Effects of autophagy inhibition by chloroquine on hepatic stellate cell activation in CCl4-induced acute liver injury mouse model.

BACKGROUND AND AIM: Hepatic stellate cells (HSCs) activation, a critical event in liver fibrosis, has been recently shown to be related to autophagy. Determine whether chloroquine (CQ) could affect (i) the activation of HSC in vivo and (ii) the hepatic damage in a mice acute liver injury model. METHODS: The acute liver injury was induced in BALB/c mice by carbon tetrachloride (CCl4 group); 24 h before and after CCl4 administration animals were treated by CQ (CCl4  + CQ group). As control, mice treated by olive oil were considered. After 48 h from CCl4 /olive oil administration, blood samples, liver tissues, and HSCs were harvested for analysis. RESULTS: In vivo, CQ attenuates CCl4 -induced acute liver damage as evidenced by (i) the reduction of liver enlargement, (ii) the reduction of liver swelling and necrosis also supported by a certain decrease of circulating transaminases level, and (iii) the reduction of liver fibrosis evaluated by collagen deposition and α-sma protein expression. In HSCs isolated from CQ treated group, we observed the inhibition of autophagy proved by the increase in p62 protein and the decrease of lc3 protein. In addition, CQ reduced the expression of the HSCs activation markers α-sma/collagen-I and down-regulated the expression of the proliferative marker ki67. CONCLUSION: The autophagy attenuation exerted by CQ together with the reduction of the expression of the proliferation marker in HSCs can lessen the acute liver damage potentially opening the way to novel therapeutic approaches for hepatic fibrosis.

Optimization of the isolation procedure and culturing conditions for hepatic stellate cells obtained from mouse.

Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC's undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco's Modified Eagle's Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 µM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture.