Analysis of oxidative processes and of myelin figures formation before and after the loss of mitochondrial transmembrane potential during 7beta-hydroxycholesterol and 7-ketocholesterol-induced apoptosis: comparison with various pro-apoptotic chemicals

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol) 7beta-hydroxycholesterol and 7-ketocholesterol are potent inducers of cell death and probably play central roles in atherosclerosis. As suggested by our previous investigations, 7-ketocholesterol might be a causative agent of vascular damage by inducing apoptosis and enhancing superoxide anion (O2*-) production. To determine the precise relationships between cytotoxicity and oxidative stress, the ability of oxysterols oxidized at C7 to induce apoptosis, to stimulate O2*- production and to promote lipid peroxidation was compared with different pro-apoptotic chemicals: antitumoral drugs (VB, Ara-C, CHX, and VP-16) and STS. All compounds, except 7alpha-hydroxycholesterol, induced apoptosis characterized by the occurrence of cells with fragmented and/or condensed nuclei, loss of mitochondrial potential, caspase-3 activation, PARP degradation, and internucleosomal DNA fragmentation. The highest proportion of apoptotic cells was found with antitumoral drugs and STS, whereas the highest overproduction of O2*- detected before and after the loss of mitochondrial potential was obtained with 7beta-hydroxycholesterol and 7-ketocholesterol. Overproduction of O2*- was always correlated with enhanced lipid peroxidation. Vit E was only capable to significantly counteract apoptosis and oxidative stress induced by 7beta-hydroxycholesterol, 7-ketocholesterol, VB and STS. By electron and fluorescence microscopy, myelin figures evocating autophagic vacuoles were barely observed under treatment with 7beta-hydroxycholesterol and 7-ketocholesterol, and their formation occurring before the loss of mitochondrial potential was reduced by Vit E. In the presence of 7alpha-hydroxycholesterol, no enhancement of O2*- production, no lipid peroxidation, and no formation of myelin figures were observed. Collectively, our data demonstrate, that there can be a more or less important stimulation of oxidative stress during apoptosis. They also suggest that enhancement of O2*- production associated with lipid peroxidation during 7beta-hydroxycholesterol and 7-ketocholesterol-induced apoptosis could contribute to in vivo vascular injury, and that myelin figures could constitute suitable markers of oxysterol-induced cell death.     

妊娠期肝内胆汁瘀积症母儿胆酸和肝功能的变化

目的:通过检测妊娠期肝内胆汁瘀积症(ICP)母儿胆汁酸和肝功能变化,探讨ICP对围产儿胆汁酸和肝功能的影响。方法:选择2004年3月~2004年12月在四川大学华西第二医院产科住院的ICP患者38例,于能量合剂治疗前后分别抽取孕妇空腹肘静脉血,分娩时(均系剖宫产)抽取脐静脉血和新生儿血查血清TBA和肝功能。结果:(1)孕妇治疗后(即手术前)TBA、肝功能(主要是ALT、AST)均较治疗前显著下降(P<0.01),治疗前后母血TBA与ALT、AST分别呈正相关(均为P<0.05);(2)孕妇术前血清TBA、ALT、AST显著高于脐静脉血(均为P<0.01);(3)新生儿血TBA显著高于脐静脉血TBA(P<0.01),而ALT、AST无显著变化(P>0.05);(4)新生儿血TBA与术前母血TBA呈正相关(P<0.05),新生儿血TBA、ALT、AST显著低于术前母血(均为P<0.01)。结论:(1)升高的母血TBA能造成孕妇肝细胞不同程度受损,且TBA值愈高,ALT、AST亦升高愈明显;(2)能量合剂治疗ICP是有益的,能降低TBA,改善肝脏功能;(3)ICP患者因胎盘胆汁酸代谢发生障碍而导致围产儿TBA明显受到影响,但肝功能未见明显变化。     

Antinociceptive,anti-inflammatory and antioxidant activities of aqueous extract from Remirea maritima (Cyperaceae)

Ethnopharmacological relevance

Remirea maritima Aubl., popularly known as “capim-da-praia”, is popularly employed in the treatment of diarrhea, kidney disease, fever, and for analgesic and anti-inflammatory purposes through the preparation of teas. Few studies have focused on the chemical composition and its biological properties.

Aim of the study

This work evaluated the antinocipetive, anti-inflammatory and antioxidant activities of the aqueous extract from Remirea maritima Aubl. as well as the isolation and identification of the chemical compounds.

Materials and methods

Compounds were isolated from aqueous extract of Remirea maritima through preparative HPLC and the structures were identified by means of NMR and MS analysis. The tests for antinociceptive, anti-inflammatory, and antioxidant activities, along with motor coordination test (Rota rod), were performed over the aqueous extract.

Results

The phytochemical investigation of aqueous extract of Remirea maritima resulted in the isolation of three flavone glycosides. The structures of these compounds were determined by means of MS and 1D and 2D NMR data as vitexin-2″–O-β-d-glucopyranoside, isovitexin-2″–O-β-d-glucopyranoside, and luteolin-7-O-glucuronide. Acute pretreatment with aqueous extract (100, 200 or 400 mg/kg, i.p.) caused a significant decrease (p<0.001) in the number of abdominal writhes. In the formalin test, higher doses significantly inhibited the late (inflammatory pain) phase of formalin-induced licking (p<0.05 or 0.001). In the hot plate test, there was no significant difference in nociceptive behavior, discarding the possible central effect of the aqueous extract. In the rota rod test, it was verified that the aqueous extract in all concentration evaluated does not alter the motor coordination of mice, such antinociceptive results were unlikely to be caused by motor abnormality. In the peritonitis test, induced by carrageenan, the treatment with aqueous extract produced a significant reduction in leukocyte migration in all concentration evaluated. Additionally, a significant reduction of lipoperoxidation (TBARS test) and in nitric oxide formation (.NO Scavenging assay) was observed in antioxidant activity assay.

Conclusion

The biological and phytochemical investigations of the aqueous extract of Remirea maritima resulted in the identification of three flavone glycosides that have been described here for the first time in Remirea and effective analgesic activity in various pain models, probably mediated via the inhibition of peripheral mediators which could be related to its strong antioxidant effect observed in vitro.     

Formation of Zinc–Peptide Spherical Microparticles During Lyophilization from <Emphasis Type="BoldItalic">tert</Emphasis>-Butyl Alcohol/Water Co-solvent System

Purpose  To understand the mechanism of spherical microparticle formation during lyophilizing a tert-Butyl alcohol (TBA)/water solution of a zinc peptide adduct. Method  A small peptide, PC-1, as well as zinc PC-1 at (3:2) and (3:1) ratios, were dissolved in 44% (wt.%) of TBA/water, gradually frozen to −50°C over 2 h (“typical freezing step”), annealed at −20°C for 6 h (“annealing step”), and subsequently lyophilized with primary and secondary drying. Zinc peptide (3:1) lyophile was also prepared with quench cooling instead of the typical freezing step, or without the annealing step. Other TBA concentrations, i.e., 25%, 35%, 54% and 65%, were used to make the zinc peptide (3:1) adduct lyophile with the typical freezing and annealing steps. The obtained lyophile was analyzed by Scanning Electron Microscopy (SEM). The zinc peptide solutions in TBA/water were analyzed by Differential Scanning Calorimeter (DSC). The surface tension of the TBA/water co-solvent system was measured by a pendant drop shape method. Results  With typical freezing and annealing steps, the free peptide lyophile showed porous network-like structure that is commonly seen in lyophilized products. However, with increasing the zinc to peptide ratio, uniform particles were gradually evolved. Zinc peptide (3:1) adduct lyophiles obtained from 25%, 35% and 44% TBA exhibit a distinctive morphology of uniform and spherical microparticles with diameters of ∼3–4 μm, and the spherical zinc peptide particles are more predominant when the TBA level approaches 20%. Adopting quench cooling in the lyophilization cycle leads to irregular shape fine powders, and eliminating the annealing step causes rough particles surface. When TBA concentration increases above 54%, the lyophiles demonstrate primarily irregular shape particles. Conclusions  A proposed mechanism of spherical particle formation of the 3:1 zinc peptide encompasses the freezing of a TBA/water solution (20–70% TBA) causing the formation of a TBA hydrate phase (“dispersed TBA hydrate”). Decreasing the temperature further causes the formation of a eutectic mixture between TBA hydrate (“eutectic TBA hydrate”) and water. Due to its low aqueous solubility, the zinc peptide adduct accumulates in both of the dispersed and eutectic TBA hydrate phases to form a hydrophobic “oil” phase. Since the eutectic TBA hydrate phase is surrounded by ice, a “solid emulsion” forms to lower the interfacial energy, and gives rise to spherical zinc peptide particles upon solvent sublimation. Possibility of liquid–liquid phase separation during freeze-drying was also investigated, and no evidence was found to support this alternative mechanism.     

血清TBA及GGT在肝病患者检测中的评价

目的:了解肝病患者血清TBA(总胆汁酸)及GGT(γ-谷氨酸转移酶)的检测对肝病的诊断价值。方法:对230例肝病患者血清及100例健康人的血清进行TBA、GGT检测,并运用统计学方法对结果进行分析和评价。结果:TBA在除慢性迁延性肝炎中升高不显著外,在其他各型肝病中均明显增高,且在慢性活动性肝炎时显著升高,其阳性率为94.2%;而GGT在肝硬化、肝癌患者中亦明显升高,但其阳性率低于TBA。结论:TBA在各型肝病中均有一定程度的升高,其为肝病检测较为敏感的指标,而GGT的持续大量升高对肝硬化向肝癌转变具有重要的诊断意义。     

Effect of garlic-derived organosulfur compounds on mitochondrial function and integrity in isolated mouse liver mitochondria

The objectives of this work were to evaluate the direct effects of diallysulfide (DAS) and diallyldisulfide (DADS), two major organosulfur compounds of garlic oil, on mitochondrial function and integrity, by using isolated mouse liver mitochondria in a cell-free system. DADS produced concentration-dependent mitochondrial swelling over the range 125–1000 μM, while DAS was ineffective. Swelling experiments performed with de-energized or energized mitochondria showed similar maximal swelling amplitudes. Cyclosporin A (1 μM), or ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 1 mM) were ineffective in inhibiting DADS-induced mitochondrial swelling. DADS produced a minor (12%) decrease in mitochondrial membrane protein thiols, but did not induce clustering of mitochondrial membrane proteins. Incubation of mitochondria with DADS (but not DAS) produced an increase in the oxidation rate of 2′,7′ dichlorofluorescein diacetate (DCFH-DA), together with depletion of reduced glutathione (GSH) and increased lipid peroxidation. DADS (but not DAS) produced a concentration-dependent dissipation of the mitochondrial membrane potential, but did not induce cytochrome c release. DADS-dependent effects, including mitochondrial swelling, DCFH-DA oxidation, lipid peroxidation and loss of mitochondrial membrane potential, were inhibited by antioxidants and iron chelators. These results suggest that DADS causes direct impairment of mitochondrial function as the result of oxidation of the membrane lipid phase initiated by the GSH- and iron-dependent generation of oxidants.     

Rescuing hepatocytes from iron-catalyzed oxidative stress using vitamins B1 and B6

In the following rescue experiments, iron-mediated hepatocyte oxidative stress cytotoxicity was found to be prevented if vitamin B1 or B6 was added 1 h after treatment with iron. The role of iron in catalyzing Fenton-mediated oxidative damage has been implicated in iron overload genetic diseases, carcinogenesis (colon cancer), Alzheimer’s disease and complications associated with the metabolic syndrome through the generation of reactive oxygen species (ROS). The objectives of this study were to interpret the cytotoxic mechanisms and intracellular targets of oxidative stress using “accelerated cytotoxicity mechanism screening” techniques (ACMS) and to evaluate the rescue strategies of vitamins B1 and B6. Significant cytoprotection by antioxidants or ROS scavengers indicated that iron-mediated cytotoxicity could be attributed to reactive oxygen species. Of the B6 vitamers, pyridoxal was best at rescuing hepatocytes from iron-catalyzed lipid peroxidation (LPO), protein oxidation, and DNA damage, while pyridoxamine manifested greatest protection against ROS-mediated damage. Thiamin (B1) decreased LPO, mitochondrial and protein damage and DNA oxidation. Together, these results indicate that added B1 and B6 vitamins protect against the multiple targets of iron-catalyzed oxidative damage in hepatocytes. This study provides insight into the search for multi-targeted natural therapies to slow or retard the progression of diseases associated with Fenton-mediated oxidative damage.     

肝硬化患者血清CA125及TBA水平变化的临床意义

目的探讨血清CA199及TBA水平变化在肝硬化患者中的临床意义。方法检测78例肝硬化患者及40例健康人血清CA125及TBA水平并进行比较分析。结果肝硬化患者血清CA125及TBA水平明显高于正常对照组,并随肝硬化进展而升高（P〈0.05）。结论肝硬化患者血清CA199及TBA水平与肝功能损害程度密切相关,可以被认为是反映肝硬化程度的敏感指标,二者联合检测对肝硬化的病情判断及预后估计具有较高的参考价值。     

Referral practices and perceived barriers to timely obstetric care among Ugandan traditional birth attendants (TBA)

Objectives

To assess current beliefs, knowledge and practices of Ugandan traditional birth attendants (TBAs) and their pregnant patients regarding referral of obstructed labors and fistula cases.

Methods

Six focus groups were held in rural areas surrounding Kampala, the capital city of Uganda.

Results

While TBAs, particularly those with previous training, appear willing to refer problematic pregnancies and labors, more serious problems exist that could lessen any positive effects of training. These problems include reported abuse by doctors and nurses, and seeing fistula as a disease caused by hospitals.

Conclusions

Training of TBAs can be helpful to standardize knowledge about and encourage timely emergency obstetric referrals, as well as increase knowledge about the causes and preventions of obstetric fistula. However, for full efficacy, training must be accompanied by greater collaboration between biomedical and traditional health personnel, and increased infrastructure to prevent mistreatment of pregnant patients by medical staff.     

胃肠道外营养的新生儿血清TBA、CG、HA水平变化及临床意义

目的：探讨胃肠道外营养的新生儿血清总胆汁酸（TBA）甘胆酸（CG）及透明质酸（HA）水平变化及临床意义。方法：52例行胃肠道外营养的新生儿（足月儿32例,早产儿20例）作为观察组,28例无需胃肠道外营养的新生儿作为对照组（足月儿16例,早产儿12例）。采用酶速率法和放射免疫分析测定胃肠道外营养前后TBA、CG、HA水平变化,比较足月儿与早产儿三项水平变化的差别。结果：足月儿胃肠道外营养前TBA、CG、HA水平与对照组比较,差异无显著性（P〉0.05）;胃肠道外营养后TBA、CG、HA水平较胃肠道外营养前明显升高,差异有非常显著性（P〈0.01）;早产儿胃肠道外营养后TBA、CG、HA水平较足月儿明显升高,差异有显著性（P〈0.05）。结论：胃肠道外营养对新生儿肝胆系统功能有影响,且以早产儿为明显。