Impact of culture medium on the interpretation of qRT-PCR data in HepG2 incubated with lactobacilli.

Recently, an increasing number of studies have investigated the mechanism of action of lactobacilli in the treatment of non-alcoholic fatty liver disease. Using four computational tools (NormFinder, geNorm, Delta Ct, and BestKeeper), six potential reference genes (RGs) were analyzed in the human liver cell line HepG2 cultivated 24 h in the presence of two strains of heat-killed lactobacilli, Limosilactobacillus reuteri E and Lactiplantibacillus plantarum KG4, respectively, in different cultivation media [Dulbecco´s Modified Eagle´s Medium (DMEM) high glucose or Roswell Park Memorial Institute (RPMI)]. The analysis revealed that the suitability of RG was similar between the two lactobacilli but quite different between the two media. The commonly used RGs, 18S rRNA and glyceraldehyde-3-phosphate dehydrogenase were the most unstable in DMEM high glucose. Normalization of the mRNA expression of the target gene encoding sterol regulatory element-binding protein 1c (SREBP-1c) to different RGs resulted in different expression profiles. This demonstrates that validation of candidate RGs under specific experimental conditions is crucial for the correct interpretation of quantitative polymerase chain reaction data. In addition, the choice of media has a profound impact on the effect of lactobacilli on lipogenesis at the gene expression level, as shown by the transcription factor SREBP-1c.

Crocus sativus L. Extract (Saffron) Effectively Reduces Arthritic and Inflammatory Parameters in Monotherapy and in Combination with Methotrexate in Adjuvant Arthritis.

Rheumatoid arthritis (RA), an autoimmune disease, is characterized by inflammation that affects not only the liver but also other organs and the musculoskeletal system. The standard therapy for RA is methotrexate (MTX), which has safety limitations. The extract from Crocus sativus L. (saffron-SF) is also known for its anti-inflammatory effects. Therefore, we decided to investigate the potential benefit of SF in monotherapy via two doses (SF1-25 mg/kg of b.w.; SF2-50 mg/kg of b.w.) and in combination with MTX (0.3 mg/kg of b.w., twice a week) using adjuvant arthritis in rats. To evaluate these therapeutic settings, we used biometric, immunological, and biochemical parameters, as well as the relative gene expression of the mRNA in the liver. Our results showed a statistically significant increase in the experimental animals' body weight and the arthritic score (AS) on day 14 for monotherapy with SF1 and SF2. The change of hind paw volume (CHPV) was significant only for SF2 monotherapy on the 14th day of the experiment. A combination of SF1 and SF2 with MTX significantly modulated all the biometric parameters during the experimental period. Additionally, AS and CHPV improved considerably compared to MTX monotherapy on day 21. Furthermore, all monotherapies and combination therapies were significant for the biochemical parameter γ-glutamyl transferase (GGT) in the joint. GGT activity in the spleen was less pronounced; only MTX in combination with SF1 significantly modified this parameter. The higher dose of SF monotherapy (SF2) was similarly significant with respect to immunological parameters, such as plasmatic IL-17A, IL-1ß, and MMP-9 on day 21. The combination of both doses of SF with MTX significantly improved these immunological parameters, except for C-reactive protein (CRP), which was influenced only by the higher dose of SF2 in combination with MTX in plasma at the end of the experiment. A different effect was found for the relative expression of CD36 mRNA, where only SF1 significantly decreased gene expression in the liver. However, the relative gene mRNA expression of IL-1ß in the liver was significantly reduced by the SF monotherapies and the combination of both SF doses with MTX. Our findings showed SF's partial antiarthritic and anti-inflammatory potential in monotherapy, but the effect was stronger in combination with MTX.

Combination Therapy of Carnosic Acid and Methotrexate Effectively Suppressed the Inflammatory Markers and Oxidative Stress in Experimental Arthritis.

BACKGROUND: Combination therapy with methotrexate (MTX) is the most common therapeutic strategy used for the treatment of patients with rheumatoid arthritis (RA). In this study, we combined the natural compound carnosic acid (CA) with MTX to reduce inflammation and oxidative stress in adjuvant arthritis (AA). METHODS: AA was induced in 6-8 rats per group. MTX was administrated twice a week at a dose of 0.3 mg/kg b.w., while CA was administered daily at a dose of 100 mg/kg both in monotherapy and in combination with MTX. Plasma samples were collected on the 14th, 21st, and 28th day. Body weight and hind paw volume were measured once a week. RESULTS: We found that, mainly, the CA + MTX combination significantly reduced the hind paw swelling, the levels of IL-17A, MMP-9, and MCP-1 in plasma, and GGT activity in joint homogenates. The mRNA expression of HO-1, catalase, and IL-1ß in the liver were significantly improved by CA + MTX only. Our results indicate that adding CA to MTX treatment could be a good therapeutic option for patients suffering from RA. CONCLUSIONS: The addition of CA to methotrexate treatment significantly improved its efficacy in decreasing the development of AA by inhibiting the markers of inflammation and oxidative stress.

Alterations of HDL's to piHDL's Proteome in Patients with Chronic Inflammatory Diseases, and HDL-Targeted Therapies.

Chronic inflammatory diseases, such as rheumatoid arthritis, steatohepatitis, periodontitis, chronic kidney disease, and others are associated with an increased risk of atherosclerotic cardiovascular disease, which persists even after accounting for traditional cardiac risk factors. The common factor linking these diseases to accelerated atherosclerosis is chronic systemic low-grade inflammation triggering changes in lipoprotein structure and metabolism. HDL, an independent marker of cardiovascular risk, is a lipoprotein particle with numerous important anti-atherogenic properties. Besides the essential role in reverse cholesterol transport, HDL possesses antioxidative, anti-inflammatory, antiapoptotic, and antithrombotic properties. Inflammation and inflammation-associated pathologies can cause modifications in HDL's proteome and lipidome, transforming HDL from atheroprotective into a pro-atherosclerotic lipoprotein. Therefore, a simple increase in HDL concentration in patients with inflammatory diseases has not led to the desired anti-atherogenic outcome. In this review, the functions of individual protein components of HDL, rendering them either anti-inflammatory or pro-inflammatory are described in detail. Alterations of HDL proteome (such as replacing atheroprotective proteins by pro-inflammatory proteins, or posttranslational modifications) in patients with chronic inflammatory diseases and their impact on cardiovascular health are discussed. Finally, molecular, and clinical aspects of HDL-targeted therapies, including those used in therapeutical practice, drugs in clinical trials, and experimental drugs are comprehensively summarised.

Silver Nanoparticles Stabilized with Phosphorus-Containing Heterocyclic Surfactants: Synthesis, Physico-Chemical Properties, and Biological Activity Determination.

Phosphorus-containing heterocyclic cationic surfactants alkyldimethylphenylphospholium bromides with the alkyl chain length 14 to 18 carbon atoms were used for the stabilization of silver nanodispersions. Zeta potential of silver nanodispersions ranges from +35 to +70 mV, which indicates the formation of stable silver nanoparticles (AgNPs). Long-chain heptadecyl and octadecyl homologs of the surfactants series provided the most intensive stabilizing effect to AgNPs, resulting in high positive zeta potential values and smaller diameter of AgNPs in the range 50-60 nm. A comparison with non-heterocyclic alkyltrimethylphosphonium surfactants of the same alkyl chain length showed better stability and more positive zeta potential values for silver nanodispersions stabilized with heterocyclic phospholium surfactants. Investigations of biological activity of phospholium-capped AgNPs are represented by the studies of antimicrobial activity and cytotoxicity. While cytotoxicity results revealed an increased level of HepG2 cell growth inhibition as compared with the cytotoxicity level of silver-free surfactant solutions, no enhanced antimicrobial action of phospholium-capped AgNPs against microbial pathogens was observed. The comparison of cytotoxicity of AgNPs stabilized with various non-heterocyclic ammonium and phosphonium surfactants shows that AgNPs capped with heterocyclic alkyldimethylphenylphospholium and non-heterocyclic triphenyl-substituted phosphonium surfactants have the highest cytotoxicity among silver nanodispersions stabilized by the series of ammonium and phosphonium surfactants.

Mucin pre-cultivated Lactobacillus reuteri E shows enhanced adhesion and increases mucin expression in HT-29 cells.

Adhesion of probiotic bacteria to the mucus layer lining the gastrointestinal tract is necessary for its effective colonisation and specific therapeutic effects. Enrichment of growth medium with mucin might stimulate bacterial adhesion, probably by increasing the expression of surface structures responsible for bacteria-gut epithelia and/or mucus interactions. The aim of this study was to determine if pre-cultivation of potentially probiotic strain Lactobacillus reuteri E (LRE) with mucin stimulates its adherence to colon cell line HT-29 and if the increased adhesion modulates mucin expression in these cells. The mucin-producing HT-29 cell line was co-cultivated for 2 h with LRE grown in MRS broth or MRS broth enriched with pig gastric mucin (LRE + M). The adherence ability of LRE was evaluated microscopically and by plate counting. The relative gene expression was measured by qPCR. Pre-cultivation of LRE in mucin enriched medium significantly increased its adhesion to 14 days HT-29 in comparison with LRE by both methods (28.64% vs. 23.83%, evaluated microscopically, and 14.31 ± 3.95 × 106 CFU ml-1 vs. 8.54 ± 0.43 × 106 CFU ml-1, evaluated by plate counting). MUC2, MUC5AC, and IL-10 were significantly upregulated after co-cultivation with LRE + M in comparison to LRE and control group (lactobacilli-free HT-29). Obtained results suggest that pre-cultivation of lactobacilli with mucin may not only stimulate their adhesion abilities but also promote their effectiveness to modulate the pathways involved in the pathophysiology of some diseases, e.g., with defective mucin synthesis in ulcerative colitis or colorectal cancer.

Ferulaldehyde Improves the Effect of Methotrexate in Experimental Arthritis.

Methotrexate (MTX) is still the gold standard for treatment of rheumatoid arthritis (RA). The therapeutic efficacy of low-dose of MTX can be increased by its combination with a natural substance, ferulaldehyde (FRA). The aim of this study was to evaluate the effect FRA and MTX administered alone or in combination in adjuvant arthritis. The disease was induced to Lewis male rats by intradermal injection, which contains a suspension of heat-inactivated Mycobacterium butyricum in incomplete Freund's adjuvant. The experiment of 28 days included: healthy animals, arthritic animals, arthritic animals with administration of FRA at the oral daily dose of 15 mg/kg, arthritic animals with administration of MTX at the oral dose of 0.3 mg/kg twice a week, and arthritic animals administered with FRA and MTX. FRA in monotherapy decreased significantly only the level of interleukin-1ß (IL-1ß) and matrix metalloproteinase-9 in plasma. Combination of FRA and low-dose MTX was more effective than MTX alone when comparing body weight, hind paw volume, arthritic score, plasmatic levels of IL-1ß, activity of γ-glutamyl transferase, and relative mRNA expression of IL-1ß in the spleen. Therefore, the combination treatment was the most effective. The obtained results are interesting for future possible innovative therapy of patients with RA.

Evaluation of liposomal carnosine in adjuvant arthritis.

Liposomal carnosine could overcome the problems associated with direct application of this peptide. Anti-inflammatory and antioxidant effects of liposomal and non-liposomal carnosine in adjuvant arthritis were compared. The experiments were done on healthy animals, untreated arthritic animals, arthritic animals with oral administration of carnosine, and with subcutaneous administration of liposomal carnosine, both administered in the same daily dose of 150 mg/kg b.w. during 28 days. Carnosine reduced hind paw volume on day 28. Both forms markedly decreased interleukin-1ß, matrix metalloproteinase-9 and monocyte chemoattractant protein-1 (MCP-1) in plasma on day 14. Only liposomal carnosine reduced significantly MCP-1. Malondialdehyde, 4-hydroxynonenal, resistance to Fe2+-induced oxidation and protein carbonyls were significantly corrected after administration of any form of carnosine. Liposomal carnosine corrected more effectively the oxidative stress in plasma than did carnosine. In brain tissue, our results showed protective ability of both forms of carnosine against oxidation of proteins and lipids. They also corrected the resistance to Fe2+-induced oxidation in arthritic animals. We found that only liposomal carnosine decreased the mRNA expression of inducible nitric oxide synthase in cartilage tissue. It can be concluded that the liposomal drug-delivery system is improving the pharmacological properties of carnosine administered in arthritis.

Effect of N-Feruloylserotonin and Methotrexate on Severity of Experimental Arthritis and on Messenger RNA Expression of Key Proinflammatory Markers in Liver.

Rheumatoid arthritis (RA) is a chronic inflammatory disease, leading to progressive destruction of joints and extra-articular tissues, including organs such as liver and spleen. The purpose of this study was to compare the effects of a potential immunomodulator, natural polyphenol N-feruloylserotonin (N-f-5HT), with methotrexate (MTX), the standard in RA therapy, in the chronic phase of adjuvant-induced arthritis (AA) in male Lewis rats. The experiment included healthy controls (CO), arthritic animals (AA), AA given N-f-5HT (AA-N-f-5HT), and AA given MTX (AA-MTX). N-f-5HT did not affect the body weight change and clinical parameters until the 14th experimental day. Its positive effect was rising during the 28-day experiment, indicating a delayed onset of N-f-5HT action. Administration of either N-f-5HT or MTX caused reduction of inflammation measured as the level of CRP in plasma and the activity of LOX in the liver. mRNA transcription of TNF-α and iNOS in the liver was significantly attenuated in both MTX and N-f-5HT treated groups of arthritic rats. Interestingly, in contrast to MTX, N-f-5HT significantly lowered the level of IL-1ß in plasma and IL-1ß mRNA expression in the liver and spleen of arthritic rats. This speaks for future investigations of N-f-5HT as an agent in the treatment of RA in combination therapy with MTX.

Cooperation between the INO80 complex and histone chaperones determines adaptation of stress gene transcription in the yeast Saccharomyces cerevisiae.

In yeast, environmental stresses provoke sudden and dramatic increases in gene expression at stress-inducible loci. Stress gene transcription is accompanied by the transient eviction of histones from the promoter and the transcribed regions of these genes. We found that mutants defective in subunits of the INO80 complex, as well as in several histone chaperone systems, exhibit extended expression windows that can be correlated with a distinct delay in histone redeposition during adaptation. Surprisingly, Ino80 became associated with the ORFs of stress genes in a stress-specific way, suggesting a direct function in the repression during adaptation. This recruitment required elongation by RNA polymerase (Pol) II but none of the histone modifications that are usually associated with active transcription, such as H3 K4/K36 methylation. A mutant lacking the Asf1-associated H3K56 acetyltransferase Rtt109 or Asf1 itself also showed enhanced stress-induced transcript levels. Genetic data, however, suggest that Asf1 and Rtt109 function in parallel with INO80 to restore histone homeostasis, whereas Spt6 seems to have a function that overlaps that of the chromatin remodeler. Thus, chromatin remodeling by INO80 in cooperation with Spt6 determines the shape of the expression profile under acute stress conditions, possibly by an elongation-dependent mechanism.