Role of apoptosis in nutritional model of hepaticsteatosis in mice

Abstract

Aims Nonalcoholic steatohepatitis (NASH) is among the mostcommon causes of liver diseases, but the mechanisms involved itspathogenesis remains unclear. Oxidative stress from lipid peroxida-tion and insulin resistance have been recognized as major mechanismsfor NASH development. Recent studies suggested that cellular apop-tosis may also be an important factor responsible for liver injury inthe progression of NASH.The aims of this study were to examine theoccurrence and extent of apoptosis in a dietary model of NASH andto investigate the possible mechanisms of apoptosis in NASH.Materials and Methods Age matched C57BL6 mice (8–10weeks) were randomized to receive a methionine and choline defi-cient (MCD) diet or the MCD diet supplemented with choline bitar-trate and DL-methionine (control diet) for 2 days, 5 days, 10 day, 3weeks and up to 8 weeks respectively. Liver tissues were fixed in 10%buffered neutral formalin for routine histology and for cell apoptosisassay by TUNEL staining. Protein expression of apoptosis-relatedgene such as Fas, Fas-ligand, Bax, Bad, and BID were detected byWestern blot and mRNA expressions were assessed by real time RT-PCR. Caspase 3 activity was measured with ApoAlert Caspase-3 Col-orimetric Assay.Results In concomitant with the severity of liver histology, apop-totic cells were frequently detected in mice feeding MCD diet for 10days as compared with the control diet. Apoptotic cells were further increased in mice feeding MCD diet for 3 weeks and for 8 weeks.Expression of apoptosis-related genes including Bad, Bax, Fas andFas-L were significantly upregulated in mice fed MCD diet duringthis study course. Significant Bid cleavage was detected between day5 and week 8. Coincidently, caspase 3 activity was also significantlyenhanced from day 10 to week 8.Conclusions Significantly more hepatic apoptosis was provoked inmice feeding MCD diet, and the apoptotic response was correlatedto the development of steatohepatitis. Upregulation of proapoptoticproteins, especially Bax, Bad, Fas, and Fas-L, as well as activatedcaspase 3 may be the underlying mechanisms for apoptotic reaction.