Liver Cell Viability Research Articles

Nepetoidin B Alleviates Liver Ischemia/Reperfusion Injury via Regulating MKP5 and JNK/P38 Pathway.

Nepetoidin B (NB) has been reported to possess anti-inflammatory, antibacterial, and antioxidant properties. However, its effects on liver ischemia/reperfusion (I/R) injury remain unclear. In this study, a mouse liver I/R injury model and a mouse AML12 cell hypoxia reoxygenation (H/R) injury model were used to investigate the potential role of NB. Serum transaminase levels, liver necrotic area, cell viability, oxidative stress, inflammatory response, and apoptosis were evaluated to assess the effects of NB on liver I/R and cell H/R injury. Quantitative polymerase chain reaction (qPCR) and Western blotting were used to measure mRNA and protein expression levels, respectively. Molecular docking was used to predict the binding capacity of NB and mitogen-activated protein kinase phosphatase 5 (MKP5). The results showed that NB significantly reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, liver necrosis, oxidative stress, reactive oxygen species (ROS) content, inflammatory cytokine content and expression, inflammatory cell infiltration, and apoptosis after liver I/R and AML12 cells H/R injury. Additionally, NB inhibited the JUN protein amino-terminal kinase (JNK)/P38 pathway. Molecular docking results showed good binding between NB and MKP5 proteins, and Western blotting results showed that NB increased the protein expression of MKP5. MKP5 knockout (KO) significantly diminished the protective effects of NB against liver injury and its inhibitory effects on the JNK/P38 pathway. NB exerts hepatoprotective effects against liver I/R injury by regulating the MKP5-mediated P38/JNK signaling pathway.

Comparative transcriptomics combined with physiological and functional analysis reveals the regulatory mechanism of rainbow trout (Oncorhynchus mykiss) under acute hypoxia stress

Hypoxia, largely triggered by global warming and water contamination, has become an environmental issue of great concern, posing a great threat to aquatic ecosystem. As one of the world's most economically important fish, rainbow trout (Oncorhynchus mykiss) is extremely intolerant of hypoxic environments, however, little is known about the roles of non-coding RNAs (ncRNAs) in the response of rainbow trout to hypoxia stress. Herein, effects of moderate (Tm12L) and severe hypoxia for 12 h (Ts12L) and 12 h reoxygenation on histology, biochemical parameters (antioxidant, metabolism and immunity) and transcriptome (lncRNA, miRNA and mRNA) in rainbow trout liver were investigated. We further validated the regulatory relationships between LOC110519952, novel-m0023-5p and glut1a via dual‑luciferase reporter, overexpression and silencing assays. Compared with Tm12L, the liver in Ts12L showed more severe oxidative damage. Anaerobic, lipid and protein metabolism was enhanced under hypoxia stress, especially in Ts12L. We also found that Tm12L could strengthen innate immune response, which was inhibited in Ts12L. Besides, several hypoxia-related genes (glut1a, vegfaa, hmox, epoa, foxo1a and igfbp1) and ceRNA networks were identified from 1824, 427 and 545 differentially expressed mRNAs, miRNAs and lncRNAs, including LOC118965299-novel-m0179-3p-epoa, LOC110519952-novel-m0023-5p-glut1a, MSTRG.7382.2-miR-184-y-hmox and LOC110520012-miR-206-y-vegfaa. Through in vitro and in vivo functional analysis, we demonstrated that glut1a is a target of novel-m0023-5p, and LOC110519952 can positively regulate glut1a by targeting novel-m0023-5p. Introduction of LOC110519952 could attenuate the promoting effects of novel-m0023-5p on rainbow trout liver cell viability and proliferation. This study highlights the differences in the regulatory mechanism of rainbow trout under different concentrations of hypoxia stress and provides valuable data for further research on the molecular mechanisms of fish adaptation to hypoxic environments.

6-Gingerol attenuates hepatic ischemia/reperfusion injury through regulating MKP5-mediated P38/JNK pathway

6-Gingerol, the main bioactive compound of ginger, has antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. However, it is unclear whether 6-Gingerol has protective effects against hepatic ischemia/reperfusion (I/R) injury. In this study, the mouse liver I/R injury model and the mouse AML12 cell hypoxia/reoxygenation (H/R) model were established by pretreatment with 6-Gingerol at different concentrations to explore the potential effects of 6-Gingerol. Serum transaminase levels, liver necrotic area, cell viability, inflammatory response, and cell apoptosis were used to assess the effect of 6-Gingerol on hepatic I/R or cell H/R injury. Quantitative polymerase chain reaction (qPCR) and Western blotting were used to detect the mRNA and protein expression. The results show that 6-Gingerol decreased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels, liver necrosis, inflammatory cytokines IL-1β, IL-6, MCP-1, TNF-α expression, Ly6g+ inflammatory cell infiltration, protein phosphorylation of NF-κB signaling pathway, Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) positive cells, cell apoptosis rate, the protein expression of pro-apoptotic protein BAX and C-Caspase3, increased cell viability, and expression of anti-apoptotic protein BCL-2. Moreover, 6-Gingerol could increase the mRNA and protein expression of mitogen activated protein kinase phosphatase 5 (MKP5) and inhibit the activation of P38/JNK signaling pathway. In MKP5 knockout (KO) mice, the protective effect of 6-gingerol and the inhibition of P38/JNK pathway were significantly weakened. Therefore, our results suggest that 6-Gingerol exerts anti-inflammatory and anti-apoptotic effects to attenuate hepatic I/R injury by regulating the MKP5-mediated P38/JNK signaling pathway.

Prognostic prediction using a gene signature developed based on exhausted T cells for liver cancer patients

BackgroundLiver hepatocellular carcinoma (LIHC) is a solid primary malignancy with poor prognosis. This study discovered key prognostic genes based on T cell exhaustion and used them to develop a prognostic prediction model for LIHC. MethodsSingleR's annotations combined with Seurat was used to automatically annotate the single-cell clustering results of the LIHC dataset GSE166635 downloaded from the Gene Expression Omnibus (GEO) database and to identify clusters related to exhausted T cells. Patients were classified using ConsensusClusterPlus package. Next, weighted gene co-expression network analysis (WGCNA) package was employed to distinguish key gene module, based on which least absolute shrinkage and selection operator (Lasso) and multi/univariate cox analysis were performed to construct a RiskScore system. Kaplan-Meier (KM) analysis and receiver operating characteristic curve (ROC) were employed to evaluate the efficacy of the model. To further optimize the risk model, a nomogram capable of predicting immune infiltration and immunotherapy sensitivity in different risk groups was developed. Expressions of genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence and Cell Counting Kit-8 (CCK-8) were performed for analyzing cell functions. ResultsWe obtained 18,413 cells and clustered them into 7 immune and non-immune cell subpopulations. Based on highly variable genes among T cell exhaustion clusters, 3 molecular subtypes (C1, C2 and C3) of LIHC were defined, with C3 subtype showing the highest score of exhausted T cells and a poor prognosis. The Lasso and multivariate cox analysis selected 7 risk genes from the green module, which were closely associated with the C3 subtype. All the patients were divided into low- and high-risk groups based on the medium value of RiskScore, and we found that high-risk patients had higher immune infiltration and immune escape and poorer prognosis. The nomogram exhibited a strong performance for predicting long-term LIHC prognosis. In vitro experiments revealed that the 7 risk genes all had a higher expression in HCC cells, and that both liver HCC cell numbers and cell viability were reduced by knocking down MMP-9. ConclusionWe developed a RiskScore model for predicting LIHC prognosis based on the scRNA-seq and RNA-seq data. The RiskScore as an independent prognostic factor could improve the clinical treatment for LIHC patients.

LONP1 ameliorates liver injury and improves gluconeogenesis dysfunction in acute-on-chronic liver failure.

Acute-on-chronic liver failure (ACLF) is a severe liver disease with complex pathogenesis. Clinical hypoglycemia is common in patients with ACLF and often predicts a worse prognosis. Accumulating evidence suggests that glucose metabolic disturbance, especially gluconeogenesis dysfunction, plays a critical role in the disease progression of ACLF. Lon protease-1 (LONP1) is a novel mediator of energy and glucose metabolism. However, whether gluconeogenesis is a potential mechanism through which LONP1 modulates ACLF remains unknown. In this study, we collected liver tissues from ACLF patients, established an ACLF mouse model with carbon tetrachloride (CCl 4 ), lipopolysaccharide (LPS), and D-galactose (D-gal), and constructed an in vitro hypoxia and hyperammonemia-triggered hepatocyte injury model. LONP1 overexpression and knockdown adenovirus were used to assess the protective effect of LONP1 on liver injury and gluconeogenesis regulation. Liver histopathology, biochemical index, mitochondrial morphology, cell viability and apoptosis, and the expression and activity of key gluconeogenic enzymes were detected to explore the underlying protective mechanisms of LONP1 in ACLF. We found that LONP1 and the expressions of gluconeogenic enzymes were downregulated in clinical ACLF liver tissues. Furthermore, LONP1 overexpression remarkably attenuated liver injury, which was characterized by improved liver histopathological lesions and decreased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in ACLF mice. Moreover, mitochondrial morphology was improved upon overexpression of LONP1. Meanwhile, the expression and activity of the key gluconeogenic enzymes were restored by LONP1 overexpression. Similarly, the hepatoprotective effect was also observed in the hepatocyte injury model, as evidenced by improved cell viability, reduced cell apoptosis, and improved gluconeogenesis level and activity, while LONP1 knockdown worsened liver injury and gluconeogenesis disorders. We demonstrated that gluconeogenesis dysfunction exists in ACLF, and LONP1 could ameliorate liver injury and improve gluconeogenic dysfunction, which would provide a promising therapeutic target for patients with ACLF.

Skimmianine attenuates liver ischemia/reperfusion injury by regulating PI3K–AKT signaling pathway-mediated inflammation, apoptosis and oxidative stress

Liver ischemia/reperfusion (I/R) injury is a common injury after liver transplantation and hepatectomy. Skimmianine (Ski) has antibacterial, antiviral pharmacological effects. However, it is not clear whether Ski has a protective effect against liver I/R injury. In the present study, we established a mouse liver I/R model and an AML12 cell hypoxia-reoxygenation (H/R) model, both pretreated with different concentrations of Ski. Serum transaminase levels, necrotic liver area, cell viability, inflammatory factors, oxidative stress and apoptosis-related levels were measured to assess the protective effect of Ski against liver I/R injury. Western blotting was used to detect apoptosis-related proteins and PI3K–AKT pathway-related proteins. Mice and cells were also treated with PI3K inhibitor LY294002 to assess changes in indicators of liver injury. The results showed that Ski significantly reduced transaminase levels, liver necrosis area, oxidative stress, and apoptosis levels in mice with I/R. Ski also inhibited cell injury and apoptosis after H/R. Moreover, Ski activated phosphorylation of PI3K–AKT pathway-related proteins after liver I/R and cell H/R. Importantly, the PI3K inhibitor LY294002 effectively reversed the alleviation of I/R injury caused by Ski. These results confirm that Ski exerts a protective effect against liver I/R injury through activation of the PI3K–AKT pathway.

Pectolinarigenin attenuates hepatic ischemia/reperfusion injury via activation of the PI3K/AKT/Nrf2 signaling pathway

Hepatic ischemia/reperfusion (I/R) injury is an unavoidable complication of liver hepatectomy, transplantation, and systemic shock. Pectolinarigenin (Pec) is a flavonoid with many biological activities, which include anti-inflammatory, anti-apoptotic, and antioxidant stress. This study explored whether Pec pretreatment could reduce hepatic I/R injury and the potential mechanisms at play. After pretreatment of mice and AML12 cells with Pec, I/R and hypoxia/reoxygenation (H/R) models were established. By examining markers related to liver injury, cell viability, oxidative stress, inflammatory response, and apoptosis, the effect of Pec on important processes involved in hepatic I/R injury was assessed. Protein levels associated with the PI3K/AKT/Nrf2 pathway were analyzed by relative quantification to investigate possible pathways through which Pec plays a role in the I/R process. Pec treatment corrected abnormal transaminase levels resulting from I/R injury, improved liver injury, and increased AML12 cell viability. Moreover, Pec treatment inhibited oxidative stress, inflammation and apoptosis and could activate the PI3K/AKT/Nrf2 pathway during I/R and H/R. Further studies found that LY294002 (PI3K inhibitor) suppressed the protective effect of Pec on hepatic I/R injury. In summary, our results show that Pec inhibits oxidative stress, inflammatory responses, and apoptosis, thereby attenuating I/R-induced liver injury and H/R-induced cell damage via activation of the PI3K/AKT/Nrf2 pathway.

Liraglutide attenuates type 2 diabetes mellitus-associated non-alcoholic fatty liver disease by activating AMPK/ACC signaling and inhibiting ferroptosis

BackgroundNon-alcoholic fatty liver disease (NAFLD) is one of the most common complications of type 2 diabetes mellitus (T2DM). The pathogenesis of NAFLD involves multiple biological changes, including insulin resistance, oxidative stress, inflammation, as well as genetic and environmental factors. Liraglutide has been used to control blood sugar. But the impact of liraglutide on T2DM-associated NAFLD remains unclear. In this study, we investigated the impact and potential molecular mechanisms of inhibiting ferroptosis for liraglutide improves T2DM-associated NAFLD.MethodsMice were fed on high-fat-diet and injected with streptozotocin to mimic T2DM-associated NAFLD and gene expression in liver was analysed by RNA-seq. The fast blood glucose was measured during the period of liraglutide and ferrostatin-1 administration. Hematoxylin and eosin staining was used to evaluate the pathological changes in the liver. The occurrence of hepatic ferroptosis was measured by lipid peroxidation in vivo. The mechanism of liraglutide inhibition ferroptosis was investigated by in vitro cell culture.ResultsLiraglutide not only improved glucose metabolism, but also ameliorated tissue damage in the livers. Transcriptomic analysis indicated that liraglutide regulates lipid metabolism related signaling including AMPK and ACC. Furthermore, ferroptosis inhibitor rather than other cell death inhibitors rescued liver cell viability in the presence of high glucose. Mechanistically, liraglutide-induced activation of AMPK phosphorylated ACC, while AMPK inhibitor compound C blocked the liraglutide-mediated suppression of ferroptosis. Moreover, ferroptosis inhibitor restored liver function in T2DM mice in vivo.ConclusionsThese findings indicate that liraglutide ameliorates the T2DM-associated NAFLD, which possibly through the activation of AMPK/ACC pathway and inhibition of ferroptosis.

The oleic/palmitic acid imbalance in exosomes isolated from NAFLD patients induces necroptosis of liver cells via the elongase-6/RIP-1 pathway

Excessive toxic lipid accumulation in hepatocytes underlies the development of non-alcoholic fatty liver disease (NAFLD), phenotypically characterized by necrosis and steato-fibrosis, whose molecular mechanism is not yet fully understood. Patients with NAFLD display an imbalanced palmitic (PA) to oleic acid (OA) ratio. Moreover, increasing experimental evidence points out a relevant involvement of the exosomal content in disease progression. Aim of the study was to highlight the PA/OA imbalance within circulating exosomes, the subsequent intracellular alterations, and the impact on NALFD. Liver cells were challenged with exosomes isolated from both healthy subjects and NAFLD patients. The exosomal PA/OA ratio was artificially modified, and biological effects were evaluated. A NAFLD-derived exosomal PA/OA imbalance impacts liver cell cycle and cell viability. OA-modified NAFLD-derived exosomes restored cellular viability and proliferation, whereas the inclusion of PA into healthy subjects-derived exosomes negatively affected cell viability. Moreover, while OA reduced the phosphorylation and activation of the necroptosis marker, Receptor-interacting protein 1 (phospho-RIP-1), PA induced the opposite outcome, alongside increased levels of stress fibers, such as vimentin and fibronectin. Administration of NAFLD-derived exosomes led to increased expression of Elongase 6 (ELOVL6), Stearoyl-CoA desaturase 1 (SCD1), Tumor necrosis factor α (TNF-α), Mixed-lineage-kinase-domain-like-protein (MLKL) and RIP-1 in the hepatocytes, comparable to mRNA levels in the hepatocytes of NAFLD patients reported in the Gene Expression Omnibus (GEO) database. Genetic and pharmacological abrogation of ELOVL6 elicited a reduced expression of downstream molecules TNF-α, phospho-RIP-1, and phospho-MLKL upon administration of NAFLD-derived exosomes. Lastly, mice fed with high-fat diet exhibited higher phospho-RIP-1 than mice fed with control diet. Targeting the Elongase 6–RIP-1 signaling pathway offers a novel therapeutic approach for the treatment of the NALFD-induced exosomal PA/OA imbalance.

A Human Ovarian Tumor & Liver Organ-on-Chip for Simultaneous and More Predictive Toxo-Efficacy Assays.

In oncology, the poor success rate of clinical trials is becoming increasingly evident due to the weak predictability of preclinical assays, which either do not recapitulate the complexity of human tissues (i.e., in vitro tests) or reveal species-specific outcomes (i.e., animal testing). Therefore, the development of novel approaches is fundamental for better evaluating novel anti-cancer treatments. Here, a multicompartmental organ-on-chip (OOC) platform was adopted to fluidically connect 3D ovarian cancer tissues to hepatic cellular models and resemble the systemic cisplatin administration for contemporarily investigating drug efficacy and hepatotoxic effects in a physiological context. Computational fluid dynamics was performed to impose capillary-like blood flows and predict cisplatin diffusion. After a cisplatin concentration screening using 2D/3D tissue models, cytotoxicity assays were conducted in the multicompartmental OOC and compared with static co-cultures and dynamic single-organ models. A linear decay of SKOV-3 ovarian cancer and HepG2 liver cell viability was observed with increasing cisplatin concentration. Furthermore, 3D ovarian cancer models showed higher drug resistance than the 2D model in static conditions. Most importantly, when compared to clinical therapy, the experimental approach combining 3D culture, fluid-dynamic conditions, and multi-organ connection displayed the most predictive toxicity and efficacy results, demonstrating that OOC-based approaches are reliable 3Rs alternatives in preclinic.

Isolongifolene alleviates liver ischemia/reperfusion injury by regulating AMPK-PGC1α signaling pathway-mediated inflammation, apoptosis, and oxidative stress

Isolongifolene (ISO) has antioxidant, anti-inflammatory, anticancer, and neuroprotective effects; however, it is unclear whether ISO has a protective effects against liver ischemia/reperfusion (I/R) injury. In this study, a mouse liver I/R injury model and a mouse AML12 cell Hypoxia reoxygenation (H/R) model were established after pretreatment with different concentrations of ISO. Serum transaminase levels, necrotic liver area, cell viability, inflammation response, oxidative stress, and apoptosis were used to evaluate the effect of ISO on liver I/R or cell H/R injury. Western blotting was used to detect Bax, Bcl-2, C-Caspase3, AMPK, P-AMPK, and PGC1α protein expression levels. The AMPK inhibitor, compound C, was used to inhibit the AMPK expression. The results showed that ISO reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, liver necrosis, inflammatory factors IL-1β, IL-6, MCP-1, and TNF-α expression, MPO+ inflammatory cell infiltration, MDA content, TUNEL-positive cell number, cell apoptosis rate, and the expression of pro-apoptotic proteins Bax and C-Caspase3, while increasing cell viability, SOD and GSH activity, and the expression of anti-apoptotic protein Bcl-2. Moreover, Western blotting results showed that ISO could increase the protein expression of P-AMPK and PGC1α. Following the addition of compound C, the protective effect of ISO was significantly weakened. Therefore, our results suggest that ISO alleviates liver I/R injury by regulating AMPK-PGC1α signaling pathway-mediated anti-inflammatory, and antioxidant and anti-apoptotic effects.

Wheat germ oil vitamin E cytoprotective effect and its nutrigenomics signature in human hepatocyte lipotoxicity

Wheat germ oil (WGO) is rich in α-tocopherol (vitamin E, VE), a vitamin that has long been suggested to exert hepatoprotective effects. In this study, this function of WGO-VE and its transcriptomics fingerprint were investigated in comparison with RRR−α-tocopherol and all-rac-α-tocopherol (nVE and sVE, respectively), in human liver cells treated with oleic acid (OA) to develop steatosis and lipotoxicity. Used in chemoprevention mode, all the VE formulations afforded significant reduction of the OA-induced steatosis and its consequent impact on lipotoxicity indicators, including ROS production and efflux (as H2O2), and apoptotic and necrotic cell death. A trend toward a better control of lipotoxicity was observed for WGO-VE and nVE compared to sVE.Gene microarray data demonstrated that these effects of VE formulations were associated with significantly different responses of the cellular transcriptome to compensate for the modifications of OA treatment, including the downregulation of cellular homeostasis genes and the induction of genes associated with defects of liver cell metabolism, fibrosis and inflammation, liver disease and cancer. Ingenuity Pathway Analysis data showed that WGO-VE modulated genes associated with liver carcinogenesis and steatosis, whereas nVE modulated genes involved in liver cell metabolism and viability biofunctions; sVE did not significantly modulate any gene dataset relevant to such biofunctions.In conclusion, WGO-VE prevents lipotoxicity in human liver cells modulating genes that differ from those affected by the natural or synthetic forms of pure VE. These differences can be captured by precision nutrition tools, reflecting the molecular complexity of this VE-rich extract and its potential in preventing specific cues of hepatocellular lipotoxicity.

Long non-coding RNA MALAT1 aggravated liver ischemia-reperfusion injury via targeting miR-150-5p/AZIN1

ABSTRACT Long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a crucial role in the process of renal ischemia-reperfusion (IR) injury and myocardial IR injury. However, its mechanism in liver IR injury is not clear. IR and hypoxia/reoxygenation (H/R) model were built on C57BL/6 mice. Blood samples were obtained from the inferior vena cava of the model mice. MALAT1 expression was detected in IR model and H/R model. Supported by experimental results, the impacts of MALAT1 on viability, apoptosis, and inflammation of H/R model cells were detected. The correlation between MALAT1 and downstream genes was analyzed by mechanism assays. MALAT1 was detected to be upregulated in IR model and H/R model. MALAT1 knockdown had inhibitory effects on apoptosis and inflammatory reaction while promoting liver cell viability in H/R condition. Meanwhile, MALAT1 targeted miR-150-5p to regulate antizyme inhibitor 1 (AZIN1) in liver cells. Finally, MALAT1 regulated viability, apoptosis, and inflammatory reaction of liver cells by targeting miR-150-5p and AZIN1. To conclude, MALAT1 targeted miR-150-5p/AZIN1 to accelerate liver IR injury, suggesting that MALAT1 might be a novel target for liver IR injury.

Punicalagin Reversed the Hepatic Injury of Tetrachloromethane by Antioxidation and Enhancement of Autophagy.

Hepatic injury is significant in the pathogenesis and development of many types of liver diseases. Punicalagin (PU) is a bioactive antioxidant polyphenol found in pomegranates. To explore its protective effect against carbon tetrachloride (CCl4)-induced liver injury and the mechanism, Institute of Cancer Research (ICR) mice and L02 cells were used to observe the changes of serum biochemical indicators, histopathological liver structure, cell viability, antioxidative indices, and autophagy-related proteins were assessed. In ICR mice, PU ameliorated the CCl4-induced increase of the serum aspartate aminotransferase, alanine aminotransferase, the activity of liver lactate dehydrogenase, and the damage of histopathological structure, and exhibited a hepatoprotective effect against CCl4. PU attenuated oxidative stress by decreasing the liver malondialdehyde level and increasing the activities of liver superoxide dismutase, glutathione peroxidase, and the expression of the liver nuclear factor E2-related factor (Nrf2) protein. Furthermore, according to the vivo and vitro experiments, PU might activate autophagy through the mediation of the Akt/FOXO3a and P62/Nrf2 signaling pathway. Taken together, these results suggest that PU may protect against CCl4-induced liver injury through the upregulation of antioxidative activities and autophagy.

Insulin-loaded mucoadhesive nanoparticles based on mucin-chitosan complexes for oral delivery and diabetes treatment

In this study, insulin-loaded nanoparticles (NPs) were prepared via self-gelation method using chitosan and aqueous soluble snail mucin as natural polymers. Herein, mucins were ionically interacted with chitosan at different concentrations to obtained insulin-loaded NPs, labelled as A1 (1:1) (i.e., chitosan 2 % w/v + mucin 2 % w/v) and A2 (2:1) (chitosan 4 % w/v + mucin 2 % w/v), using poloxamer and poly vinyl alcohol as solid surfactant. Such formulation was selected to provide the necessary dynamics for the formation of the nanoparticles while maintaining the surface properties that will favor the encapsulation of insulin. Each system was characterized in terms of their particle size distribution, morphology, zeta potential, and polydispersity index. In vitro release of insulin was evaluated in acidic solution (pH 1.2) and phosphate buffer solution (pH 7.4), and the hypoglycaemic activity was evaluated in diabetes rats. The prepared insulin-loaded NPs displayed particles with relatively smooth surfaces and an average particle size of 479.6 and 504.1 nm for A1 and A2, respectively. Zeta potential and polydispersity index, ranged from 22.1 to 31.2 mV and 0.155–0.185, respectively. The encapsulating efficiency for the systems A1 and A2 were 88.6 and 92.5, respectively, and a self-sustained release of encapsulated insulin was observed for over a period of 8 h. In vivo studies revealed a pronounced hypoglycaemic effect in diabetic rats after peroral administration of the insulin-loaded NPs compared to the effect caused by free oral insulin solution. In addition, both the pharmacokinetic and toxicity results showed low plasma clearance of insulin and no signs of toxicity on the liver enzyme and cell viability, which suggested good biocompatibility of the NPs formulations. Overall, the formation of NPs of insulin with chitosan and snail mucin represents a potentially safe and promising approach to protect insulin and enhance its peroral delivery.

Auranofin Releasing Antibacterial and Antibiofilm Polyurethane Intravascular Catheter Coatings.

Intravascular catheter related bloodstream infections (CRBSIs) are a leading cause of hospital-acquired infections worldwide, resulting not only in the burden of cost and morbidity for patients but also in the over-consumption of medical resources for hospitals and health care organizations. In this study, a novel auranofin releasing antibacterial and antibiofilm polyurethane (PU) catheter coating was developed and investigated for future use in preventing CRBSIs. Auranofin is an antirheumatic drug with recently identified antimicrobial properties. The drug carrier, PU, acts as a barrier surrounding the antibacterial agent, auranofin, to extend the drug release profile and improve its long-term antibacterial and antibiofilm efficacy and potentially the length of catheter implantation within a patient. The PU+auranofin coatings developed here were found to be highly stretchable (exhibiting ~500% percent elongation), which is important for the compliance of the material on a flexible catheter. PU+auranofin coated catheters were able to inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA) for 8 to 26 days depending on the specific drug concentration utilized during the dip coating process. The PU+auranofin coated catheters were also able to completely inhibit MRSA biofilm formation in vitro, an effect that was not observed with auranofin or PU alone. Lastly, these coatings were found to be hemocompatible with human erythrocytes and maintain liver cell viability.

Protective effect of 7,3′,4′-flavon-3-ol (fisetin) on acetaminophen-induced hepatotoxicity in vitro and in vivo

BackgroundAcetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure in clinic. Fisetin (FST) is a phenolic compound that has been isolated from many natural products. PurposeOur aim is to study the protection effect and mechanisms of FST on APAP-induced hepatotoxicity in endogenous metabolism and metabolomics in vitro and in vivo. MethodsFST was i.g. administered to mice at 10, 20 and 40 mg/kg for 7 days and a single dose of APAP (400 mg/kg) was given on the last day. Serum and tissue were collected for biochemical analysis. L-02 cells were used to assess cell viability. LC–MS was used to study the metabolic fingerprinting in vivo and vitro. PCA and OPLS-DA were used to search the potential biomarkers (VIP > 1, p < 0.05). The pathway analysis was conducted on Metaboanalyst 4.0. Then liver oxidative stress indices and glutathione markers were examined using PCR and kits. ResultsALT, AST, liver histological observation and cell viability results showed that FST could reverse APAP induced toxicology in mice and L-02 cells. In metabolomics study, 26 metabolites in vitro and 60 metabolites in vivo were identified by searching in the library and most of them decreased to normal level in FST treatment. It is observed in pathway analysis that the most significant pathway was glutathione metabolism. Furthermore, the results of mRNA and immunofluorescence showed that FST suppressed ROS formation in liver tissue and L-02 cells, as well as restored the expression of GPX1, GST and other antioxidative enzymes genes. ConclusionOur results indicate that FST prevented APAP-induced hepatotoxicity by regulating glutathione metabolism and the expression of related antioxidative signals.

Experimental Applications of in situ Liver Perfusion Machinery for the Study of Liver Disease.

The liver is involved in a wide range of activities in vertebrates and some other animals, including metabolism, protein synthesis, detoxification, and the immune system. Until now, various methods have been devised to study liver diseases; however, each method has its own limitations. In situ liver perfusion machinery, originally developed in rats, has been successfully adapted to mice, enabling the study of liver diseases. Here we describe the protocol, which is a simple but widely applicable method for investigating the liver diseases. The liver is perfused in situ by cannulation of the portal vein and suprahepatic inferior vena cava (IVC), with antegrade closed circuit circulation completed by clamping the infrahepatic IVC. In situ liver perfusion can be utilized to evaluate immune cell migration and function, hemodynamics and related cellular reactions in each type of hepatic cells, and the metabolism of toxic or other compounds by changing the composition of the circulating media. In situ liver perfusion method maintains liver function and cell viability for up to 2 h. This study also describes an optional protocol using density-gradient centrifugation for the separation of different types of hepatic cells, allowing the determination of changes in each cell type. In summary, this method of in situ liver perfusion will be useful for studying liver diseases as a complement to other established methods.

Humulus japonicus Extracts Protect Against Lipopolysaccharide/d-Galactosamine-Induced Acute Liver Injury in Rats.

It has been described that Humulus japonicus has potential antituberculosis and anti-inflammatory effects. The antiaging activity of H. japonicus extract (HJE) was also examined not only in yeast but also in human fibroblast cells. We evaluated the protective effect of HJE on hepatotoxicity in this study. We demonstrated the expression of antioxidative enzyme and cytokines in plasma. The vehicle control group received orally administered normal saline. Acute hepatotoxicity rat model was induced by lipopolysaccharide (LPS) (30 μg/kg) and d-galactosamine (D-GalN) (500 mg/kg) by intraperitoneal injection. The positive control group received orally administered silymarin (10 mg/kg). Three HJE groups received 8, 16, and 32 mg/kg. The blood samples were prepared to evaluate malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) level to examine oxidative stress, and hepatic tissue was assayed for myeloperoxidase (MPO). Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and tumor necrosis factor-α (TNF-α) activities were also assayed to examine liver cell viability. Pretreatment with HJE decreased levels of AST, ALT, MDA, MPO, and TNF-α and increased levels of SOD and CAT compared with the LPS/D-GalN-treated group. These results suggested that HJE has the potential to reduce oxidative damage and inflammatory reactions in LPS/D-GalN-induced liver injury in the rat model. It can also increase survival rate in LPS/D-GalN-induced hepatotoxicity rat models.

Effect of Garlic, Zinc Oxide and CTAB Nanocomposite against Hepatocellular Carcinoma in Vitro and Rats on Cell Cycle, Caspase 3and DNA Fragmentation Percent

Nanoparticles plans to overcome the problems related to human diseases at the nanoscale level and making significant contributions to the improvement of new approaches of drug delivery in cancer and can provide a stage for combined therapeutics with subsequent monitoring of response. (Garlic and zinc oxide (ZnO) coated by cetyl trimethyl ammonium bromide) nanoparticles have been applying treatment action against hepatocellular carcinoma in vitro and in vivo which was done on 60 rats and was divided into four groups, each groups have (15 rats), (Group 1) NTBR (Negative control) group; (Group 2) (TBR) HCC (DEN) group (Group 3) (TBRT) (garlic / Zinc oxide / CTAB) nanoparticles; (Group 4) (TBRT Doxorubicin) .The changes of the biochemical indices of serum, and the levels of the Cell cycle, Caspase 3and DNA fragmentation percent of liver were analyzed to evaluate the treatment activity. At the end of the study (10 weeks), DEN induced elevation of the serum biochemical indices alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gammaglutamyl transferase, Alpha feto-protein, (garlic/Zinc oxide/ CTAB) nanoparticles administration significantly inhibited the increase of the ALT, AST, ALP, GGT and AFP .The mechanistic studies demonstrated that (garlic/Zinc oxide/CTAB) nanoparticles counteracted DEN induced protein levels of Caspase 3and DNA fragmentation percent were significantly increased. We observed that nanocompsite have different effects on liver cell viability via killing cancer cells, while posing no effect on normal cells of the liver. The marked difference in cytotoxicity between cancer cells and normal cells suggests an exciting potential for nanocompsite as novel alternatives to hepatocellular carcinoma therapy