Preliminary Exploration Of Non-alcoholic Fatty Liver Disease Pathogenesis And Serum Biomarker

Background and aim:Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver diseases worldwide with unclear mechanism. In order to better understand pathogenesis of NAFLD as well as to provide new insights into NAFLD diagnosis and treatment, three main areas of NAFLD were focused in this article, including protective effect and corresponding mechanism of omega 3 fatty acids on NAFLD, lncRNA profiling in NAFLD mice model and diagnositic role of complement C3 in NAFLD.Methods:In this stusy, NAFLD animal model and cellular model were constructed according to previous reports that mice fed with high fat diet and cells treated with free fatty acids were classical NAFLD models in vivo and in vitro, respectively. These NAFLD models were evaluated through oil red staining and serum chemical analysis.Effect of various medications including omega 3 fatty acids on hepatocytes in vitro, were tested by Oil red O staining as well as flow cytometry. Level of autophagy was assessed with Western Blot by autophagy flux LC3 II/LC3 I, and further detected by immunefluroscense. Expression levels of certain genes were identified with quantitive realtime PCR.In NAFLD lncRNA profiling, liver sample of NAFLD mouse were harvested and total RNA were extracted, purified, hybridized and then scanned accrding to manufacture’s protocals.In cross-sectional study, baseline demographic data, serum biochemical analysis and liver sonography were collected and logistic analysis was adopted in quantifying association between NAFLD and other factors, including Complement C3.Results:In this study, results showed that free fatty acids (FFA) induced lipid accumulation and apoptosis in hepatocytes. Inhibition or genetic defect of autophagy increases lipid accumulation induced by FFA, while induction acts inversely. Omega 3 fatty acids reduced lipid accumulation and inhibited apoptosis induced by FFA. Omega 3 fatty acids induced autophagy by down-regulating Stearoyl-CoA Desaturasel expression in hepatocytes.In lncRNA profiling,89 up-regulated and 177 down-regulated mRNAs were identified, together with 291 dysregulated lncRNAs. Bioinformatic analysis of these RNAs has categorized these RNAs into pathways including arachidonic acid metabolism, circadian rhythm, linoleic acid metabolism, Peroxisome Proliferator Activated Receptor signaling pathway, sphingolipid metabolism, steroid biosynthesis, tryptophan metabolism and tyrosine metabolism were compromised. Quantative PCR of 9 mRNAs and 8 lncRNAs of interest (named as fatty liver related lncRNA, FLRL) was conducted and testified previous microarray results. Several lncRNAs, such as FLRL1, FLRL6 and FLRL2 demonstrated to be likely a key player in circadian rhythm targeting Per3, Per2 and Arntl, clock-associated proteins. While FLRL8, FLRL3 and FLRL7 showed their potential role in PPAR signaling pathway through interaction with Fabp5, Lpl and Fads2.In cross-sectional study among adults who took their annual health examinations at Zhenhai Lianhua Hospital during 2014, a total of 7540 participants (5069 men and 2471 women) were included; NAFLD patients had higher serum complement C3 levels (P< 0.001), and these levels were positively associated with both NAFLD prevalence and severity (P< 0.001). The association remains among lean and metabolic syndrome-free participants and further verified by multivariable regression analysis that showed that serum complement C3 was independently associated with risk for NAFLD (Odds Ratio =5.231; 95% Confidence Interval:3.169-8.635).Conclusion:Current study revealed protective effect of Omega 3 fatty acids on hepatocyte against lipotoxicity through induction of autophagy, and a complete lncRNA and corresponding mRNA profiling in NAFLD as well as positive association of serum complement C3 level with prevalence and severity of NAFLD in a large Chinese cohort, providing new insight into pathogenesis of NAFLD as well as diagnosis and prognosis.