Compoziția acizilor grași în laptele de vacă de băut din rețelele comerciale din or. Kiev, Ucraina

Introduction. There are several important aspects requiring a control of fatty acid composition in drinking milk distributed across the retail stores: milk is one of the main consumer products; fatty acids are the main energy substrates, involved in the synthesis of cellular structural components, whereas their interaction might impact the intensity of body growth and development; the fatty acid composition of milk varies depending on the diet and the animal's specific physiological state; in case of udder diseases, a change milk composition may occur; prevention of product counterfeiting distributed across the retail stores. Material and methods. The present research examined milk samples from five dairy producers that distribute their products across the retailing chains in Kyiv. Milk samples were collected to determine the fatty acid composition. Milk fat was extracted following the Folch method. The milk fatty acid content assessment was carried out by gas chromatography. Results. 20 fatty acids were identified in the tested milk samples. A higher level of saturated fatty acids was recorded in milk from "Ferma" and "Selyanskoye for kids" dairy producers. The highest content of unsaturated fatty acids was registered in products of “Ukrainskoe” and “Molokia” dairy trademarks. Conclusions. The fatty acid composition of the drinking cow’s milk distributed across trading networks in Kiev was found to be heterogeneous, by differing in the content of both long-chain saturated fatty acids and unsaturated fatty acids.Introducere. Există câteva aspecte importante care necesită un control riguros al compoziției acizilor grași din laptele de băut distribuit în magazinele de vânzare cu amănuntul: laptele este unul dintre produsele esențiale de consum; acizii grași sunt principalele substraturi energetice implicate în sinteza componentelor structurale celulare, în timp ce interacțiunea lor ar putea afecta intensitatea dezvoltării organismului uman; compoziția acizilor grași din lapte variază în funcție de alimentație și de starea fiziologică specifică animalului; în cazul bolilor ugerului poate surveni modificarea compoziției laptelui; prevenirea contrafacerii produselor lactate distribuite în magazinele de vânzare cu amănuntul. Material si metode. Prin prezenta cercetare s-au examinat mostrele de lapte de la cinci producători de lactate care își distribuie produsele prin lanțurile de vânzare cu amănuntul din or. Kiev pentru a determina compoziția acizilor grași. Grăsimea din lapte a fost extrasă prin metoda Folch. Evaluarea conținutului de acizi grași din lapte a fost efectuată prin cromatografie gazoasă. Rezultate. În probele de lapte testate au fost identificați 20 de acizi grași. Un nivel mai ridicat de acizi grași saturați a fost înregistrat în laptele de la producătorii de lactate „Ferma” și „Selyanskoye pentru copii”. Cel mai mare conținut de acizi grași nesaturați a fost înregistrat în produsele mărcilor comerciale de lactate „Ukrainskoe” și „Molokia”. Concluzii. Compoziția de acizi grași din laptele de vacă distribuit în rețelele comerciale din or. Kiev s-a dovedit a fi heterogenă, prin diferența în conținutul atât de acizi grași saturați cu lanț lung, cât și de acizi grași nesaturați

Comparison of the Therapeutic Effects of Human and Mouse Adipose-Derived Stem Cells in a Murine Model of Lipopolysaccharide-Induced Acute Lung Injury

Introduction. Adipose-derived stem cells (ASCs) have emerged as important regulators of inflammatory/immune responses in vitro and in vivo and represent attractive candidates for cell-based therapies for diseases that involve excessive inflammation. Acute lung injury (ALI) is an inflammatory condition for which treatment is mainly supportive due to lack of effective therapies. In this study, the therapeutic effects of ASC-based therapy were assessed in vivo by comparison of the anti-inflammatory properties of both human and murine ASCs in a mouse model of lipopolysaccharide (LPS)-induced ALI. Methods. Human ASCs (hASCs) or mouse ASCs (mASCs) were delivered to C57Bl/6 mice (7.5 x 105 total cells/mouse) by oropharyngeal aspiration (OA) four hours after the animals were challenged with lipopolysaccharide (15 mg/kg). Mice were sacrificed 24 and 72 hours after LPS exposure, and lung histology examined for evaluation of inflammation and injury. Bronchoalveolar lavage fluid (BALF) was analyzed to determine total and differential cell counts, total protein and albumin concentrations, and myeloperoxidase (MPO) activity. Cytokine expression in the injured lungs was measured at the steady-state mRNA levels and protein levels for assessment of the degree of lung inflammation. Results: Both human and mouse ASC treatments provided protective anti-inflammatory responses. There were decreased levels of leukocyte (for example neutrophil) migration into the alveoli, total protein and albumin concentrations in BALF, and MPO activity after the induction of ALI following both therapies. Additionally, cell therapy with both cell types effectively suppressed the expression of proinflammatory cytokines and increased the anti-inflammatory cytokine interleukin 10 (IL-10). Overall, the syngeneic mASC therapy had a more potent therapeutic effect than the xenogeneic hASC therapy in this model. Conclusions: Treatment with hASCs or mASCs significantly attenuated LPS-induced acute lung injury in mice. These results suggest a potential benefit for using an ASC-based therapy to treat clinical ALI and may possibly prevent the development of acute respiratory distress syndrome (ARDS)

Tubastatin ameliorates pulmonary fibrosis by targeting the TGFβ-PI3K-Akt pathway

<div><p>Background</p><p>Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal disease. Histone deacetylase 6 (HDAC6) alters function and fate of various proteins via deacetylation of lysine residues, and is implicated in TGF-β1-induced EMT (epithelial-mesenchymal transition). However, the role of HDAC6 in pulmonary fibrosis is unknown.</p><p>Methods</p><p>HDAC6 expression in IPF and control lungs was assessed by quantitative real-time PCR (qRT-PCR) and immunoblots. Lung fibroblasts were treated with TGF-β1 ± HDAC6 inhibitors (Tubacin, Tubastatin, ACY1215, or MC1568), and fibrotic markers such as type I collagen were assessed using qRT-PCR and immunoblots. Mice were treated with bleomycin (oropharyngeal aspiration; single dose) ± Tubastatin (intraperitoneally injection; daily for 21 days), and lung collagen expression was gauged using immunoblots and trichrome staining. In a separate experiment, HDAC6 wild-type (WT) and knockout (KO) mice were administered bleomycin, and lungs were evaluated in the same manner.</p><p>Results</p><p>HDAC6 expression was deregulated in IPF lungs. Among the HDAC6 inhibitors tested, only Tubastatin significantly repressed TGF-β1-induced expression of type-1 collagen in lung fibroblasts, and this finding was coupled with decreased Akt phosphorylation and increased Akt-PHLPP (PH domain and Leucine rich repeat Protein Phosphatase) association. Tubastatin repressed TGF-β1-induced S6K phosphorylation, HIF-1α expression, and VEGF expression. Tubastatin also repressed TGF-β1-induced inhibition of LC3B-II (a marker of autophagosome formation). In bleomycin-treated mouse lungs, HDAC6 expression was increased, and Tubastatin repressed type-1 collagen expression. However, in HDAC6 KO mice, bleomycin-induced type-1 collagen expression was not repressed compared to WT mice. Knockdown of HDAC6, as well as HDAC10, another potential Tubastatin target, did not inhibit TGF-β1-induced collagen expression in lung fibroblasts.</p><p>Conclusions</p><p>HDAC6 expression is altered during lung fibrogenesis. Tubastatin represses TGF-β1-induced collagen expression, by diminishing Akt phosphorylation and regulating downstream targets such as HIF-1α-VEGF axis and autophagy. Tubastatin-treated WT mice are protected against bleomycin-induced fibrosis, but HDAC6 KO mice are not. Our data suggest that Tubastatin ameliorates pulmonary fibrosis, by targeting the TGFβ-PI3K-Akt pathway, likely via an HDAC6-independent mechanism.</p></div

Tubastatin decreases TGF-β1-induced phosphorylation of Akt, likely by restoring the association of Akt and PHLPP.

<p>(A) Subconfluent NHLFs were pretreated with Tubastatin for 6 hours and then co-treated with TGF-β1 and Tubastatin. Protein expression levels were assessed using immunoblots at the designated time points. Tubastain repressed TGF-β1-induced phosphorylation of Akt, but not Smad2 or Smad3. (B) Subconfluent NHLFs were pretreated with Tubastatin for 6 hours and then co-treated with TGF-β1 and Tubastatin for 12 hours. <i>Left</i>: Cell lysates were immunoprecipitated with an anti-Akt antibody, and protein expression levels for PHLPP and Akt were assessed using immunoblots. IP denotes immunoprecipitation. The fourth lane (*) represents IP using isotype control IgG antibody and the combined samples from the three treatment groups (i.e. an equal amount of the sample from each treatment group was mixed). <i>Right</i>: “Input” refers immunoblots without IP. TGF-β1 disrupted Akt-PHLPP association, and this was ameliorated by Tubastatin.</p

HDAC6 knockdown does not ameliorate TGF-β1-induced type-1 collagen expression in NHLFs.

<p>Subconfluent NHLFs were pretreated with siRNA for 24 hours and then co-treated with TGF-β1 for 48hours. ++ denotes double concentration. The immunoblots show that knockdown of HDAC6 and/or HDAC10 failed to repress TGF-β1-induced type-1 collagen expression in NHLFs.</p

Tubastatin decreases TGF-β1-induced expression of type-1 collagen.

<p>(A-E) Subconfluent NHLFs (A, C, D, E) or IPF fibroblasts (B) were pretreated with an HDAC6 inhibitor (Tubacin, Tubastatin, ACY1215, or MC1568) for 6 hours and then co-treated with TGF-β1 and the HDAC6 inhibitor. (A, B) Protein expression levels were assessed using immunoblots at 48 hours. D: DMSO, Tc: Tubacin, Ts: Tubastatin, A: ACY1215, M: MC1568; Col-1: type-1 collagen, α-SMA: α-smooth muscle actin (C) Gene expression levels for collagen-1 (col1a1) were assessed using qRT-PCR at 24 hours. * p<0.05 compared to the control; # p<0.05 compared to the TGF-β1. (D) Correlation between the degree of α-tubulin acetylation and collagen-1 protein expression in experiments involving Tubastatin treatment. There was a modest inverse correlation between the degree of α-tubulin acetylation and collagen-1 expression (r² = 0.7602 [p<0.0001] for pooled results of six independent experiments.). (E) An MTT assay showing that Tubastatin does not affect NHLF viability. Values for cell viability were normalized to the control group (= DMSO treatment).</p

HDAC6 knockout mice are not protected against bleomycin-induced pulmonary fibrosis.

<p>(A, B) HDAC6 wild-type (WT) and knockout (KO) mice were treated with PBS or bleomycin (2units/kg; by oropharyngeal aspiration) on Day 0 and then lungs were harvested on Day 21. (A) Collagen expression was assessed using trichrome staining of lung sections. Representative images (taken at 40x magnification) are shown. Scale bars: 3mm (left panels), 300μm (middle panels), 200μm (right panels). (B) Protein expression levels were assessed using immunoblots of lung homogenates.</p

Tubastatin-treated mice are protected against bleomycin-induced pulmonary fibrosis.

<p>Mice were treated with PBS or bleomycin (2units/kg; by oropharyngeal aspiration) on Day 0, followed by daily intraperitoneal injection of DMSO or Tubastatin (80 mg/kg/day) on Day 1 through Day 21 (n = 4-5/group). (A) Collagen expression was assessed using trichrome staining of lung sections. Representative images (taken at 40x magnification) are shown. Scale bars: 3mm (left panels), 300μm (middle panels), 200μm (right panels). (B) Protein expression levels were assessed using immunoblots of lung homogenates. Densitometry analysis of type-1 collagen expression is shown in the bar graphs. * p<0.05 compared to the control (= PBS+DMSO); # p<0.05 compared to Bleomycin+DMSO.</p