Inhibition of fatty acid amide hydrolase suppresses referred hyperalgesia induced by bladder inflammation

Study Type – Aetiology (case series) Level of Evidence 4

OBJECTIVE

? To determine (i) the presence of fatty acid amide hydrolase (FAAH) in the urinary bladder; (ii) whether or not endogenous fatty acid ethanolamides are synthesized by the bladder; (iii) the effects of FAAH inhibition on referred hyperalgesia associated with acute bladder inflammation in rats.

MATERIALS AND METHODS

? Immunohistochemistry and immunoblotting were performed to detect FAAH in the bladder. Acrolein (1 mM, 400 µL) was instilled into bladders of female Wistar rats to induce cystitis. Referred mechanical hyperalgesia was assessed by application of Von Frey monofilaments to the hind paws. ? Animals were killed 4, 24, 48 and 72 h after acrolein instillation, and the fatty acid ethanolamide content of bladders was measured using isotope‐dilution liquid chromatography/mass spectrometry. ? Other rats were treated with the FAAH inhibitor URB597 (0.3 mg/kg, i.p.) after the induction of cystitis, and the mechanical sensitivity of the hind paws was determined.

RESULTS

? Immunohistochemistry and immunoblotting showed the presence of FAAH in the bladder, with greatest abundance in the urothelium. ? Acrolein‐induced cystitis increased fatty acid ethanolamide content (including anandamide) in the bladder in a time‐dependent manner. Inhibition of FAAH diminished referred hyperalgesia associated with acute bladder inflammation.

CONCLUSIONS

? The results obtained in the present study indicate that (i) FAAH is present in the urinary bladder; (ii) fatty acid ethanolamides are increased during bladder inflammation; (iii) inhibition of FAAH could be an effective therapeutic approach for the treatment of bladder pain. ? These results raise the possibility that inhibitors of enzymes responsible for metabolism of fatty acid ethanolamides could inhibit pain associated with bladder inflammation.     

微粒体环氧化酶与谷胱甘肽S转移酶的遗传多态性对新生儿出生体重的影响

「目的」探讨母亲微粒体环氧化酶（ＥＰＨＸ１）与谷胱甘肽Ｓ转移酶（ＧＳＴＭ１，ＧＳＴＴ１）的遗传多态笥对新生儿出生体重的影响。「方法」采用回顾性流行病学调查方法，使用统一调查表，由经过培训的调查员在北京燕山某医院对入院分娩的６３０个母亲婴儿对进行调查。「结果」单因素分析发现：ＥＰＨＸ１和ＧＳＴＴ１、ＧＳＴＭ１基因多态对新生儿出生体重的影响无显著意义。经母亲年龄、文化程度、生育史、芳香烃溶剂暴露、在家     

Detoxication Strategy of Epoxide Hydrolase��The Basis for a Novel Threshold for Definable Genotoxic Carcinogens

From our recent work on the three-dimensional structure of epoxide hydrolases we theoretically deduced the likelihood of a two-step catalytic mechanism that we and others have subsequently experimentally confirmed. Analysis of the rate of the two steps by us and by others show that the first step—responsible for removal of the reactive epoxide from the system—works extraordinarily fast (typically three orders of magnitude faster than the second step), sucking up the epoxide like a sponge. Regeneration of the free enzyme (the second step of the catalytic mechanism) is slow. This becomes a toxicological problem only at doses of the epoxide that titrate the enzyme out. Our genotoxicity work shows that indeed this generates a practical threshold below which no genotoxicity is observed. This shows that—contrary to old dogma—practical thresholds exist for definable genotoxic carcinogens.     

泛素羧基末端水解酶-1抑制剂对多巴胺能神经元的神经毒性作用

Objective To investigate the neurotoxic effects ofLDN-57444, a specific ubiquitin C-termiual hydrolase L1 (UCH-L1) inhibitor, on dopaminergic neurons and the possible mechanism. Methods The viability of SK-N-SH cells exposed to 5, 10, 25, 50, 75 or 100 μmol/L LDN-57444 for 24 h was assessed using MTT assay, and the cell apoptosis was detected with Hoechst staining. Western blot was performed to identify the expressions of UCH-L1 protein, ubiquitin monomer and polyubiquitinated proteins, and the activity of the ubiquitin-proteasome system (UPS) was evaluated with fluorometry. Results After exposure to UCH-LI inhibitor for 24 h, the cell process-like structures of SK-N-SH cells diminished, and the cell body shrank and became spherical. Exposure to LDN-57444 resulted in concentration-dependent reduction of the cell viability, and the reduction became statistically significant following the exposure to 50 μmol/L LDN-57444, as compared with that in the control group (P<0.05). The exposure also resulted in obvious cell apoptosis as shown by nuclear fragmentation and presence of the apoptotie bodies. Western blot detected no obvious changes in UCH-L1 protein expression but identified reduced ubiquitin monomer and increased polyubiquitinated protein expression in the cells. Fluorometry showed reduced activity of UPS in the exposed cells. Conclusion UCH-L1 inhibitor produces neurotoxicity to dopaminergie neurons and induces cell apoptosis possibly as the result of impaired UPS activity and intracellular accumulation of polyubiquitinated proteins following the exposure.     

ACT and UCH-L1 polymorphisms in Parkinson's disease and age of onset.

alpha1-Antichymotrypsin (ACT) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have been suggested as susceptibility factors for Parkinson's disease (PD). We replicated these findings in a Chinese case-control sample consisting of 160 PD cases and 160 carefully matched control subjects. Genotypes were determined using polymerase chain reaction and BstN1 or Rsa1 restriction enzyme assay. Analysis showed no significant difference between PD patients and controls for genotype or allele frequencies of the ACT and UCH-L1 S18Y polymorphisms. UCH-L1 S18Y polymorphism carriers, however, were found to be significantly less frequent in early-onset PD patients with a reduced risk of 0.557 (95% C.I. = 0.314-0.985; P = 0.043). These data suggest that ACT polymorphism does not influence the risk for developing PD. UCH-L1 S18Y polymorphism, however, may be a weak protective factor against early-onset PD.     

室旁核内脂肪酸酰胺水解酶上调介导慢性心衰大鼠交感神经兴奋亢进

目的:探讨慢性心衰大鼠室旁核(PVN)内脂肪酸酰胺水解酶(FAAH)表达失衡导致中枢交感传出增加的机制。方法:雄性Wistar大鼠左冠状动脉结扎8周后,用超声心动图检测心功能,并通过组织病理学测定心肌梗死面积,鉴定大鼠心衰模型构建成功;酶联免疫吸附法测血浆去甲肾上腺素(NE)水平;Western blot测定PVN内FAAH蛋白表达量;高效液相色谱法测定PVN内N-花生四烯酰乙醇胺(AEA)的生成量;PVN微量注射AEA、FAAH抑制剂PF3845或r AAV2-FAAH sh RNA病毒,记录交感驱动和心功能指标的变化。结果:与假手术组相比,心衰组心功能显著降低,血浆NE显著增加,PVN内FAAH表达量显著上调,AEA生成量显著减少(P0.05);PVN微量灌注PF3845、AEA或r AAV2-FAAH sh RNA病毒后,心衰组交感驱动指标显著降低,心功能明显改善。结论:心衰状态下PVN内FAAH蛋白表达上调可能引起AEA生成量的减少,从而增强交感神经兴奋性,恶化心衰。     

Association between polymorphisms of microsomal epoxide hydrolase and COPD: results from meta-analyses

Background and objective: COPD is a complex polygenic disease in which gene–environment interactions are very important. The gene encoding microsomal epoxide hydrolase (EPHX1) is one of several candidate loci for COPD pathogenesis and is highly polymorphic. Based χ on the polymorphisms of EPHX1 gene (tyrosine/histidine 113, histidine/arginine 139), the population can be classified into four groups of putative EPHX1 phenotypes (fast, normal, slow and very slow). A number of studies have investigated the association between the genotypes and phenotypes of EPHX1 and COPD susceptibility in different populations, with inconsistent results. A systematic review and meta‐analysis of the published data was performed to gain a clearer understanding of this association. Methods: The MEDLINE database was searched for case–control studies published from 1966 to August 2007. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. Results: Sixteen eligible studies, comprising 1847 patients with COPD and 2455 controls, were included in the meta‐analysis. The pooled result showed that the EPHX1 113 mutant homozygote was significantly associated with an increased risk of COPD (OR 1.59, 95% CI: 1.14–2.21). Subgroup analysis supported the result in the Asian population, but not in the Caucasian population. When the analysis was limited to only the larger‐sample‐size studies, studies in which controls were in Hardy–Weinberg equilibrium and studies in which controls were smokers/ex‐smokers, the pooled results supported the conclusion. The EPHX1 139 heterozygote protected against the development of COPD in the Asian population, but not in the Caucasian population. The other gene types of EPHX1 113 and EPHX1 139 were not associated with an increased risk of COPD. The slow activity phenotype of EPHX1 was associated with an increased risk of COPD. The fast activity phenotype of EPHX1 was a protective factor for developing COPD in the Asian population, but not in the Caucasian population. However, the very slow activity phenotype of EPHX1 was a risk for developing COPD in the Caucasian population, but not in the Asian population. Conclusions: The polymorphisms of EPHX1 113 and EPHX1 139 are genetic contributors to COPD susceptibility in Asian populations. The phenotypes of EPHX1 were contributors to overall COPD susceptibility.     

Peroxisomal translocation of soluble epoxide hydrolase protects against ischemic stroke injury

Soluble epoxide hydrolase (sEH) contributes to cardiovascular disease, including stroke, although the exact mechanism remains unclear. While primarily a cytosolic enzyme, sEH can translocate into peroxisomes. The relevance of this for stroke injury is not understood. We tested the hypothesis that sEH-mediated injury is tied to the cytoplasmic localization. We found that a human sEH variant possessing increased affinity to peroxisomes reduced stroke injury in sEH-null mice, whereas infarcts were significantly larger when peroxisomal translocation of sEH was disrupted. We conclude that sEH contributes to stroke injury only when localized in the cytoplasm, while peroxisomal sEH may be protective.     

泛素羧基末端水解酶L1在年龄相关性白内障晶状体上皮细胞中的表达及其抗氧化作用

背景 氧化损伤是年龄相关性白内障发生的主要原因,而泛素蛋白酶系统参与晶状体的分化发育,研究发现其关键酶泛素羧基末端水解酶L1( UCHL1)参与帕金森病和阿尔茨海默病等年龄相关性疾病的发生发展,且与氧化应激有关. 目的 研究UCHL1在年龄相关性白内障发病过程中的作用.方法 收集24例单纯年龄相关性白内障患者术后获得的晶状体囊膜(皮质性白内障12例、核性白内障12例)、5例正常人晶状体前囊膜上皮和人晶状体上皮细胞(LECs)系SRA01/04细胞,采用免疫荧光法检测UCHL1在各组人晶状体前囊膜上皮细胞中的表达情况.构建UCHL1真核表达质粒,鉴定后采用脂质体转染法转染SRA01/04细胞作为UCHL1过表达组,同时采用绿色荧光蛋白(GFP)真核表达质粒转染SRA01/04细胞作为GFP过表达组,使用梯度过氧化氢叔丁醇(TBHB)处理24h后,采用MTT法检测各组人LECs的活性变化.结果 免疫荧光检测表明,UCHL1在各组人LECs中均有表达,但在正常晶状体囊膜、皮质性白内障以及核性白内障晶状体囊膜上皮细胞表达量的总体差异有统计学意义(F=13.441,P=0.000).皮质性白内障组以及核性白内障组晶状体囊膜上皮细胞中UCHL1的表达量均低于正常晶状体组(P=0.000、0.000),但皮质性白内障组和核性白内障组之间UCHL1的表达量差异无统计学意义(P=0.164).Western blot鉴定结果表明,UCHL1真核表达质粒转染后可见SRA01/04细胞中UCHL1的强表达.MTT检测结果显示,0.3 mol/L TBHB处理24 h后,UCHL1过表达组细胞活性吸光度(A570/630)值与GFP过表达组比较有增高的趋势,而0.2、0.4、0.5 mol/LTBHP均导致SRA01/04细胞的耐受或者大量凋亡. 结论 UCHL1具有抗氧化作用,且可能在年龄相关性白内障的发生发展过程中起抑制作用.     

弓形虫三磷酸核苷水解酶基因克隆、表达与鉴定

目的 对刚地弓形虫三磷酸核苷水解酶基因（NTPase）进行克隆、表达和鉴定。 方法 采用PCR扩增刚地弓形虫RH株的NTPase基因，克隆入pGEM?-T Easy载体，经酶切与测序鉴定后亚克隆至表达质粒pBAD-HisB，并转入大肠埃希菌(E.coli)BL21（DE3）中进行诱导表达。用镍-次氮基三乙酸亲和层析柱纯化重组质粒pBAD-HisB-NTPase表达产生的含组氨酸的重组蛋白，用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质印迹（Western blotting）分析蛋白表达产物。 结果 PCR扩增得到特异的刚地弓形虫NTPase-Ⅱ基因序列，经测序鉴定无基因突变。SDS-PAGE结果表明NTPase-Ⅱ基因在E.coli BL21（DE3）中获得高效表达，其融合蛋白相对分子质量（Mr）约70 000，与理论值相近。Western blotting分析结果显示，纯化的重组蛋白可被弓形虫感染的鼠血清及鼠抗重组蛋白血清识别。 结论 所克隆表达的弓形虫NTPase-Ⅱ重组蛋白具有良好的抗原性。