸能力和抗氧化能力产生变化。冷冻干燥是菌株常用的保藏方法,对菌体转录水平影响较大,能够临时改变菌株的多种特性。文章以植物乳杆菌M547、M621、M748、戊糖乳杆菌M750为研究对象,探究冷冻干燥对菌株传代稳定性的影响。结果表明,冷冻干燥影响菌株的耐胆盐稳定性,对产酸能力、耐酸能力、抗氧化能力无显著影响。

一株具有缓解酒精性肝损伤的发酵粘液乳杆菌及其功效评价研究

该研究从新疆奶疙瘩中分离鉴定一株发酵粘液乳杆菌(Limosilactobacillus fermentum)WHH2438,以商业菌株鼠李糖乳酪杆菌(Lacticaseibacillus rhamnosus)GG为对照,从抗氧化特性、改善肠道屏障功能、耐受性和黏附性等方面,进行体外功效评价。之后以C57BL/6 J小鼠为实验对象,使用液体酒精饲料建立慢性酒精性肝损伤模型,验证菌株的缓解酒精性肝损伤功能。结果显示,该发酵粘液乳杆菌清除DPPH自由基和羟自由基能力显著优于LGG,在保护肠道细胞和肠道屏障方面也有优秀的能力。通过酒精性肝损伤功能动物剂量效价实验,发现菌株WHH2438高剂量组和灭活组的血清谷丙转氨酶和谷草转氨酶相比模型组明显下降、肝脏甘油三脂和还原型谷胱甘肽下降且接近对照,总体有较明显的改善效果,说明该菌株降低酒精对肝脏的氧化损伤,延缓酒精性脂肪肝的形成,具有良好的缓解酒精性肝损伤功能。综上,发酵粘液乳杆菌WHH2438具有良好的缓解慢性酒精性肝损伤功能,且菌株特性优良,能顺利到达肠道,并黏附于肠道上皮细胞,发挥益生功效。

Influence of gut microbiome composition on effect variations of anti-cholesterol treatment among individual mice

This study investigated if the variation in the effect of anti-cholesterol(AC)treatment on individual mice are related to gut microbiome composition.The bile salt hydrolase(BSH)activity of 23 commercial fermented milk products was examined to select a fermented milk product for AC treatment.Mice were fed to different diets for 6 weeks:high-fat(60%of total calories from fat;D1),high-dietary fibre(20%cellulose;D2),and low-fat(17.2%of total calories from fat;D3)diets to change their gut microbiomes.Subsequently,faecal microbiome was transplanted(FMT)into mice treated with high cholesterol diet contained 2%cholesterol,followed by AC or non-AC(sterile tap water,STW)treatments.Control groups with normal(NC)and highcholesterol diets(PC)were prepared for both AC and STW treatment.All experimental groups were subjected to serum and liver cholesterol,cholesterol metabolism-related(CMR)gene expression,and intestinal microbiome analyses.D3-FMT mice showed the most significant enhancements in cholesterol ratio and decreased hepatic cholesterol levels with AC treatment.Moreover,upregulation of the Cyp7a1 gene expression was observed in this group.Furthermore,the intestinal microbiome analysis indicated higher abundances of BSH-producing Eubacterium,Bifidobacterium,and Parabacteroides in the D3-FMT+AC group compare to others,potentially contributing to increased bile acid synthesis.

酸汤中产酸酵母的筛选及性能测定

产酸酵母作为酸汤发酵过程中的优势菌株,是影响酸汤产品品质的关键因素。本文通过形态学、生理生化和分子生物学,对从酸汤样品中分离出的产酸菌株进行鉴定,并测定分析其产酸、耐酸和耐胆盐能力。结果表明,RS^(-1)4为盔状毕赤酵母(Pichia galeiformis),CS^(-1)6和CS^(-1)9为膜醭毕赤酵母(Pichia membranaefaciens),45、RS^(-1)3、RS^(-1)5、CS-21和CS-22为满洲毕赤酵母(Pichia manshurica)。其中,菌株45的产酸能力最强,总酸含量达到41.47 g·L^(-1),且8株酵母菌均能在pH值为1.5和牛胆盐浓度为0.70%的条件下生长,具有较高的耐酸性能和耐牛胆盐性能。本研究通过对酸汤中产酸酵母的筛选及其性能测定,为开发酸汤纯种发酵菌株和提升酸汤发酵产品品质提供理论支撑。

EDTAK_(2)抗凝真空采血管质量分析

目的 通过对EDTAK_(2)真空采血管公称液体容量、泄漏、添加剂的实际量3个关键性能的检验,评价真空采血管的质量。方法 实验样品共27批。样品分布在6个省份的10家生产企业,其中山东4批(14.8%),河北1批(3.7%),广东1批(3.7%),湖北1批(3.7%),湖南18批(66.7%),贵州2批(7.4%)。按照行业标准YY/T 0314—2007《一次性使用人体静脉血样采集容器》检测27批真空采血管公称液体容量、泄漏和添加剂的实际量3个关键性能。结果 公称液体容量合格率为85.2%,泄漏合格率为100%,添加剂的实际量合格率为60%。通过产品技术要求与行业标准比对,发现部分企业产品技术要求检验方法存在缺陷或检验方法不具操作性,甚至方法错误,存在不合理、不科学。产品标签上未写明具体添加剂的种类、未明确具体使用海拔等问题。不合格的主要原因有采血管生产中抽真空工艺不稳定、添加剂的配制和添加剂的加注量不准确、质量控制不严、产品技术要求编写不严谨,文件编写审核机制不健全等。结论 部分厂家生产的真空采血管存在质量问题,各方应高度重视真空采血管质量控制因素,提高产品的整体质量。

Physiological and molecular biochemical mechanisms of bile formation

This review considers the physiological and molecular biochemical mechanisms of bile formation.The composition of bile and structure of a bile canaliculus,biosynthesis and conjugation of bile acids,bile phospholipids,formation of bile micellar structures,and enterohepatic circulation of bile acids are described.In general,the review focuses on the molecular physiology of the transporting systems of the hepatocyte sinusoidal and apical membranes.Knowledge of physiological and biochemical basis of bile formation has implications for understanding the mechanisms of development of pathological processes,associated with diseases of the liver and biliary tract.

黏豆包发酵液中乳酸菌分离鉴定及其益生性能

从农家自然发酵黏豆包谷物发酵液中分离乳酸菌,对菌株进行鉴定,并对益生性能进行研究。利用梯度稀释法从黏豆包中筛选获得6株乳酸菌,经形态学分析并结合16S rRNA序列分析分别为棒状乳杆菌(Lactobacillus coryniformis)、植物乳杆菌(Lactobacillus plantarum)、短杆菌(Levilactobacillus brevis)、发酵乳杆菌(Lactobacillus fermentans)、清酒乳杆菌(Latilactobacillus sakei)和曲氏乳杆菌(Lactobacillus curvatus)。对菌株的益生性能评价结果表明:菌株HSDF3(曲氏乳杆菌)的抗逆性和耐酸性能较好,pH3.0的环境中存活率为100%;耐盐测试中,菌株HSDF9(清酒乳杆菌)的耐盐效果最好,在盐含量10%的MRS肉汤中仍可存活,活菌数可达8.93×106 CFU/mL;耐胆盐测试中,菌株HSDF12(棒状乳杆菌)存活率最高,在0.3%胆盐浓度的MRS肉汤培养基中培养3 h之后的存活率为7.171%;耐人工胃液测试中,经过3 h的模拟胃液培养之后,菌株HSDF3(曲氏乳杆菌)的存活率最高,达到82.91%;耐人工肠液测试中,经过6 h的模拟肠液培养之后,HSDF12(棒状乳杆菌)存活率最高,达到93.42%;抗生素敏感性试验中,菌株HSDF15(短杆菌)对四环素、羧苄西林、头孢氨苄、米诺环素、头孢唑啉、氨苄西林、头孢拉定等抗生素均敏感。

华南虎源乳酸菌的分离筛选及体外益生特性

乳酸菌对动物肠道健康具有非常重要的作用。为了筛选出具有益生特性的优良菌株,对健康华南虎(Panthera tigris amoyensis)粪便中的乳酸菌进行分离培养、纯化及鉴定,并通过研究菌株对大肠埃希氏杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、沙门氏菌(Salmonella sp.)和迟钝爱德华氏菌(Edwardsiella tarda)的抑菌性及菌株的耐酸、耐胆盐、溶血性和对24种药敏纸片的耐药性,对菌株生物学特性进行评价。结果显示:共分离得到78株乳酸菌,其中15株具有较好的抑菌性能,对以上4种致病菌的抑菌圈均大于15 mm,含6株罗伊氏乳杆菌(Lactobacillus reuteri,22YY08、22DC20、22XX19、22Hui09、22LL14和22XX03)、3株卷曲乳杆菌(L. crispatus,22HH02、22YY13和22YY09)、2株动物乳杆菌(Ligilactobacillus animalis,22Hui15、22LL07)、1株戊糖片球菌(Pediococcus pentosaceus,22DC18)、1株黏膜乳杆菌(Lactobacillus mucosae,22DC10)、1株约氏乳杆菌(L. johnsonii,22DC09)和1株格氏乳球菌(Lactococcus garvieae,GaL110)。通过进一步试验筛选出8株乳酸菌,在pH=2.5和胆盐浓度为0.1%时表现出较好的耐酸、耐胆盐性能,均不产生溶血性,但其中3株罗伊氏乳杆菌22Hui09、22YY08和22LL14对多种药物耐药,其他菌株对多种药物敏感。本试验筛选获得的3株罗伊氏乳杆菌(22DC20、22XX03和22XX19)、1株戊糖片球菌(22DC18)和1株格氏乳球菌(GaL110)有希望作为具有益生特性的菌株应用到华南虎益生菌制剂的开发。

胆汁酸与非酒精性脂肪性肝病的关系

非酒精性脂肪性肝病(NAFLD)患病率的迅速增加亟需新的治疗方法来预防疾病进展为肝纤维化、肝硬化及肝癌。尽管已经为阐明NAFLD疾病进展的病理机制做出了不懈努力,但目前尚无有效的治疗方法。胆汁酸(BA)通过激活核受体和G蛋白偶联受体调控全身代谢,已被确定为参与脂质、葡萄糖和能量代谢的重要信号分子。BA稳态的失调与NAFLD疾病的严重程度有关。本文总结了BA代谢中的重要配体和其在NAFLD进展中的作用,以期为靶向BA信使治疗NAFLD提供依据。

胆盐水解酶筛选及酶法提制鸡胆汁酸的研究

试验旨在从常见双歧杆菌筛选获得高活性胆盐水解酶的菌株并用于鸡胆汁酸的提制。从5种常见双歧杆菌菌株(两歧双歧杆菌、婴儿双歧杆菌、青春双歧杆菌、乳双歧杆菌、长双歧杆菌)中,通过平板Ca2+沉淀法及茚三酮法对活性双歧杆菌进行酶活评价,将高活性菌株的粗酶用于鸡胆汁酸的提制,使用薄层色谱法和高效液相法对所制胆汁酸进行定性和定量检测。结果显示:4种双歧杆菌(两歧双歧杆菌、婴儿双歧杆菌、青春双歧杆菌和乳双歧杆菌)对甘氨型和牛磺型鹅去氧胆酸均具有水解活性,婴儿双歧杆菌对甘氨鹅去氧胆酸和牛磺鹅去氧胆酸比酶活均显著高于其他3种双歧杆菌(P<0.05),比酶活分别为0.48 U/mg和0.27 U/mg。酶法工艺提制鸡胆汁酸的得率为5.34%,提取物中鹅去氧胆酸(CDCA)含量为70.7%。结果表明:婴儿双歧杆菌具有高活性胆盐水解酶,酶法工艺提制鸡胆汁酸反应条件温和,提取物纯度较高,较为节能环保。

发酵蔬菜中乳酸菌分离及其耐酸耐胆盐特性分析

为获得优良的耐酸耐胆盐乳酸菌菌株,从市售发酵蔬菜样品中分离乳酸菌,通过形态学观察以及糖发酵试验对其进行初步鉴定,并开展耐酸耐胆盐初筛及人工胃肠液复筛试验。结果表明:分离出的18株乳酸菌菌株中,有11株具有较高的耐酸耐胆盐能力;经过人工胃肠液复筛后,存活率高于30%的有4株,其中cf12菌株在人工胃液中存活率达到95%,人工肠液处理4、8 h后的存活率分别为173%、194%,菌液浓度未有下降反而有所增加,说明该菌株对低酸性环境及高胆盐环境均具有较强的耐受能力。4株乳酸菌经16S rDNA分子生物学鉴定表明,菌株cf10为植物乳杆菌Lactobacillus plantarum,菌株cf9、cf12为短乳杆菌Lactobacillus brevis,菌株cf18为副短乳杆菌Lactobacillus parabrevis。