Mouse Liver Sections Research Articles

Multi-time point transcriptomics and metabolomics reveal key transcription and metabolic features of hepatic ischemia-reperfusion injury in mice

Hepatic ischemia-reperfusion injury is an unavoidable surgical complication of liver transplantation and the leading cause of poor graft function and increased mortality post-transplantation. Multiple mechanisms have been implicated in ischemia-reperfusion injury; however, the characteristic changes at the transcriptional and metabolic levels in the early, intermediate, and late phases of ischemia-reperfusion injury remain unclear. In the study, mice underwent laparotomy following anesthesia, and the blood vessels of the liver were clipped using a vascular clamp to form 70% warm ischemia of the liver. Mouse liver sections and serum samples were collected and divided into the Sham, I1R12, I1R24, and I1R48 groups. Transcriptomics and metabolomics analyses were performed to study characteristic alterations during the early, intermediate, and late phases of ischemia-reperfusion injury. Quantitative real-time PCR was used to validate the critical differentially expressed genes. The differentially expressed genes and metabolites were identified by transcriptomics and metabolomics analyses. Moreover, GO and KEGG enrichment analyses indicated that glucose metabolism remodeling, inflammatory response activation, and lipid metabolism remodeling were characteristic changes in the early, intermediate, and late phases of ischemia-reperfusion injury, respectively. In summary, our study revealed the importance of glucolipid metabolism in ischemia-reperfusion injury and provided potential therapeutic intervention targets and a new perspective to explore the underlying mechanisms of ischemia-reperfusion injury.

Unraveling the molecular links between benzopyrene exposure, NASH, and HCC: an integrated bioinformatics and experimental study

Benzopyrene (B[a]P) is a well-known carcinogen that can induce chronic inflammation and fibrosis in the liver, leading to liver disease upon chronic exposure. Nonalcoholic steatohepatitis (NASH) is a chronic liver condition characterized by fat accumulation, inflammation, and fibrosis, often resulting in hepatocellular carcinoma (HCC). In this study, we aimed to investigate the intricate connections between B[a]P exposure, NASH, and HCC. Through comprehensive bioinformatics analysis of publicly available gene expression profiles, we identified differentially expressed genes (DEGs) associated with B[a]P exposure, NASH, and liver cancer. Furthermore, network analysis revealed hub genes and protein–protein interactions, highlighting cellular metabolic dysfunction and disruption of DNA damage repair in the B[a]P-NASH-HCC process. Notably, HSPA1A and PPARGC1A emerged as significant genes in this pathway. To validate their involvement, we conducted qPCR analysis on cell lines and NASH mouse liver tissues and performed immunohistochemistry labeling in mouse and human HCC liver sections. These findings provide crucial insights into the potential regulatory mechanisms underlying benzopyrene-induced hepatotoxicity, shedding light on the pathogenesis of B[a]P-associated NASH and HCC. Moreover, our study suggests that HSPA1A and PPARGC1A could serve as promising therapeutic targets. Enhancing our understanding of their regulatory roles may facilitate the development of targeted therapies, leading to improved patient outcomes.

Decellularized Mouse Liver as a Small-Scale Scaffold for the Creation of a Miniaturized Human Liver

The liver is the main organ responsible for drug metabolism; hepatocyte-based culture models play a fundamental role in understanding liver physiology. While different types of two-dimensional and three-dimensional liver models have been developed, the failure to accurately mimic native liver, including extracellular matrix (ECM) composition, complex structure, and rich vascular network as well as shortage of liver donors, impede their clinical translation. Herein, we have realized a “miniature human liver” based on the infusion of primary human hepatocytes into a decellularized liver template (DC-liver) made from the right liver lobe of mouse. We performed detergent-based decellularization of the mouse liver sections via portal vein (PV) perfusion and confirmed the successful removal of cell content, and the preservation of the vascular network. Subsequently, the DC-liver templates were recellularized at varying cell densities, including 3 × 106 and 1 × 107 cells/mL, and liver specific gene expression was assessed. Overall, hepatocytes in the recellularized liver constructs (RC-liver) expressed liver-specific mRNA expression markers (Hnf4α, Alb) at a level comparable to the unseeded, freshly isolated hepatocytes and to hepatocytes seeded inside collagen gels. However, the RC-liver with the highest cell density exhibited poor cell distribution and blockage of the PV, in general, hepatocytes showed elevated levels of Cyp1a1. Further analysis hinted at hypoxic conditions inside the constructs, showing higher mRNA expression levels of hypoxia-related genes (Hif-1α, Casp3, Zo-1). Fortunately, oxygen supplementation appeared to alleviate hypoxia, which markedly reduced expression levels of Hif-1α and Cyp1α1. As a proof-of-concept, primary human hepatocytes were also seeded into the DC-liver templates, and we could confirm liver specific mRNA expression (HNF4A, ALB, CYP3A4). Altogether, the above results indicate a profound potential in the use of DC-liver tissue for the fabrication of a miniature human liver, which may have potential application prospects for regenerative medicine, tissue engineering (TE) and other related disciplines.

Desorption Electrospray Ionization Mass Spectrometry Imaging Allows Spatial Localization of Changes in Acetaminophen Metabolism in the Liver after Intervention with 4-Methylpyrazole.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the US, and hepatotoxicity is initiated by a reactive metabolite which induces characteristic centrilobular necrosis. The only clinically available antidote is N-acetylcysteine, which has limited efficacy, and we have identified 4-methylpyrazole (4MP, Fomepizole) as a strong alternate therapeutic option, protecting against generation and downstream effects of the cytotoxic reactive metabolite in the clinically relevant C57BL/6J mouse model and in humans. However, despite the regionally restricted necrosis after APAP, our earlier studies on APAP metabolites in biofluids or whole tissue homogenate lack the spatial information needed to understand region-specific consequences of reactive metabolite formation after APAP overdose. Thus, to gain insight into the regional variation in APAP metabolism and study the influence of 4MP, we established a desorption electrospray ionization mass spectrometry imaging (DESI-MSI) platform for generation of ion images for APAP and its metabolites under ambient air, without chemical labeling or a prior coating of tissue which reduces chemical interference and perturbation of small molecule tissue localization. The spatial intensity and distribution of both oxidative and nonoxidative APAP metabolites were determined from mouse liver sections after a range of APAP overdoses. Importantly, exclusive differential signal intensities in metabolite abundance were noted in the tissue microenvironment, and 4MP treatment substantially influenced this topographical distribution.

Effects of Rice-Husk Silica Liquid in Streptozotocin-Induced Diabetic Mice.

Type 2 diabetes mellitus is a complex multifactorial disease characterized by poor glucose tolerance and insulin resistance. Rice-husk silica liquid (RHSL) derived from rice husk has the ability to improve the dysfunction of pancreatic β-cells. This study aimed to confirm the potential protective effects of RHSL in streptozotocin (STZ)-induced diabetic mice. Diabetes was induced in male C57BL/6J mice by intraperitoneal administration of STZ (200 mg/kg BW). RHSL, food-grade silica liquid (FDSL), and rosiglitazone (RSG) were administered to diabetic mice for 12 weeks after successful induction of diabetes. During the experiment, fasting blood glucose, serum insulin, and organ weights were measured. The histopathology of liver tissue was evaluated by H&E staining. Western blotting was performed to assess protein expression levels. The results showed that RHSL significantly reversed the serum insulin levels and improved oral glucose tolerance test (OGTT) results (p < 0.05). In addition, liver sections of STZ-induced diabetic mice after RHSL treatment showed neatly arranged and intact hepatocytes. Furthermore, RHSL was more effective than FDSL in increasing the expression of SIRT1 and decreasing the expression of the PPAR-γ and p-NF-κB proteins. Taken together, this study demonstrated that RHSL ameliorated STZ-induced insulin resistance and liver tissue damage in C57BL/6J mice.

RESPONSE OF FIBROBLASTS, MAST CELLS AND LIVER COLLAGEN ON FORMATION OF EXPERIMENTAL AMYLOIDOSIS

The aim of the study was to quantify the response of fibroblasts, mast cells and liver collagen to formation of experimental amyloidosis. Material and methods. All mice were kept on a standard vivarium diet. Experimental mice underwent formation of amyloidosis by subcutaneous administration of soy cream substitute. Intact mice (IG – 3 specimen) and the internal control group for amyloidosis development (KM – 3 specimen) received water in a free access mode. For mice of the second (VM – 3) and third groups (VFM – 3), water was replaced with dry red grape wine "Cabernet Sauvignon" (Crimea) with an ethyl alcohol content of 10–12°, sugar – 5–10 mg/dm³ against the background of amyloidosis. In addition, fructose 5 g/100 ml of wine was added to the wine of VFM group. To assess the relative area of amyloid-free collagen fibers, dewaxed liver sections were stained using the tricolor technique developed by V.A. Kozlov et al. (2017), to assess the relative area of amyloid deposits and to count fibroblasts (FB) – staining with hematoxylin and Congo red, to identify mast cells (TC) and to assess their functionality – Unna's method. The functional state of mast cells was assessed by the degranulation index (ID) and the sulfatation index (IC) which we proposed earlier. The obtained numerical data were used to calculate a new statistical value – the TC/FB index. The data are presented in the form of mean values and median values indicating the percentiles 10 and 90. Differences of the groups were determined using z‑test. Differences in median values were determined using F-test. Results and their discussion. Liver sections of intact mice corresponded to the histological norm. The relative area of amyloid in the liver sections in CM group was 15.2±2.26%, VM group – 1.9±2.1 and VMF group – 2.5±2.9% (p= 0.0000). The relative area of amyloid–free collagen fibers in IG was 0.50±0.18%, in KM is 0.11±0.03 (p = 0.0151), in VM is 0.51±0.16 and in VMF – 0.69±0.18%. ID in IG – 0.38±0.19, KM – 0.39±0.2, VM – 0.55±0.09, VMF – 1.03±0.19 (p = 0.0065 to IG). IS in IG – 0.57±0.19, KM – 0.38±0.2, VM – 0.54±0.09, VMF – 1.23±0.17 (p = 0.0051 to IG). The median values of TC number in IG were 0.8 (0.5¸1.3), KM – 0.4 (0.3¸0.5), VM – 3.0 (2.0¸3.7), VMF – 2.0 (1.3¸3.0), to IG p &lt; 0.05 in all cases. The median values of the FB number in IG were 35.3 (29.9¸39.9), KM – 40.1 (26.1¸55.4), VM – 28.6 (20.3¸33.7), to IG p &lt; 0.05 in all cases, VMF – 51.3 (46.2¸55.4), p &gt; 0.5. The median values of the TC/FB index in IG were 0.022 (0.013¸0.038), KM – 0.009 (0.007¸0.012), p = 0.0000, VM – 0.11 (0.063¸0.167), p = 0.0012, VMF – 0.037 (0.024¸0.06). Conclusions. When forming experimental amyloidosis in the liver of mice, there is a decrease in the number of TC with a parallel increase in the number of FB per field of vision. Substitution of water in the diet of mice by dry red grape wine inhibits the development of changes characteristic of experimental amyloidosis induced by soy cream substitute. A new statistical value – the TC/FB index reflects both the severity of amyloid liver damage and the effectiveness of preventive measures in the form of substituting water by wine.

Identification and Spatial Visualization of Dysregulated Bile Acid Metabolism in High-Fat Diet-Fed Mice by Mass Spectral Imaging.

Changes in overall bile acid (BA) levels and specific BA metabolites are involved in metabolic diseases, gastrointestinal, and liver cancer. BAs have become established as important signaling molecules that enable fine-tuned inter-tissue communication within the enterohepatic circulation. The liver, BAs site of production, displayed physiological and functional zonal differences in the periportal zone versus the centrilobular zone. In addition, BA metabolism shows regional differences in the intestinal tract. However, there is no available method to detect the spatial distribution and molecular profiling of BAs within the enterohepatic circulation. Herein, we demonstrated the application in mass spectrometry imaging (MSI) with a high spatial resolution (3 μm) plus mass accuracy matrix-assisted laser desorption ionization (MALDI) to imaging BAs and N-1-naphthylphthalamic acid (NPA). Our results could clearly determine the zonation patterns and regional difference characteristics of BAs on mouse liver, ileum, and colon tissue sections, and the relative content of BAs based on NPA could also be ascertained. In conclusion, our method promoted the accessibility of spatial localization and quantitative study of BAs on gastrointestinal tissue sections and demonstrated that MALDI-MSI was a valuable tool to investigate and locate several BA molecules in different tissue types leading to a better understanding of the role of BAs behind the gastrointestinal diseases.

Atheroprotective and hepatoprotective effects of trans-chalcone through modification of eNOS/AMPK/KLF-2 pathway and regulation of COX-2, Ang-II, and PDGF mRNA expression in NMRI mice fed HCD.

The effects of trans-chalcone on atherosclerosis and NAFLD have been investigated. However, the underlying molecular mechanisms of these effects are not completely understood. This study aimed to deduce the impacts of trans-chalcone on the eNOS/AMPK/KLF-2 pathway in the heart tissues and the expression of Ang-II, PDFG, and COX-2 genes in liver sections of NMRI mice fed HCD. Thirty-two male mice were divided into four groups (n = 8): control group; fed normal diet. HCD group; fed HCD (consisted of 2% cholesterol) (12weeks). TCh groups; received HCD (12weeks) besides co-treated with trans-chalcone (20mg/kg and 40mg/kg b.w. dosages respectively) for 4weeks. Finally, the blood samples were collected to evaluate the biochemical parameters. Histopathological observations of aorta and liver sections were performed by H&E staining. The real-time PCR method was used for assessing the expression of the aforementioned genes. Histopathological examination demonstrated atheroma plaque formation and fatty liver in mice fed HCD which were accomplished with alteration in biochemical factors and Real-time PCR outcomes. Administration of trans-chalcone significantly modulated the serum of biochemical parameters. These effects were accompanied by significant increasing the expression of eNOS, AMPK, KLF-2 genes in heart sections and significant decrease in COX-2, Ang-II, and PDGF mRNA expression in liver sections. Our findings propose that the atheroprotective and hepatoprotective effects of trans-chalcone may be attributed to the activation of the eNOS/AMPK/KLF-2 pathway and down-regulation of Ang-II, PDFG, and COX-2 genes, respectively.

Beneficial Activities of Alisma orientale Extract in a Western Diet-Induced Murine Non-Alcoholic Steatohepatitis and Related Fibrosis Model via Regulation of the Hepatic Adiponectin and Farnesoid X Receptor Pathways.

The hepatic adiponectin and farnesoid X receptor (FXR) signaling pathways play multiple roles in modulating lipid and glucose metabolism, reducing hepatic inflammation and fibrosis, and altering various metabolic targets for the management of non-alcoholic fatty liver disease (NAFLD). Alisma orientale (AO, Ze xie in Chinese and Taeksa in Korean) is an herbal plant whose tubers are enriched with triterpenoids, which have been reported to exhibit various bioactive properties associated with NAFLD. Here, the present study provides a preclinical evaluation of the biological functions and related signaling pathways of AO extract for the treatment of NAFLD in a Western diet (WD)-induced mouse model. The findings showed that AO extract significantly reversed serum markers (liver function, lipid profile, and glucose) and improved histological features in the liver sections of mice fed WD for 52 weeks. In addition, it also reduced hepatic expression of fibrogenic markers in liver tissue and decreased the extent of collagen-positive areas, as well as inhibited F4/80 macrophage aggregation and inflammatory cytokine secretion. The activation of adiponectin and FXR expression in hepatic tissue may be a major mechanistic signaling cascade supporting the promising role of AO in NAFLD pharmacotherapy. Collectively, our results demonstrated that AO extract improves non-alcoholic steatohepatitis (NASH) resolution, particularly with respect to NASH-related fibrosis, along with the regulation of liver enzymes, postprandial hyperglycemia, hyperlipidemia, and weight loss, probably through the modulation of the hepatic adiponectin and FXR pathways.

Evaluation of the Effect of Hydromethanolic Seed Extract of Lepidium sativum L. (Fetto) on Deep-fried Palm Oil Diet Induced Nonalcoholic Fatty Liver Disease on Male Swiss Albino Mice.

IntroductionNonalcoholic fatty liver disease (NAFLD) is the most prevalent disease due to a dramatic change in dietary habits, especially an increase in consumption of fat and carbohydrates in deep-fried foods.ObjectiveThe objective was to evaluate the effect of hydromethanolic seed extract of Lepidium sativum on deep-fried palm oil diet induced NAFLDon male mice.MethodsAn experimental study design was conducted. Twenty-four male mice aged 8 to 10 weeks, weighing 32–42 g were divided into four groups. The four groups were divided into two controls and two treatments. Mice in normal control (C0) were administered only with the basal diet whereas negative control (C1) provided only with the deep-fried palm oil diet. The treatment groups T1, and T2 were administered with deep-fried palm oil diet and HMSELS at dose of 200 and 400 mg/kg/day, respectively for 28 days. Then on day 29, the mice were fasted overnight, anaesthetized and sacrificed by cervical dislocation after blood was taken by cardiac puncture for liver function tests while liver tissues were taken for histopathology investigation.ResultsThe serum ALT and total bilirubin showed significant decrement whereas the serum albumin levels showed significant increment in T2 group. However, serum AST and ALP levels were decreased significantly in both T1 and T2 groups. Besides, the T2 group liver sections of mice were showed better effect of HMSELS on restoring the damaged liver histopathology almost toward normal.ConclusionThe HMSELS at a dose of 400 mg/kg/day (T2) was more effective on the liver function tests and liver histopathology that altered by feeding deep-fried palm oil diet. The good protective effect of HMSELS against deep-fried palm oil diet-induced NAFLD might be due to its antioxidant content.

THE EFFECT OF DIFFERENT FREE FATTY ACID FRACTIONS FROM HYDROLYZED VIRGIN COCONUT OIL ON CHANGES OF LIPID PROFILE AND LIVER MORPHOLOGY INDUCED BY HIGH FAT DIET: AN IN VIVO STUDY

This study investigated effect of virgin coconut oil (VCO)-derived fatty acids on the alteration of lipid profiles and liver tissues of high fat diet (HFD) fed mice. The initial fatty acids mixture was successfully obtained via hydrolysis process using Candida rugosa lipase (CRL). Enrichment of medium chain fatty acids (MCFA) from the initial fatty acid mixture using distillation process was developed to achieve 3 fractions of fatty acids mixture including Fraction I (MCFA mainly from C8 to C10), Fraction II of lauric acid (C12), and Fraction III (long chain fatty acids from C14 to C20). Among these agents, VCO only diminished ALT level but the initial FFA mixture decreased both ALT and AST levels compared with HFD-induced mice. Moreover, Fraction I and Fraction II showed an obvious difference in decreasing ALT, AST, total cholesterol, and LDL cholesterol levels as well as increasing HDL cholesterol level compared to other agents. It was observed a microvesicular steatosis and mild inflammation in liver section of mice fed with HFD whereas VCO, initial fatty acids, and Fraction II showed the beneficial effect on liver damage.

Deletion of p38γ attenuates ethanol consumption- and acetaminophen-induced liver injury in mice through promoting Dlg1.

Acetaminophen (APAP) is one of the major causes of drug-induced acute liver injury, and ethanol may aggravate APAP-induced liver injury. The problem of ethanol- and APAP-induced liver injury becomes increasingly prominent, but the mechanism of ethanol- and APAP-induced liver injury remains ambiguous. p38γ is one of the four isoforms of P38 mitogen activated protein kinases, that contributes to inflammation in different diseases. In this study we investigated the role of p38γ in ethanol- and APAP-induced liver injury. Liver injury was induced in male C57BL/6 J mice by giving liquid diet containing 5% ethanol (v/v) for 10 days, followed by gavage of ethanol (25% (v/v), 6 g/kg) once or injecting APAP (200 mg/kg, ip), or combined the both treatments. We showed that ethanol significantly aggravated APAP-induced liver injury in C57BL/6 J mice. Moreover, the expression level of p38γ was up-regulated in the liver of ethanol-, APAP- and ethanol+APAP-treated mice. Knockdown of p38γ markedly attenuated liver injury, inflammation, and steatosis in ethanol+APAP-treated mice. Liver sections of p38γ-knockdown mice displayed lower levels of Oil Red O stained dots and small leaky shapes. AML-12 cells were exposed to APAP (5 mM), ethanol (100 mM) or combined treatments. We showed that P38γ was markedly increased in ethanol+APAP-treated AML-12 cells, whereas knockdown of p38γ significantly inhibited inflammation, lipid accumulation and oxidative stress in ethanol+APAP-treated AML-12 cells. Furthermore, we revealed that p38γ could combine with Dlg1, a member of membrane-associated guanylate kinase family. Deletion of p38γ up-regulated the expression level of Dlg1 in ethanol+APAP-treated AML-12 cells. In summary, our results suggest that p38γ functions as an important regulator in ethanol- and APAP-induced liver injury through modulation of Dlg1.

Fibroblast growth factor 7 releasing particles enhance islet engraftment and improve metabolic control following islet transplantation in mice with diabetes

Transplantation of islets in type 1 diabetes (T1D) is limited by poor islet engraftment into the liver, with two to three donor pancreases required per recipient. We aimed to condition the liver to enhance islet engraftment to improve long‐term graft function. Diabetic mice received a non‐curative islet transplant (n = 400 islets) via the hepatic portal vein (HPV) with fibroblast growth factor 7‐loaded galactosylated poly(DL‐lactide‐co‐glycolic acid) (FGF7‐GAL‐PLGA) particles; 26‐µm diameter particles specifically targeted the liver, promoting hepatocyte proliferation in short‐term experiments: in mice receiving 0.1‐mg FGF7‐GAL‐PLGA particles (60‐ng FGF7) vs vehicle, cell proliferation was induced specifically in the liver with greater efficacy and specificity than subcutaneous FGF7 (1.25 mg/kg ×2 doses; ~75‐µg FGF7). Numbers of engrafted islets and vascularization were greater in liver sections of mice receiving islets and FGF7‐GAL‐PLGA particles vs mice receiving islets alone, 72 h posttransplant. More mice (six of eight) that received islets and FGF7‐GAL‐PLGA particles normalized blood glucose concentrations by 30‐days posttransplant, versus zero of eight mice receiving islets alone with no evidence of increased proliferation of cells within the liver at this stage and normal liver function tests. This work shows that liver‐targeted FGF7‐GAL‐PLGA particles achieve selective FGF7 delivery to the liver‐promoting islet engraftment to help normalize blood glucose levels with a good safety profile.

Establishment and validation of highly accurate formalin-fixed paraffin-embedded quantitative proteomics by heat-compatible pressure cycling technology using phase-transfer surfactant and SWATH-MS

The purpose of this study was to establish a quantitative proteomic method able to accurately quantify pathological changes in the protein expression levels of not only non-membrane proteins, but also membrane proteins, using formalin-fixed paraffin-embedded (FFPE) samples. Protein extraction from FFPE sections of mouse liver was increased 3.33-fold by pressure cycling technology (PCT) and reached the same level as protein extraction from frozen sections. After PCT-assisted processing of FFPE liver samples followed by SWATH-MS-based comprehensive quantification, the peak areas of 88.4% of peptides agreed with those from matched fresh samples within a 1.5-fold range. For membrane proteins, this percentage was remarkably increased from 49.1 to 93.8% by PCT. Compared to the conventional method using urea buffer, the present method using phase-transfer surfactant (PTS) buffer at 95 °C showed better agreement of peptide peak areas between FFPE and fresh samples. When our method using PCT and PTS buffer at 95 °C was applied to a bile duct ligation (BDL) disease model, the BDL/control expression ratios for 80.0% of peptides agreed within a 1.2-fold range between FFPE and fresh samples. This heat-compatible FFPE-PCT-SWATH proteomics technology using PTS is suitable for quantitative studies of pathological molecular mechanisms and biomarker discovery utilizing widely available FFPE samples.

Design of dye and superparamagnetic iron oxide nanoparticle loaded lipid nanocapsules with dual detectability in vitro and in vivo.

Lipid nanocapsules are treasured nanoparticulate systems, although they lack detectability in biological environments. To overcome this, we designed LNCs loaded simultaneously with fluorescent dye and superparamagnetic iron oxide nanoparticles (Dual LNCs). The introduction of both labels did not alter nanoparticle characteristics such as size (50nm), size distribution (polydispersity index<0.1) or surface modifications, including the effectiveness of targeting ligands. Furthermore, the colloidal stability, particle integrity and biocompatibility of the nanoparticles were not negatively affected by label incorporation. These Dual LNCs are concomitantly visualizable via fluorescence and transmitted light imaging after either the internalization by cells or systemic administration to mice. Importantly, they are detectable in liver sections of mice using transmission electron microscopy without additional enhancement. The iron content of 0.24% (m/m) is sufficiently high for precise quantification of nanoparticle concentrations via inductively coupled plasma optical emission spectroscopy. Dual LNCs are precious tools for the investigation of in vitro and in vivo performances of lipid nanocapsule formulations, since they allow for the use of complementary imaging methods for broad range detectability.

Kupffer Cell and Monocyte-Derived Macrophage Identification by Immunofluorescence on Formalin-Fixed, Paraffin-Embedded (FFPE) Mouse Liver Sections.

Kupffer cells are the liver-resident macrophages and represent the first line of defense between the pathogens circulating from the intestines through the portal vein and systemic circulation. Recent works have highlighted the complex heterogeneity of macrophage functions and origins, thus raising awareness on the need for a better characterization of macrophage populations. The immunohistochemistry method here described, allows for a rapid distinction between Kupffer cells and monocyte-derived macrophages present on formalin-fixed, paraffin-embedded mouse liver samples. This protocol has been optimized for its reproducibility, reliability, and simplicity.

Peroxiredoxin 5 ameliorates obesity-induced non-alcoholic fatty liver disease through the regulation of oxidative stress and AMP-activated protein kinase signaling

Non-alcoholic fatty liver disease (NAFLD) is becoming the most common chronic liver disease globally. NAFLD—which can develop into liver fibrosis, nonalcoholic steatohepatosis, cirrhosis, and hepatocellular carcinoma—is defined as an excess accumulation of fat caused by abnormal lipid metabolism and excessive reactive oxygen species (ROS) generation in hepatocytes. Recently, we reported that Peroxiredoxin 5 (Prx5) plays an essential role in regulating adipogenesis and suggested the need to further investigation on the potential curative effects of Prx5 on obesity-induced fatty liver disease. In the present study, we focused on the role of Prx5 in fatty liver disease. We found that Prx5 overexpression significantly suppressed cytosolic and mitochondrial ROS generation. Additionally, Prx5 regulated the AMP-activated protein kinase pathway and lipogenic gene (sterol regulatory element binding protein-1 and FAS) expression; it also inhibited lipid accumulation, resulting in the amelioration of free fatty acid-induced hepatic steatosis. Silence of Prx5 triggered de novo lipogenesis and abnormal lipid accumulation in HepG2 cells. Concordantly, Prx5 knockout mice exhibited a high susceptibility to obesity-induced hepatic steatosis. Liver sections of Prx5-deletion mice fed on a high-fat diet displayed Oil Red O-stained dots and small leaky shapes due to immoderate fat deposition. Collectively, our findings suggest that Prx5 functions as a protective regulator in fatty liver disease and that it may be a valuable therapeutic target for the management of obesity-related metabolic diseases.

Antioxidant and anticancer activity of synthesized 4-amino-5-((aryl substituted)-4H-1,2,4-triazole-3-yl)thio-linked hydroxamic acid derivatives

The antioxidant and anticancer activity of twelve 5-substituted-4-amino-1,2,4-triazole-linked hydroxamic acid derivatives were evaluated. Previously synthesized 2-((4-amino-5-substituted-4H-1,2,4-triazol-3-yl)thio)-N-hydroxyacetamide and 3-((4-amino-5-substituted-4H-1,2,4-triazol-3-yl)thio)-N-hydroxypropanamide (6a-6l) were evaluated for in vitro antioxidant and in vivo anticancer activity. MDA-MB-231, MCF-7 and HCT 116 cell lines were used to evaluate IC50 values, in vitro. Ehrlich ascites carcinoma (EAC)-induced mice model was used to evaluate in vivo anticancer potential. Different biological markers were examined for drug-related toxicities. Compound 6b revealed more potent antioxidant property among all tested compounds, even than the ascorbic acid. The IC50 values of compound 6b were found to be 5.71±2.29μg/ml (DPPH assay) and 4.12±0.5μg/ml (ABTS assay). Histopathology of liver sections of drug-treated mice was evaluated. Survival analysis showed that compound 6b could increase the life span as of the standard drug. After the assessment of all in vivo anticancer study related data, it was found that compound 6b possess superior anticancer potency in terms of efficacy and toxicity. From this experimental design, it could be concluded that further modification of this prototypical structure will lead to develop more potent antioxidant as well as an anticancer agent in the future.

Growth retardation and apoptotic death of tumor cells by Artemisia herba-alba oral administration in Ehrlich solid carcinoma bearing mice

At present, there is a rapidly growing interest in studying the cytotoxic effects of Artemisia herba alba Asso, Asteraceae, in various cancer cell lines. However, its antitumor effectiveness has not been investigated. Therefore, the current study was conducted to study the effect of A. herba alba extract on the proliferation and growth of solid tumor cells in Ehrlich Solid Carcinoma bearing mice. Oral administration of A. herba alba extract resulted in significant reductions in tumor size, tumor weight and mice body weight, as well as caused concurrent significant increases in the DNA breakages and apoptotic DNA damage induction in a time-dependent manner. A. herba alba extract also raised the expression level of p53 gene and reduced of K-ras expression in a time-dependent manner. Minor histological lesions were observed in the liver and kidney tissues sections of mice administered A. herba alba extract compared with the high histological lesions observed in the liver and kidney tissues of artesunate and cisplatin treated groups. Thus, we concluded that A. herba alba extract exhibited promising potential antitumor efficacy with greater safety than artesunate and the commercially used anticancer drug cisplatin in mice.

Imaging and Quantitative Detection of Lipid Droplets by Yellow Fluorescent Probes in Liver Sections of Plasmodium Infected Mice and Third Stage Human Cervical Cancer Tissues.

The diagnosis and prognosis of the disease associated with lipid irregularity are areas of extreme significance. In this direction, fluoranthene based yellow fluorescent probes (FLUN-550, FLUN-552, FLUN-547) were designed and synthesized by conjugating the ethanolamine headgroup of the phospholipid phosphatidyl-ethanolamine present in biological membranes. Owing to unique photophysical properties and aqueous compatibility, these probes were successfully employed for staining lipid droplets (LDs) in preadipocytes and Leishmania donovani promastigotes. Furthermore, using the fluorescent probes FLUN-550 and FLUN-552 we successfully imaged and quantitatively detected the excess accumulation of lipids in a liver section of Plasmodium yoelii MDR infected mice (3- to 4-fold) and the tissue sections of third stage human cervical cancer patients (1.5- to 2-fold) compared to normal tissues. To the best of our knowledge, this is the first report of yellow fluorescent probes for imaging and quantitative detection of LDs in human cervical cancer tissues. These new yellow fluorescent lipid probes (FLUN-550 and FLUN-552) showed great potential for diagnosis of cervical cancer patients.