Research ArticleLiver Bid suppression for treatment of fibrosis associated with non-alcoholic steatohepatitis
Graphical abstract
Introduction
Metabolic non-alcoholic fatty liver disease (NAFLD) has become one of most common forms of chronic liver disease worldwide. Growing evidence demonstrates that patients within the NAFLD spectrum who have progressed to non-alcoholic steatohepatitis (NASH), in particular NASH and fibrosis, are at a higher risk for disease-related morbidity and mortality [1], [2], [3]. The development of novel, effective therapies for patients with more advanced forms of the disease are urgently needed [4]. Hepatocellular apoptosis is emerging as an important, if not critical, mechanism contributing to the progression of fibrotic NASH [5]. In hepatocytes, certain lipids, such as free fatty acids (FFAs), can upregulate the expression of cell death receptors, as well as induce organelle stress, in particular mitochondrial dysfunction (commonly referred to as lipotoxicity), which may lead to apoptosis [5]. Fibrosis is based on the activation of hepatic stellate cells (HSCs) and experimental studies suggest that hepatocyte apoptosis and the resulting apoptotic bodies are important activators of HSCs [6]. Indeed, apoptotic bodies from hepatocytes are engulfed by HSCs, stimulating the fibrogenic activity of these cells; DNA fragments from apoptotic hepatocytes can also activate HSCs [6]. Notably, attenuation of hepatocyte apoptosis by inhibition of caspases, in particular caspase 3 and 8, reduces fibrogenesis in animal models of NASH [7], [8] thus establishing the proof of concept for anti-apoptotic NASH therapy.
BID is a BH3-only BCL-2 family member that is cleaved by caspase-8 into its active form, truncated BID (tBID), which links the extrinsic and intrinsic apoptosis pathways. tBID formation is crucial for the amplification of apoptotic death signals in cells like hepatocytes (called type 2 cells), where activation of the mitochondrial pathway is essential for cell death to occur. BID, however, is dispensable for apoptosis in most other cell types (called type 1 cells). We recently demonstrated that hepatocyte-specific Bid deficient mice are resistant to the lethal effects of Fas activation in vivo [9]. Here we tested the hypothesis that selective ablation of BID in hepatocytes can effectively reduce liver injury and fibrosis associated with NASH. To test this hypothesis in this study, we used two different approaches: Bid knockdown in wild-type (WT) mice via RNAi technology, and hepatocyte-specific Bid deficient (BidΔhep) mice, both animal groups were fed a chorine-deficient L-amino acid defined (CDAA) diet.
Section snippets
siRNA screening
Ten different stealth RNAiTM siRNAs were synthesized from Life Technologies (Life Technologies, Carlsbad, USA). Bid target sequences;
Bid1: 5′-AGCACAUCACAGACCUGCUGGUGUU-3′
Bid2: 5′-CCGCUCCUUCAACCAAGGAAGAAUA-3′
Bid3: 5′-AGGAAGAAUAGAGCCAGAUUCUGAA-3′
Bid4: 5′-CAGAUUCUGAAAGUCAGGAAGAAAU-3′
Bid5: 5′-GAAAGUCAGGAAGAAAUCAUCCACA-3′
Bid6: 5′-CAGCUAGCCGCACAGUUCAUGAAUG-3′
Bid7: 5′-GAGAACGACAAGGCCAUGCUGAUAA-3′
Bid8: 5′-GCCAUGCUGAUAAUGACCAUGCUGU-3′
Bid9: 5′-CACCAUCUUUGCUCCGUGAUGUCUU-3′
Bid10:
Bid suppression in NASH mice using RNAi technology
In order to achieve efficient gene knockdown using RNAi technology, we initially concentrated our efforts on identifying and selecting a target sequence. For this study we synthesized 10 different target sequences and checked the liver Bid mRNA expression level using a low dose RNAi treatment (4 mg/kg) for the short time point at day 2 or a high dose RNAi treatment (7 mg/kg) for the long time point at day 14. We selected three siRNAs – Bid3, Bid4, and Bid10 – from the short time point, and then
Discussion
The main findings of the present study relate to the role of RNA-based therapy to modulate hepatic Bid, a key pro-apoptotic protein that triggers mitochondrial dysfunction during lipotoxicity, as a potential novel therapeutic strategy for NASH. Bid siRNA suppression via next generation siRNA technology lead to a reduction of fibrosis associated with a reduction in liver inflammation and apoptotic cell death. Bid knockdown in hepatocytes, rather than NPC in the liver, is crucial for the
Financial support
This work was supported by NIH grants U01AA022489 and DK082451 to AEF. UCSD Neuroscience Core for microscopy is supported by a grant P30 CA23100.
Conflict of interest
The authors state no conflict of interest, except Xavier De Mollerat Du Jeu and Andronikou Nektaria are employees of Life Technologies
Authors’ contributions
A.Eguchi designed and performed experiments, analyzed data, and wrote the manuscript; X.De Mollerat Du Jeu and A.Nektaria synthesized siRNA delivery vesicles; C. Johnson performed experiments; A.E.F. conceived the idea, helped design the experiments, provided the funding for the study, and helped draft and critically revise the manuscript.
Acknowledgements
We thank the UCSD Neuroscience Core, especially Jennifer Santini for microscopy assistance.
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