Decline In Viability Research Articles

Metformin relieves H/R-induced cardiomyocyte injury through miR-19a/ACSL axis - possible therapeutic target for myocardial I/R injury

This study proposed to investigate the function of miR-19a/ACSL axis in hypoxia/reoxygenation (H/R)-induced myocardial injury and determine whether metformin exerts its protective effect via miR-19a/ACSL axis. Firstly, bioinformatics analysis of data from Gene Expression Omnibus (GEO) database indicated that miR-19a was downregulated in patients with myocardial infarction (MI) compared to that in control group. H/R model was constructed with AC16 cells in vitro. qRT-PCR assay revealed that miR-19a was downregulated in H/R-treated AC16 cells. Then, CCK-8 assay demonstrated that upregulation of miR-19a significantly alleviated H/R-induced decline of cell viability. Moreover, bioinformatics prediction, western blotting and dual-luciferase reporter assays were performed to check the target genes of miR-19a, and ACSL1 was determined as a downstream target gene of miR-19a. Besides, the analysis based on Comparative Toxicogenomics Database (CTD) suggested that metformin targeting ACSL1 can be used as a potential drug for further research. Biological function experiments in vitro revealed that H/R markedly declined the viability and elevated the apoptosis of AC16 cells, while metformin can significantly mitigate these effects. Furthermore, overexpression of miR-19a significantly strengthened the beneficial effect of metformin on H/R-induced AC16 cells injury, which can be reversed by upregulation of ACSL1. In conclusion, metformin can alleviate H/R-induced cells injury via regulating miR-19a/ACSL axis, which lays a foundation for identifying novel targets for myocardial I/R injury therapy.

Breast cancer radioresistance may be overcome by osteopontin gene knocking out with CRISPR/Cas9 technique

PurposeOsteopontin (OPN) is a phosphoglycoprotein, with a wide range of physiological and pathological roles. High expression of OPN promotes aggressive behavior, causes poor prognosis in tumor cells, and reduces the survival of patients. Since overexpression of OPN gives rise to radioresistance, the effects of the gene knock out using the CRISPR/Cas9 system in combination with radiation are emphasized. Material and methodsWe used the CRISPR/Cas9 technique to knock out the OPN gene in the MDA-MB-231 cell line. After transfection, the cells were irradiated. The changes of the OPN mRNA levels, the apoptosis, and the differences in cell viability were assessed. ResultsA significant reduction in the OPN expression was observed alone or along with irradiation. The knocked out gene alone increased apoptosis rate. The cell viability decreased to after knocking out of the OPN gene. The gene knocking-out combined with irradiation led to more decline of cell viability. ConclusionOur results demonstrated that after knocking out the OPN gene, the MDA-MB-231 cells showed a significant radiosensitivity. Therefore, the OPN knock out in combination with conventional radiotherapy, may become an efficient therapeutic target in the future.

MIR210HG Aggravates Sepsis-Induced Inflammatory Response of Proximal Tubular Epithelial Cell via the NF-κB Signaling Pathway.

PurposeAcute kidney injury (AKI) is a serious complication of sepsis and is characterized by inflammatory response. MicroRNA-210 host gene (MIR210HG) is upregulated in human proximal tubular epithelial cells under treatment of inflammatory cytokines. This study aimed to explore the role of MIR210HG in sepsis-induced AKI.Materials and MethodsCell viability was detected by a cell counting kit 8 assay. The levels of proinflammatory cytokines were detected by enzyme-linked immunosorbent assay kits. The protein levels of p65, IκBα, and p-IκBα were examined by western blot analysis. The nuclear translocation of nuclear factor kappa B (NF-κB) was detected by immunofluorescence assay. The histological changes of kidneys were analyzed by hematoxylin and eosin staining assay.ResultsLipopolysaccharide (LPS) treatment significantly inhibited cell viability and increased productions of proinflammatory cytokines in proximal tubular epithelial cells (HKC-8). Additionally, MIR210HG levels in HKC-8 cells were increased by LPS treatment. MIR210HG silencing inhibited the LPS-induced cell inflammatory response. MIR210HG activated the NF-κB signaling pathway by promoting the phosphorylation of IκBα and nuclear translocation of p65. Rescue assays revealed that the MIR210HG-induced increase of cytokines levels and decline of cell viability were rescued by QNZ treatment. Knockdown of MIR210HG decreased blood urea nitrogen, serum creatinine, and proinflammatory cytokine levels in AKI rats. Moreover, the knockdown of MIR210HG protected against AKI-induced histological changes of kidneys in rats.ConclusionMIR210HG promotes sepsis-induced inflammatory response of HKC-8 cells by activating the NF-κB signaling pathway. This novel discovery may be helpful for the improvement of sepsis-induced AKI.

Hsa_circ_0002060 Knockdown Ameliorates Osteoporosis by Targeting MiR-198-5p.

Osteoporosis (OP) is increasingly becoming one of a major health concerns all over the world. However, the limitations of current therapeutic drugs for OP are including considerable side effects and low efficacy. Therefore, it is required to develop new therapeutic drugs for OP. This study aimed to investigate the role of hsa_circ_0002060 in the regulation of osteoporosis. Osteoblast cells (hFOB 1.19) were transfected with hsa_circ_0002060 small interfering RNA (siRNA), following by stimulated with dexamethasone (DEX) to mimic OP in vitro. Cell counting kit-8, apoptosis, and JC-1 mitochondrial membrane potential assays were used to evaluate the cell viability, apoptosis, and mitochondrial membrane potential, respectively. Western blot was conducted to detect the expression of proteins. In addition, the levels of reactive oxygen species, superoxide dismutase, glutathione and malondialdehyde were measured with enzyme-linked immunosorbent assay (ELISA). The putative target of hsa_circ_0002060 was verified by dual luciferase reporter assay and RNA pull down. At last, the role of hsa_circ_0002060 in the progression of OP was investigated with an ovariectomy (OVX)-induced OP mouse model. The results indicated DEX could induce cell viability decline in hFOB 1.19 cells, which was ameliorated by hsa_circ_0002060 knockdown. Consistently, DEX-induced cell apoptosis of hFOB 1.19 was ameliorated by hsa_circ_0002060 knockdown as well. As for the underlying mechanisms study, hsa_circ_0002060 was proved to regulate the viability of hFOB 1.19 cells through targeting miR-198-5p/Bax axis. Additionally, hsa_circ_0002060 knockdown alleviated ovariectomy-induced OP in a mouse model. Taken together, hsa_circ_0002060 knockdown alleviated the progression of OP by targeting miR-198-5p. Hsa_circ_0002060 might possibly be served as a therapeutic target for treating OP.

Effect of forkhead box O1 in renal tubular epithelial cells on endotoxin-induced acute kidney injury.

Mitochondrial damage in renal tubular epithelial cells (RTECs) is a hallmark of endotoxin-induced acute kidney injury (AKI). Forkhead box O1 (FOXO1) is responsible for regulating mitochondrial function and is involved in several kidney diseases. Here, we investigated the effect of FOXO1 on endotoxin-induced AKI and the related mechanism. In vivo, FOXO1 downregulation in mouse RTECs and mitochondrial damage were found in endotoxin-induced AKI. Overexpression of FOXO1 by kidney focal adeno-associated virus (AAV) delivery improved renal function and reduced mitochondrial damage. Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1-α), a master regulator of mitochondrial biogenesis and function, was reduced in endotoxin-induced AKI, but the reduction was reversed by FOXO1 overexpression. In vitro, exposure to LPS led to a decline in HK-2 cell viability, mitochondrial fragmentation, and mitochondrial superoxide accumulation, as well as downregulation of FOXO1, PGC1-α, and mitochondrial complex I/V. Moreover, overexpression of FOXO1 in HK-2 cells increased HK-2 cell viability and PGC1-α expression, and it alleviated the mitochondrial injury and superoxide accumulation induced by LPS. Meanwhile, inhibition of FOXO1 in HK-2 cells by siRNA treatment decreased PGC1-α expression and HK-2 cell viability. Chromatin immunoprecipitation assays and PCR analysis confirmed that FOXO1 bound to the PGC1-α promoter in HK-2 cells. In conclusion, downregulation of FOXO1 in RTECs mediated endotoxin-induced AKI and mitochondrial damage. Overexpression of FOXO1 could improve renal injury and mitochondrial dysfunction, and this effect occurred at least in part as a result of PGC1-α signaling. FOXO1 might be a potential target for the prevention and treatment of endotoxin-induced AKI.

Condition of plants and populations of Trientalis europaea L. within forest phytocenoses of Ukrainian Left Bank

Based on the use of morphometric and vitality analysis, the condition of plants and populations of Trientalis europaea L. in 10 phytocenoses of coniferous, mixed, small-leaved and deciduous forests, typical for the Left Bank of Ukraine, was assessed. The morphometric analysis took into account seven static and two allometric indicators. Vitality analysis was conducted according to the method of Yu.A. Zlobin, based on the height of plants, total phytomass and leaf surface area. It is shown that plants from different forest vegetation conditions are statistically significantly different in the values of all studied morphoparameters and in each phytocenosis the formation of Trientalis europaea ramets of characteristic size and morphostructure takes place, which is clearly proved on the basis of morphograms. That is, the adaptation of plants of this species to habitat conditions occurs with the widespread implementation of morphoadaptations, which are accompanied by the manifestation of both morphological variability and morphological plasticity.
 The consequence and manifestation of the adaptation of Trientalis europea ramets to habitat conditions is the differentiation of plants according to the level of vitality, and, as a result, the formation of populations of different qualitative types. In the forest phytocenoses of the Left Bank of Ukraine, populations of all three vitality categories are represented: depressed, balanced and prosperous.
 Among the studied groups, the share of depressed populations is only 10 %. So, the results of vitality analysis indicate a fairly high level of viability of populations of Trientalis europea in forest phytocenoses of Ukraine. Prosperous populations with a high capacity for sustainable functioning are formed under the tent of coniferous, small-leaved and deciduous forests. The decline in plant viability and size is mainly due to an increase in the general closure of the upper tiers of the forest, an increase in the density and projective cover of species forming grass-shrub tiers, as well as on wet soils.

N-BuOH extract of Bletilla striata exerts chemopreventive effects on lung against SiO2 nanoparticles through activation of Nrf2 pathway

BackgroundSiO2 nanoparticles (nm SiO2) are ubiquitous in daily life and are acknowledged to be detrimental to human health. Bletilla striata is a traditional medicine used for generations in China and its polysaccharide has the anti-pulmonary fibrosis effect. PurposeTo investigate the lung protective effect of the small molecules (n-BuOH extract) of B. striata and clarify the underlying mechanism. Study design and methodsC57BL/6 mice were subjected to intratracheal instillation with nm SiO2 nanoparticle suspension (7 mg/kg) to construct the in vivo model of nm SiO2-induced lung injury. The chemical profile of the n-BuOH extract of B. striata was investigated by HPLC analysis using authentic samples isolated from B. striata. Gymnoside II with the most potent chemoprotective capacity in the n-BuOH extract was used to clarify the potential bio-active molecular basis of the n-BuOH extract using in vitro experiments. The cytotoxicity, apoptosis, oxidative stress, and the Nrf2 signaling pathway were examined in SiO2-induced A549 cells. ML385 was adopted to down-regulate the Nrf2 expression. ResultsThe n-BuOH extract of B. striata (40 mg/kg) could alleviate the SiO2-induced lung injury by increasing Nrf2 expression and thereby suppressing Bax/Bcl-2 pathway in the nm SiO2-induced mice model. The chemical profile study showed that militarine, gymnoside II, and 4-allyl-2, 6-dimethoxyphenol glucoside were the main constituents of n-BuOH extract. Studies on gymnoside II revealed that it could partially restore the SiO2-induced decline in cell viability while did not affect the growth of normal A549 cells within the concentration range of 1-50 μM, suggesting a protective effect against nm SiO2 in lung A549 cells. The hoechst 33258 staining, flow cytometry, and western blot experiments demonstrated that gymnoside II (25 μM) could partially reverse the SiO2-induced cell apoptosis and ROS production by enhancing Nrf2, HO-1, and γ-GCSc expressions and Nrf2 silencing by ML385 abrogated the effects of gymnoside II (25 μM) on apoptosis and ROS production in A549 cells. ConclusionThe present study suggests that in addition to the polysaccharide, small molecules (n-BuOH extract) of B. striata can also elicit a protective effect on lung injuries through the Nrf2-dependent mechanism and gymnoside II is one of the main bio-active constituents contributing to the n-BuOH extract-elicited lung protective effect against nm SiO2.

α-Lipoic acid alleviates ferroptosis in the MPP+ -induced PC12 cells via activating the PI3K/Akt/Nrf2 pathway.

Parkinson's disease (PD) is a typical neurodegenerative disease. α-Lipoic acid (α-LA) can reduce the incidence of neuropathy. The present study explored the role and mechanism of α-LA in 1-methyl-4-phenylpyridinium (MPP+ )-induced cell model of PD. The PD model was induced via treating PC12 cells with MPP+ at different concentrations. MPP+ and α-LA effects on PC12 cells were assessed from cell viability and ferroptosis. Cell viability was detected using the cell counting kit-8 assay. Malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), iron, reactive xygen species (ROS), and glutathione (GSH) concentrations, and ferroptosis-related protein SLC7A11 and GPx4 expressions were used for ferroptosis evaluation. p-PI3K, p-Akt, and nuclear factor erythroid 2-related factor 2 (Nrf2)protein levels were detected. The PI3K/Akt/Nrf2 pathway inhibitors were applied to verify the role of the PI3K/Akt/Nrf2 pathway in α-LA protection against MPP+ -induced decreased cell viability and ferroptosis. MPP+ -reduced cell viability and induced ferroptosis as presented by increased MDA, 4-HNE, iron, and ROS concentrations, and reduced levels of GSHand ferroptosis marker proteins (SLC7A11 and GPx4). α-LA attenuated MPP+ -induced cell viability decline and ferroptosis. The PI3K/Akt/Nrf2 pathway was activated after α-LA treatment. Inhibiting the PI3K/Akt/Nrf2 pathway weakened the protection of α-LA against MPP+ treatment. We highlighted that α-LA alleviated MPP+ -induced cell viability decrease and ferroptosis in PC12 cells via activating the PI3K/Akt/Nrf2 pathway.

A Proteomics- and Metabolomics-Based Study Revealed That Disorder of Palmitic Acid Metabolism by Aconitine Induces Cardiac Injury.

Currently, research on cardiac injury by aconitine focuses on its effect to directly interfere with the function of cardiac ion channels. Further, abnormal lipid metabolism could cause cardiac injury via inflammatory signaling pathway. In our preliminary study, we discovered that aconitine could alter the metabolism processes of various substances, including palmitic acid. Inspired by these studies, we investigated how elevation of palmitic acid by aconitine causes cardiac injury. Aconitine induced cardiac injury in rats (0.32 mg/kg, d = 7), and the cardiac injury was confirmed by electrocardiogram and serum biochemical study. The proteomic and metabolomic results showed that the palmitic acid level increases in heart tissue, and the NOD-like receptor (NLR) signaling pathway showed a strong effect of cardiac injury. The palmitic acid results in cell viability decline and activates NLR signaling in vitro. The shRNA-mediated knockdown of NLRP3 and NOD1/2 attenuates palmitic acid-induced inhibitory effect on cells and inhibited activation of the NLR signaling pathway. Collectively, this study reveals that aconitine provoked palmitic acid elevation could aggravate cardiac injury via the NLR signaling pathway. This study suggests that drug triggered disorder of the metabolism process could evoke cardiac injury and could propose a new strategy to study drug cardiac injury.

A quantitative test for heat-induced cell necrosis in vascular cambium and secondary phloem of Eucalyptus obliqua stems

Abstract Aims Exposure of Eucalyptus tree stems to the radiant heat of forest fires can kill cambial cells and their embedded regenerative meristems, thus preventing epicormic resprouting and recovery of the tree. Currently, there is no tissue-level method to quantify the viability of cambial cells in Eucalyptus following heat exposure. The first aim of this study was to adapt and validate the tetrazolium reduction method of testing for cell viability in Eucalyptus. The second aim was to apply the method to establish a threshold level of cambium cell viability in Eucalyptus obliqua to enable the identification of a critical temperature. Methods The study used the tetrazolium reduction method to quantitatively determine phloem–cambium cell viability in Eucalyptus. Circular sections of bark with underlying phloem and cambium were cut from mature E. obliqua and samples ranging in mass from 1 to 30 mg were exposed for 1 min to temperature treatments ranging from 20 to 85 °C and kept for 20–22 h at room temperature in 0.8% 2,3,5-triphenyl tetrazolium chloride (TTC) to test for cell viability. The 1,3,5-triphenyl tetrazolium formazan (TPF) formed was cold extracted with ethanol and quantified as absorbance at 485 nm. Important Findings The TTC reduction method reliably quantified a decline in cell viability with rising temperature in tissue sections that included vascular cambium, and identified 60 °C as the critical temperature for cambium–phloem cells of Eucalyptus species. Cell viability, calculated as [TPF Treatment °C]/[TPF 20 °C], declined by 90% between 20 and 85 °C. The cell viability results confirmed that significant tissue necrosis occurred in Eucalyptus at temperatures between 50 and 70 °C, after 1 min of in vitro tissue heating. The decline in cell viability with increasing temperature shown by the TTC method was consistent with an independently derived count of live cells following temperature treatment and neutral red staining.

Culturing Articular Cartilage Explants in the Presence of Autologous Adipose Tissue Modifies Their Inflammatory Response to Lipopolysaccharide.

The purpose of the current study was to explore the effect of autologous adipose tissue on cartilage responses to lipopolysaccharide (LPS). We hypothesized that LPS elicits an inflammatory response in cartilage, and that response is augmented in the presence of adipose tissue. Furthermore, we hypothesized that this augmented inflammatory response is due, at least in part, to increased exposure of cartilage to adipose tissue-derived c3a. Porcine cartilage explants from market-weight pigs were cultured in the presence or absence of autologous adipose tissue for 96 hours, the final 48 hours of which they were stimulated with LPS (0 or 10 μg/mL). Adipose tissue explants were also cultured alone, in the presence or absence of LPS. Media from all cartilage treatments was assayed for c3a/c3a des Arg, PGE2, GAG, and NO, and the viability of cartilage tissue was determined by differential fluorescent staining. Media from adipose tissue explants was assayed for c3a/c3a des Arg and PGE2. LPS produced a significant increase in PGE2, GAG, and NO production when cartilage was cultured in the absence of adipose tissue. Coculture of adipose tissue prevented a significant increase in PGE2 in cartilage explants. There was no effect of adipose tissue on LPS-induced GAG or NO, but the presence of adipose tissue significantly reduced cell viability in LPS-stimulated cartilage explants. Adipose tissue explants from lean animals reduced inflammatory responses of cartilage to LPS via a c3a/c3a des Arg-independent mechanism and were associated with a significant decline in cell viability. Thus, contrary to our hypothesis, adipose tissue from lean animals does not augment the inflammatory response of cartilage to stimulation by LPS. The mechanism of modulatory effects of adipose tissue on LPS-induced increase in PGE2 and decline in chondrocyte viability requires further research but appears to have occurred via a mechanism that is independent of adipocentric c3a/c3a des Arg.

Establishment and characterization of a gill cell line from pearl gentian grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀) and its application in cadmium toxicology

A novel gill cell line from pearl gentian grouper (Epinephelus lanceolatus♂×Epinephelus fuscoguttatus♀, PGGG cell line) was established, its application in cadmium (Cd) toxicology was demonstrated in this study. Primary cultures and PGGG subcultures were carried out at 25 °C in Dulbecco’s Modified Eagle medium/F12 medium (1:1; pH 7.2) supplemented with 15% fetal bovine serum (FBS). Primary PGGG cells were spindle-shaped, proliferated into a confluent monolayer within two weeks and were continuously subcultured over passage 60. The growth of cells at passages 20, 40, and 60 was examined. Chromosome analysis revealed that the chromosomal number of normal PGGG cells was 48, but the number of cells with the normal chromosomes number decreased during the passaging process. Cadmium is one of the most toxic metals in aquatic systems and has been associated with multiple animal and human health problems. To interpret the cytotoxicity and related mechanisms of cadmium, PGGG cells were used as an in vitro model. After treatment with cadmium at concentrations ranging from 1 µM to 500 µM, PGGG cells demonstrated dose- and time-dependent cytotoxicity, manifested as morphological abnormalities and a viability decline. Further, it was found that the reactive oxygen species (ROS) and malondialdehyde (MDA) levels were elevated following cadmium exposure, and related genes involved in the antioxidant system, including those encoding catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and Kelch-like- ECH-associated protein 1 (Keap1), were regulated differently. In addition, PGGG cells treated with cadmium had the typical features associated with apoptosis, including phosphatidylserine (PS) externalization; upregulated expression of caspase-3, −8, and −9; and apoptotic body formation. In general, the PGGG cell line may serve as a useful tool for studying the toxic mechanisms of cadmium or other toxicants or for toxicity testing and environment monitoring.

Seed Germiability, viability and longevity of Rhododendron arboreum var. arboreum Sm.

The present study has been undertaken with the objective to assess the germiability, viability and longevity of R. arboreum seeds at laboratory conditions as these factors are very important to promote the process of natural regeneration of species in wild which ultimately responsible for the better survival and existence of species. The seeds for experiment were collected from Craignaino forest in Mashobara Range of Shimla forest division. Seed parameters like Germination per cent, germination energy and germination value were calculated to test the germiability from the freshly collected seeds continuously for the period of six years. Seed viability was assessed through germination method and TTZ (Tetrazolium salt) method. A storage trial was designed by using polythene bags and cloth bags as a medium of storage of seeds, to check the longevity of the seeds under three different temperature gradients (deep freezer, Multi cooling chamber and normal room temperature). The results are diverse, having common observation with higher viability in freshly collected seeds as compared to the previous years. The overall Results showed tendency of decline in germiability as well as loss in viability over a period of time. The deep freeze (-20°C) is the best suitable temperature gradient for the storage of R. arboreum seeds as the maximum viability of the seeds has been observed in this medium with the 50% decline in viability in the period of 3 years.

Temperature Stratification and Monochromatic Light Break Dormancy and Facilitate On-Demand In Situ Germination in the Seagrass Halophila ovalis, with Seed Viability Determined by a Novel X-Ray Analysis

The seagrass Halophila ovalis rapidly colonizes marine sediments from seed across a range of depth, light, and temperature conditions, making it ideal for restoration projects. Yet, presently, it is not a targeted restoration species as the biology of seed dormancy and germination is poorly understood. This study addresses that knowledge gap by experimenting with temperature and light pre-treatments with the aim to determine optimal conditions for high rates of seed germination. H. ovalis seeds were pre-treated ex situ by applying nine combinations of temperature and monochromatic light. Two thirds of the pre-treated seeds were placed at separate field sites in conditions suboptimal for natural germination while one third remained in ex situ culture under suboptimal germination conditions (17 °C). Seed viability was monitored to determine if storage conditions impacted viability. X-ray analysis revealed a general decline in viability with time in storage. To analyze the effectiveness of pre-treatments, we compared the results of ex situ and in situ germination. Pre-treating seeds with red light (673 nm) and a stepwise temperature increase (15–20–25 °C) yielded the highest germination success across field and laboratory culture conditions. This indicates that we can use temperature and monochromatic light pre-treatments to break seed dormancy in the laboratory and induce “on-demand” germination of seeds after delivery to field sites potentially leading to more efficient and successful restoration practices with this species. Similarly, the conditions required for breaking dormancy and germination in H. ovalis (red spectrum light and a stepwise temperature increase) represent conditions associated with spring river freshwater runoff and warming of estuaries and coastal seas in temperate Australia.

Effects of genetically modified human skin fibroblasts, stably overexpressing hepatocyte growth factor, on hepatic functions of cocultured C3A cells.

Diseases leading to terminal hepatic failure are among the most common causes of death worldwide. Transplant of the whole organ is the only effective method to cure liver failure. Unfortunately, this treatment option is not available universally due to the serious shortage of donors. Thus, alternative methods have been developed that are aimed at prolonging the life of patients, including hepatic cells transplantation and bridging therapy based on hybrid bioartificial liver devices. Parenchymal liver cells are highly differentiated and perform many complex functions, such as detoxification and protein synthesis. Unfortunately, isolated hepatocytes display a rapid decline in viability and liver-specific functions. A number of methods have been developed to maintain hepatocytes in their highly differentiated state in vitro, amongst them the most promising being 3D growth scaffolds and decellularized tissues or coculture with other cell types required for the heterotypic cell-cell interactions. Here we present a novel approach to the hepatic cells culture based on the feeder layer cells genetically modified using lentiviral vector to stably produce additional amounts of hepatocyte growth factor and show the positive influence of these coculture conditions on the preservation of the hepatic functions of the liver parenchymal cells' model-C3A cells.

Characterization of primary mouse hepatocyte spheroids as a model system to support investigations of drug-induced liver injury

Primary mouse hepatocytes isolated from genetically defined and/or diverse lines and disease models are a valuable resource for studying the impact of genetic and environmental factors on drug response and disease. However, standard monolayer cultures result in a rapid decline in mouse hepatocyte viability and functionality. Therefore, we evaluated 3D spheroid methodology for long-term culture of primary mouse hepatocytes, initially to support investigations of drug-induced liver injury (DILI). Primary hepatocytes isolated from male and female C57BL/6J mice were used to generate spheroids by spontaneous self-aggregation in ultra-low attachment plates. Spheroids with well-defined perimeters were observed within 5 days after seeding and retained morphology, ATP, and albumin levels for an additional 2 weeks in culture. Global microarray profiling and quantitative targeted proteomics assessing 10 important drug metabolizing enzymes and transporters demonstrated maintenance of mRNA and protein levels in spheroids over time. Activities for 5 major P450 enzymes were also stable and comparable to activities previously reported for human hepatocyte spheroids. Time- and concentration-dependent decreases in ATP and albumin were observed in response to the DILI-causing drugs acetaminophen, fialuridine, AMG-009, and tolvaptan. Collectively, our results demonstrate successful long-term culture of mouse hepatocytes as spheroids and their utility to support investigations of DILI.

Genotoxicity of Noscapine Nanosuspension Prepared by Microfluidic Reactors on HepG2 Cell Line

Noscapine is an antispasmodic alkaloid used as antitussive and anti-cough obtained from plants in the Papaveraceae family which this benzylisoquinoline alkaloid and its synthetic subsidiaries (called noscapinoids) are being assessed for their anticancer potential. The present research aimed to investigate the induction of DNA destruction and viability of HepG2 tumor spheroid culture influenced by noscapine and nanosuspension of noscapine. Culture of HepG2 cells as spheroids was treated with different concentrations of noscapine for 24 h on Day 11. Afterward, viability assay and alkaline comet assay methods were applied to examine the viability and induced DNA destruction, respectively. Based on the results, no significant impact was observed from Tween 40 on the viability and DNA damage levels in comparison with the control (p > 0.05). Moreover, increasing noscapine concentration resulted in a dose-dependent reduction in viability of hepatic cancer cells and elevation of DNA damages, showing a correlation between rises of DNA damages and viability decline.

Phytochemical composition and in vitro safety evaluation of Ziziphora clinopodioides Lam. ethanolic extract: Cytotoxicity, genotoxicity and mutagenicity assessment

Ethnopharmacological relevanceThe application of the herb Ziziphora clinopodioides Lam. in folk medicine and as a food additive has been recommended due to its many claimed bioactivities. Regardless of the plant benefits, its safety considerations are largely unknown. Aim of the studyThe aim of the present research was to determine the chemical compositions and cytotoxicity, genotoxicity, and mutagenicity potentials of the ethanolic extract of Ziziphora clinopdioides Lam. (EEZC). Materials and methodsGC-MS and LC-MS analysis were used for chemical composition determination. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trypan blue exclusion dye assays were used for cytotoxicity and the Comet assay was employed for genotoxicity assessment on human blood lymphocytes. Also, the Ames Salmonella/microsome test was carried out for the evaluation of mutagenicity. ResultsPulegone was the main component of the n-hexane fraction. Different phenolic acids and flavonoids were detected by LC-MS. The cytotoxicity study indicated a conspicuous decline in human lymphocyte viability ranging from 52% to 100% as showed by the MTT assay and 67% up to 100% by the trypan blue assay, at 1 and 10 mg/mL, respectively. The Comet assay results revealed a dose dependent genotoxicity, in so much as 90% and 98% of the cells were screened as damaged at concentrations of 5 and 10 mg/mL, respectively. An incidence rate of 8% and 13% of grade 4 damage was observed at 5 and 10 mg/mL, respectively. Additionally, the DNA damage index (DI) was elevated dose-dependently by a rising concentration of the extract, wherein the DI at 10 mg/mL concentration was 2.22, which was 22 times greater than that of negative control, and even more than positive control. The Ames test exhibited no signs of mutagenicity for neither Salmonella typhimurium TA98 nor TA100 strains, accompanied or unaccompanied by S9 metabolic activation. ConclusionResults indicated a dose-dependent cytotoxicity and genotoxicity potential of the EEZC on human lymphocytes, suggesting that this plant should be used with caution by consumers, even in the food and pharmaceutical industries. Since the plant usage in daily life continues to increase due to its ever growing phytotherapical and phytonutritional properties, it may pose a health risk by its high concentration's uptake. Although no mutagenicity of this extract was observed in this study, further research is recommended to clarify the mutagenic risks of this herb.

Ginger Extract Loaded into Chitosan Nanoparticles Enhances Cytotoxicity and Reduces Cardiotoxicity of Doxorubicin in Hepatocellular Carcinoma in Mice

This study aimed to investigate the impact of ginger extract (GE) loaded into chitosan nanoparticles (CNPs) in enhancing cytotoxicity and reducing cardiotoxicity of doxorubicin (DXN) in hepatocellular carcinoma (HCC) induced mice. DXN and GE were loaded into CNPs and cytotoxicity of loaded and unloaded drugs against HepG2 cells was evaluated. HCC was induced in male albino mice by injection of diethylnitrosamine (DINA). Mice were divided into eight groups (n = 15): (1) normal control, (2) DINA, (3) CNPs, (4) free DXN, (5) CNPs DXN, (6) free GE, (7) CNPs GE, and (8) CNPs DXN + CNPs GE. Both GE and DXN loaded into CNPs showed a greater decline in cell viability of HepG2 cells than the unloaded forms. GE CNPs displayed pronounced anticancer activity In Vivo through apoptosis, greater down-regulation of multidrug resistance 1, enhancement of anti-oxidant activity and depletion of vascular endothelial growth factor content in liver tissues. GE CNPs in combination with DXN CNPs showed nearly normal hepatic lobule architecture and the greatest increase in apoptotic cell count. Co-treatment group had decreased cardiac malondialdehyde, tumor necrosis factor-α and serum activity of creatine kinase and lactate dehydrogenase. Combination of GE CNPs and DXN CNPs might be a potentially effective therapeutic approach for HCC.

Abnormal Ferroptosis in Myelodysplastic Syndrome.

BackgroundFerroptosis is a form of iron-dependent non-apoptotic cell death, with characteristics of loss of the activity of the lipid repair enzyme, glutathione (GSH) peroxidase 4 (GPX4), and accumulation of lethal reactive lipid oxygen species. The mechanism of ferroptosis in myelodysplastic syndrome (MDS) is unclear.MethodsCell viability assay, reactive oxygen species (ROS) assay, GSH assay, and GPX activity assay were performed to study the regulation of ferroptosis in MDS cells obtained from MDS patients, the iron overload model mice, and cell lines.ResultsThe growth-inhibitory effect of decitabine could be partially reversed by ferrostatin-1 and iron-chelating agent [desferrioxamine (DFO)] in MDS cell lines. Erastin could increase the cytotoxicity of decitabine on MDS cells. The level of GSH and the activity of GPX4 decreased, whereas the ROS level increased in MDS cells upon treatment with decitabine, which could be reversed by ferrostatin-1. The concentration of hemoglobin in peripheral blood of iron overload mice was negatively correlated with intracellular Fe2+ level and ferritin concentration. Iron overload (IO) led to decreased viability of bone marrow mononuclear cells (BMMNCs), which was negatively correlated with intracellular Fe2+ level. Ferrostatin-1 partially reversed the decline of cell viability in IO groups. The level of GSH and the activity of GPX4 decreased, whereas the ROS level increased in BMMNCs of IO mice. DFO could increase the level of GSH. Ferrostatin-1 and DFO could increase the GPX4 activity of BMMNCs in IO mice. Ferrostatin-1 could significantly reverse the growth-inhibitory effect of decitabine in MDS patients. Decitabine could significantly increase the ROS level in MDS groups, which could be inhibited by ferrostatin-1 or promoted by erastin. Ferrostatin-1 could significantly reverse the inhibitory effect of decitabine on GSH levels in MDS patients. Erastin combined with decitabine could further reduce the GSH level. Erastin could further decrease the activity of GPX4 compared with the decitabine group.ConclusionFerroptosis may account for the main mechanisms of how decitabine induced death of MDS cells. Decitabine-induced ROS raise leads to ferroptosis in MDS cells by decreasing GSH level and GPX4 activity.