Liver Homogenates Research Articles

Discovery of new non-covalent and covalent inhibitors targeting SARS-CoV-2 papain-like protease and main protease

Global coronavirus disease 2019 (COVID-19) pandemic still threatens human health and public safety, and the development of effective antiviral agent is urgently needed. The SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) are vital proteins in viral replication and promising therapeutic targets. Additionally, PLpro also modulates host immune response by cleaving ubiquitin and interferon-stimulated gene product 15 (ISG15) from ISGylated host proteins. In this report, we identified [1,2]selenazolo[5,4-c]pyridin-3(2H)-one and benzo[d]isothiazol-3(2H)-one as attractive scaffolds of PLpro and Mpro inhibitors. The representative compounds 6c and 7e exhibited excellent PLpro inhibition with percent inhibition of 42.9% and 44.9% at 50 nM, respectively. The preliminary enzyme kinetics experiment and fluorescent labelling experiment results determined that 6c was identified as a covalent PLpro inhibitor, while 7e was a non-covalent inhibitor. Molecular docking and dynamics simulations revealed that 6c and 7e bound to Zn-finger domain of PLpro. Compounds 6c and 7e were also identified to potent Mpro inhibitors, and they exhibited potent antiviral activities in SARS-CoV-2 infected Vero E6 cells, with EC50 value of 3.9 μM and 7.4 μM, respectively. In addition, the rat liver homogenate half-life of 6c and 7e exceeded 24 h. These findings suggest that 6c and 7e are promising led compounds for further development of PLpro/Mpro dual-target antiviral drugs.

Применение молекулярно-генетических предикторов и машинного обучения в оценке токсичности химических веществ

Toxic hepatites is an important public health issue in Russia. They have diverse etiologies, including industrial toxins, drugs, and ethanol, leading to significant differences in pathogenesis at the molecular and genetic level. This study aimed to analyze genes involved in the response to toxic liver injury of different origins and to establish a model to predict the etiology of toxic hepatitis. To model acute toxic hepatitis, outbred white male rats weighing 180–200 g were used. They were divided into four groups: control group, tetrachloromethane group, paracetamol group, and ethanol group. 24 h and 72 h after toxicant administration rats were anesthetized and mRNA levels of Chek1, Gclc, Gstm1, Gstp1, Gstt1, Nfe2l2, Nqo1, and Ripk1 genes in liver homogenate were studied. Analysis of the expression of these genes showed that the expression profile varies depending on the etiology of toxic hepatitis. In tetrachloromethane poisoning, there was a marked increase in gene expression of Nqo1 (p?=?0.001), Gstm1 (p?=?0.037), and a decrease in gene expression of Nfe2l2 (p?=?0.004), Ripk1 (p?=?0.004). However, liver exposure to paracetamol and its metabolites resulted in decreased Gstm1 gene expression (p?=?0.001) and increased Nfe2l2 (p?=?0.009), Gclc (p?=?0.001), and Chek1 (p?=?0.011) gene expression. No significant changes in the expression of these genes are found in alcohol intoxication. A model was constructed to predict the etiology of toxic hepatitis on the basis of gene expression indices. Keywords: acute toxic hepatitis, paracetamol, tetrachloromethane, ethanol, gene expression.

Triphenylphosphine Thiolate Gold(I) Complexes with Redox-Active Schiff Bases: Synthesis, Electrochemical Properties, and Biological Activity

New gold(I) phosphine thiolate complexes [(Ph3P)Au(SLn)] I–V with Schiff bases LnSH containingredox-active catechol, phenol, or quinone methide moieties were synthesized and characterized. Themolecular structure of compound I in the crystalline state was established by X-ray diffraction (CCDCno. 2237815). The electrochemical behavior of compounds I–V was studieв by cyclic voltammetry. The proposedelectrooxidation mechanism of the complexes involves the Au–S bond cleavage, the disulfide formation,as well as the oxidation of the redox active group of the ligand. In the cathode region, complexes I–IIItend to form relatively stable monoanionic species. The radical scavenging activity of complexes decreases incomparison to free ligands in the reactions with synthetic radicals and the CUPRAC test. Compounds I, II,IV, and V have no clear-cut effect on the promoted DNA damage; however, they show antioxidant actionin the non-enzymatic lipid peroxidation of rat liver homogenate. Compounds I–V demonstrate a weak antibacterialactivity against Staphylococcus aureus strains. The gold(I) complexes cytotoxicity was studied againstA-549, MCF-7, and HTC-116 cancer cell lines using MTT assay. The test compounds are characterized byhigher selectivity to certain types of cells than the sulfur-containing Schiff bases. The presence of quinonemethide moiety in the ligand in case of V significantly increases the cytotoxicity against all of the cell lines.

Exogenous Melatonin Protects against Oxidative Damage to Membrane Lipids Caused by Some Sodium/Iodide Symporter Inhibitors in the Thyroid.

The thyroid gland is the primary site of sodium/iodide symporter (NIS), an intrinsic plasma membrane protein responsible for the active uptake of iodine, which is indispensable for thyroid hormone synthesis. Since exposure of the thyroid to NIS inhibitors can potentially have harmful effects on the entire organism, it is important to investigate the potential protective effects of known antioxidants, such as melatonin and indole-3-propionic acid (IPA), against pro-oxidative action of classic NIS inhibitors. The study aimed to check if and to what extent melatonin and IPA interact with some confirmed NIS inhibitors regarding their effects on oxidative damage to membrane lipids in the thyroid. For comparison with the thyroid gland, in which NIS is typically present, the liver tissue-not possessing NIS-was applied in the present study. Thyroid and liver homogenates were incubated in the presence of tested NIS inhibitors (i.e., NaClO3, NH4SCN, KSeCN, KNO3, NaF, KClO4, and BPA) in different ranges of concentrations with/without melatonin (5 mM) or IPA (5 mM). The malondialdehyde+4-hydroxyalkenals (MDA + 4-HDA) concentration (LPO index) was measured spectrophotometrically. NaClO3 increased LPO in the thyroid and in the liver, but these pro-oxidative effects were not prevented by either melatonin or IPA. Instead, pro-oxidative effects of NH4SCN observed in both tissues were prevented by both indole substances. KSeCN and NaF increased LPO only in the thyroid, and these pro-oxidative effects were prevented by melatonin and IPA. KNO3, KClO4, and BPA did not increase LPO, which can be due to their low concentrations resulting from restricted solubility. In conclusion, as melatonin prevented oxidative damage to membrane lipids in the thyroid caused by some sodium/iodide symporter inhibitors, this indoleamine shoud be considered as a potential protective agent when produced appropriately in living organisms but also as an exogenous substance recommended to individuals overexposed to NIS inhibitors.

Electrochemical Activation of Metabolic Enzymes in Human Liver S9 Fractions

Human liver subcellular fractions such as liver microsomes, liver cytosol fractions, or S9 fractions are widely used in in-vitro assays to predict liver metabolism. The S9 fractions are the supernatants of human liver homogenates consist of both human liver microsomes and the cytosol. Therefore, these fractions contain majority of cytochrome P450 (CYP) enzymes and soluble phase II enzymes like glucuronosyltransferases, N-acetyl transferases, sulfotransferases, and glutathione-S-transferases. Here we report the direct electrochemistry and biocatalytic features of redox active enzymes impregnated in S9 fractions. We investigated the electrochemical properties of S9 films by immobilizing them onto a high purity graphite electrode and performing cyclic voltammetry in anaerobic (Ar saturated) and aerobic (O2 saturated) conditions. The electron transfer between the S9 film and the high purity graphite electrode was observed at the formal potential of -0.48 V, with respect to Ag/AgCl reference electrode, which confirmed the activation of CYP450 reductase enzyme in the film. These films also demonstrated a catalytic oxygen reduction with a maximum current density of ~0.5 mA cm-2 at the scan rate of 0.050V/s. We investigated the CYP activity in the S9 biofilm for phase 1 metabolism using diclofenac hydroxylation as a probe reaction and could able to identify metabolic products using liquid chromatography-mass spectrometry (LC-MS) technique. Eventually, we envision to develop a platform to perform both phase I and II metabolic reactions in a single electrochemical assay.

A New Avenue for Enhanced Treatment of Hyperuricemia and Oxidative Stress: Design, Synthesis and Biological Evaluation of Some Novel Mutual Prodrugs Involving Febuxostat Conjugated with Different Antioxidants

Febuxostat (FEB) is the first non-purine xanthine oxidase inhibitor (XOI) used for the treatment of hyperuricemia and gout. The oxidative stress induced by reactive oxygen species (ROS) which accompany purine metabolism by XO, could contribute to cellular damage and several pathological conditions. In this view, the present work addresses the evaluation of combining the hypouricemic effect of FEB and the free radical scavenging potential of various natural antioxidants in a single chemical entity by implementing the “mutual prodrug” strategy. Accordingly, a series of five ester prodrugs containing FEB together with different naturally occurring antioxidants namely, thioctic acid (4), thymol (5), menthol (6), vanillin (7), and guaiacol (8) was synthesized. Prominently, all the chemically conjugated prodrugs (4 – 8) revealed an obvious increase in the hypouricemic and antioxidant potentials when compared with their corresponding promoieties and physical mixtures. Moreover, they showed a potential protective effect against CCl4-induced hepatotoxicity and oxidative stress, together with no cytotoxicity on normal breast cells (MCF10A). Furthermore, the in vitro chemical and enzymatic stability studies of the prodrugs (4 – 8) using a developed HPLC method, verified their stability in different pHs, and rapid hydrolysis in rabbit plasma and liver homogenate to their parent metabolites. Moreover, the prodrugs (4 – 8) showed higher lipophilicity and lower aqueous solubility when compared to the parent drugs. Finally, the obtained merits from the implementation of the mutual prodrug strategy would encourage further application in the development of promising candidates with high therapeutic efficacy and improved safety profiles.

Hepatoprotective and in vivo antioxidant effects of granulometric classes and decoction of Ficus dicranostyla Mildbread leaves powders against carbon tetrachloride-induced hepatotoxicity in Wistar rats.

Ficus dicranostyla is a plant from the Moraceae family commonly used in African countries for its nutritional value and its believed medicinal properties. Its antioxidant in vitro capacity and its richness in phenolic compounds have been previously demonstrated. This work aimed at evaluating the hepatoprotective and in vivo antioxidant activities of different granulometric fractions of the F. dicranostyla leaves against carbon tetrachloride-induced hepatotoxicity in rats. Powdery fractions (<125, 250-125, and ≥250 μm), and the unsieved powder, obtained from the F. dicranostyla leaves were water-dissolved and given orally to rats at the same dose (250 mg/kg body weight) before administering carbon tetrachloride intraperitoneally (1 mL/Kg bw). The lipid status parameters (total cholesterol, triglycerides, HDL-cholesterol, and LDL-cholesterol), hepatic toxicity through aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) in blood plasma, and antioxidant status by measuring the malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) in liver homogenate were performed. The activities of all parameters registered a significant (p < .05) alteration in CCl4-treated rats, which were significantly recovered toward an almost normal level in coadministered with Ficus dicranostyla leaf powder samples in a particle size-dependent manner. Results suggest that the smaller particle size of the powder fraction, as well as the decoction powder of Ficus dicranostyla, may be used as hepatoprotective and antioxidant agents.

Correlation between the level of sulfane sulfur and the expression/activity of sulfurtransferases in chicken tissues – a possible ways of cyanide detoxification

Cyanogenic glycosides are a large group of secondary metabolites that are widely distributed in the many plants commonly consumed by humans, birds, and other animals. Thiosulfate sulfurtransferase (TST) and 3-mercaptopyruvate sulfurtransferase (MPST), are two evolutionary-related enzymes that constitute the defense against cyanide toxication and participate in the production of sulfane sulfur-containing compounds. The expression and activity of TST and MPST as well as the level of sulfane sulfur in chicken tissue homogenates of the liver, heart, and gizzard were investigated. The highest expression/activity of TST and MPST was noticed in liver homogenates which was associated with the high sulfane sulfur level. Both the expression and activity of TST as well as the sulfane sulfur level in chicken gizzard homogenates were significantly lower than in the liver and heart. Both TST and MPST enzymes can play an important role in cyanide detoxification in chicken tissues. Maintaining appropriate sulfane sulfur level together with the high activity of these enzymes is essential to protect tissues from the toxic effects of cyanide, released from certain nutrients.

Antimalarial Efficacy and Antioxidant Activity of Lophira lanceolata Stem Bark Ethanol Extract Using Plasmodium berghei Induced-Malaria in Swiss Albino's Mice.

Malaria remains a major public health problem in the tropical and subtropical regions. This study aimed of investigating the antimalarial and antioxidant activities of ethanol extract of Lophira lanceolata stem bark. Methodology. The antimalarial activity was determined using the Peter 4-days' suppressive and Rane's curative tests on Swiss albino: these mice were infected with 1 × 107 parasitized red blood cells. The percentage reduction of parasitemia was related to each test, and the liver homogenate was used to assay malondialdehyde, superoxide dismutase, nitrogen monoxide, catalase, and glutathione for the evaluation of oxidative stress. During the curative test, blood was collected for hematological parameters, alanine aminotransferase and aspartate aminotransferase to evaluate liver function. The ethanol extract of L. lanceolata showed a dose-dependent suppressive activity with the highest suppression of 88.22% at 500 mg/kg. Suppression produced by the extract was not significantly higher than that of the reference drug with 96.1%. Similarly, the extract at doses 125, 250, and 500 mg/kg showed significant decreases (P < 0.05) in a dose-dependent manner during the curative test. The ethanol extract of L. lanceolata caused a reduction of tissue markers, such as hepatic oxidative stress, as it increased the enzymatic activity of antioxidant enzymes. The ethanol extract of L. lanceolata possesses both antimalarial and antioxidant activities. However, further in vivo toxicity tests are required to guarantee their safety.

Antidiabetic activity of eupafolin through peroxisome proliferator-activated receptor-gamma and PI3K/Akt signaling in Type 2 diabetic rats.

Eupafolin is a phyto compound of flavone that exerts anti-inflammatory, antioxidant, and antiproliferative properties. The main purpose of this study is to examine the antidiabetic effect of eupafolin on nicotinamide-streptozotocin (STZ)-induced Type 2 diabetes (T2D) rats. After nicotinamide (120 mg/kg) treatment, STZ (60 mg/kg) was administrated intravenously to induce T2D. Rats with fasting blood glucose (FBG) > 200 mg/dL are chosen for the study 7 days after T2D induction. The eupafolin treatment was continued for another 15 days. FBG and an oral glucose tolerance test (OGTT) were measured on the 21st day after T2D induction. The blood lipid, serum insulin, and homeostatic model assessment (HOMA-IR) were determined. In liver homogenate, oxidative stress indicators were measured. In addition, the effect of eupafolin on the expression of the proteins InsR, insulin receptor substrate (IRS)-2, GLUT4, PPARγ, and phosphatidylinositol 3-kinase (PI3K)/Akt was investigated using a western blot. As measured by OGTT and HOMA-IR, eupafolin treatment reduced FBG and insulin resistance (IR). Furthermore, when compared to diabetic rats, liver antioxidant enzymes were dramatically normalized. The level of glycogen in the liver of diabetic rats was increased by eupafolin treatment. In T2D rats, eupafolin dramatically increased the InsR, IRS-2, GLUT4, and PPARγ. Further, the eupafolin treatment activated the PI3K/Akt signaling in T2D rats. These findings imply that the antidiabetic mechanism of eupafolin may be related to the activation of the PPARγ and the PI3K/Akt signaling pathway in T2D rats. As a result, the flavonoid eupafolin could be an antidiabetic medication for T2D after a comprehensive clinical investigation.

Phosphorylation of insulin receptor substrates (IRS-1 and IRS-2) is attenuated following cecal ligation and puncture in mice

BackgroundSepsis is characterized as an insulin resistant state. However, the effects of sepsis on insulin’s signal transduction pathway are unknown. The molecular activity driving insulin signaling is controlled by tyrosine phosphorylation of the insulin receptor β-subunit (IRβ) and of insulin receptor substrate molecules (IRS) -1 and IRS-2.HypothesisCecal ligation and puncture (CLP) attenuates IRβ, IRS-1 and IRS-2 phosphorylation.MethodsIACUC-approved studies conformed to ARRIVE guidelines. CLP was performed on C57BL/6 mice; separate cohorts received intraperitoneal insulin at baseline (T0) or at 23 or 47 h. post-CLP, 1 h before mice were euthanized. We measured levels of (1) glucose and insulin in serum, (2) IRβ, IRS-1 and IRS-2 in skeletal muscle and liver homogenate and (3) phospho-Irβ (pIRβ) in liver and skeletal muscle, phospho-IRS-1 (pIRS-1) in skeletal muscle and pIRS-2 in liver. Statistical significance was determined using ANOVA with Sidak’s post-hoc correction.ResultsCLP did not affect the concentrations of IRβ, IRS-1or IRS-2 in muscle or liver homogenate or of IRS-1 in liver. Muscle IRS-1 concentration at 48 h. post-CLP was higher than at T0. Post-CLP pIRS-1 levels in muscle and pIRβ and pIRS-2 levels in liver were indistinguishable from T0 levels. At 48 h. post-CLP pIRβ levels in muscle were higher than at T0. Following insulin administration, the relative abundance of pIRβ in muscle and liver at T0 and at both post-CLP time points was significantly higher than abundance in untreated controls. In T0 controls, the relative abundance of pIRS-1 in muscle and of pIRS-2 in liver following insulin administration was higher than in untreated mice. However, at both post-CLP time points, the relative abundance of pIRS-1 in muscle and of pIRS-2 in liver following insulin administration was not distinguishable from the abundance in untreated mice at the same time point. Serum glucose concentration was significantly lower than T0 at 24 h., but not 48 h., post-CLP. Glucose concentration was lower following insulin administration to T0 mice but not in post-CLP animals. Serum insulin levels were significantly higher than baseline at both post-CLP time points.ConclusionsCLP impaired insulin-induced tyrosine phosphorylation of both IRS-1 in muscle and IRS-2 in liver. These findings suggest that the molecular mechanism underlying CLP-induced insulin resistance involves impaired IRS-1/IRS-2 phosphorylation.

Ameliorative effects of Orlistat and metformin either alone or in combination on liver functions, structure, immunoreactivity and antioxidant enzymes in experimentally induced obesity in male rats

BackgroundPrevalence of obesity is increasing worldwide. Obesity is associated with incidences of metabolic disorders and cardiovascular diseases and the risk of having it rose sharply during the COVID-19 pandemic. Obesity is associated with oxidative stress, inflammatory markers and hepatic disorders and has become one of the silent killer diseases affecting global health. MethodsThis study examined the effects of obesity on liver functions (ALT, AST and LDH), lipid profile (TG, TC, HDL-c, LDL-c and vLDL-c), tumour necrosis factor alpha (TNF-α), inflammatory marker, C-reactive protein (CRP), leptin hormone and antioxidant enzymes (CAT, SOD and GPx) and lipid peroxidation marker (MDA) in liver homogenates besides histological structure of the liver tissues and assessment of DNA damage. Fifty male Wistar rats were used and they were divided into five treatment groups: I-Control group, II-high-fat diet (HFD) treated group (Obesity) group, III-HFD plus Orlistat (ORL), IV-HFD plus metformin (Met) and V- HFD plus ORL plus Met. ResultsExperimentally-induced obesity caused a significant increase in liver enzymes including lipid markers (triglycerides and total cholesterol), inflammatory markers, tumour markers and lipid peroxidation markers and a concurrent decline in antioxidant enzymes and damage of liver main structures characterised by presence of congestion and accumulation of mononuclear inflammatory cells in blood sinusoids. In contrast, groups treated with either ORL or Met or both group, we recorded restoration of normal hepatic structures and a decline in DNA damage, liver enzymes and antioxidant levels. The best restoration and amelioration were observed in the group treated with a combination of ORL and Met. ConclusionOur findings indicated the synergistic effect of ORL and Met in ameliorating hepatic functions and lipid profile, alleviating inflammation, genotoxicity and side effects of experimentally-induced obesity.

Pharmacological effect of Argyrolobium roseum (Camb.) Jaub & Spach extracts against lead-induced toxicity in rats.

Argyrolobium roseum (Camb.) Jaub & Spach (Papilionaceae) is a medicinal plant, cultivated in northern areas of Pakistan. The consumption of trace minerals (lead) is very toxic to the vital organs of the body, therefore the overcome of these minerals is very necessary. In this regard, this study aimed to assess the potential pharmacological effect of aqueous and ethanolic extract of Argyrolobium roseum (Camb.) Jaub & Spach against pb-induced oxidative stress, histological changes in Pb-induced rats' liver and kidney, and anti-inflammatory effect. The metal concentrations in liver and kidney homogenates were measured through atomic absorption spectrophotometer. The antioxidant activity was measured through DPPH and FRAP assay. Pb concentrations were significantly higher in liver and kidney homogenates after injection of Pb acetate was given intraperitoneally (45.2 ± 6.8 and 58.8 ± 7.9, respectively; p < .0001). The level of Pb in liver and kidney homogenates was significantly reduced by aqueous and ethanolic extracts of Argyrolobium roseum (Camb.) Jaub & Spach. The Pb + Aq-600 mg/kg-treated rats exhibited a protective effect on hepatocytes cells against Pb-induced liver injury and restored the cells of the kidney. Pb + Aq-600 mg/kg showed higher antioxidant activity as compared to other treated groups. The highest decreased MDA level was found in liver and kidney homogenate of Pb + Aq-600 mg/kg rats (11.2 ± 1.51 nmol/mg; p < .001) and GSH and CAT levels tended to normal after treatment of Pb + Aq-600 mg/kg in rats. The ALAD, ALT, AST, and ALP level were enhanced and tended to be normal after the Aq-400 and Aq-600 mg/kg treatment in Pb-exposed rats. The result showed that 600 mg/kg Aq + Pb exhibited significant (p < .001) anti-inflammatory activity. The findings of this study concluded that treatment of the aqueous extract of Argyrolobium roseum (Camb.) Jaub & Spach reduces the renal and hepatic damage in Pb-induced rats and it also decreases oxidative stress via improving antioxidant components.

Genotoxicity assessment of eight nitrosamines using 2D and 3D HepaRG cell models.

N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates. There are only limited data on the genotoxicity of nitrosamines in human cell systems. In this study, we used metabolically competent human HepaRG cells, whose metabolic capability is comparable to that of primary human hepatocytes, to evaluate the genotoxicity of eight nitrosamines [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. Under the conditions we used to culture HepaRG cells, three-dimensional (3D) spheroids possessed higher levels of CYP activity compared to 2D monolayer cells; thus the genotoxicity of the eight nitrosamines was investigated using 3D HepaRG spheroids in addition to more conventional 2D cultures. Genotoxicity was assessed as DNA damage using the high-throughput CometChip assay and as aneugenicity/clastogenicity in the flow-cytometry-based micronucleus (MN) assay. Following a 24-h treatment, all the nitrosamines induced DNA damage in 3D spheroids, while only three nitrosamines, NDBA, NDEA, and NDMA, produced positive responses in 2D HepaRG cells. In addition, these three nitrosamines also caused significant increases in MN frequency in both 2D and 3D HepaRG models, while NMBA and NMPA were positive only in the 3D HepaRG MN assay. Overall, our results indicate that HepaRG spheroids may provide a sensitive, human-based cell system for evaluating the genotoxicity of nitrosamines.

"Yajieshaba" prevents acute alcoholic liver injury and repairs the intestinal mucosal barrier

Ethnopharmacological relevanceAn essential factor related to the acute alcoholic liver injury is damage to the intestinal mucosal barrier. Yajieshaba (YJSB) is a commonly used formulation of Dai people in China and protects the liver. Aim of the studyThis study investigated whether YJSB can prevent acute alcoholic liver injury by regulating the intestinal mucosal barrier. Materials and methodsThe mice received 0.39 g/kg, 1.17 g/kg, and 3.51 g/kg dose YJSB for 7 days, a mouse model of acute alcoholic liver injury was established by a single instillation of 56% alcohol. Plasma biochemical markers were analyzed, liver injury was identified by histopathology, lipopolysaccharide (LPS), nuclear factor-k-gene binding (NF-κB), hepatic inflammatory factors, oxidative stress factors and reactive oxygen species (ROS) content was analyzed. The morphological changes of intestinal histology were observed by H&E staining, and the ultrastructure of ileal cells was observed by transmission electron microscopy. Immunofluorescence and Western blot was used to determine the expression levels of transporters and enzymes involved in Claudin 1, Occludin and zona occludens 1 (ZO-1) homeostasis in the liver and intestine. ResultsThe findings showed that YJSB reduced the levels of aspartate aminotr ansferase (AST), alanine aminotransferase (ALT) and total bile acid (TBA), both of which are indicators of liver function and had a protective effect against liver injury. In the liver homogenate, YJSB reduced the level of LPS, NF-κB, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), decreased the level of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-PX) and catalase (CAT) and ROS. The results of hematoxylin and eosin (H&E) staining and transmission electron microscopy analysis revealed that YJSB reduced the degree of damage to intestinal tissue and intracellular organelles, implying that YJSB can reduce the "attack factor" that causes intestinal barrier damage, increase the "defense factor" that protects the intestinal barrier. The results of immunohistochemistry and Western blotting analysis showed that YJSB could increase the expression of claudin 1, occludin, and ZO-1 proteins, suggesting that the mechanism of action of YJSB against acute alcohol liver injury involves the upregulation of the expression of the intestinal barrier-related proteins and the repair of the damaged intestinal barrier. ConclusionsYJSB can block LPS, oxidative stress factors, and other harmful substances in the blood and protect the liver resisting acute alcoholic liver injury.

Investigation of the Bioactivity of Escin in Hydrogen Peroxide-Induced Oxidative Stress

Escin is a triterpene saponin obtained from the horse chestnut tree Aesculus hippocastanum L. (Hippocastanaceae). The aim of this study is to investigate the bioactivity of escin against hydrogen peroxide-induced oxidative stress in quails. The study was conducted in male Japanese quails (n=10) with similar pre-experimental weights (Coturnix coturnix japonica) on average. In order to create oxidative stress in quails, hydrogen peroxide was given to drinking water ad libitum for 1 week. At the end of the period, escin was injected intraperitoneally to the quails twice, every other day. The weights of the experimental animals were measured at the beginning and the end of the experiment and compared. In addition, the 10-day live weight change rate and feed consumption rates of the experimental animals were also compared. Total antioxidant (TAC), total oxidant (TOC) and oxidative stress index (OSI) were measured colorimetrically from serum and liver homogenates of animals in the experimental groups. In addition, IL-1β translation levels from serum samples were investigated by ELISA method. According to the data obtained from the study, 10-day body weight change rates increased by 33% in the hydrogen peroxide added group compared to the control group, while an increase of 133% was observed in the escin-treated group. Besides, in serum and liver samples, it was found that escin showed strong antioxidant properties by decreasing the amount of TOC and increasing TAC levels in the group escin administered with hydrogen peroxide, compared to the group administered only with hydrogen peroxide. It was also observed that escin significantly suppressed the levels of IL-1β induced by hydrogen peroxide. As a result, escin was thought to be an alternative molecule in improving growth performance by showing significant antioxidant activity in the prevention of oxidative stress and inflammation in quails. However, multiple trials and detailed molecular analyzes are needed to obtain more detailed information on the subject.

Rational design of highly stabilized and selective adrenomedullin analogs.

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids with a disulfide bond and an amidated C-terminus. Due to the vasodilatory and cardioprotective effects, the agonistic activity of the peptide on the adrenomedullin 1 receptor (AM1 R) is of high pharmacological interest. However, the wild-type peptide shows low metabolic stability leading to rapid degradation in the cardiovascular system. Previous work by our group has identified proteolytic cleavage sites and demonstrated stabilization of ADM by lipidation, cyclization, and N-methylation. Nevertheless, these ADM analogs showed reduced activity and subtype selectivity toward the closely related calcitonin gene-related peptide receptor (CGRPR). Here, we report on the rational development of ADM derivatives with increased proteolytic stability and high receptor selectivity. Stabilizing motifs, including lactamization and lipidation, were evaluated regarding AM1 R and CGRPR activation. Furthermore, the central DKDK motif of the peptide was replaced by oligoethylene glycol linkers. The modified peptides were synthesized by Fmoc/t-Bu solid-phase peptide synthesis and receptor activation of AM1 R and CGRPR was measured by cAMP reporter gene assay. Peptide stability was tested in human blood plasma and porcine liver homogenate and analyzed by RP-HPLC and MALDI-ToF mass spectrometry. Combination of the favorable lactam, lipidation, ethylene glycol linker, and previously described disulfide mimetic resulted in highly stabilized analogs with a plasma half-life of more than 144 h. The compounds display excellent AM1 R activity and wild-type-like selectivity toward CGRPR. Additionally, dose-dependent vasodilatory effects of the ADM derivatives lasted for several hours in rodents. Thus, we successfully developed an ADM analog with long-term in vivo activity.

The effect of fucoxanthin on the development of CCl&lt;sub&gt;4&lt;/sub&gt;-induced liver fibrosis

Background. According to current concepts regarding hepatic fibrosis, myofibroblast differentiation from stellate cells, regulated by transforming growth factor-β (TGF-β), is a key step in its pathogenesis. Hence, inhibition of TGF-β-dependent activation of hepatic stellate cells has been suggested as a promising strategy for preventing the disease development.Aim. To explore whether the administration of fucoxanthin at a dose of 30 mg/kg is efficient in suppressing hepatic fibrosis.Materials and Methods. The experiments were carried out on 30 outbred ICR/CD1 mice which have been divided into three groups: intact animals, animals with untreated hepatic fibrosis which has been induced by intraperitoneal injections of CCl4 (2 μl/g, 6 weeks, twice per week), and animals which received fucoxanthin per os (30 mg/kg daily for 5 weeks) after inducing hepatic fibrosis as described above. Histological examination was performed by Sirius Red staining using the METAVIR fibrosis and activity score. Immunohistochemical analysis was performed by quantitation of α-SMA-positive myofibroblasts, CD45-positive leukocytes, and TIMP-1-positive regions. Further, we quantified TGF-β in liver homogenate as well as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the serum by means of enzyme-linked immunosorbent assay. An assessment of liver function was conducted by measuring serum alanine aminotransferase, aspartate aminotransferase, and albumin levels.Results. Fucoxanthin decreased the number of myofibroblasts and leukocytes, the volume of connective tissue and TIMP-1-positive regions, and the level of TGF-β in the liver homogenate, altogether indicative of ameliorated hepatic fibrosis. In accord, treatment with fucoxanthin reduced serum IL-1β, TNF-α, alanine aminotransferase and aspartate aminotransferase, and increased serum albumin.Conclusion. Treatment with fucoxanthin at a dose of 30 mg/kg has an antifibrotic effect and diminishes liver fibrosis.

Gut microbiota-based pharmacokinetic-pharmacodynamic study and molecular mechanism of specnuezhenide in the treatment of colorectal cancer targeting carboxylesterase

Specnuezhenide (SNZ) is among the main components of Fructus Ligustri Lucidi, which has anti-inflammation, anti-oxidation, and anti-tumor effect. The low bioavailability makes it difficult to explain the mechanism of pharmacological effect of SNZ. In this study, the role of the gut microbiota in the metabolism and pharmacokinetics characteristics of SNZ as well as the pharmacological meaning were explored. SNZ can be rapidly metabolized by the gut microbiome, and two intestinal bacterial metabolites of SNZ, salidroside and tyrosol, were discovered. In addition, carboxylesterase may be the main intestinal bacterial enzyme that mediates its metabolism. At the same time, no metabolism was found in the incubation system of SNZ with liver microsomes or liver homogenate, indicating that the gut microbiota is the main part involved in the metabolism of SNZ. In addition, pharmacokinetic studies showed that salidroside and tyrosol can be detected in plasma in the presence of gut microbiota. Interestingly, tumor development was inhibited in a colorectal tumor mice model administered orally with SNZ, which indicated that SNZ exhibited potential to inhibit tumor growth, and tissue distribution studies showed that salidroside and tyrosol could be distributed in tumor tissues. At the same time, SNZ modulated the structure of gut microbiota and fungal group, which may be the mechanism governing the antitumoral activity of SNZ. Furthermore, SNZ stimulates the secretion of short-chain fatty acids by intestinal flora in vitro and in vivo. In the future, targeting gut microbes and the interaction between natural products and gut microbes could lead to the discovery and development of new drugs.

Molecular mechanism of anti-obesity effect of total lutein oxidized products (LOPs) in diet-induced obese mice

The present study explores the molecular mechanism of anti-obesity effect of total lutein oxidized products (LOPs) in high-fat diet-induced obese C57BL/6 mice. Total LOPs (50, 100, 200 mg/kg b.wt.) were intubated for 22 weeks. Lipid profile, peroxides, AST, ALT, GST activities, lipid metabolizing enzymes/molecules, 3T3-L1 cell differentiation, TGF-β1 and p38 MAPK expressions were studied. Total LOPs at 100 mg/kg b.wt. reduced plasma and hepatic cholesterol, triglycerides, phospholipids, ALT and AST activities. FAS and CPT activities were inhibited in the liver homogenate, and epididymal WAT (eWAT). Total LOPs at 200 mg/kg b.wt. reduced HMGCoA:Mevalonate in eWAT, lipid peroxides in eWAT and liver. GST activity was high in the liver and eWAT. Total LOPs (100μg/ml) suppressed adipogenesis and decreased the accumulation of lipid droplets from two to eight days. Adiponectin was upregulated, PPAR γ, lipoprotein lipase, TGF-β1, and p38 MAPK were down-regulated. The results have shown that total LOPs are potent anti-obese molecules. ABBREVIATIONS: AIN 93G: American institute of nutrition 93 for growth; ALT: alanine transaminase; AST: aspartate transaminase; CEBPα: ccaat/enhancer binding protein α; C-DNA: complementary deoxyribonucleic acid; CPT: carnitine palmitoyl transferase; GST: glutathione-S-transferase; ELISA: enzyme-linked immune sorbent assay; FAS: fatty acid synthase; HDL: high-density lipoprotein; HFD: high-fat diet; HMG CoA: β-Hydroxy β-methylglutaryl-Coenzyme A; LDL: low-density lipoprotein; LOPs: total lutein oxidized products; LPL: lipoprotein lipase; MDA-Malonaldehyde; MTT: 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide; MVA: mevalonate; p38MAPK: p38 mitogen-activated protein kinases; RNA: ribonucleic acid; RTq-PCR: quantitative reverse transcription polymerase chain reaction; PPAR-γ: peroxisome proliferator-activated receptor-gamma; TGF-β1: transforming growth factor beta 1; WAT: white adipose tissue.