RESUMO
Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic disease worldwide, consisting of a broad spectrum of diseases such as simple steatosis (NAFL), non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. Hepatic inflammation plays a key role in the pathophysiology of NAFLD. Inflammatory mediators such as cytokines and chemokines are considered as contributing factors to NAFLD development and progression. In the present study, we aimed to investigate the inflammatory protein signatures as predictive disease-specific markers for non-alcoholic fatty liver disease (NAFLD). This cross-sectional study included healthy control (n = 64), NAFL (n = 109), and NASH (n = 60) human subjects. Serum concentrations of various cytokines and chemokines were evaluated using sensitive multiplex assays. We used principal component analysis (PCoA) to reveal distinct differences in the levels of cytokines and chemokines between each of the study groups. Further, a random forest classification model was developed to identify the panel of markers that could predict diseases. The protein-protein network analysis was performed to determine the various signaling pathways associated with the disease-specific panel of markers. Serum concentrations of TNF-α, IL-1ß, IL-1ra, G-CSF, PDGF-BB, MCP-1, MIP-1a, MIP-1b, RANTES, eotaxin, IL-8 and IP-10 were significantly increased in NASH group as compared to control group. Furthermore, serum concentrations of IL-9 and IL-13 were significantly lower in the NASH group, whereas IL-2 levels were significantly decreased in the NAFL group when compared to the control group. PCoA results demonstrated statistically significant differences in cytokines and chemokines between each of the study groups (PERMANOVA p = 0.001; R2 = 0.102). RANTES, IL-1ra, MIP-1b, IL-2, and G-CSF could differentiate the NAFL group from the controls; G-CSF, IL-1ra, TNF-α, RANTES, and IL-9 could differentiate the NASH group from the controls; and G-CSF, IL-9, IL-13, eotaxin, and TNF- α could differentiate the NASH group from the NAFL group. Our protein-protein network revealed that these markers are involved in cytokine-cytokine receptor interaction, Th1 and Th2 cell differentiation, TNF, chemokine, JAK/STAT, P13K/Akt, TLR, NOD-like receptor, NF-kB, and adipocytokine signaling pathways which might be responsible for disease pathogenesis. Our study findings revealed a set of distinct cytokine and chemokine markers and they might be considered as biomarkers in distinguishing NASH from NAFL. Future multicentre studies with larger sample size are recommended to determine the potential utility of these panels of markers.
RESUMO
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a complex disease which is characterized by the deposition of fats in the hepatocytes. Further, it progresses to nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma. The increasing prevalence of NAFLD urges to find the non-invasive predictive biomarkers. In this study, we sought to determine increased BMP8B levels as predictors for the progression of NAFLD. METHODS: In the present cross-sectional study, circulatory BMP8B levels were measured in healthy controls (n = 56), NAFL patients (n = 72) and NASH patients (n = 77) by using an ELISA kit. Human hepatic BMP8B mRNA expression was measured in the liver tissue of control and NASH patients. In addition, BMP8B expression was confirmed by immunohistochemistry analysis. Furthermore, hepatic BMP8B mRNA expression was measured in wild type (WT) mice, WT mice fed with choline deficient high fat diet (WT+CDHF), iNOS (inducible nitric oxide synthase) knockout (iNOS-/-) mice, iNOS-/- fed with CDHF diet (iNOS-/-+CDHF). RESULTS: Increased circulatory BMP8B levels and BMP8B mRNA expression in hepatic tissue were significantly higher in NASH patients as compared with the control subjects. BMP8B expression was increased parallel to the fibrosis score in the hepatic tissues of NASH patients. It was observed that increased BMP8B levels have shown a significant positive correlation between aspartate aminotransferase (r = 0.31, p = 0.005), alanine aminotransferase (r = 0.23, p = 0.045), APRI (r = 0.30, p = 0.009), and Fib-4 score (r = 0.25, p = 0.036) in NASH patients. BMP8B has maintained a significant association with NASH and shown high sensitivity (92.91%) and specificity (92.73%) in NASH patients. Furthermore, increased BMP8B mRNA expression levels were observed in iNOS-/-+CDHF mice. CONCLUSION: Our study findings confirmed that BMP8B increases with the severity of the disease and BMP8B shows potential as a non-invasive predictive biomarker to identify NAFLD progression. However, future studies should investigate circulatory BMP8B levels in a large number of patients and also its impact on liver during NAFLD progression.
