Proteomic alteration in lung tissue of rats exposed to cigarette smoke

Cigarette smoke has been widely investigated in terms of epidemiological and pathological studies in relation to human lung diseases. In this study, we conducted a proteomic analysis to characterize the differential protein expression in lung tissue of rats exposed to cigarette smoke. Wistar rats were exposed to cigarette smoke twice a day, 30 min each for 1, 2 and 4 months, respectively. The total protein of lung tissue was extracted for two-dimensional electrophoresis (2-DE) and analyzed with ImageMaster 2D Platinum software. A total of 28 differentially expressed proteins between the control and the smoke-exposed groups were screened and of which 18 were identified by matrix assistant laser desorption ion-top of flight-mass spectrometry (MALDI-TOF-MS) or MALDI- TOF-TOF analysis, revealing 10 up-regulated and 8 down-regulated proteins. The up-regulated expression of two proteins, receptor for advanced glycation endpoints (RAGE) and thioredoxin (Trx), were validated by immunoblotting and found to be consistent with the proteomic analysis. The results presented in this study demonstrate the identification of proteomic pattern as an early indicator of lung damages induced by cigarette smoke. The differentially expressed proteins may be applied as exposure biomarkers in future experimental as well as epidemiologic investigations upon confirmation by a greater sample size and more validate study design for the proteomic research.     

宫颈癌与非宫颈癌组织的差异蛋白表达

目的：用蛋白质组学的方法,获得宫颈癌组织和非宫颈癌组织的差异蛋白表达图谱。寻找与宫颈癌发生、发展相关的特异蛋白,为探讨宫颈癌发病机制提供实验室依据。方法利用双向电泳、蛋白质串联质谱（MS／MS）分析宫颈癌的差异蛋白表达图谱,并于 NCBI 数据库中检索明确蛋白身份。结果经比对分析差异蛋白表达图谱及数据库检索,初步鉴定了12种蛋白,分别为：膜联蛋白 V 、膜联蛋白 I 、膜联蛋白 A1、膜联蛋白 A4、锰超氧化物歧化酶、转羟乙醛酶、假定蛋白、核不均一性核糖核蛋白 H2、载脂蛋白 A‐I 、热休克蛋白、L1蛋白、复杂的 T‐蛋白亚基伽玛亚型。结论这些蛋白可能与宫颈癌的发生、发展有关。     

Differential effects of methylmercury on the synthesis of protein species in dorsal root ganglia of the rat

Dorsal root ganglia from control and methylmercury(MeHg)-treated rats were incubated in vitro with ³⁵S-methionine ant the proteins synthesized were analyzed by two-dimensional electrophoresis. The double labelling method, in which proteins of control dorsal root ganglia labelled in vitro with ³H-leucine were added to each of the two samples as an internal standard, was used to minimize unavoidable errors arising from the resolving procedure itself. The results obtained showed that the effect of MeHg on the synthesis of proteins in dorsal root ganglia was not uniform for individual protein species in the latent period of MeHg intoxication. Among 200 protein species investigated, 157 showed inhibition of synthesis close to that of the total proteins in the tissue (68% of the control). Among the remaining protein species, 20 showed real stimulation of synthesis, whereas 7 were moderately inhibited and 16 were inhibited more strongly than the total proteins in the tissue. These results suggest that the effect of MeHg on the synthetic rates for protein species in dorsal root ganglia differs with the species, and that unusual elevation or reduction of the synthesis of some protein species caused by MeHg may lead to impairment of normal nerve functions.This work was supported in part by a grant from the Japanese Environmental Agency     

Chronic intoxication with acetaldehyde stimulates collagen biosynthesis in rat liver

It was found that chronic intoxication of rats with acetaldehyde results in a distinct, progressive increase of 5³H-proline incorporation into collagen synthesized by liver. At the same time, biosynthesis of other proline-containing (noncollagenous) proteins does not change significantly. The effects are similar to those induced by chronic intoxication of rats with ethanol. Since acetaldehyde is an intermediary metabolite formed during ethanol oxidation in liver, it may be concluded that acetaldehyde is a factor responsible for alcohol-induced liver fibrosis.     

LP（a）在糖尿病肾病患者中的变化及氟伐他汀对其的影响

目的：观察2型糖尿病肾病患者血清LP（a）水平变化,分析LP（a）水平与肾损害程度的相关性,并探讨氟伐他汀对LP（a）的影响及防治糖尿病肾病进展中的意义。方法：选择192例不同尿微量清蛋白排泄率的2型糖尿病患者和50例对照者,测定其血清中TG、TC、HDL-C、VLD-C,LP（a）的水平及Scr、UAER。分析血清Lp（a）质量浓度变化与DN进展之间的关系。同时测定氟伐他汀在早期糖尿病肾病组中治疗后上述指标。结果：2型糖尿病肾病患者LP（a）随肾功能的减退呈升高趋势,两者呈正相关（P〈0.05）,而血糖与肾损害程度无明显相关性;氟伐他丁能降低糖尿病肾病患者LP（a）水平而对血糖水平无影响。结论：LP（a）水平与2型糖尿病肾病肾损害有关,可作为糖尿病肾病的危险性预测因素,氟伐他丁能下调LP（a）水平,具有肾脏保护作用。     

Induction of monooxygenases and growth in rat liver by progesterone

Female Wistar rats were treated with various doses of progesterone orally via the diet or via the SC route. Oral treatment resulted in enhanced progesterone levels in the liver as measured by radioimmunoassay. There were up to 3-fold increases in activity of ethylmorphine demethylation by isolated microsomes; metabolism of aminopyrine and benzphetamine was less enhanced, that of aniline and P-nitroanisol showed no distinct increases. Progesterone also caused increases in liver size and total liver protein by up to 50%; total liver DNA showed only slight, insignificant increments. These studies suggest that hepatic effects of progesterone are similar to those previously described with synthetic steroids such as pregnenolone-16-carbonitrile (PCN) and cyproterone acetate.Abbreviations A aniline - AP aminopyrine - BPA Benzphetamine - CPA cyproterone acetate - EM ethylmorphine - PCN pregnenolone-16-carbonitrile - p-NA P-nitroanisole     

Proteomic profiling and protein identification by 2-DE-MS in the liver of Carassius auratus exposed to Yongcheng coalmine subsidence area

Proteomic profile of the Carassius auratus liver was constructed to evaluate contamination degree of aquatic organism exposed to coalmine subsidence area. The results revealed that 55 proteins were up-regulated and 51 down-regulated among 160 differentially expressed proteins. These proteins were mainly involved in the physiological activities, such as granzyme B signaling, dermatan sulfate biosynthesis and cytotoxic T lymphocyte mediated apoptosis of target cells. Meanwhile, they were related to two network interaction modules. One was cell adhesion and migration, angiogenesis, DNA repair, and the other was small molecule metabolism, structural molecule activity and defense response. Ingenuity Pathway Analysis (IPA) was further utilized to predict differential proteins molecular activation, including tnf, tp53, Anxa3, fgf2 and mvp. The result sheds light on liver protein expression profiles of C. auratus, and provides important data for controlling and managing water pollution from heavy metals.     

丙烯腈染毒对大鼠肝脏钙稳态某些指标的影响

本研究观察了丙烯腈对大鼠肝脏 Ca2 - ATPase、Mg2 - ATPase、Na / K - ATPase和磷酸化酶 a( P- a)活性的影响 ,探讨其对大鼠肝脏钙稳态的影响。结果表明 ,随着染毒剂量的增大和染毒时间的延长 ,各 ATPase活性均逐渐降低 ,而 P- a的活性却逐渐升高 ( P<0 .0 1) ,其中高剂量( 5 0 mg/ kg)组的各观察时段和染毒 42天时各染毒组酶活性的变化均具有显著性意义 ( P<0 .0 5 ) ,并具有较好的剂量 -反应关系 ( P<0 .0 1)。结果提示 ,丙烯腈可影响大鼠肝脏钙稳态某些指标变化 ,并可能导致肝脏钙稳态的失调     

Imbalance in redox system of rat liver following permethrin treatment in adolescence and neonatal age

Abstract

1. The effect of different permethrin treatments on the redox system of rat liver, is presented. Two types of oral administration were chosen: (i) sub-chronic treatment (1/10 of LD₅₀ for 60 days) during adolescence (5 weeks old) and (ii) sub-acute treatment (1/44 of LD₅₀ for 15 days) during early life (from postnatal days 6–21).

2. The results show that adolescent permethrin treatment induces damage to the liver redox system, increasing lipid and protein peroxidation and reducing membrane fluidity in the hydrophilic--hydrophobic region of the bilayer. In addition, glutathione peroxidase (GPx) and GSH levels resulted decreased, while glutathione transferase (GST) and catalase (CAT) levels increased.

3. The rats treated in early life with permethrin and sacrificed in adult age, showed less signs of damage compared to those exposed during adolescence in which lipid peroxidation was increased by 32%, whereas for the first group the raise was only 11%. Moreover, fluidity improved in the deeper hydrophobic membrane region of the treated group, while the level of CAT was significantly lower compared to the control one.

4. Although sub-chronic treatment increased CAT and GST and decreased GPx and GSH levels, the present data suggest that a shorter exposure to permethrin during neonatal age decreased CAT level and it could represent an important risk factor for the onset of long-term liver damage.     

Effect of dithiocarb and dimethyl sulfoxide on irreversible binding of 14C-bromobenzene to rat liver microsomal protein

The irreversible binding of [¹⁴C]-bromobenzene to rat liver microsomal protein in vitro was inhibited by dithiocarb and DMSO. Dithiocarb suppressed this binding in a time- and concentration-dependent manner (I₅₀ = 4.5 10^–5 M). DMSO reduced the degree of covalent binding by 61% from 5 x 10^–5 M to 8 × 10^–4 M. Dithiocarb was also effective in inhibiting irreversible binding of bromobenzene to liver protein in vivo. Our results are consistent with the hypothesis that dithiocarb exerts its antihepatotoxic efficacy by depressing microsomal mixed-function oxidase activity.     

Nortriptyline influences protein pathways involved in carbohydrate metabolism and actin-related processes in a rat gene-environment model of depression

Although most available antidepressants increase monoaminergic neurotransmission, their therapeutic efficacy is likely mediated by longer-term molecular adaptations. To investigate the molecular changes induced by chronic antidepressant treatment we analysed proteomic changes in rat pre-frontal/frontal cortex and hippocampus after nortriptyline (NT) administration. A wide-scale analysis of protein expression was performed on the Flinders Sensitive Line (FSL), a genetically-selected rat model of depression, and the control Flinders Resistant Line (FRL). The effect of NT treatment was examined in a gene-environment interaction model, applying maternal separation (MS) to both strains.In the forced swim test, FSL rats were significantly more immobile than FRL animals, whereas NT treatment reduced immobility time. MS alone did not modify immobility time, but it impaired the response to NT in the FSL strain.In the proteomic analysis, in FSL rats NT treatment chiefly modulated cytoskeleton proteins and carbohydrate metabolism. In the FRL strain, changes influenced protein polymerization and intracellular transport. After MS, NT treatment mainly affected proteins in nucleotide metabolism in FSL rats and synaptic transmission and neurite morphogenesis pathways in FRL rats. When the effects of NT treatment and MS were compared between strains, carbohydrate metabolic pathways were predominantly modulated.     

Inhibition of the hepatotoxicity of paracetamol and its irreversible binding to rat liver microsomal protein

Dithiocarb and (+)-cyanidanol-3 prevented paracetamol-induced liver injury in rats in vivo. Both, as well as two other antihepatotoxic agents, deanol and DMSO, inhibited covalent binding of [³H]-paracetamol to rat liver microsomal proteins in vitro. Dithiocarb and (+)-cyanidanol-3 were the most effective inhibitors. The concentrations of the antidotes yielding 50% inhibition (I₅₀) valued 1 · 8 × 10^–5 M for dithiocarb and 2 · 1 × 10^–5 M for (+)-cyanidanol-3.     

Effects of N-cyclopentyladenosine and caffeine on sleep regulation in the rat

To study the role of adenosine in sleep regulation, the adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) and the antagonist caffeine were administered to rats. Intraperitoneal (i.p.) CPA 1 mg/kg but not 0.1 mg/kg, suppressed rapid-eye-movement (REM) sleep and enhanced electroencephalographic (EEG) slow-wave activity (power density 0.75–4.0 Hz) in non-REM sleep. The latter effect was remarkably similar to the response to 6-h sleep deprivation. The effects persisted when CPA-induced hypothermia was prevented. Caffeine (10 and 15 mg/kg i.p.) elicited a dose-dependent increase in waking followed by a prolonged increase of slow-wave activity in non-REM sleep. The combination of caffeine (15 mg/kg) and sleep deprivation caused less increase in slow-wave activity than sleep deprivation alone, indicating that caffeine may reduce the buildup of sleep pressure during waking. The results are consistent with the involvement of adenosine in the regulation of non-REM sleep.     

乙醇诱导大鼠肝脏超微结构改变与护肝灵的干预

目的 观察护肝灵对乙醇诱导的大鼠肝脏超微结构改变的影响.方法 以乙醇灌胃法诱导大鼠肝损伤模型,护肝灵高、低剂量干预,易善复为阳性对照,连续42 d,H-600Ⅳ型透视电镜观察肝细胞超微结构和糖原.结果 乙醇灌胃各组大鼠的肝细胞出现不同程度的核固缩、线粒体肿胀、内质网扩张,偶见酒精小体(Mallory小体);细胞内出现脂滴;细胞间出现胶原纤维;糖原减少或消失.护肝灵低剂量组与阳性药相当,肝细胞核轻微固缩;线粒体肿胀明显,嵴断裂消失,粗面内质网扩张;偶见糖原.高剂量组肝细胞超微结构清晰,优于阳性药,与阴性对照组比较无显著差异.结论 护肝灵对乙醇诱导的大鼠肝细胞超微结构破坏有保护作用.     

吴茱萸碱调节 Nod样受体蛋白 3信号通路对非酒精性脂肪性肝病大鼠肝组织损伤的影响

目的探讨吴茱萸碱调节 Nod样受体蛋白 3(NLRP3)信号通路影响非酒精性脂肪性肝病( NAFLD)大鼠肝组织损伤情况。方法 2022年 5—11月选取大鼠喂食高脂饲料 8周后,通过随机数字表法将大鼠分为模型组、吴茱萸碱低剂量组(吴茱萸碱 4 mg/kg)、吴茱萸碱中剂量组(吴茱萸碱 8 mg/kg)、吴茱萸碱高剂量组(吴茱萸碱 16 mg/kg)、阳性药组(多烯磷脂酰胆碱 200 mg/kg)和 NLRP3组(吴茱萸碱 16 mg/kg +1 mg/kg NLRP3信号通路激活剂尼日利亚菌素)每组各 10只,另有 10只大鼠喂食普通饲料作为对照组,所有大鼠均给予相对应药物干预,给药 4周;生化分析仪检测血清谷丙,转氨酶( GPT)、谷草转氨酶( GOT)、胆固醇、三酰甘油( TG); HE染色观察肝组织病理;油红 O染色观察肝组织脂肪沉积;酶联免疫吸附测定( ELISA)检测大鼠血清肿瘤坏死因子 α(TNF-α)、白细胞介素( IL)-18、IL-1β水平;实时荧光定量逆转录聚合酶链反应( qRT-PCR)和蛋白质印迹法检测大鼠肝脏组织 NLRP3、凋亡相关斑点样蛋白( ASC)、胱天蛋白酶 -1(caspase-1)表达水平。结果与对照组相比,模型组大鼠肝组织肝小叶结构紊乱,肝细胞增大,可见明显颗粒状脂滴以及脂质积累,并伴有炎症细胞浸润,肝组织脂肪变性明显,大鼠体质量［( 582.65±16.25)g比( 476.29±10.62)g］、肝指数［( 3.79±0.25)%比( 2.48±0.18)%］、血清 GPT［( 79.62±3.59)U/L比(36.22±2.15)U/L］、 GOT［( 185.25±5.18)U/L比( 71.69±3.56)U/L］、胆固醇［( 2.93±0.19)mmol/L比( 1.72±0.12)mmol/L］、 TG［( 1.19±0.11) mmol/L比( 0.56±0.07)mmol/L］、 TNF-α［( 162.48±4.25)ng/L比( 41.76±2.49)ng/L］、 IL-18［( 135.75±3.37)ng/L比( 23.52±2.02)ng/L］、 IL-1β［(201.88±4.67)ng/L比( 92.64±2.99)ng/L］水平,肝组织 NLRP3、ASC、caspase-1 mRNA表达以及 NLRP3、ASC、活化胱天蛋白酶 -1(cleaved caspase-1)蛋白表达均明显升高( P<0.05);与模型组相比,吴茱萸碱低、中、高剂量组和阳性药组大鼠肝组织损伤、肝脏脂肪变性、炎症细胞浸润和脂滴积累明显减轻,大鼠体质量［( 551.37±15.72)g、(530.52±15.49)g、(509.48±15.18)g、(517.71±12.73)g比( 582.65±16.25)g］、肝指数［( 3.41±0.20)%、(3.13±0.16)%、(2.81±0.17)%、(3.02±0.213)%比( 3.79±0.25)%］、血清 GPT［( 68.16±3.41)U/L、(55.94±3.05)U/L、(46.45±2.72)U/L、(48.71±2.34)U/L比( 79.62±3.59)U/L］、 GOT［( 161.32±4.73)U/ L、(138.64±4.50)U/L、(113.57±4.05)U/L、(121.48±4.11)U/L比( 185.25±5.18)U/L］、胆固醇［( 2.58±0.17)mmol/L、(2.25±0.16) mmol/L、(1.91±0.13)mmol/L、(1.99±0.15)mmol/L比( 2.93±0.19)mmol/L］、 TG［( 1.01±0.10)mmol/L、(0.83±0.08)mmol/L、(0.62±0.07)mmol/L、(0.70±0.09)mmol/L比( 1.19±0.11)mmol/L］、 TNF-α［( 137.15±3.69)ng/L、(113.53±3.34)ng/L、(79.37±2.92)ng/L、(85.61±3.07)ng/L比( 162.48±4.25)ng/L］、 IL-18［( 111.34±3.05)ng/L、(72.98±2.66)ng/L、(47.61±2.438)ng/L、(51.59±2.55)ng/L比(135.75±3.37)ng/L］、 IL-1β［(171.52±4.34)ng/L、(152.23±4.02)ng/L、(129.95±3.51)ng/L、(517.71±12.73)ng/L比( 136.76±3.73)ng/L］水平,肝组织NLRP3、ASC、caspase-1 mRNA表达以及 NLRP3、ASC、cleaved caspase-1蛋白表达均明显降低( P<0.05); NLRP3信号通路激活剂尼日利亚菌素的加入明显减弱了吴茱萸碱对 NAFLD大鼠肝组织的保护作用。结论吴茱萸碱可通过抑制 NLRP3信号通路明显改善 NAFLD大鼠肝组织损伤、脂肪病变和炎症反应。     

Changes in cytochrome P450 enzymes by 1,1-dichloroethylene in rat liver and kidney

We examined the effect of 1,1-dichloroethylene (1,1-DCE) on microsomal cytochrome P450 (P450) enzymes in rat liver and kidney. Rats were treated intraperitoneally with 1,1-DCE daily for 4 days, at doses of 200, 400, and 800 mg/kg. Among the P450-dependent monooxygenase activities in liver microsomes, testosterone 2α-hydroxylase (T2AH), which is associated with CYP2C11 activity, was remarkably decreased by 800 mg/kg 1,1-DCE. The level relative to control activity was <10%. Furthermore, immunoblotting showed that 1,1-DCE (≥400 mg/kg) significantly decreased CYP2C11/6 protein levels in liver microsomes. In addition, 7-methoxyresorufin O-demethylase (MROD), 7-ethoxycoumarin O-deethylase (ECOD), benzphetamine N-demethylase (BZND), chlorzoxazone 6-hydroxylase (CZ6H), and testosterone 6β-hydroxylase (T6BH) activities were significantly decreased by the highest dose of 1,1-DCE (by 40–70%). However, the activities of other P450-dependent monooxygenases, namely 7-ethoxyresorufin O-deethylase (EROD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND), erythromycin N-demethylase (EMND), lauric acid ω-hydroxylase (LAOH), and testosterone 7α-hydroxylase (T7AH) were not affected by 1,1-DCE at any dose. Immunoblotting showed CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A2/1 protein levels were significantly decreased by 60–66% by 1,1-DCE (800 mg/kg), whereas that of CYP4A1/2 was not affected by any dose of 1,1-DCE. By contrast, among the P450-dependent monooxygenase activities in kidney microsomes, only CZ6H activity was increased by 1,1-DCE (1.6-fold at 800 mg/kg). Also, it was␣observed that 1,1-DCE (800 mg/kg) significantly increased CYP2E1 protein levels by immunoblotting (∼1.5-fold). These results suggest that 1,1-DCE changes the constitutive P450 isoforms in the rat liver and kidney, and that these changes closely relate to the toxicity of 1,1-DCE. Received: 28 January 1997 / Accepted: 18 August 1997     

Role of ATP and glycogen reserves in both paracetamol sulfation and glucuronidation by cultured precision-cut rat liver slices

Precision-cut rat liver slices (PCLS) were used to investigate the formation of paracetamol conjugates. The time course of biochemical markers such as ATP and GSH content, glycogen levels and protein synthesis rates was recorded over a period of time of 26 h and taken as index of slices viability. Low values of ATP (3.6 nmol/mg prot), GSH (7.1 nmol/mg prot) and protein synthesis rates (94.1 pmol leu/mg prot×min⁻¹) were initially observed. Thereafter, they gradually recovered up to 6 h but decreased values were seen after 20 h. Glycogen, however, dropped rapidly during the first 6 h, being no longer detected after 20 h of incubation. The reincubation of PCLS in a fresh medium for 6 h allowed a strong recovery of GSH, ATP and protein synthesis rates, but no gluconeogenesis was observed. Meanwhile, paracetamol sulfate formation was fairly constant (about 3 μg/mg protein) while glucuronide gradually disappeared. The amount of both UGT1A1 and ST1A1 did not correlate with their respective enzymatic activities. We suggest that loss of glycogen impair glucuronide conjugation by decreasing the availability of UDPGA, and that low values of ATP are largely enough to support sulfotransferase activity.     

以CDNB为探针测定大鼠肝微粒体中GST活性并优化反应体系

目的以1-氯-2,4-二硝基苯(CDNB)为探针,优化大鼠肝微粒体中谷胱甘肽硫转移酶(GST)催化反应的条件,为准确测定GST活性提供依据。方法采用Welch Materials Ultimate TM XB C18反相柱(250 mm×4.6 mm,5μm),流动相为乙腈-水(7∶3),流速0.8 mL.min-1,检测波长238 nm。首先于0.05 mg.mL-1蛋白浓度下,孵育10 min,检测CDNB能否被GST催化发生反应,然后分别在CDNB及CDNB+GSH两种反应条件下,比较不同浓度蛋白、孵育时间、底物浓度的反应差异,选出合理反应条件。结果 CDNB在选定色谱条件下实现了快速分离,且无内源性干扰。CDNB在GST催化下发生了反应;在不同浓度蛋白、孵育时间、底物浓度下,两种反应条件下CDNB反应存在明显差异(P<0.05):CDNB及GSH作为起始条件时反应较少,此外,CDNB反应量与蛋白浓度、孵育时间分别呈线性关系,是优化反应条件的重要依据。结论采用CDNB测定GST活性时,选择合适的CDNB及GSH反应条件能准确地测定GST活性,可用于GST活性测定及相关动力学分析。