The content of nitric oxide and S-nitrosothiols in rats’ liver cells under the different supplementation of macronutrient

This paper presents studies of nitric oxide and low-molecular S-nitrosothiols in the mitochondrial and cytosolic fractions of the rats' liver under the conditions of, alimentary protein deprivation, consumption of excess sucrose content and combined action of two adverse factors. In order to model the low-protein diet of the animal for 28 days received an isocaloric diet containing 4.7% protein, 10% fat, 81,3% carbohydrates (starch – 37%, sucrose – 30%, cellulose – 5%) and was calculated in accordance with the recommendations of the American Institute of Nutrition. The high-sugar diet consisted of 14% protein, 10% fat, 72% carbohydrates (starch – 37%, sucrose – 30%, cellulose – 5%). The mitochondrial and cytosolic fraction of rat liver cells were obtained by the method of differential centrifugation. Nitrogen oxide content was assessed by a unified method by determining the NO2- content, which is a stable metabolite of nitric oxide. Since NO is inactivated into an oxidase reaction with the conversion into nitrite or nitrate that is quickly metabolized, the nitrogen oxide content was assessed by the change in NO2-. The concentration of S-nitrosothiols was recorded, respectively, by determining the concentration of nitrite anion before and after the addition of Hg2+ ions, which by modifying the S – N bonds catalyzes the release of S-nitrosyl thiols of nitric oxide. An increase in NO content in both hepatic subcellular fractions of the rats’ experimental groups compared to control values was found. However, a lack of protein in the diet (protein deficiency in the diet leads to an increase in nitric oxide levels in 3-4 times) can be considered as a key factor in the recorded changes in the mitochondria of the animals’ liver, while in the cytosol - excessive consumption of sucrose (3-5 times increase). Regarding the level of S-nitrosothiols, in the studied fractions, multidirectional changes in their concentration were found. Thus, an increase in the content of nitrosyl derivatives in the mitochondria of rat’s liver cells with a simultaneous decrease in their level in the cytosol indicates dysmetabolic disorders in the transport system and deposition of nitric oxide, which can lead to the development of nitrosative stress under the experimental conditions.

Antioxidant Constituents from the Stem and Leaf of Helicteres hirsuta Loureiro

Background: Helicteres hirsuta has been used traditionally as a useful agent for hepatoprotective treatment. The aim of the current study is to isolate chemical constituents from the EtOAc extract of Helicteres hirsuta stem and evaluate the capacity of the isolated compounds against hydroperoxide damaged rat liver cells Methods: Column chromatography was used for phytochemical isolation whereas MTT method was applied for intracellular antioxidative assay Results: Phytochemical analysis of the EtOAc extract of Helicteres hirsuta stem led to the isolation and determination of four triterpenoids betulin (1), bentulinic acid (2), alphitolic acid (3), and oleanolic acid (4), together with two steroids stigmast-4-ene-6β-ol-3-one (5), and β-sitostenone (6), whereas EtOAc extract of its leaf composed of three steroids cucurbitacin D (11), cucurbitacin I (12), and simiarenol (13), four flavonoids tiliroside (14), potengriffioside A (15), kaempferide (16), and isokaempferide (17), along with two carotenoids lutelin (18), and β-carotene (19). Isolated compounds 3, 5, 6, 16, and 17 were found in genus Helicteres for the first time, while carotenoids 18, and 19 were never isolated from family Sterculiaceae before. Phytochemicals derived H. hirsuta species are also useful agents for antioxidative drugs, e.g, flavonol 17 induced the significant EC50 value of 22.24 ± 0.14 μg/mL, as compared with that of positive control curcumin (EC50 19.33 ± 0.77 μg/mL), against hydroperoxide damaged rat liver cells Conclusion: Antioxidative activity of the EtOAc extract of Helicteres hirsuta stem against hydroperoxide is mostly based on the role of flavonoids.

Niacin downregulates chemokine (c-c motif) ligand 2 (CCL2) expression and inhibits fat synthesis in rat liver cells

Purpose: To elucidate the role of chemokine (c-c motif) ligand 2 (CCL2) in fat metabolism in hepatocytes. Methods: Following partial hepatectomy, regenerated rat liver cells were isolated and cultured for 24 h were transfected with recombinant plasmid pEGFP-N1-CCL2 using liposomes. Niacin was added to the culture medium to inhibit fat synthesis. CCL2 expression was measured using western blot, while the expression of acly-coa synthetase long chain family 4 (ACSL4) and apolipoprotein E (ApoE) were assessed using real-time PCR. Results: At 12 h after transfection, GFP-positive rates in the pEGFP-N1 and pEGFP-N1-CCL2 transfection groups were 42.4 ± 5.6 % and 45.1 ± 3.5 %, respectively. Expression levels of CCL2 increased over time in pEGFP-N1 transfection group, pEGFP-N1-ccl2 transfection group, and niacin and pEGFP-N1-ccl2 transfection co-treatment group; however, CCL2 expression levels in the niacin and pEGFP-N1-ccl2 transfection co-treatment groups were similar to that of pEGFP-N1 transfection group, which were significantly lower than those of the pEGFP-N1-ccl2 transfection group. Expressionlevel trends of fat-related genes ACSL4 and ApoE were similar to that of CCL2. Conclusion: Niacin downregulates the expression of CCL2, thereby inhibiting lipid synthesis in liver cells. Keywords: Chemokine 2, Niacin, Hepatectomy, Lipid synthesis, Transfection

Pyrazinamide enhances lipid peroxidation and antioxidant levels to induce liver injury in rat models through PI3k/Akt inhibition

Pyrazinamide (PZA) is an anti-tuberculosis drug known to causes liver injury. phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling protects against liver injury by promoting cellular antioxidant defenses and reducing intracellular reactive oxygen species (ROS) and lipid peroxidation. The regulatory mechanisms and functions of PI3K/Akt signaling during the hepatotoxicity of PZA are however not fully understood. Rats were administered PZA or/and the PI3K activator (740Y-P) for 7 days. The levels of serum parameters were examined via standard enzymatic techniques and the pathological status of the liver was confirmed by H & E staining. The levels of lipid peroxidation and antioxidant production were determined using commercial kits. Liver apoptosis was assessed by TUNEL staining. The expression of apoptosis-related proteins and PI3K/Akt signaling were assessed by western blot analysis. PZA treatment significantly increased serum alanine transaminase, aspartate transaminase, gamma-glutamyl transpeptidase and tall bilirubin leading to liver damage in rats. PZA also facilitated lipid peroxidation and suppressed antioxidant defenses. PZA led to apoptotic induction in rat liver cells through the downregulation of Bcl-2 and the upregulation of Bax and caspase-3. PZA also dramatically inhibited PI3K/Akt signaling in rat liver cells. We further verified that PI3K/Akt signaling in response to 740Y-P could attenuate hepatic injury, lipid peroxidation and apoptosis in rat liver cells in response to PZA. We reveal that PZA-induced liver injury in rats occurs through PI3k/Akt signaling, the recovery of which prevents liver injury in rat models.

On-Chip Fabrication of Cell-Attached Microstructures using Photo-Cross-Linkable Biodegradable Hydrogel

We developed a procedure for fabricating movable biological cell structures using biodegradable materials on a microfluidic chip. A photo-cross-linkable biodegradable hydrogel gelatin methacrylate (GelMA) was used to fabricate arbitrary microstructure shapes under a microscope using patterned ultraviolet light. The GelMA microstructures were movable inside the microfluidic channel after applying a hydrophobic coating material. The fabricated microstructures were self-assembled inside the microfluidic chip using our method of fluid forcing. The synthesis procedure of GelMA was optimized by changing the dialysis temperature, which kept the GelMA at a suitable pH for cell culture. RLC-18 rat liver cells (Riken BioResource Research Center, Tsukuba, Japan) were cultured inside the GelMA and on the GelMA microstructures to check cell growth. The cells were then stretched for 1 day in the cell culture and grew well on the GelMA microstructures. However, they did not grow well inside the GelMA microstructures. The GelMA microstructures were partially dissolved after 4 days of cell culture because of their biodegradability after the cells were placed on the microstructures. The results indicated that the proposed procedure used to fabricate cell structures using GelMA can be used as a building block to assemble three-dimensional tissue-like cell structures in vitro inside microfluidic devices.