Fig 1: Summary figure representing the signaling cascades. We suppose that activation by PRRs triggers RIG1 expression. In addition, FFA-accumulation, and ROS formation leads to Keap1-NRF2-ARE activation, whereby it is likely that many biological processes take place in the organism in a parallel manner (for example NFκB activation is known to induce ROS formation). Abbreviations: PRRs: pattern recognition receptors; FFAs: free fatty acids; ROS: reactive oxygen species; Keap1: Kelch-like ECH-associated protein 1; ARE: antioxidant response elements.
Fig 2: Semi-quantitative immunohistochemical analysis of RIG-1, pNRF2, and SOCS3 and protein expressions. Images show representative cases of our NAFLD cohort. (A–C) We detected stronger cytoplasmic RIG1 expression in NASH than in NAFL. (A–C) Boxplots depict that the percentage of RIG1 stained tissue to total tissue is higher in NASH than in NAFL; ** p = 0.01. (A) In the IHC images, representative cytoplasmic RIG1 staining in NAFL (B) and NASH (C) is shown (asterisk). Further, the ratio of pNRF2 stained nuclei to total nuclei was higher in NASH than in NAFL; *** p = 0.001 (D). Representative pNRF2 IHC images of NAFL (E) and NASH (F) demonstrate stronger nuclear staining (arrows) in NASH than in NAFL. Also, cytoplasmic SOCS3 expression was stronger in NASH than in NAFL (G–I). Boxplots depict the ratio of the area with cytoplasmic SOCS3 immunopositivity to the total area with increased SOCS3 expression in NASH compared with NAFL; *** p < 0.001 (G). Representative SOCS3 IHC images of NAFL (H) and NASH (I) show representative areas of cytoplasmic SOCS3 immunopositivity (asterisk). Differences between groups were analyzed using Mann–Whitney U tests; bold lines inside the box plot represent median levels. Results are significant at * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001; bars = 200 μm.
Fig 3: SOCS3 protein expressions are positively associated with RIG1, Ki67, syntaxin, and SGLT2 expressions. (A,C) High degree of correlations were detected to RIG1 (r = 0.765; p < 0.001) and syntaxin (r = 0.717; p < 0.001). (B,D) The degree of correlations to Ki67 and to SGLT2 were significant but moderate (r = 0.463; p < 0.001 and r = 0.333; p = 0.010). Pearson’s correlation coefficient was used to evaluate protein expression results with significance set at p = 0.05.
Fig 4: pNRF2 protein expressions are positively associated with RIG1, syntaxin, Ki67, M30, and SOCS3 expressions. (A,B) We observed a high degree of correlations between pNRF2 and syntaxin (r = 0.604; p < 0.001) and Ki67 (r = 0.645; p < 0.001). (C,D) The degree of correlation between pNRF2 and M30 was moderate (r = 0.431; p = 0.001) and between pNRF2 and SOCS3 was high (r = 0.560; p < 0.001). Pearson’s correlation coefficient was used to evaluate protein expression results with significance set at p ≤ 0.05. High degree of correlation: Pearson’s correlation coefficient, also known as Pearson’s r = 0.5–1.0; moderate degree of correlation: Pearson’s r = 0.3–0.49; low degree of correlation: Pearson’s r < 0.3.
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