十味蒂达胶囊对动物胆汁分泌及胆石溶解作用的研究

目的观察十味蒂达胶囊对大鼠胆汁分泌及对兔植入人胆石的溶石作用.方法将Wistar大鼠麻醉后,在胆总管插入引流管,比较给药前后胆汁分泌量及各组给药后胆红素含量的变化.新西兰大耳白兔麻醉后,植入人胆石,比较给药组与对照组胆石溶解的程度.结果十味蒂达胶囊2.4%,1.2%,0.6%三个剂量组均能增加大鼠胆汁分泌,胆红素含量,胆汁分泌依次为0.66%±0.19%,0.62%±0.24%,0.35mL±0.25mL.与生理盐水组0.08mL±0.054mL相比有非常显著差异t检验P<0.001,胆红素含量依次为124.48μmol/L±14.81μmol/L,78.20μmol/L±17.13μmol/L,71.49μmol/L±15.97μmol/L,与生理盐水组54.14μmol/L±21.67μmol/L相比,高、中剂量组均有非常显著性差异t检验P<0.001和P<0.05.体外溶石实验显示,十味蒂达胶囊在1%,0.5%,0.1%三个不同浓度均有明显的溶石作用,高、中、低三个不同浓度组胆石变化率分别为4.41%,3.7%,3.5%.蒸馏水组为1.93%;胆固醇含量;蒸馏水组为0.85±0.16,三个不同浓度组分别为0.65±0.15,0.63±0.13,0.59±0.11,经统计学处理,t检验有显著性差异,P<0.05.体内溶石实验显示,给药后植入兔胆囊内的胆石重量较植入前自身比较明显减轻或溶解消失,经统计学处理,t检验有非常显著的差异,P<0.001.结论十味蒂达胶囊对动物有明显的利胆、溶石作用.     

胆道排石胶囊的药效学研究——防治食饵性胆结石作用

胆道排石胶囊 １００、５００ｍ ｇ／ｋｇ 　ｐｏ 能显著降低豚鼠食饵性胆结石的发生率,降低胆汁中游离胆红素的百分含量;２００、５００ｍ ｇ／ｋｇ ｉｇ 可明显增加大鼠胆汁流量,抑制醋酸所致小鼠的扭体反应次数。提示该方具有预防食饵性胆结石的形成、利胆和镇痛等作用。     

胆固醇和脂肪酸对CYP7A1的调节

胆固醇7α-羟化酶(CYP7A1)是胆固醇转化为胆酸过程中的关键酶,对维持机体的胆固醇内环境稳定起着重要作用。胆固醇对CYP7Al有诱导作用,在种属之间有很大差异。新近发现胆固醇并不单独决定该酶的活性,膳食中的脂肪酸影响胆固醇的组合,最终决定该酶的活性。这些调节作用主要通过肝X受体α(LXRα)、视黄醇X受体α(RXRα)和过氧化物酶体增殖物激活受体α(PPARα)对CYP7Al在转录水平的调节来实现。     

大黄灵仙胶囊调控兔胆石症模型肝功能及超微病理的作用机制研究

目的探讨大黄灵仙胶囊对兔胆石症模型的肝肾功能干预调节作用及超微病理结构改变。方法将新西兰大白兔80只随机分为正常对照组、药物对照组、模型组及治疗组,每组20只。正常对照组予常规饲料饲养,模型组和治疗组予致石饲料饲养;药物对照组和治疗组予大黄灵仙胶囊640 mg/kg溶水灌胃,1次/d,持续8周。采静脉血行生化检测,采肝组织行病理及电镜观察肝组织病理结构改变。结果与正常对照组比较,模型组肝功能指标均明显升高;与模型组相比,治疗组的肝功能指标明显下降(P0.01)。肝脏病理显示,正常对照组未见明显变化;模型组肝组织纤维化明显增多,间隙变窄,部分呈脂肪样变或者坏死;治疗组肝组织纤维化不明显,只有轻微样改变。超微病理显示,模型组肝细胞核不规则,部分细胞核出现核固缩、凋亡,内质网扩张、增生,粗面内质网可见脱颗粒现象,线粒体嵴融合并伴有空泡水肿,成纤维细胞成梭形伴有脂滴,胞浆溶解;治疗组细胞核较圆,与正常对照组相比,核周隙增宽,核胞浆中见脂滴少甚至没有,线粒体仍有空泡,细胞质中细胞器丰富。结论大黄灵仙胶囊可减轻胆石症所导致的肝脏纤维化、肝功能损伤,修复受损肝细胞,维持肝细胞结构稳定。     

胆石消胶囊消石利胆作用的实验研究

[目的]研究胆石消胶囊对小鼠胆结石模型和大鼠利胆实验的消石利胆作用.[方法]采用致石饲料饲喂小鼠复制胆结石模型,通过成石率、血清总胆固醇(TC)、丙氨酸氨基转移酶(ALT)水平和病理组织学等指标,检测胆石消胶囊的舒肝解瘀、软坚散结作用;应用大鼠胆管引流方法验证其排石利胆疗效.[结果]与模型组比较,各给药组胆结石成石率、血清TC、ALT水平明显降低(P＜0.01),肝组织病变明显减轻,胆汁流量明显增加(P＜0.05,＜0.01).[结论]胆石消胶囊能降低小鼠胆结石成石率,具有降脂、护肝、利胆的药理作用.     

蛹虫草多糖降胆固醇作用的机制探讨

目的 提取并纯化蛹虫草多糖(CMPS),分析相对分子质量分布,初步探讨其降胆固醇的作用机制。方法 通过热水提取-醇沉法分离CMPS,采用Sevage试剂、AB-8大孔吸附树脂进行纯化,应用高性能凝胶渗透色谱明确CMPS相对分子质量分布。模拟机体胃肠道环境,测定不同质量浓度CMPS结合胆酸盐能力;培养HepG2细胞,观察CMPS对肝细胞增殖率及细胞内胆固醇水平的影响,蛋白免疫印迹分析肝细胞内3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMG-CoA-R)及甾醇调节元件结合蛋白2(SREBP-2)表达变化。结果 CMPS中相对分子质量为3 119 kDa的多糖含量最高;CMPS体外结合胆酸盐能力较强,与胆酸钠、牛磺胆酸钠及甘氨胆酸钠的结合率分别达到63%、62%和50%。细胞实验数据表明低于200 mg/L的CMPS干预HepG2细胞24 h没有抑制其增殖率。同时,在安全质量浓度范围内(＜200 mg/L),CMPS可显著下调肝细胞内SREBP-2和HMG-CoA-R蛋白表达,降低肝细胞内总胆固醇水平。结论 CMPS具有良好的体外降胆固醇活性。     

烟酸对小鼠体内胆固醇逆转运的影响及其机制

目的观察烟酸干预后对小鼠体内胆固醇逆转运的影响,并探讨其机制。方法14只C57BL/6小鼠随机分为两组,分别给予普通饲料、烟酸添加饲料喂养4周后,腹腔注射经乙酰化-低密度脂蛋白及3H-胆固醇处理过的小鼠巨噬细胞悬液(0.5mL/鼠,细胞数为5.0×106),单独笼养24h后测定粪便中的3H-胆固醇含量(占注射总量的百分比);逆转录聚合酶链反应测定小鼠肝脏和小肠三磷酸腺苷结合盒转运体G5、肝X受体α mRNA及肝脏胆固醇7α羟化酶mRNA表达并用Western blot印迹检测肝脏和小肠的三磷酸腺苷结合盒转运体G5和肝X受体α蛋白表达。结果与对照组比较,烟酸组小鼠肝脏和小肠的三磷酸腺苷结合盒转运体G5、肝X受体α,肝脏胆固醇7α羟化酶mRNA水平和肝肠的三磷酸腺苷结合盒转运体G5、肝X受体α蛋白质水平均增高,粪便中的胆固醇流出率增加21%。结论烟酸可能通过上调体内肝X受体α,从而刺激三磷酸腺苷结合盒转运体G5、胆固醇7α羟化酶表达来发挥促进小鼠体内胆固醇逆转运作用。     

清胆胶囊对胆固醇结石小鼠肝脏中PPAR-γ、CYP7A1及NF-κB表达的影响

[目的]观察清胆胶囊对胆固醇结石小鼠肝脏过氧化物酶体增殖物激活受体(PPAR)-γ、胆固醇7α-羟化酶(CYP7A1)及核因子(NF)-κB表达的影响。[方法]38只C57BL/6雌性小鼠随机分为正常对照组(n=10)、模型组(n=15)和清胆胶囊组(n=13)。除正常对照组外,模型组和清胆胶囊组小鼠采用高脂饮食诱发法建立胆固醇结石模型。造模过程中,清胆胶囊组小鼠予清胆胶囊0.36 g/(kg.d)灌胃治疗。8周后观察各组小鼠的成石率,并用Western Blotting法检测肝脏中PPAR-γ、CYP7A1及NF-κB的表达。[结果]清胆胶囊组成石率显著降低(P0.01),小鼠肝脏PPARγ及CYP7A1表达增强,NF-κB表达降低。[结论]清胆胶囊可通过增强肝脏PPARγ及CYP7A1的表达,抑制NF-κB核转位来发挥防治胆固醇结石的作用。     

胆通胶囊预防色素性结石形成的机制研究

[目的]用林可霉素皮下注射诱发豚鼠胆囊色素性结石动物模型,观察胆通胶囊对色素性结石形成的预防作用及其机制.[方法]实验组(分大、小剂量)在注射林可霉素的同时灌服胆通胶囊提取物,模型组灌服等量0.85%氯化钠溶液,正常组单纯灌服0.85%氯化钠溶液.[结果]胆通胶囊大剂量组成石率为23.0%,模型组成石率为100%,两组间差异有统计学意义(P＜0.01).两组间胆汁细菌培养阳性率(15.0%和91.7%)、间接胆红素与直接胆红素比值、总胆酸以及钙离子水平、肝功能等指标差异均有统计学意义(P＜0.01).[结论]胆通胶囊具有预防色素性结石形成的作用,这种作用可能与其保护肝细胞和胆囊组织、维持胆汁有形成分代谢的正常化以及抑菌抗菌等多重作用有关.     

Hepatobiliary transport of bile acids and organic anions

Recent studies have elucidated the mechanism and regulation of hepatic transport of bile acids and organic anions. Bile acids are taken up into hepatocytes by basolateral transporters, Na⁺‐dependently by Na⁺/taurocholate cotransporting polypeptide (NTCP) and Na⁺‐independently by organic anion transporting polypeptide (OATP) families. Organic anions are taken up into hepatocytes by OATP families. These compounds are then transported in hepatocytes bound to cytosolic binders, and subjected to transport by ATP binding cassette (ABC) transporters at the canalicular membrane. Amidated bile acids are excreted into bile by bile salt export pump (BSEP), and organic anions and bile acid sulfates and glucuronides are excreted by multidrug resistance protein 2 (MRP2). Hepatic transporters are downregulated under cholestasis in rats and humans, except for MRP3, a basolateral ABC transporter, which is upregulated and may have a role in removing bile acids and organic anions from hepatocytes to the blood under cholestatic conditions. Nuclear receptors, which bind bile acids, have been shown to regulate the expression of hepatic transporters. Farnesoid X receptor (FXR), which downregulates CYP7A1, the rate‐limiting enzyme of bile acid biosynthesis, upregulates BSEP and downregulates NTCP. MRP2 is upregulated by both FXR and pregnane X receptor (PXR), which upregulates CYP3A.     

ApoB-100, ApoE and CYP7A1 gene polymorphisms in Mexican patients with cholesterol gallstone disease

AIM: To determine the possible association of the ApoB100 (Xba Ⅰ ), ApoE (Hha Ⅰ ) and CYP7A1 (Bsa Ⅰ ) gene polymorphisms, with the development of cholesterol gallstone disease (GD) in a Mexican population. METHODS: The polymorphisms were analyzed by polymerase chain reaction followed by restriction fragment length polymorphism, in two groups matched by ethnicity, age and sex: patients with GD (n = 101) and stone-free control subjects (n = 101). RESULTS: Allelic frequencies in patients and controls were: 34....     

Effects of bile duct ligation and cholic acid treatment on fatty liver in two rat models of non-alcoholic fatty liver disease

Background

Non-alcoholic fatty liver disease, one of the most prevalent liver disorders in Western countries, is characterized by hepatic accumulation of triglycerides. Bile acids have long been known to affect triglyceride homeostasis through a not completely understood mechanism.

Aim

To analyse the effects of two different manipulations of bile acid circulation on non-alcoholic fatty liver disease.

Methods

Two animal models of non-alcoholic fatty liver disease were developed by either feeding rats with a choline deficient or with a high fat diet. After 4 weeks, rats were randomized to undergo either bile duct ligation, sham operation or cholic acid administration.

Results

During cholestasis there was an increased CYP7A1 expression, the rate limiting enzyme in bile acid synthesis, and a reduction of hepatic concentration of oxysterols, ligands of the liver X receptors. Target genes of the liver X receptors, involved in fatty acid and triglyceride synthesis, were down-regulated in association with decreased hepatic triglyceride content and improvement of fatty liver. Administration of cholic acid, ligand of farnesoid X receptor, also had a beneficial effect on fatty liver in rats on choline deficient diet.

Conclusion

These results indicate that pharmacological approaches increasing the expression of CYP7A1 or stimulating farnesoid X receptor pathway could represent a promising treatment for non-alcoholic fatty liver disease.     

Suppression of bile acid synthesis by thyroid hormone in primary human hepatocytes

AIM: It is known that thyroid hormones alter the bile acid metabolism in humans, however the effect on individual enzymes has been difficult to elucidate. This is mainly due to the lack of human liver cell lines producing bile acids. We used cultures of primary human hepatocytes to study the effects of triiodothyronine (T3) on bile acid synthesis. METHODS: Primary hepatocytes were isolated from liver tissue obtained from three different patients undergoing liver resection due to underlying malignancy. The hepatocytes were cultured under serum-free conditions and treated from d 1 to d 5 with culture containing 0.1 -1000 nmol/L of T3. Bile acid formation and mRNA levels of key enzymes were analysed. RESULTS: The lowest concentration of T3 decreased cholic acid (CA) formation to 43%-53% of controls and chenodeoxycholic acid (CDCA) to 52%-75% of controls on d 5. The highest dose further decreased CA formation to 16%-48% of controls while CDCA formation remained at 50%-117% of controls. Expression of mRNA levels of cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) dose-dependently decreased. Sterol 27-hydroxylase (CYP27A1) levels also decreased, but not to the same extent. CONCLUSION: T3 dose-dependently decreased total bile acid formation in parallel with decreased expression of CYP7A1 and CYP8B1. CA formation is inhibited to a higher degree than CDCA, resulting in a marked decrease in the CA /CDCA ratio.     

Prenatal diagnosis of progressive familial intrahepatic cholestasis type 2

Background and Aim:  Progressive familial intrahepatic cholestasis type 2 (PFIC2) results from genetic defects of the hepatobiliary bile salt export pump (BSEP, ABCB11 ) at chromosome 2q24. Patients with progressive cholestasis and liver cirrhosis usually need liver transplantation in the first decade. Mutations in ABCB11 are also associated with benign recurrent intrahepatic cholestasis type 2 and intrahepatic cholestasis of pregnancy in adult patients. We aimed to make the prenatal diagnosis of PFIC2.
Methods:  Genetic diagnosis was performed by genomic DNA analysis. Prenatal genetic diagnosis was made by fetal amniotic DNA and chorionic DNA analysis.
Results:  We report on two families of PFIC2 with inherited compound heterozygous mutations of ABCB11 (M183V and R303K in Family 1, V284L and 1145delC in Family 2) from the parents. An infant with heterozygous M183V mutation was later born healthy in Family 1. A fetus with compound heterozygous missense mutation V284L and 1145delC was terminated in Family 2.
Conclusion:  Prenatal diagnosis of PFIC2 was helpful to prevent further affected children in families with this fatal disease.     

The role of interleukin-1 receptor antagonist in the prevention and treatment of disease

Abstract

?Interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α) play key proinflammatory roles in a variety of human diseases, including rheumatoid arthritis (RA). IL-1 receptor antagonist (IL-1Ra) is a naturally occurring structural variant of IL-1 that competitively inhibits receptor binding of IL-1. Four forms of IL-1Ra have been described: secretory IL-1Ra (sIL-1Ra) and three intracellular molecules (icIL-1Ra1, 2, and 3). Excess amounts of IL-1Ra are necessary to inhibit the biological effects of IL-1. The endogenous production of IL-1Ra plays an anti-inflammatory role, but the level of production of IL-1Ra in inflamed tissues may not be adequate to block IL-1 effectively. An allelic polymorphism in the IL-1Ra gene is associated with a variety of human diseases, largely of epithelial or endothelial cell origin. The disease associated allele IL1RN*2 may lead to a decreased production of icIL-1Ra1 by these cells, predisposing the patient to an imbalance in the IL-1 system. The therapeutic administration of IL-1Ra was found to be safe and efficacious in the treatment of RA. Intraarticular delivery of the IL-1Ra cDNA by ex vivo gene therapy in patients with RA was effective in enhancing local IL-1Ra production. This unique form of therapy is under further evaluation.     

胰升糖素样肽GLP－1（7－36）NH_2对血糖和血糖调节激素的影响

采用ＧＬＰ－１（７－３６）ＮＨ２外周灌流及脑室注射到禁食大鼠的方法，观察ＧＬＰ－１（７－６）ＮＨ２对大鼠血糖及血糖调节激素的影响。结果表明：静脉输入ＧＬＰ－１（７－３６）ＮＨ２使糖耐量曲线明显低平，血糖浓度恢复实验前水平比对照组提前。一次性推注葡萄糖后，外周输入ＧＬＰ－１（７－３６）ＮＨ２使Ｃ肽特别是胰岛素浓度明显增高，而不影响胰升糖素的水平。脑室注射ＧＬＰ－１（７３６）ＮＨ２对血糖无明显影响。本实验表明ＧＬＰ－１（７－３６）ＮＨ２在一次性推注葡萄糖后是通过刺激胰岛β细胞的分泌来调节血糖水平的。ＧＬＰ－１（７－３６）ＮＨ２可能在糖尿病的致病机理中起重要作用。本研究为研制ＧＬＰ－１（７－３６）ＮＨ２高活性类似物奠定了基础。     

Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases

Recent advances in metabolic engineering have demonstrated that microbial biosynthesis can provide a viable alternative to chemical synthesis for the production of bulk and fine chemicals. Introduction of a new biosynthetic pathway typically requires the expression of multiple heterologous enzymes in the production host, which can impose stress on the host cell and, thereby, limit performance of the pathway. Unfortunately, analysis and treatment of the host stress response can be difficult, because there are many sources of stress that may interact in complex ways. We use a systems biological approach to analyze the stress imposed by expressing different enzyme variants from a lineage of soluble P450 monooxygenases, previously evolved for heterologous activity in Saccharomyces cerevisiae. Our analysis identifies patterns of stress imposed on the host by heterologous enzyme overexpression that are consistent across the evolutionary lineage, ultimately implicating heme depletion as the major stress. We show that the monooxygenase evolution, starting from conditions of either high or low stress, caused the cellular stress to converge to a common level. Overexpression of rate-limiting enzymes in the endogenous heme biosynthetic pathway alleviates the stress imposed by expression of the P450 monooxygenases and increases the enzymatic activity of the final evolved P450 by an additional 2.3-fold. Heme overexpression also increases the total activity of an endogenous cytosolic heme-containing catalase but not a heterologous P450 that is membrane-associated. This work demonstrates the utility of combining systems and synthetic biology to analyze and optimize heterologous enzyme expression.     

Effect of bile acids on the biliary excretion of pravastatin in rats

Aim: The biliary excretion of pravastatin, an HMG‐CoA reductase inhibitor, is mediated by the multidrug resistance protein 2, but a recent report suggests that pravastatin is also a substrate of the bile salt export pump, which transports bile acids. We examined the effects of bile acids on biliary pravastatin excretion in rats. Methods: The effect of taurocholate on biliary pravastatin excretion, and that of pravastatin on biliary taurocholate excretion was examined in bile‐drained rats. Results: Taurocholate had no effect on biliary pravastatin excretion, whereas pravastatin with a dose to cause biliary excretory maximum significantly inhibited biliary taurocholate excretion. Conclusion: These data indicate that increased serum bile acids will not affect the pharmacokinetics of pravastatin in patients with hepatobiliary diseases. Although pravastatin inhibited biliary taurocholate excretion, it is unlikely that pravastatin significantly inhibits biliary bile acid excretion by its therapeutic doses.     

Increased susceptibility for intrahepatic cholestasis of pregnancy and contraceptive-induced cholestasis in carriers of the 1331T〉C polymorphism in the bile salt export pump

AIM： To study the association of three common ABCB11 and ABCC2 polymorphisms （ABCB11： 1331T〉C→V444A; ABCC2： 3563T〉A → V1188E and 4544G 〉A → C1515Y） with intrahepatic cholestasis of pregnancy （ICP） and contraceptive-induced cholestasis （CIC）. METHODS： ABCB11 and ABCC2 genotyping data were available from four CIC patients and from 42 and 33 ICP patients, respectively. Allele-frequencies of the studied polymorphisms were compared with those in healthy pregnant controls and Caucasian individuals. Furthermore, serum bile acid levels were correlated with the presence or absence of the 1331 C allele. RESULTS： The ABCB11 1331T〉C polymorphism was significantly more frequent in cholestatic patients than in pregnant controls： C allele 76.2% （CI, 58.0-94.4） vs 51.3% （CI 35.8-66.7）, respectively （P = 0.0007）; and CC allele 57.1% （CI 36.0-78.3） vs 20% （CI 7.6-32.4）, respectively （P = 0.0065）. All four CIC patients were homozygous carriers of the C allele. In contrast, none of the studied ABCC2 polymorphism was overrepresented in ICP or CIC patients. Higher serum bile acid levels were found in carriers of the 1331CC genotype compared to carriers of the TT genotype. CONCLUSION： Our data support a role for the ABCB11 1331T〉C polymorphism as a susceptibility factor for the development of estrogen-induced cholestasis, whereas no such association was found for ABCC2. Serum bile acid and 7-glutamyl transferase levels might help to distinguish ABCB4- and ABCB11-related forms of ICP and CIC.