scholarly journals Genipin Reverses HFD-Induced Liver Damage and Inhibits UCP2-Mediated Pyroptosis in Mice

2018 ◽  
Vol 49 (5) ◽  
pp. 1885-1897 ◽  
Author(s):  
Hong Zhong ◽  
Mengting Liu ◽  
Yaya Ji ◽  
Minjuan Ma ◽  
Kun Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Lactate Dehydrogenase ◽  
Liver Damage ◽  
Lipid Accumulation ◽  
Water Soluble ◽  
Morbidly Obese ◽  
Ldh Release

Background/Aims: Liver damage is a typical manifestation of nonalcoholic fatty liver disease (NAFLD). It originates from excessive fat accumulation, leading to hepatocyte death, inflammation, and fibrosis. Nonalcoholic steatohepatitis (NASH) is a type of NAFLD with a prevalence of 49% in morbidly obese patients. Pyroptosis plays an important role in the development of NASH; thus, it is important to elucidate the effect of lipid accumulation on pyroptosis. Genipin (GNP), a natural water-soluble cross-linking agent, has hepatoprotective effects and decreases lipid accumulation in the liver; however, the mechanisms underlying these effects are unknown. Methods: In this study, qPCR and Western blot were used to examine pyroptotic gene expression in high-fat diet (HFD) induced obese mice and free fatty acids (FFAs) treated hepatocytes. At the same time, relative lactate dehydrogenase (LDH) release and Hoechst & propidium iodide (PI) staining were done to verify cell death. To explore the molecular mechanism, cell transfection were constructed with siRNA or plasmid to obtain knockdown or overexpression hepatocytes. Results: We found that HFD-fed mice and FFAs-treated hepatocytes had obvious pyroptosis, and addition of GNP reversed liver damage and inhibited pyroptosis both in vitro and in vivo. Besides, UCP2 knockdown cells showed suppressed FFAs-mediated pyroptosis, as determined by decreased pyroptotic gene expression, reduced lactate dehydrogenase (LDH) release, and reduced cell death. Consistent with this, cells transfected with UCP2 had upregulated pyroptotic gene expression, increased LDH release, and increased cell death. Conclusion: GNP reverses HFD-induced liver damage and inhibits UCP2-mediated pyroptosis. Thus, GNP may serve as a potential therapeutic candidate for NAFLD.

scholarly journals Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Mandy Rauschner ◽  
Luisa Lange ◽  
Thea Hüsing ◽  
Sarah Reime ◽  
Alexander Nolze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Western Blot ◽  
Tumor Cell ◽  
Low Ph ◽  
Strong Increase ◽  
Acidic Ph ◽  
The Impact

Abstract Background The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. Methods Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. Results NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. Conclusions These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

scholarly journals TB-4 Antitumor effects of a novel curcumin derivative curcumin monoglucuronide on glioblastoma cells in vitro and in vivo

2021 ◽  
Vol 3 (Supplement_6) ◽  
pp. vi6-vi6
Author(s):  
Takashi Fujii ◽  
Shun Yamamuro ◽  
Masamichi Takahashi ◽  
Akihide Kondo ◽  
Yoshitaka Narita ◽  
...  
Keyword(s):  
Cell Death ◽  
Water Soluble ◽  
Dependent Manner ◽  
Antitumor Effects ◽  
Multiple Cell ◽  
Human Gbm ◽  
Dose Dependent ◽  
Novel Therapeutic

Abstract The therapeutic outcome of glioblastomas (GBMs) is still very poor. Therefore, invention of novel therapeutic methods against GBM cases is considered urgent. The antitumor effects of naturally-derived compounds are attracting attention recently, and therapeutic efficacy of curcumin, a plant-derived compound previously used for multiple purpose, has been indicated in many cancer systems; however, clinical application of curcumin is considered difficult because of its poor bioavailability (under 1 %). Curcumin monoglucuronide (CMG), a water-soluble prodrug of curcumin recently developed for overcoming this weakness, has been demonstrated excellent antitumor effects for several malignancies in vitro and in vivo; therefore, we investigated the effects of CMG against GBM cells. CMG induced cell death of human GBM cells lines (T98G, U251MG, and U87MG) by dose dependent manner by triggering multiple forms of cell death such as apoptosis and perthanatos. Immunoblotting of CMG-treated GBM cell lysates demonstrated activation of multiple cell death signaling. Furthermore, immunodeficiency mice harboring intracerebral U87MG cell xenografts systemically treated by CMG showed significantly prolonged survival compared with control mice. These results suggest CMG would be a novel therapeutic agent against GBM cases.

scholarly journals The neutrophil-mobilizing cytokine interleukin-26 in the airways of long-term tobacco smokers

2018 ◽  
Vol 132 (9) ◽  
pp. 959-983 ◽  
Author(s):  
Karlhans Fru Che ◽  
Ellen Tufvesson ◽  
Sara Tengvall ◽  
Elisa Lappi-Blanco ◽  
Riitta Kaarteenaho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Bronchitis ◽  
Pathogenic Bacteria ◽  
Cell Model ◽  
Water Soluble ◽  
Chronic Obstructive ◽  
Neutrophil Accumulation

Long-term tobacco smokers with chronic obstructive pulmonary disease (COPD) or chronic bronchitis display an excessive accumulation of neutrophils in the airways; an inflammation that responds poorly to established therapy. Thus, there is a need to identify new molecular targets for the development of effective therapy. Here, we hypothesized that the neutrophil-mobilizing cytokine interleukin (IL)-26 (IL-26) is involved in airway inflammation amongst long-term tobacco smokers with or without COPD, chronic bronchitis or colonization by pathogenic bacteria. By analyzing bronchoalveolar lavage (BAL), bronchail wash (BW) and induced sputum (IS) samples, we found increased extracellular IL-26 protein in the airways of long-term smokers in vivo without further increase amongst those with clinically stable COPD. In human alveolar macrophages (AM) in vitro, the exposure to water-soluble tobacco smoke components (WTC) enhanced IL-26 gene and protein. In this cell model, the same exposure increased gene expression of the IL-26 receptor complex (IL10R2 and IL20R1) and nuclear factor κ B (NF-κB); a proven regulator of IL-26 production. In the same cell model, recombinant human IL-26 in vitro caused a concentration-dependent increase in the gene expression of NF-κB and several pro-inflammatory cytokines. In the long-term smokers, we also observed that extracellular IL-26 protein in BAL samples correlates with measures of lung function, tobacco load, and several markers of neutrophil accumulation. Extracellular IL-26 was further increased in long-term smokers with exacerbations of COPD (IS samples), with chronic bronchitis (BAL samples ) or with colonization by pathogenic bacteria (IS and BW samples). Thus, IL-26 in the airways emerges as a promising target for improving the understanding of the pathogenic mechanisms behind several pulmonary morbidities in long-term tobacco smokers.

3 IN VITRO MATURATION ALTERS GENE EXPRESSION IN MOUSE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 82
Author(s):  
M. Paczkowski ◽  
C. Bidwell ◽  
D. Spurlock ◽  
J. Waddell ◽  
R. L. Krisher
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Dna Methyltransferase ◽  
In Vitro Maturation ◽  
Fold Increase ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Dna Methyltransferase 1

The in vitro culture environment significantly impacts nuclear maturation, fertilization, embryonic development, and epigenetic competence; however, our knowledge of the effects of in vitro maturation on oocyte developmental competence, and specifically cytoplasmic maturation, is limited. The objective of this experiment was to identify alterations in the transcriptome of oocytes matured in vitro compared to those matured in vivo that correlate to developmental competence. Immature oocytes were collected from Day 26 and 7-8-week-old B6D2F1 mice 48 h post-pregnant mare serum gonadotropin (PMSG) administration and matured for 16 h in Gmat supplemented with 0.5 mm citric acid, 0.5 mm cysteamine, 100 ng mL–1 epidermal growth factor (EGF), 0.05% insulin-transferrin-selenium (ITS; v/v), 0.01% recombumin (v/v) and 2 mg mL–1 fetuin. In vivo-matured oocytes from females of the same ages were collected from the oviducts 62 h post-PMSG and 14 h post-hCG and mating to vasectomized males. In vivo- and in vitro-matured oocytes were identified visually by the presence of the first polar body. Mature oocytes were pooled into three groups of 150 oocytes per treatment and lysed; poly A+ RNA was extracted. Samples were processed through two cycles of linear amplification and hybridized to the GeneChip� Mouse Genome 430 2.0 Array (Affymetrix, Inc., Santa Clara, CA, USA), with three arrays per treatment. Microarray data were sorted and filtered to include genes that were classified as having two present calls per treatment. The data were then normalized to the chip median and analyzed using a one-way analysis of variance; the level of significance was calculated at P < 0.01. In total, 2.17% (482/22170) and 1.61% (358/22170) of genes were differentially expressed between in vitro- and in vivo-matured oocytes in Day 26 and 7–8-week-old mice, respectively. However, 72.82% (351/482) and 67.87% (243/358) of differentially expressed genes had increased abundance in the in vitro- and in vivo-matured oocytes, respectively. Transcripts involved in gene expression, cellular growth and proliferation, and cellular development were increased in in vivo-matured oocytes from both age groups compared to those matured in vitro. Cell death was one of the higher ranking functional groups increased in the 7–8-week-old in vitro-matured oocytes compared to the 7–8-week-old in vivo-matured oocytes. Specific genes altered by in vitro maturation conditions in Day 26 oocytes were DNA methyltransferase 1 (>7-fold increase in vivo), caspase 8 (>4-fold increase in vivo), and eukaryotic translation initiation factor 1B (>4-fold increase in vivo). DNA methyltransferase 1 and ubiquitin-conjugating enzyme E2T were significantly increased in in vivo-matured 7–8-week-old oocytes (>3-fold and >5-fold, respectively). These results indicate that gene expression is altered in oocytes matured in vitro compared to those matured in vivo. Based on the functional annotations of genes differentially expressed, dysregulation of gene expression in the oocyte resulting in altered DNA methylation and an up-regulation in cell death pathways are potential developmental mechanisms influenced by in vitro culture conditions that correlate to reduced embryonic developmental potential.

197 Hepcidin and hemojuvelin gene expression in rat liver damage: In vivo and in vitro studies

2006 ◽  
Vol 44 ◽  
pp. S81-S82
Author(s):  
N. Sheikh ◽  
K. Tron ◽  
J. Dudas ◽  
B. Saile ◽  
D. Batusic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rat Liver ◽  
Liver Damage ◽  
In Vitro Studies

211 IN VITRO CULTURE CONDITIONS AFFECT GENE EXPRESSION PATTERN OF BOVINE BLASTOCYST IN A STAGE-SPECIFIC MANNER

2013 ◽  
Vol 25 (1) ◽  
pp. 254 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
In Vitro Culture ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Specific Manner

The aim of this study was to examine the effect of in vitro culture conditions at specific phases of early embryonic development on the transcriptome profile of bovine blastocysts. Simmental heifers were superovulated and artificially inseminated 2 times with the same frozen–thawed commercial bull semen. Using nonsurgical endoscopic oviductal flushing technology (Besenfelder et al. 2001 Theriogenology 55, 837–845), 6 different blastocyst groups were flushed out at different time points (2-, 4-, 8-, 16-, 32-cell and morula). After flushing, embryos cultured under in vitro conditions until the blastocyst stage. Blastocysts from each group were collected and pooled in groups of 10. Complete in vivo blastocysts were produced and used as control. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group v. the in vivo control group to examine the transcriptome profile of blastocysts. A clear difference in terms of the number of differentially expressed genes (DEG, fold change ≥2, false discovery rate ≤0.05) has been found between groups flushed out at 2-, 4-, and 8-cell (1714, 1918, 1292 DEG, respectively) and those flushed out at 16-, 32-cell and morula stages and cultured in vitro until blastocyst stage (311, 437, 773 DEG, respectively) compared with the complete vivo group. Ontological classification of DEG showed cell death to be the most significant function in all groups. However, the longer time embryos spent under in vitro conditions, the more the percentage of DEG involved in cell death and apoptosis processes are represented in those groups. In addition, genes related to post-translational modification and gene expression processes were significantly dysregulated in all groups. Pathway analysis revealed that protein ubiquitination pathway was the dominant pathway in the groups flushed out at 2-, 4-, and 8-cells but not in the other groups flushed at later stages compared with the in vivo control group. Moreover, retinoic acid receptor activation and apoptosis signalling pathways followed the same pattern. Embryos flushed out before the time of embryonic genome activation and subsequently cultured in vitro were highly affected by culture conditions. Overall, the results of the present study showed that despite the fact that embryos originated from the same source, in vitro culture condition affected embryo quality, measured in terms of gene expression, in a stage-specific manner.

scholarly journals Role of LpL (Lipoprotein Lipase) in Macrophage Polarization In Vitro and In Vivo

2019 ◽  
Vol 39 (10) ◽  
pp. 1967-1985 ◽  
Author(s):  
Hye Rim Chang ◽  
Tatjana Josefs ◽  
Diego Scerbo ◽  
Namrata Gumaste ◽  
Yunying Hu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lipoprotein Lipase ◽  
Lipid Accumulation ◽  
Macrophage Polarization ◽  
Myeloid Cell ◽  
Acid Uptake ◽  
Lipid Uptake ◽  
Very Low Density Lipoproteins

Objective: Fatty acid uptake and oxidation characterize the metabolism of alternatively activated macrophage polarization in vitro, but the in vivo biology is less clear. We assessed the roles of LpL (lipoprotein lipase)-mediated lipid uptake in macrophage polarization in vitro and in several important tissues in vivo. Approach and Results: We created mice with both global and myeloid-cell specific LpL deficiency. LpL deficiency in the presence of VLDL (very low-density lipoproteins) altered gene expression of bone marrow–derived macrophages and led to reduced lipid uptake but an increase in some anti- and some proinflammatory markers. However, LpL deficiency did not alter lipid accumulation or gene expression in circulating monocytes nor did it change the ratio of Ly6C high /Ly6C low . In adipose tissue, less macrophage lipid accumulation was found with global but not myeloid-specific LpL deficiency. Neither deletion affected the expression of inflammatory genes. Global LpL deficiency also reduced the numbers of elicited peritoneal macrophages. Finally, we assessed gene expression in macrophages from atherosclerotic lesions during regression; LpL deficiency did not affect the polarity of plaque macrophages. Conclusions: The phenotypic changes observed in macrophages upon deletion of Lpl in vitro is not mimicked in tissue macrophages.

A water-soluble analog of triptolide induces ovarian cancer cell death in vitro and in vivo

2013 ◽  
Vol 130 (1) ◽  
pp. e130
Author(s):  
C. Rivard Hunt ◽  
M. Geller ◽  
C. Evans ◽  
R. Vogel ◽  
S. Ramakrishnan ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Death ◽  
Cancer Cell ◽  
Ovarian Cancer Cell ◽  
Water Soluble ◽  
Cancer Cell Death

Apoptosis and gene expression of collagenases but not gelatinases in rabbit disc fragment cultures

2008 ◽  
Vol 8 (6) ◽  
pp. 552-560 ◽  
Author(s):  
Daniel Haschtmann ◽  
Stephen J. Ferguson ◽  
Jivko V. Stoyanov
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Intervertebral Disc ◽  
Environmental Changes ◽  
Apoptotic Cell ◽  
Biological Response ◽  
Swelling Properties ◽  
Lactate Dehydrogenase Activity

Object The object of this study was to characterize the biological response of isolated intervertebral disc fragments to in vitro culture conditions with respect to cell death and inflammatory and catabolic changes. The acquired data could help to gain a better understanding of the biological reaction of disc tissue when exposed to environmental changes along with altered nutritional and osmotic conditions, as are encountered in different in vitro disc models or disc diseases in vivo. Methods Intervertebral disc anulus fragments were isolated from Burgundy rabbits and cultured in standard media for 3 days. The disc fragments were analyzed for their swelling properties, proteoglycan loss on histological studies, lactate dehydrogenase activity, apoptosis, gene expression of collagenases and gelatinases, and for proinflammatory (MCP-1, IL-8, and IL-6) and apoptosis-associated (TNF-α, Fas-L, and caspase 3) genes. Results The results demonstrate that disc specimens were swelling, and a loss of proteoglycans with disarrangement of anulus architecture was observed. The disc cells underwent rapid apoptosis with upregulation of various proinflammatory genes. Both collagenases, matrix metalloproteinase (MMP)–1 and MMP-13, were increasingly transcribed, whereas the gelatinases MMP-2 and MMP-9 did not respond or were downregulated. Conclusions Cultured disc fragments swell and undergo necrotic and apoptotic cell death combined with a catabolic gene response and gene expression of proinflammatory and chemoattractant proteins. Some of these findings have been demonstrated before in various spinal disorders. In addition, disc fragments are not suitable for long-term culture if a stable disc metabolism is desired, and the described changes have to be considered when using isolated disc material for in vitro cultures.

scholarly journals Bioactivity of Recombinant Humanized Monoclonal Antibody Against HER2 and Its Mechanism of Action on Ovarian Cancer

2021 ◽  
Author(s):  
Jinfeng Zeng ◽  
Rui Zhang ◽  
Rui Guan
Keyword(s):  
Gene Expression ◽  
Ovarian Cancer ◽  
Monoclonal Antibody ◽  
Lactate Dehydrogenase ◽  
Transient Gene Expression ◽  
Animal Experiments ◽  
Growth Factor Receptor ◽  
Lactate Dehydrogenase Release

Abstract BACKGROUND: Human epidermal growth factor receptor 2 (HER2) protein is overexpressed on the surface of various epithelial ovarian cancer tissues, which mediates the proliferation, differentiation, metastasis and signal transduction of tumor cells and is a potential cancer therapeutic target.METHODS: In this paper, the recombinant anti-HER2 humanized IgG1 monoclonal antibody (rhHer2-mAb) was expressed in HEK293F cells by constructing mammalian cell expression vector and optimizing transfection conditions. The antibody was purified by rProtein A affinity chromatography, and its mediated antibody dependent cytotoxicity (ADCC) was identified by lactate dehydrogenase(LDH) lactate dehydrogenase release assay. The anti-tumor activity of rhHer2-mAb was evaluated in NOD/SCID mice. RESULTS: The expression of rhHer2-mAb in HEK293F cells was at the highest level (100.5 mg/L) when the ratios of DNA/PEI and light chain/heavy chain was 1:4 and 1:2, respectively. The IC50 on ADCC of antibodies against SK-OV-3, OVCAR-3 and A-2780 cells were 12.36, 5.43 and 102.90ng/ml, respectively. Animal experiments in mice showed that rhHer2-mAb could effectively inhibit the growth of SK-OV-3 tumor at the dose of 10 mg/kg.CONCLUSIONS: The recombinant monoclonal antibody was obtained by transient gene expression technology and its bioactivity was studied in vitro and in vivo , providing a novel insight for the development and production of future biotechnology-based drugs using transient gene expression technology of HEK293F.

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