scholarly journals Apoptosis of rat hepatic stellate cells induced by diallyl trisulfide and proteomics profiling in vitro

2017 ◽  
Vol 95 (5) ◽  
pp. 463-473
Author(s):  
Yajie Zhang ◽  
Xiaoming Zhou ◽  
Lipeng Xu ◽  
Lulu Wang ◽  
Jinling Liu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Hepatic Stellate Cells ◽  
Colorimetric Assay ◽  
Expression Patterns ◽  
Stellate Cells ◽  
Control Group ◽  
Dependent Manner ◽  
Diallyl Trisulfide ◽  
Proliferation And Apoptosis

Diallyl trisulfide (DATS), a major garlic derivative, inhibits cell proliferation and triggers apoptosis in a variety of cancer cell lines. However, the effects of DATS on hepatic stellate cells (HSCs) remain unknown. The aim of this study was to analyze the effects of DATS on cell proliferation and apoptosis, as well as the protein expression profile in rat HSCs. Rat HSCs were treated with or without 12 and 24 μg/mL DATS for various time intervals. Cell proliferation and apoptosis were determined using tetrazolium dye (MTT) colorimetric assay, bromodeoxyuridine (5-bromo-2′-deoxyuridine; BrdU) assay, Hoechst 33342 staining, electroscopy, and flow cytometry. Protein expression patterns in HSCs were systematically studied using 2-dimensional electrophoresis and mass spectrometry. DATS inhibited cell proliferation and induced apoptosis of HSCs in a time-dependent manner. We observed clear morphological changes in apoptotic HSCs and dramatically increased annexin V-positive – propidium iodide negative apoptosis compared with the untreated control group. Twenty-one significant differentially expressed proteins, including 9 downregulated proteins and 12 upregulated proteins, were identified after DATS administration, and most of them were involved in apoptosis. Our results suggest that DATS is an inducer of apoptosis in HSCs, and several key proteins may be involved in the molecular mechanism of apoptosis induced by DATS.

Norepinephrine induces calcium spikes and proinflammatory actions in human hepatic stellate cells

2006 ◽  
Vol 291 (5) ◽  
pp. G877-G884 ◽  
Author(s):  
Pau Sancho-Bru ◽  
Ramón Bataller ◽  
Jordi Colmenero ◽  
Xavier Gasull ◽  
Montserrat Moreno ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Portal Hypertension ◽  
Liver Fibrosis ◽  
Hepatic Stellate Cells ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Stellate Cells ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Cell Contraction

Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through α1-adrenoceptors. However, the underlying cellular and molecular mechanisms are largely unknown. Here, we investigated the effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert vasoactive, inflammatory, and fibrogenic actions in the injured liver. Adrenoceptor expression was assessed in human HSC by RT-PCR and immunocytochemistry. Intracellular Ca2+ concentration ([Ca2+]i) was studied in fura-2-loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin light chain II (MLC II) phosphorylation. Cell proliferation and collagen-α1(I) expression were assessed by [3H]thymidine incorporation and quantitative PCR, respectively. NF-κB activation was assessed by luciferase reporter gene and p65 nuclear translocation. Chemokine secretion was assessed by ELISA. Normal human livers expressed α1A-adrenoceptors, which were markedly upregulated in livers with advanced fibrosis. Activated human HSC expressed α1A-adrenoceptors. NE induced multiple rapid [Ca2+]i oscillations (Ca2+ spikes). Prazosin (α1-blocker) completely prevented NE-induced Ca2+ spikes, whereas propranolol (nonspecific β-blocker) partially attenuated this effect. NE caused phosphorylation of MLC II and cell contraction. In contrast, NE did not affect cell proliferation or collagen-α1(I) expression. Importantly, NE stimulated the secretion of inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner. Prazosin blocked NE-induced chemokine secretion. NE stimulated NF-κB activation. BAY 11-7082, a specific NF-κB inhibitor, blocked NE-induced chemokine secretion. We conclude that NE stimulates NF-κB and induces cell contraction and proinflammatory effects in human HSC. Catecholamines may participate in the pathogenesis of portal hypertension and liver fibrosis by targeting HSC.

MicroRNA-126-5p Regulates Proliferation and Apoptosis of IL-22-Stimulated Human Keratinocytes Through Regulating Caspase 1

2021 ◽  
Vol 11 (5) ◽  
pp. 1010-1016
Author(s):  
Weifeng Zha ◽  
Bo Guo ◽  
Shuyue Chen ◽  
Junwei Lu ◽  
Yunyun Shan
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Cell Model ◽  
Luciferase Reporter ◽  
Control Group ◽  
Hacat Cells ◽  
Luciferase Reporter Gene ◽  
Proliferation And Apoptosis

Objective: The study was aimed to explore the roles of miR-126-5p in psoriasis and the underlying molecular mechanisms. Methods: In vitro cell model of psoriasis was established by IL-22 induction. CASP1, the target gene of miR-126-5p, was predicted by TargetScan and verified through the dual luciferase reporter gene system. qRT-PCR was used to measure the mRNA expression of miR-126-5p and CASP1 in IL-22 stimulated HaCaT cells. The protein expression of CASP1, cleaved-caspase3 and caspase3 were measured by Western blot analysis. MTT assay and flow cytometry analysis were performed to detect the cell proliferation and apoptosis. A Caspase3 Activity Assay kit was used to detect the activity of Caspase3. Results: miR-126-5p was high expressed in IL-22 stimulated HaCaT cells compared with normal HaCaT cells. We predicted and verified that CASP1 was a direct target of miR-126-5p, and the mRNA and protein expression of CASP1 were reduced in IL-22 stimulated HaCaT cells compared with the normal HaCaT cells. miR-126-5p inhibitor and CASP1-siRNA significantly decreased the expression of miR-126-5p and CASP1 in HaCaT cells respectively. miR-126-5p inhibitor up-regulated the expression of CASP1 in HaCaT cells, and the effect was reversed by the transfection with CASP1-siRNA. In comparison with the control group, miR-126-5p inhibitor decreased the cell proliferation, induced apoptosis, and improved the activity of Caspase3, enhanced cleaved-caspase3/caspase3 ratio in IL-22 stimulated HaCaT cells, and all the effects were reversed by down-regulating CASP1. Conclusion: We demonstrated that miR-126-5p inhibitor played a protective role in psoriasis by targeting CASP1, evidenced by inhibiting IL-22-induced HaCaT cell proliferation and inducing apoptosis.

20(S)-Ginsenoside Rg3 Inhibits Human Glioma Cell Growth through the Suppression of Voltage-Gated K+ Channels

2014 ◽  
Vol 998-999 ◽  
pp. 160-163
Author(s):  
Qin Ru ◽  
Xiang Tian ◽  
Yu Xiang Wu ◽  
Kai Yue ◽  
Lin Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Underlying Mechanism ◽  
Control Group ◽  
Dependent Manner ◽  
Human Glioma ◽  
Voltage Gated ◽  
New Findings ◽  
Proliferation And Apoptosis ◽  
M Phase ◽  
Channel Currents

Previous studies demonstrated that 20(S)-ginsenoside Rg3 (20S-Rg3) could effectively inhibit tumor cell proliferation as well as K+ channel currents expressed in xenopus oocytes. However, the effect of 20S-Rg3 on the growth of human glioma cells and the ion channels expressed in tumor cells was rarely reported in the literature. In the present study, we investigated the effect and the underlying mechanism of 20S-Rg3 on cell proliferation and apoptosis of human glioma U87-MG cells. In vitro results exhibited that 20S-Rg3 had potent cytotoxic effect and significantly inhibited the proliferation of U87-MG cells in a dose-and time-dependent manner. Typical arrest at G2/M phase was induced, and the apoptosis rate of U87-MG cells was significantly higher in the 20S-Rg3 treatment group than in the control group. Electrophysiological results showed that 80 μmol/L 20S-Rg3 substantially inhibited voltage-gated K+ currents of U87-MG cells. Together, these results suggest that the suppression of voltage-gated K+ currents might play an important role in the 20S-Rg3-induced cell death, and these new findings provide useful data for further study of the antitumor effect of 20S-Rg3.

Roles of vitamin D and its receptor in the proliferation and apoptosis of luteinised granulosa cells in the goat

2020 ◽  
Vol 32 (3) ◽  
pp. 335 ◽  
Author(s):  
Xiaolei Yao ◽  
Zhibo Wang ◽  
M. A. El-Samahy ◽  
Caifang Ren ◽  
Zifei Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Vitamin D ◽  
Granulosa Cells ◽  
Expression Patterns ◽  
Follicular Development ◽  
Proliferation Rate ◽  
Control Group ◽  
Dependent Manner ◽  
Proliferation And Apoptosis ◽  
Dose Dependent

The objective of this study was to investigate the dose-dependent effect of 1α,25-(OH)2VD3 (Vit D3) on invitro proliferation of goat luteinised granulosa cells (LGCs) and to determine the underlying mechanisms of its action by overexpressing and silencing vitamin D receptor (VDR) in LGCs. Results showed that VDR was prominently localised in GCs and theca cells (TCs) and its expression increased with follicle diameter, but was lower in atretic follicles than in healthy follicles. The proliferation rate of LGCs was significantly higher in the Vit D3-treated groups than in the control group, with the highest proliferation rate observed in the 10nM group; this was accompanied by changes in the expression of cell cycle-related genes. These data indicate that Vit D3 affects LGC proliferation in a dose-dependent manner. Contrary to the VDR knockdown effects, its overexpression upregulated and downregulated cell cycle- and apoptosis-related genes respectively; moreover, supplementation with 10nM of Vit D3 significantly enhanced these effects. These results suggest that changes in VDR expression patterns in LGCs may be associated with follicular development by regulation of cell proliferation and apoptosis. These findings will enhance the understanding of the roles of Vit D3 and VDR in goat ovarian follicular development.

scholarly journals COX-2 Regulates the Proliferation and Apoptosis of Activated Hepatic Stellate Cells through CDC27

2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Yang Hu ◽  
Nian Fu ◽  
Li Xian Chen ◽  
Jian Hua Xiao ◽  
Xue Feng Yang
Keyword(s):  
Cell Proliferation ◽  
Hepatic Fibrosis ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Cycle Arrest ◽  
Proliferation And Apoptosis ◽  
Cox 2 ◽  
Activated Hepatic Stellate Cells

Cyclooxygenase-2 (COX-2) is an important rate-limiting enzyme in the synthesis of prostaglandins (PGs), which can be upregulated by various stimuli. COX-2 has been shown to be involved in the occurrence and development of hepatic fibrosis by regulating the proliferation and apoptosis of hepatic stellate cells (HSCs) in previous studies. The aims of the study are to study the mechanism of how COX-2 regulates the proliferation and apoptosis of HSCs and to provide new targets for the prevention and treatment of hepatic fibrosis. A short hairpin RNA targeting COX-2 was constructed, and the changes in proliferation and apoptosis of liver tissue cells and HSCs were observed, respectively. COX-2-shRNA-1 significantly suppressed the proliferation of HSCs in vivo. Moreover, knockdown of COX-2 significantly suppressed cell proliferation and accelerated cell cycle arrest and apoptosis in vitro. Among those differential genes related to cell proliferation and apoptosis, CDC27 and Sh3kbp1 were upregulated, but Plcd4 was suppressed. Mechanistically, the influence of COX-2 on HSCs partly depends on upregulating CDC27. Our results demonstrated that COX-2 regulates the proliferation and apoptosis of activated hepatic stellate cells through the CDC27 pathway. This study contributes to our understanding of the effect of COX-2 for the treatment of hepatic fibrosis.

scholarly journals The Epigenetically-Regulated microRNA-378a Targets TGF-β2 in TGF-β1-Treated Hepatic Stellate Cells

2016 ◽  
Vol 40 (1-2) ◽  
pp. 183-194 ◽  
Author(s):  
Fujun Yu ◽  
Jianhuan Yang ◽  
Kate Huang ◽  
Xiaodong Pan ◽  
BiCheng Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Promoter Methylation ◽  
Hepatic Stellate Cells ◽  
Transforming Growth Factor ◽  
Methylation Status ◽  
Stellate Cells ◽  
Luciferase Reporter ◽  
Type I ◽  
Dependent Manner ◽  
Tgf Β1

Background/Aims: In liver fibrosis, the activation of hepatic stellate cells (HSCs) is considered as a pivotal event. It is well known that transforming growth factor-β1 (TGF-β1) is the main stimuli factor responsible for HSC activation. microRNAs (miRNAs), regulating various biological processes, have recently been shown to be involved in HSC activation. A recent study reported that deficiency of miR-378a contributes to cardiac fibrosis via TGF-β1-dependent paracrine mechanism. However, the involvement of miR-378a and its roles in TGF-β1-induced HSC activation remains largely unknown. Methods: miR-378a expression was detected in TGF-β1-treated cells and patients with cirrhosis. Then, effects of miR-378a overexpression on cell proliferation and HSC activation were analyzed. We also analyzed the binding of miR-378a to the 3′-untranslated region of TGF-β2. Results: In response to TGF-β1, miR-378a expression was down-regulated in a dose-dependent manner. miR-378a overexpression suppressed both cell proliferation and cell cycle in TGF-β1-treated LX-2 cells. Moreover, miR-378a overexpression inhibited TGF-β1-induced HSC activation including the reduction of α-smooth muscle actin (α-SMA) and type I collagen. Similarly, miR-378a resulted in a reduction in cell proliferation, and the expressions of α-SMA and Col1A1 in TGF-β1-treated primary HSCs. Notably, TGF-β2 was confirmed as a target of miR-378a by luciferase reporter assays. Interestingly, miR-378a promoter methylation may be responsible for miR-378a down-regulation in TGF-β1-treated LX-2 cells and TGF-β1-treated primary HSCs. Further studies confirmed that reduced miR-378a was associated with promoter methylation in patients with cirrhosis compared with healthy controls. Conclusion: Our results demonstrate that miR-378a expression is associated with its methylation status in TGF-β1-treated cells, and epigenetically-regulated miR-378a inhibits TGF-β1-induced HSC activation, at least in part, via TGF-β2.

scholarly journals Transcriptional repression of SIRT1 by protein inhibitor of activated STAT 4 (PIAS4) in hepatic stellate cells contributes to liver fibrosis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Lina Sun ◽  
Zhiwen Fan ◽  
Junliang Chen ◽  
Wenfang Tian ◽  
Min Li ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Hepatic Stellate Cells ◽  
High Glucose ◽  
Interstitial Fibrosis ◽  
Stellate Cells ◽  
Quiescent State ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Sirt1 Expression ◽  
Primary Mouse

Abstract Interstitial fibrosis represents a key pathological process in non-alcoholic steatohepatitis (NASH). In the liver, fibrogenesis is primarily mediated by activated hepatic stellate cells (HSCs) transitioning from a quiescent state in response to a host of stimuli. The molecular mechanism underlying HSC activation is not completely understood. Here we report that there was a simultaneous up-regulation of PIAS4 expression and down-regulation of SIRT1 expression accompanying increased hepatic fibrogenesis in an MCD-diet induced mouse model of NASH. In cultured primary mouse HSCs, stimulation with high glucose activated PIAS4 while at the same time repressed SIRT1. Over-expression of PIAS4 directly repressed SIRT1 promoter activity. In contrast, depletion of PIAS4 restored SIRT1 expression in HSCs treated with high glucose. Estrogen, a known NASH-protective hormone, antagonized HSC activation by targeting PIAS4. Lentivirus-mediated delivery of short hairpin RNA (shRNA) targeting PIAS4 in mice ameliorated MCD diet induced liver fibrosis by normalizing SIRT1 expression in vivo. PIAS4 promoted HSC activation in a SIRT1-dependent manner in vitro. Mechanistically, PIAS4 mediated SIRT1 repression led to SMAD3 hyperacetylation and enhanced SMAD3 binding to fibrogenic gene promoters. Taken together, our data suggest SIRT1 trans-repression by PIAS4 plays an important role in HSC activation and liver fibrosis.

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