Search

Purpose : Our previous studies indicate that phorbol 12-myristate 13-acetate (PMA)-treated U937 cells cultured on collagen I-coated dishes express lowered production of pro-inflammatory mediators in parallel through reduced reactive oxygen species (ROS) levels. By contrast, PMA-treated U937 cells on gelatin, the denatured collagen, show enhanced production of pro-inflammatory mediators, mediated by up-regulating autophagy levels. The present study is aimed to investigate the effect of ROS levels in PMA-treated U937 cells cultured on gelatin-coated surface. Material and methods : MTT assay, flow cytometric analysis of ROS and autophagy, biochemical detection of antioxidant levels, enzyme-linked immunosorbent assay, and western blot were used. Results : Gelatin-coating increased ROS levels in PMA-treated U937 cells. Increased ROS levels are involved in the regulation of cell aggregation and the release of pro-inflammatory mediators in gelatin-coated culture. These results lead to the query about the crosstalk between the two positive regulators, the autophagy and ROS. Autophagy induction is attenuated by N -acetyl-L-cysteine treatment, but the treatment with autophagy inhibitor, 3-methyladenine, does not affect ROS levels, suggesting ROS are upstream of autophagy in the regulation axis of differentiated U937 cells on gelatin-coated surface. Further study confirmed that upregulation of autophagy was responsible for ROS-induced cell aggregation and production of pro-inflammatory mediators. Conclusion : The results suggest that gelatin-coating promotes the aggregation of PMA-treated U937 cells and the production of pro-inflammatory mediators by ROS-autophagy signaling pathway.

Preadipocyte migration is a fundamental and important process for the development of tissue organization, especially in the development of primitive adipose tissue and adipocyte tissue wound healing. However, excessive migration may result in abnormal development and fibrosis-related diseases such as hypertrophic scar. We previously reported that type I collagen (collagen I) enhanced migration of 3T3-L1 preadipocytes via phosphorylation and/or acetylation of NF-κB p65, and the enhanced cell migration is repressed by silibinin treatment through sirt1. It is known that sirt1 has an ability to deacetylate acetylated NF-κB p65, but little is known about the effect of sirt1 on phosphorylated NF-κB p65. This study aims to examine the potential effect of sirt1 on the regulation of phosphorylated NF-κB p65 and the underlying mechanism. Autophagy is involved in many physiological and pathological processes, including regulation of cell migration as well as in cellular homeostasis. The present study demonstrates that silibinin induces autophagy in a dose-dependent manner in 3T3-L1 cells. Autophagy is under the regulation of sirt1/AMPK pathway, and inhibits collagen I-enhanced migration of 3T3-L1 cells through negative regulation of NF-κB p65 phosphorylation but not acetylation. The expression of peroxisome proliferator-activated receptor α (PPARα) is up-regulated with silibinin accompanying up-regulation of autophagy through activating sirt1 in 3T3-L1 cells. Taken together, these findings indicate that silibinin-induced autophagy is mediated by up-regulation of PPARα-sirt1-AMPK, contributing to repression of type I collagen-enhanced migration in murine 3T3-L1 preadipocytes through down-regulation of phosphorylated NF-κB p65.

Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling, inflammation or tissue damage. The present study is aimed to investigate possible biological roles of gelatin by examining its effects on monocyte-like histiocytic lymphoma cell line U937. Once stimulated by phorbol 12-myristate 13-acetate (PMA), U937 cells differentiate into macrophage-like cells, changing from non-adherent to adherent cells with extended pseudopodia. Here we pre-treated the cell dishes with gelatin solution for cell culture. Interestingly, we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes, accompanying NF-κB-mediated production of pro-inflammatory cytokines, whereas cell aggregation was not detected on non-coated dishes. Moreover, differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis. Surprisingly, formation of multicellular aggregates and pro-inflammatory cytokine production were both attenuated by either down-regulation of autophagy with inhibitors, such as 3-methyladenine (3MA) or chloroquine (CQ), or repression of endocytosis with siRNA targeting Endo180. Moreover, autophagy was inhibited by si-Endo180, and endocytosis was suppressed by 3MA, suggesting a positive feedback loop between autophagy and endocytosis. The results revealed that gelatin-coating induced differentiated U937 cells to form cell aggregates and promote NF-κB-mediated pro-inflammatory cytokine production at least partially through an endocytosis-autophagy pathway., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Primary cilia are microtubule-based organelles that extend from nearly all vertebrate cells. Abnormal ciliogenesis and cilia length are suggested to be associated with hypertension and obesity as well as diseases such as Meckel-Gruber syndrome. Extracellular matrix (ECM), comprising cellular microenvironment, influences cell shape and proliferation. However, influence of ECM on cilia biogenesis has not been well studied. In this study we examined the effects of type I collagen (col I), the major component of ECM, on primary cilia growth. When cultured on collagen-coated dishes, confluent 3T3-L1 cells were found to exhibit fibroblast-like morphology, which was different from the cobblestone-like shape on non-coated dishes. The level of autophagy in the cells cultured on col I-coated dishes was attenuated compared with the cells cultured on non-coated dishes. The cilia of the cells cultured on col I-coated dishes became longer, accompanying increased expression of essential proteins for cilia assembly. Transfection of the siRNA targeting microtubule-associated protein light chain 3 (LC3) further enhanced the length of primary cilia, suggesting that col I positively regulated cilia growth through inhibition of autophagy. Histone deacetylase 6 (HDAC6), which was suggested as a mediator of autophagy in our previous study on primary cilia, was down-regulated with col I. 3T3-L1 cells treated with the siRNA against HDAC6 reduced the autophagy level and enhanced collagen-induced cilia elongation, implying that HDAC6 was involved in mediating autophagy. In conclusion, col I promotes cilia growth through repressing the HDAC-autophagy pathway that can be involved in the interaction between primary cilia and col I., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Purpose: Type I collagen is one of the most abundant components of extracellular matrix. We previously illustrated that murine fibrosarcoma L929 cells grew well on type I collagen gel and escaped from TNFα-induced cell death. In this study, we investigated the mechanism underlying the protective effect of collagen gel., Material and Methods: We used western blot, confocal microscopy, MTT assay and flow cytometry by introducing fluorescence staining to determine the expression levels of nuclear factor kappa B (NF-κB), inhibitory ratio and autophagy., Results: L929 cells on collagen gel showed higher expression of NF-κB in the nucleus. Inhibition of NF-κB with pyrrolidine dithiocarbamate hydrochloride (PDTC) or knockdown by NF-κB-siRNA canceled the protective effect of collagen gel on L929 cells from TNFα-induced death, suggesting for the role of NF-κB in the protection from cell death. We found a new aspect of the effect of PDTC on L929 cells cultured on collagen gel. PDTC alone without TNFα induced apoptosis in the L929 cells cultured on collagen gel but not the cells on plastic dish. The apoptosis induction of the L929 cells cultured on collagen gel with PDTC was repressed by inhibiting autophagy with chloroquine, an autophagy inhibitor, suggesting that autophagy contributes to the death induced by the treatment with PDTC. Possible underlying mechanism of this finding is discussed., Conclusion: NF-κB played an important role in protecting the L929 cells cultured on collagen gel from TNFα-induced death.

Alzheimer's disease (AD) is a progressive, neurodegenerative disease. Accumulating evidence suggests that inflammatory response, oxidative stress and autophagy are involved in amyloid β (Aβ)-induced memory deficits. Silibinin (silybin), a flavonoid derived from the herb milk thistle, is well known for its hepatoprotective activities. In this study, we investigated the neuroprotective effect of silibinin on Aβ 25-35 -injected rats. Results demonstrated that silibinin significantly attenuated Aβ 25-35 -induced memory deficits in Morris water maze and novel object-recognition tests. Silibinin exerted anxiolytic effect in Aβ 25-35 -injected rats as determined in elevated plus maze test. Silibinin attenuated the inflammatory responses, increased glutathione (GSH) levels and decreased malondialdehyde (MDA) levels, and upregulated autophagy levels in the Aβ 25-35 -injected rats. In conclusion, silibinin is a potential candidate for AD treatment because of its anti-inflammatory, antioxidant and autophagy regulating activities.

Besides comprising scaffolding, extracellular matrix components modulate many biological processes including inflammation and cell differentiation. We previously found precoating cell plates with extracellular matrix collagen I, or its denatured product gelatin, causes aggregation of macrophage-like human lymphoma U937 cells, which are induced to differentiation by phorbol myristate treatment. In the present study, we investigated the influence of gelatin or collagen I precoating on the bacteria phagocytosis in PMA-stimulated U937 cells. Colony forming units of phagocytosed bacteria, Giemsa-staining of cells with phagocytosed bacteria, confocal microscopic and flow cytometric analysis of cells with phagocytosed FITC-labeled bacteria and non-bioactive latex beats were conducted. Gelatin precoating enhances the phagocytosis of both Gram-negative and positive bacteria, as shown by the increased colony forming units of bacteria phagocytosed by cells, and increased intracellular bacteria observed after Giemsa-staining. But collagen I has no marked influence. Confocal microscopy reveals that both live and dead FITC-bacteria were phagocytosed more in the cells with gelatin-coating but not collagen-coating. Of note, both gelatin and collagen I coating had no influence on the phagocytosis of non-bioactive latex beads. Since gelatin-coating increases autophagy but collagen I has no such impact, we are curious about the role of autophagy. Inhibiting autophagy reduced the phagocytosis of bacteria, in cells with gelatin-coating, while stimulating autophagy enhanced phagocytosis. This study finds the bacteria-phagocytosis stimulatory effect of gelatin in PMA-treated U937 cells and reveals the positive regulatory role of autophagy, predicting the potential use of gelatin products in anti-bacterial therapy. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive, neurodegenerative disease. Accumulating evidence suggests that inflammatory response, oxidative stress and autophagy are involved in amyloid β (Aβ)-induced memory deficits. Silibinin (silybin), a flavonoid derived from the herb milk thistle, is well known for its hepatoprotective activities. In this study, we investigated the neuroprotective effect of silibinin on Aβ-injected rats. Results demonstrated that silibinin significantly attenuated Aβ-induced memory deficits in Morris water maze and novel object-recognition tests. Silibinin exerted anxiolytic effect in Aβ-injected rats as determined in elevated plus maze test. Silibinin attenuated the inflammatory responses, increased glutathione (GSH) levels and decreased malondialdehyde (MDA) levels, and upregulated autophagy levels in the Aβ-injected rats. In conclusion, silibinin is a potential candidate for AD treatment because of its anti-inflammatory, antioxidant and autophagy regulating activities. [ABSTRACT FROM AUTHOR]

Purpose: Gelatin has been considered to exist as intermediate substance of collagen catabolism in tissue remodeling or under inflammatory conditions. We have initiated the study on possible biological functions of gelatin that can exist temporally and locally under the conditions of remodeling and inflammationMaterials and methods: To this purpose, we investigated cell proliferation and survival on gelatin-coated dishes and the response to tumor necrosis factor α (TNFα)–induced cytotoxicity in L929 cells. Autophagy level, ATP level, and ROS generation are examined.Results: L929 cells detached from the gelatin-coated dishes and formed multicellular aggregates. TNFα-induced cytotoxicity in L929 cells was inhibited by gelatin-coating culture. The cells on gelatin-coated dishes showed reduced cellular ATP levels and increased adenosine 5′-monophosphate (AMP)–activated protein kinase (AMPK) phosphorylation, leading to increased ROS generation and autophagy.Conclusion: This study showed that gelatin-coated culture protected L929 cells from TNFα-induced cytotoxicity and suggested for a possible pathophysiological function of gelatin in regulating cellular functions. [ABSTRACT FROM PUBLISHER]

Depression is one of the most frequent psychiatric disorders of Alzheimer's disease (AD). Depression and anxiety are associated with increased risk of developing AD. Silibinin, a flavonoid derived from milk thistle ( Silybum marianum ), has been used as a hepato-protectant in the clinical treatment of liver diseases. In this study, the effect of silibinin on Aβ-induced anxiety/depression-like behaviors in rats was investigated. Silibinin significantly attenuated anxiety/depression-like behaviors caused by Aβ1-42-treatment as shown in tail suspension test (TST), elevated plus maze (EPM) and forced swimming tests (FST). Moreover, silibinin was able to attenuate the neuronal damage in the hippocampus of Aβ1-42-injected rats. Silibinin-treatment up-regulated the function through BDNF/TrkB pathway and attenuated autophagy in the hippocampus. Our study provides a new insight into the protective effects of silibinin in the treatment of anxiety/depression. [ABSTRACT FROM AUTHOR]

The extracellular matrix (ECM) regulates cell behavior through signal transduction and provides a suitable place for cell survival. As one of the major components of the extracellular matrix, type I collagen is involved in regulating cell migration, proliferation and differentiation. We present a system in which 3T3-L1 preadipocyte cells are induced for adipogenic differentiation on type I collagen coated dishes. Our previous study has found that type I collagen inhibits adipogenic differentiation via YAP activation. Here we further reveal that type I collagen inactivates autophagy by up-regulating mTOR activity via the YAP pathway. Under collagen-coating conditions, co-localization of lysosomes with mTOR was increased and the level of downstream protein p-S6K was elevated, accompanied by a decrease in the level of autophagy. Autophagy is negatively correlated with adipogenesis under type I collagen coating. Through the YAP-autophagy axis, type I collagen improves glycolipid metabolism accompanied by increased mitochondrial content, enhanced glucose uptake, reduced release of free fatty acids (FFAs) and decreased intracellular lipid accumulation. Our findings provide insight into the strategy for dealing with obesity: Type I collagen or the drugs with inhibitory effects on autophagy or YAP, have a potential to accelerate the energy metabolism of adipose tissue, so as to better maintain the homeostasis of glucose and lipids in the body, which can be used for achieving weight loss. [Display omitted] • Type I collagen inhibits autophagy by up-regulating YAP-mTOR pathway. • Autophagy is negatively correlated with adipogenesis under type I collagen coating. • Type I collagen improves glucose and lipid metabolism through the YAP-autophagy axis. [ABSTRACT FROM AUTHOR]

Alcoholic liver disease has become one of the main causes of liver injury, and its prevention and cure are important medical tasks. Silibinin, a natural flavonoid glycoside, is a conventional hepatic protectant. This study elucidates the modulation of ferroptosis in silibinin's protective effects on ethanol- or acetaldehyde-induced liver cell damage by using human carcinomatous liver HepG2 cells and immortalized liver HL7702 cells. Our results show that ferroptosis is induced in the cells treated with ethanol or acetaldehyde, as evidenced by the increased ROS stress and iron level. Silibinin resolves the oxidative stress and reduces iron level. Ferroptosis induced by ethanol- or acetaldehyde involving nuclear receptor co-activator 4 (NCOA4)-dependent autophagic degradation of ferritin, a protein for storing iron is rescued by silibinin. PINK1 and Parkin-mediated mitophagy is arrested in ethanol- or acetaldehyde-treated cells but reversed by silibinin. Ferritin degradation and ROS level are further increased when PINK1 or Parkin is silenced in the cells treated with ethanol or acetaldehyde. Collectively, our study reveals that silibinin inhibits ethanol- or acetaldehyde-induced ferroptosis in two liver cell lines, HepG2 and HL7702 cells, providing new therapeutic strategies for alcoholic liver injury. [Display omitted] • Ethanol or acetaldehyde triggers ferroptosis in hepatocytes. • Autophagy is involved in ferroptosis treated with ethanol or acetaldehyde. • Ethanol or acetaldehyde reduces PINK1/Parkin-mediated mitophagy in ferroptosis. • Silibinin inhibits ethanol- or acetaldehyde-induced ferroptosis in two liver cell lines. • Silibinin enhances mitophagy in ethanol- or acetaldehyde-induced liver injury. [ABSTRACT FROM AUTHOR]

Murine fibrosarcoma L929 cells have been used to test efficacy of proinflammatory cytokine TNFα. In the present study, we reported on protective effect of type I collagen gel used as L929 cell culture. L929 cell grew and proliferated well on collagen gel. However, the L929 cells exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional cell dishes as well as the cells tended to aggregate. On conventional cell culture dishes, the cells treated with TNFα became round in shape and eventually died in a necroptotic manner. The cells cultured on collagen gel, however, were completely unaffected. TNFα treatment was reported to induce autophagy in L929 cells on the plastic dish, and therefore we investigated the effect of collagen gel on induction of autophagy. The results indicated that autophagy induced by TNFα treatment was much reduced when the cells were cultured on collagen gel. In conclusion, type I collagen gel protected L929 cell from TNFα-induced cell death. [ABSTRACT FROM AUTHOR]

Silibinin, one of the flavonoids isolated from milk thistle seeds of Silybum marianum , has hepatoprotective properties against toxins in clinical. However, the detailed mechanisms have remained unclear. This study investigates the underlying mechanism of silibinin in the protection against ethanol- or acetaldehyde-induced damage of neonatal mouse primary hepatocytes in vitro. The results show that ethanol inhibited proliferation of hepatocytes in a time (12, 24, 36 h) and dose-dependent (0–800 mM) manner. However, silibinin did not show protective effect on ethanol (500 mM)-induced suppression of hepatocyte proliferation. Acetaldehyde, the toxic metabolite of ethanol, appearing immediately in individuals after drink also inhibited the proliferation of hepatocytes in a dose-dependent (0–12 mM) manner. Surprisingly, silibinin significantly increased the cell viability and reduced the leakage of alanine amino transferase (ALT) and aspartate amino transferase (AST) in acetaldehyde-treated hepatocytes, suggesting that silibinin protected cell injury caused by acetaldehyde treatment. The apoptosis-inducing effect of acetaldehyde was demonstrated by the increased number of cells in sub-G1 phase as well as caspase-3 activation. Further study shows that acetaldehyde induced autophagy in the hepatocytes. The autophagy inhibitors, 3-Methyladenine (3-MA) and chloroquine (CQ), further decreased the viability of cells treated with acetaldehyde, suggesting that autophagy plays a protective role against apoptosis. Consistently, silibinin (20 μM) significantly reduced the activation of caspase 3 or apoptosis and increased the conversion of LC3-I to LC3-II or autophagy. Taken together, it is concluded that silibinin does not repress the ethanol- induced hepatocyte injury, whereas silibinin reduces acetaldehyde-caused hepatocyte injury through down-regulation of apoptosis and up-regulation of autophagy. • Acetaldehyde (ACH) induced apoptosis in liver cells; • AcH induced autophagy in liver cells; • Silibinin protects AcH-induced death, but not ethanol-induced death of hepatocytes. [ABSTRACT FROM AUTHOR]