RESUMEN
BACKGROUND: The nasal vaccine HB-ATV-8 has emerged as a promising approach for NAFLD (non-alcoholic fatty liver disease) and atherosclerosis prevention. HB-ATV-8 contains peptide seq-1 derived from the carboxy-end of the Cholesteryl Ester Transfer Protein (CETP), shown to reduce liver fibrosis, inflammation, and atherosclerotic plaque formation in animal models. Beyond the fact that this vaccine induces B-cell lymphocytes to code for antibodies against the seq-1 sequence, inhibiting CETP's cholesterol transfer activity, we have hypothesized that beyond the modulation of CETP activity carried out by neutralizing antibodies, the observed molecular effects may also correspond to the direct action of peptide seq-1 on diverse cellular systems and molecular features involved in the development of liver fibrosis. METHODS: The HepG2 hepatoma-derived cell line was employed to establish an in vitro steatosis model. To obtain a conditioned cell medium to be used with hepatic stellate cell (HSC) cultures, HepG2 cells were exposed to fatty acids or fatty acids plus peptide seq-1, and the culture medium was collected. Gene regulation of COL1A1, ACTA2, TGF-ß, and the expression of proteins COL1A1, MMP-2, and TIMP-2 were studied. AIM: To establish an in vitro steatosis model employing HepG2 cells that mimics molecular processes observed in vivo during the onset of liver fibrosis. To evaluate the effect of peptide Seq-1 on lipid accumulation and pro-fibrotic responses. To study the effect of Seq-1-treated steatotic HepG2 cell supernatants on lipid accumulation, oxidative stress, and pro-fibrotic responses in HSC. RESULTS AND CONCLUSION: Peptide seq-1-treated HepG2 cells show a downregulation of COLIA1, ACTA2, and TGF-ß genes, and a decreased expression of proteins such as COL1A1, MMP-2, and TIMP-2, associated with the remodeling of extracellular matrix components. The same results are observed when HSCs are incubated with peptide Seq-1-treated steatotic HepG2 cell supernatants. The present study consolidates the nasal vaccine HB-ATV-8 as a new prospect in the treatment of NASH directly associated with the development of cardiovascular disease.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Vacunas , Animales , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/farmacología , Metaloproteinasa 2 de la Matriz , Proteínas de Transferencia de Ésteres de Colesterol/metabolismo , Regulación hacia Abajo , Hepatocitos/metabolismo , Fibrosis , Cirrosis Hepática/patología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Ácidos Grasos/metabolismo , Lípidos/farmacología , Hígado/metabolismoRESUMEN
The modulation of inflammation is pivotal for uterine homeostasis. Here we evaluated the effect of the oestrus cycle on the expression of pro-inflammatory and anti-inflammatory markers in a cellular model of induced fibrosis. Mare endometrial stromal cells isolated from follicular or mid-luteal phase were primed with 10 ng/mL of TGFß alone or in combination with either IL1ß, IL6, or TNFα (10 ng/mL each) or all together for 24 h. Control cells were not primed. Messenger and miRNA expression were analyzed using real-time quantitative PCR (RT-qPCR). Cells in the follicular phase primed with pro-inflammatory cytokines showed higher expression of collagen-related genes (CTGF, COL1A1, COL3A1, and TIMP1) and mesenchymal marker (SLUG, VIM, CDH2, and CDH11) genes; p < 0.05. Cells primed during the mid-luteal overexpressed genes associated with extracellular matrix, processing, and prostaglandin E synthase (MMP2, MMP9, PGR, TIMP2, and PTGES; p < 0.05). There was a notable upregulation of pro-fibrotic miRNAs (miR17, miR21, and miR433) in the follicular phase when the cells were exposed to TGFß + IL1ß, TGFß + IL6 or TGFß + IL1ß + IL6 + TNFα. Conversely, in cells from the mid-luteal phase, the treatments either did not or diminished the expression of the same miRNAs. On the contrary, the anti-fibrotic miRNAs (miR26a, miR29b, miR29c, miR145, miR378, and mir488) were not upregulated with treatments in the follicular phase. Rather, they were overexpressed in cells from the mid-luteal phase, with the highest regulation observed in TGFß + IL1ß + IL6 + TNFα treatment groups. These miRNAs were also analyzed in the extracellular vesicles secreted by the cells. A similar trend as seen with cellular miRNAs was noted, where anti-fibrotic miRNAs were downregulated in the follicular phase, while notably elevated pro-fibrotic miRNAs were observed in extracellular vesicles originating from the follicular phase. Pro-inflammatory cytokines may amplify the TGFß signal in the follicular phase resulting in significant upregulation of extracellular matrix-related genes, an imbalance in the metalloproteinases, downregulation of estrogen receptors, and upregulation of pro-fibrotic factors. Conversely, in the luteal phase, there is a protective role mediated primarily through an increase in anti-fibrotic miRNAs, a decrease in SMAD2 phosphorylation, and reduced expression of fibrosis-related genes.