|Support type:||Scholarships in Brazil - Doctorate|
|Effective date (Start):||January 01, 2017|
|Effective date (End):||February 29, 2020|
|Field of knowledge:||Health Sciences - Nutrition|
|Principal researcher:||Luís Fernando Barbisan|
|Grantee:||Guilherme Ribeiro Romualdo|
|Home Institution:||Instituto de Biociências (IBB). Universidade Estadual Paulista (UNESP). Campus de Botucatu. Botucatu , SP, Brazil|
Recently, hepatic fibrosis and carcinogenesis have been linked to altered expression of several miRNAs. In contrast, studies indicate that coffee drinking is associated with a 40% lower risk of developing fibrosis or liver cancer. Thus, the present project will evaluate whether caffeine alone or associated with trigoneline and/or chlorogenic acid (CGA): (A) attenuates fibrosis-associated hepatocarcinogenesis (FAH) in mice; (B) attenuates the fibrosis in co-culture of malignant hepatocytes and hepatic stellate cells (HSCs) and (C) alters the miRNAs global expression profile and the expression of target genes of differentially expressed miRNAs in the liver. In vivo: for FAH model, C3H/He male mice will receive single dose of diethylnitrosamine (intraperitoneal [i.p.], 50 mg/Kg body weight [b.w.]) at the postnatal day (PND) 14. From the 8th to 16th week, the animals receive three weekly doses of carbon tetrachloride (i.p., initial dose 0.25 mg/Kg b.w., and maximum dose 1.25 mg/Kg b.w.). In addition, from the 7th to the 17th week (1x/day, 5x/week), animals will receive by gavage caffeine (50 mg/Kg b.w./day); caffeine and trigonelline (50 and 25 mg/Kg b.w./day , respectively); caffeine and CGA (50 and 25 mg/Kg b.w./day); caffeine, trigonelline and CGA (50, 25 and 25 mg/Kg b.w./day) or just vehicle (distilled water). The animals will be euthanized at 17th week. Liver samples will be collected for histological analysis: histopathological evaluation (HE), analysis of collagen content (Picrosirius Red) and immunohistochemistry to activated HSCs (desmin and ±-SMA), macrophage (F4/80), preneoplastic liver lesions (CK8/18), cell proliferation (Ki-67) and apoptosis (cleaved caspase-3) markers. Other liver samples will be used for global miRNA expression (nCounter miRNA Expression Assay). After identification of differentially expressed miRNAs, bioinformatics prediction of targets genes will be performed. The list of predicted genes will be used to evaluate the expression of target mRNAs by RT-qPCR. In vitro: Co-culture of malignant hepatocytes (C3A) and human HSCs (LX-2) will be performed. Initially, the activation of HSCs will be performed by exposing them to lipopolysaccharide (LPS). In sequence, the co-culture will be exposed to caffeine (10, 5 or 2.5 mM), trigonelline (5, 2.5 or 25.1 mM) or CGA (5, 2.5 or 25.1 mM) alone or in combination for 24 to 48 hours. Samples of co-culture will be collected for histological analysis: HE, Picrosirius Red and immunofluorescence for HSCs counting. Other samples will be used for collagen I determination and quantification of pro-inflammatory cytokines (IL-6, TNF-±, TGFb-1, I-17A and IL-23) by ELISA. Moreover, the gene expression of collagen ±1(I), ±-SMA, TGF²-1, MMP-2, MMP-9, MMP-1 and TIMP-1 will be evaluated by qRT-PCR. The data will be statistically analyzed by ANOVA or Kruskal-Wallis test. Significant differences will be assumed when p<0.05.