Bruno Souza Basso

Capsaicin induces de-differentiation of activated hepatic stellate cell

Hepatic stellate cells (HSC) play a key role in liver fibrogenesis. Activation of PPARγ and inhibition of fibrogenic molecules are potential strategies to block HSC activation and differentiation. A number of natural products have been suggested to have antifibrotic effects for the de-activation and de-differentiation of HSCs. The purpose of this study was to investigate the in vitro effects of capsaicin on HSC de-activation and de-differentiation. The results demonstrated that capsaicin induced quiescent phenotype in GRX via PPARγ activation. Significant decrease in COX-2 and type I collagen mRNA expression was observed in the first 24 h of treatment. These events preceded the reduction of TGF-β1 and total collagen secretion. Thus, capsaicin promoted down-regulation of HSC activation by its antifibrotic and anti-inflammatory actions. These findings demonstrate that capsaicin may have potential as a novel therapeutic agent for the treatment of liver fibrosis.

Capsaicin modulates proliferation, migration, and activation of hepatic stellate cells.

Capsaicin, the active component of chili pepper, has been reported to have antiproliferative and anti-inflammatory effects on a variety of cell lines. In the current study, we aimed to investigate the effects of capsaicin during HSC activation and maintenance. Activated and freshly isolated HSCs were treated with capsaicin. Proliferation was measured by incorporation of EdU. Cell cycle arrest and apoptosis were investigated using flow cytometry. The migratory response to chemotactic stimuli was evaluated by a modified Boyden chamber assay. Activation markers and inflammatory cytokines were determined by qPCR, immunocytochemistry, and flow cytometry. Our results show that capsaicin reduces HSC proliferation, migration, and expression of profibrogenic markers of activated and primary mouse HSCs. In conclusion, the present study shows that capsaicin modulates proliferation, migration, and activation of HSC in vitro.

Gallic acid reduces cell growth by induction of apoptosis and reduction of IL-8 in HepG2 cells.

Hepatocellular carcinoma is the most prevalent primary liver tumor and is among the top ten cancer that affect the world population. Its development is related, in most cases, to the existence of chronic liver injury, such as in cirrhosis. The knowledge about the correlation between chronic inflammation and cancer has driven new researches with anti-inflammatory agents that have potential for the development of antitumor drugs. Gallic acid is a phenolic acid found in many natural products and have shown anti-inflammatory, anti-tumor, anti-mutagenic and antioxidant actions. The purpose of this study was to investigate the effect of gallic acid on acute and chronic cell proliferation and inflammatory parameters of hepatocellular carcinoma cells (HepG2), as well as to investigate the mechanisms involved. Results showed that the gallic acid decreased the proliferation of HepG2 cells in a dose-dependent manner (Trypan blue exclusion assay), without causing necrosis (LDH assay). We observed a significant increase in the percentage of small and regular nuclei (Nuclear Morphometric Analysis assay), a significant induction of apoptosis by Annexin V-FITC and PI assay and no interference with the cell cycle using the FITC BrdU Flow Kit. We observed a significant reduction in the levels of IL-8 and increased levels of IL-10 and IL-12 (Cytometric Bead Array Human Inflammation Assay). Furthermore, gallic acid caused no cancer cells regrowth at a long term (Cumulative Population Doubling assay). According to these results, gallic acid showed a strong potential as an anti-tumor agent in hepatocellular carcinoma cells.

Liraglutide, a glucagon-like peptide-1 analog, induce autophagy and senescence in HepG2 cells

&NA; It has been reported that glucagon‐like peptide‐1 (GLP‐1) agents have been associated with both the increased risk of cancer and inhibition of tumor growth and metastases. The aim of this study is to evaluate the effect of liraglutide on hepatocellular carcinoma cells – HepG2. Cytometry was used to evaluate mechanism related to decreased cell proliferation. Nuclear staining and morphometric analysis were also used to verify the process that was taking place after treatment with liraglutide, and in order to better understand the mechanism, TGF‐&bgr;1 was performed. HepG2 cells decreased proliferation after liraglutide treatment without altering oxidative stress levels. Liraglutide was able to induce autophagy and senescence through the increase of TGF‐&bgr;1 which possibly explains the growth decrease. We have demonstrated that liraglutide has an antiproliferative effect in HepG2 cells inducing autophagy and senescence by the increase of TGF‐&bgr;1.

Therapeutic ultrasound stimulates MC3T3-E1 cell proliferation through the activation of NF-κB1, p38α, and mTOR

As the population ages, osteometabolic diseases and osteoporotic fractures emerge, resulting in substantial healthcare resource utilization and impaired quality of life. Many types of mechanical stimulation have the potential of being recognized by bone cells after a mechanical sign is transformed into a biological one (a process called mechanotransduction). The therapeutic ultrasound (TU) is one of several resources capable of promoting bone cell mechanical stimulation. Therefore, the main purpose of present study was to evaluate the effect of TU on the proliferation of pre‐osteoblasts using in vitro bioassays.

Catechin attenuates activation of hepatic stellate cells

(+)‐Catechin is a type of catechin present in large amounts in açaí fruits and cocoa seeds. Besides its antioxidant and anti‐inflammatory activities, little is known about its effects in the liver, especially during hepatic fibrosis. We report here the effects of (+)‐catechin on hepatic stellate cells. (+)‐Catechin induced quiescent phenotype in GRX cells, along with an increase in lipid droplets. Proliferator‐activated receptor γ mRNA expression was upregulated, whereas type I collagen mRNA expression was downregulated. Pro‐inflammatory cytokines were not influenced by (+)‐catechin, whereas the levels of interleukin 10 were significantly increased. The data provide evidence that (+)‐catechin can reduce hepatic stellate cell activation.

Fructose-1,6-bisphosphate decreases IL-8 levels and increases the activity of pro-apoptotic proteins in HepG2 cells

Hepatocellular carcinoma (HCC) is the most prevalent primary liver tumor that affects the world population. Liver cancer inevitably causes great harms and its treatment is extremely difficult. Its development is related to the existence of chronic liver injury, such as in cirrhosis. Cancer is a disease related to the process of inflammation so, research with anti-inflammatory agents has been performed for the development of anti-tumor drugs. Fructose-1,6-bisphosphate (FBP), a metabolite of the glycolytic route, has shown anti-inflammatory actions. The purpose of this study is to investigate the effect of FBP on HepG2 cells growth and inflammatory parameters. Results showed that FBP decreased the proliferation of HepG2 cells through trypan blue assay, without causing necrosis, shown by the intracellular release of LDH. By flow cytometry, we observed a significant IL-8 decrease which is closely related to the tumoral progression and chemotherapeutic resistance, especially in HCC. Then, we found, by RT-PCR, a high expression level of pro-apoptotic protein, such as Bax and p53, and decreased the expression levels of anti-apoptotic proteins, like Bcl-2 suggesting apoptosis. Finally, our results showed that FBP can be a potential therapeutic agent to slow the progress of HCC.

Low-intensity pulsed ultrasound (LIPUS) stimulates mineralization of MC3T3-E1 cells through calcium and phosphate uptake

HighlightsLIPUS can stimulate the pre‐osteoblast bone cell mineralization.LIPUS stimulates mineralization through calcium and phosphate uptake.LIPUS promote HA formation deposited in a framework of collagen fibers. ABSTRACT The present study aimed to evaluate the effect of low‐intensity pulsed ultrasound (LIPUS) on pre‐osteoblast mineralization using in vitro bioassays. Pre‐osteoblastic MC3T3‐E1 cells were exposed to LIPUS at 1 MHz frequency, 0.2 W/cm2 intensity and 20% duty cycle for 30 min. The analyses were carried out up to 336 h (14 days) after exposure. The concentration of collagen, phosphate, alkaline phosphatase, calcium and transforming growth factor beta 1 (TGF‐&bgr;1) in cell supernatant and the presence of calcium deposits in the cells were analyzed. Our results showed that LIPUS promotes mineralized nodules formation. Collagen, phosphate, and calcium levels were decreased in cell supernatant at 192 h after LIPUS exposure. However, alkaline phosphatase and TGF‐&bgr;1 concentrations remained unchanged. Therapeutic pulsed ultrasound is capable of stimulating differentiation and mineralization of pre‐osteoblastic MC3T3‐E1 cells by calcium and phosphate uptake with consequent hydroxyapatite formation.

Leucine reduces the proliferation of MC3T3-E1 cells through DNA damage and cell senescence

Leucine (Leu) is an essential branched-chain amino acid, present in dairy products, which has been investigated for its important role in cell signaling. The effects of Leu on several kinds of cells have been studied, altough little is known on its action upon bone cells and cell proliferation. Thus, the aim of this study is to investigate the effects of Leu supplementation on the proliferation of pre-osteoblasts from MC3T3-E1 lineage. MC3T3-E1 cells were kept in Alpha medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimitotic. Cells were treated during 48h by adding 50μM of Leu, which corresponds to a 12.5% increase of the amino acid in the culture medium. The evaluation of viability and proliferation of cultured cells was performed using Trypan Blue dye. In order to identify the mechanisms related to the decreased cellular proliferation, assays were performed to assess cytotoxicity, apotosis, oxidative stress, inflammation, autophagy, senescence and DNA damage. Results showed that Leu supplementation decreased cell proliferation by 40% through mechanisms not related to cell necrosis, apoptosis, oxidative stress, autophagy or inhibition of the mTORC1 pathway. On the other hand, Leu supplementation caused DNA damage. In conclusion, Leu caused a negative impact on bone cell proliferation by inducing cell senescence through DNA damage.

Erratum to: Fructose-1,6-bisphosphate reverts iron-induced phenotype of hepatic stellate cells by chelating ferrous ions

Hepatic fibrosis is an extracellular matrix deposition by hepatic stellate cells (HSC). Fibrosis can be caused by iron, which will lead to hydroxyl radical production and cell damage. Fructose-1,6-bisphosphate (FBP) has been shown to deliver therapeutic effects in many pathological situations. In this work, we aimed to test the effects of FBP in HSC cell line, GRX, exposed to an excess of iron (Fe). The Fe-treatment increased cell proliferation and FBP reversed this effect, which was not due to increased necrosis, apoptosis or changes in cell cycle. Oil Red-O staining showed that FBP successfully increased lipid content and lead GRX cells to present characteristics of quiescent HSC. Fe-treatment decreased PPAR-γ expression and increased Col-1 expression. Both effects were reversed by FBP which also decreased TGF-β1 levels in comparison to both control and Fe groups. FBP, also, did not present scavenger activity in the DPPH assay. The treatment with FBP resulted in decreased proliferation rate, Col-1 expression and TGF-β1 release by HSC cells. Furthermore, activated PPAR-γ and increased lipid droplets induce cells to become quiescent, which is a key event to reversion of hepatic fibrosis. FBP also chelates iron showing potential to improve Cell redox state