Applications like drug development need simple and streamlined methods to process samples from 96-well cell culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well cell culture plates while being relatively cheap and simple. We developed a method based on the qPCR 'Cells-to-cDNA' approach and validated it against commercially available kits using the same approach or spin columns-based RNA purification. For this purpose, we conducted a series of comparisons of gene expression from peripheral blood mononuclear cells, SK-HEP-1 and U-87 cell cultures in 96-well plates. Our final method involved lysing cells with 25-100 µl solution of 0.5% SDS, 10 mM DTT, 1 mg ml-1 proteinase K dissolved in water, 1 h incubation at 50°C, followed by proteinase K inactivation at 90°C for 5 min and lysate neutralization with 1 : 1 dilution by 20% Tween 20 solution. Reverse transcription and qPCR were carried out using standard methods. This method showed a mean reduction of Ct ± s.d. value by 2.4 ± 1.3 compared with the 'Cells-to-cDNA' kit and by 1.4 ± 0.5 compared with the RNA purification kit with lower variability.

• Klíčová slova
• RNA isolation, cell lysis, in vitro, mRNA, peripheral blood mononuclear cells, proteinase k, qPCR,
• MeSH
• analýza nákladů a výnosů MeSH
• buněčné kultury metody   ekonomika   MeSH
• komplementární DNA * genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• leukocyty mononukleární cytologie   metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• stanovení celkové genové exprese metody   ekonomika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• komplementární DNA * MeSH
• messenger RNA MeSH

BACKGROUND: The complexity of the galaninergic system is still not fully understood, especially under specific pre-existing comorbidities related to metabolic dysfunction. A plant-derived triterpenoid celastrol was demonstrated to exert a complex effect on the galaninergic system and to have hepatoprotective and anti-obesity properties. However, the exact molecular mechanisms responsible for these effects remain unclear. Specifically, there are no data on the impact of celastrol on the heart and liver galaninergic system. Therefore, this study aimed to investigate the effects of celastrol on the galaninergic system expression in the heart and liver of mice suffering from diet-induced obesity and metabolic dysfunction-associated steatotic liver disease and steatohepatitis (MASLD/MASH). METHODS: The male mice C57BL/6J were fed a Western-type high-fat diet for 16 and 20 weeks to induce obesity and MASLD/MASH. Celastrol was administered along with a specific diet for the last 4 weeks to evaluate its impact on the progression of these conditions. Moreover, the inhibitor of sterol regulatory element-binding protein 1/2 (SREBP1/2), fatostatin, was also tested to compare its influence on the galaninergic system with celastrol. RESULTS: The study demonstrates that celastrol treatment was safe and led to a reduction in food and energy intake, body fat and liver weights, and MASLD-to-MASH progression and improved glucose tolerance, serum biochemistry markers, and hepatic lipid peroxidation in mice. Quantitative gene expression originally showed significant regulation of galanin and all three of its receptors (GalR1/2/3) in the heart ventricles and only GalR2 in the liver of obese mice. Celastrol influenced the gene expression of galanin receptors: it downregulated Galr1 in the heart and upregulated Galr2 in the liver and Galr3 in the heart ventricles, potentially affecting energy metabolism, oxidative stress, and inflammation. Fatostatin suppressed gene expression of all the detected members of the galaninergic system in the heart ventricles, depicting the role of SREBP in this process. CONCLUSION: These findings suggest that celastrol may beneficially modulate the galaninergic system under obesity and MASLD-to-MASH progression, indicating its potential as a therapeutic agent for disorders associated with metabolic dysfunction.

• Klíčová slova
• MASH, MASLD, celastrol, fatostatin, galanin receptor, heart, mouse, obesity,
• Publikační typ
• časopisecké články MeSH

A laboratory-scale bioreactor was re-evaluated, with the aim of improving its use for the perfused culture of rat hepatocytes. In contrast to conventional culture systems, the flat membrane bioreactor (FMB) showed good functionality and biochemical competence during 2-3 days. Hepatocytes cultured in the FMB, specifically in a "sandwich" configuration, were functionally stable, as shown by a high rate of urea biosynthesis after challenge with NH4Cl, a low alanine-aminotransferase leakage and suppressed spontaneous nitric oxide (NO) production. Moreover, the time-course of the disappearance of cyclosporin A (CsA) from the perfusate demonstrated the high biotransformation capacity of cells in the FMB. The effect of CsA on the modulation of urea and spontaneous NO production demonstrated flexibility, in that minor changes could be observed at diverse time intervals and in a non-destructive way. The monitoring of nitrite levels during various steps of isolation and culture suggested that spontaneously produced NO has a negative impact on hepatocyte metabolic and functional integrity. In spite of the sophisticated techniques that are being used for the preparation of bioreactors, with hepatocytes surviving for longer periods, our data have shed light on some factors that could be important for the successful use of similar models for pharmacotoxicological and other biomedical applications.

• MeSH
• alternativy výzkumu na zvířatech metody   MeSH
• analýza rozptylu MeSH
• bioreaktory * MeSH
• biotransformace MeSH
• časové faktory MeSH
• cyklosporin metabolismus   MeSH
• hepatocyty metabolismus   MeSH
• hodnotící studie jako téma MeSH
• krysa rodu Rattus MeSH
• membrány metabolismus   MeSH
• močovina metabolismus   MeSH
• oxid dusnatý biosyntéza   MeSH
• testy toxicity metody   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• cyklosporin MeSH
• močovina MeSH
• oxid dusnatý MeSH

Effects of endotoxemia-induced NO production on rat liver and hepatocytes in culture were investigated. Rats were treated intraperitoneally with saline, lipopolysaccharide (LPS, 10 mg/kg), L-nitroarginine methyl ester (L-NAME)+LPS, aminoguanidine (AG)+LPS, FK 506+LPS, S-nitroso-N-acetyl penicillamine (SNAP)+L-NAME+LPS and SNAP+FK 506+LPS. Mortality, hepatocyte viability and liver function test were estimated. Liver morphology was observed by light and electron microscopy. Hepatocyte cultures were treated with LPS, cytokine mixture (CM) with or without FK 506, L-NAME or AG. Hepatocyte function and inducible form of NOS (iNOS) expression were evaluated. Twenty-four hours after treatments with saline, LPS, L-NAME+LPS, AG+LPS, FK 506+LPS, SNAP+L-NAME+LPS and SNAP+FK 506+LPS, rat mortalities were 0%, 10%, 48%, 8%, 20%, 38% and 0%, and hepatocyte viabilities were 93+/-3%, 80+/-3%, 52+/-8%, 88+/-1%, 70+/-3%, 80+/-4% and 82+/-3%, respectively. AG+LPS or L-NAME+LPS administration was followed by excessive vacuolization of hepatocytes with lesions in the intermediary lobule zone characterized by features of secondary necrosis as a continuation of apoptotic processes. SNAP+L-NAME+LPS resulted in a well-preserved structure of central vein lobules with sparse signs of apoptosis. Treatment with LPS or CM increased iNOS expression in hepatocyte culture, which was inhibited by L-NAME, FK 506 or AG. AG reduced LPS-induced rise in alanine aminotransferase leakage. LPS-induced NO exerts cytoprotective effects in vivo, while LPS-induced NO in vitro appears to be toxic. Based on the data of this report, one cannot use in vitro results to predict in vivo responses to LPS-induced NO production. The pharmacological modulation of iNOS expression or NO production in vivo or in vitro, therefore, by the development of specific NO donors or inhibitors is promising for improvement of hepatocyte functions under the two experimental conditions, respectively.

• MeSH
• alanintransaminasa krev   MeSH
• dusitany analýza   MeSH
• guanidiny farmakologie   MeSH
• hepatocyty účinky léků   metabolismus   patologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• interferon gama farmakologie   MeSH
• interleukin-1 farmakologie   MeSH
• játra účinky léků   metabolismus   patologie   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lipopolysacharidy farmakologie   toxicita   MeSH
• messenger RNA účinky léků   genetika   metabolismus   MeSH
• NG-nitroargininmethylester farmakologie   MeSH
• penicilamin analogy a deriváty   farmakologie   MeSH
• potkani Wistar MeSH
• regulace genové exprese enzymů účinky léků   MeSH
• synthasa oxidu dusnatého, typ II MeSH
• synthasa oxidu dusnatého antagonisté a inhibitory   genetika   MeSH
• takrolimus farmakologie   MeSH
• TNF-alfa farmakologie   MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanintransaminasa MeSH
• dusitany MeSH
• guanidiny MeSH
• inhibitory enzymů MeSH
• interferon gama MeSH
• interleukin-1 MeSH
• lipopolysacharidy MeSH
• messenger RNA MeSH
• NG-nitroargininmethylester MeSH
• Nos2 protein, rat MeSH Prohlížeč
• penicilamin MeSH
• pimagedine MeSH Prohlížeč
• S-nitro-N-acetylpenicillamine MeSH Prohlížeč
• synthasa oxidu dusnatého, typ II MeSH
• synthasa oxidu dusnatého MeSH
• takrolimus MeSH
• TNF-alfa MeSH

The goals of the present study were to provide information into the controversy about nitric oxide (NO) status of the liver during endotoxemia and to assess the role of the phosphatase inhibitor cyclosporin A (CsA) during the insult. Rats were injected with saline, lipopolysaccharide (LPS, 10 mg/kg i.p.) or cyclosporin A (CsA, 5 mg/kg. i.p.) + LPS, S-nitroso-N-acetyl penicillamine (SNAP, 0.1 mMikg) + CsA + LPS or molsidomine (molsid, 0.2 mg/kg) + CsA + LPS. Rat hepatocytes were isolated and tested for metabolic competence by the rate of urea synthesis and for lipid peroxidation. Hepatocytes were cultured under various treatments as LPS or cytokine mixture (CM, TNF-alpha 500 U/ml, INF-gamma 100 U/ml, IL-1beta 200 U/ ml) with or without CsA and iNOS expression was evaluated by NO productivity and by RT-PCR. Twenty-four hours after LPS dosing in vivo, the mortality rate was 15%, while CsA pretreatment increased mortality rate to 30% and reduced hepatocyte viability, increased ALT leakage and reduced urea synthesis. SNAP and Molsid resulted in complete survival of rats, increased urea synthesis, increased cell viability and reduced alanine aminotransferase leakage. LPS or CM increased iNOS expression while CsA pretreatment reduced iNOS expression. There was no correlation between lipid peroxide levels in hepatocytes and functional status of hepatocytes under various treatments. This study demonstrates that NO produced during endotoxemia and under the present conditions is protective to the liver and may function as an adaptive mechanism and that the inhibition of iNOS by compounds like CsA produce unfavorable effects.

• MeSH
• alanintransaminasa metabolismus   MeSH
• chromatografie agarózová MeSH
• cyklosporin farmakologie   MeSH
• donory oxidu dusnatého farmakologie   MeSH
• endotoxemie enzymologie   MeSH
• hepatocyty účinky léků   imunologie   patologie   MeSH
• imobilizované buňky MeSH
• imunosupresiva farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• krysa rodu Rattus MeSH
• kultivační média MeSH
• močovina metabolismus   MeSH
• nízká teplota MeSH
• peroxidace lipidů účinky léků   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• potkani Wistar MeSH
• regulace genové exprese enzymů účinky léků   MeSH
• separace buněk MeSH
• synthasa oxidu dusnatého, typ II MeSH
• synthasa oxidu dusnatého antagonisté a inhibitory   biosyntéza   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alanintransaminasa MeSH
• cyklosporin MeSH
• donory oxidu dusnatého MeSH
• imunosupresiva MeSH
• inhibitory enzymů MeSH
• kultivační média MeSH
• močovina MeSH
• Nos2 protein, rat MeSH Prohlížeč
• synthasa oxidu dusnatého, typ II MeSH
• synthasa oxidu dusnatého MeSH