Long chain non-coding RNA-EN_181 potentially contributes to the protective role of N-acetylcysteine against non-alcoholic fatty liver disease in mice

Background: N-acetylcysteine (NAC), a scavenger of free radicals, possesses strong capability to improve high fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in mice. However, the underlying mechanism(s) of NAC-ameliorated NAFLD are still not fully illustrated. This study was designed to clarify the involvement of long chain non-coding RNAs (lncRNAs) in the beneficial effects of NAC on NAFLD in HFD-induced obese mice. Methods: C57BL/6J mice were arranged into normal fat diet (10% energy from fat), HFD (45% energy from fat), and HFD plus NAC (2 g/L in drinking water) groups, respectively. After 14-week intervention, the role of NAC was determined by immunohistochemistry and biochemical indexes in both plasma and liver. The profiles of hepatic lncRNAs were analyzed by whole transcriptome sequencing, and were further verified by quantitative real-time polymerase chain reaction (qRT-PCR). Meanwhile, the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses were carried out to identify the functions of candidate lncRNAs. Furthermore, lncRNA-related ceRNA network was constructed in combination with miRNA and mRNA profiles by Cytoscape software. Results: NAC treatment obviously rescued the deleterious alterations induced by HFD, including body and liver weight, hepatic triglyceride (TG), plasma alanine aminotransferase (ALT), plasma aspartate transaminase (AST), and liver histomorphology (H&E and Oil red O staining). Through whole transcriptome sequencing technology, 52167 (50758 known and 1409 novel) lncRNAs were detectable in liver samples. Our data revealed that the expressions of 175 lncRNAs were significantly changed by HFD but reversed by NAC treatment through cross-comparison. Among those lncRNAs, 5 lncRNAs, including lncRNA-NONMMUT148902.1 (NO_902.1), lncRNA-XR_001781798.1 (XR_798.1), lncRNA-NONMMUT141720.1 (NO_720.1), lncRNA-XR_869907.1 (XR_907.1), and lncRNA-ENSMUST00000132181 (EN_181), were selected based on the absolute value of log2 fold change (FC) being greater than 4, P-value ＜ 0.01, and P-adjust ＜ 0.01. Further qRT-PCR identified that lncRNA-NO_902.1, lncRNA-XR_798.1, and lncRNA-EN_181 were dramatically decreased by HFD while rescued by NAC treatment, consistent with the RNA-sequencing. Finally, we further screened and constructed a ceRNA network, including lncRNA-EN_181, 3 miRNAs, and 13 mRNAs, which was associated with the beneficial role of NAC in NAFLD. Conclusions: This study identified for the first time that lncRNA-EN_181 might be a potential target in NAC-ameliorated NAFLD. The finding enhanced our understanding of biological mechanisms in the beneficial role of NAC.