Linking Antibodies Against Apolipoprotein A-1 to Metabolic Dysfunction-Associated Steatohepatitis in Mice

Figures

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  Figure 1. Overview of the experimental approach.

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  Figure 2. Aggravation of hepatic steatosis in CDAHFD mice by AAA-1 immunization. (a) No difference in liver weight was observed between the two groups after the PI protocol. Results are presented as individual values, with bars indicating the mean and standard deviation (b) The abnormal accumulation of fat in hepatic steatosis appears on H&E staining as clear vacuoles within hepatocytes, displacing the nucleus to the cell’s periphery. These vacuoles, formed by lipid droplets that do not stain with H&E, appear as clear spaces in the pink-stained cytoplasm. Representative H&E staining of liver sections from CDAHFD mice immunized with AAA-1s or Ctl IgG. ** p= 0.007. Mann–Whitney (M-W) test. The results are presented as median and interquartile range. Yellow arrows indicate liver structures. Abbreviations, IF: inflammatory foci; PT: portal triad; CV: centrilobular vein; MaS: macrosteatosis; MiS: microsteatosis.

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  Figure 3. Comparison of inflammatory status between AAA-1-immunized and control IgG-treated mice. (a) Effects of AAA-1-immunization on plasma level of cytokines compared to control IgG-treated mice. * p = 0.035, ** p = 0.007, # p = 0.032, M-W test. The median and interquartile range are indicated by red bars. (b) Effects of AAA-1 immunization on hepatic production of cytokines mRNAs compared to control IgG-treated mice. The results are expressed as a fold change in AAA-1 values relative to control IgG values. * p = 0.04, p = 0.68 (IL-6), p = 0.9 (TNF-α), M-W test. Non significant values are denoted as “ns”. Quantification of mRNA transcripts was performed by NanoString technology.

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  Figure 4. AAA-1s induced a distinct expression of CK-18 in mouse liver tissue compared to the circulating plasma protein levels. (a) Mice receiving AAA-1s show higher levels of CK-18 protein compared to those receiving control IgG. * p = 0.03, M-W test. (b) Representative microphotographs of dissected livers stained for CK-18. Area positive for CK-18 expressed as a percentage and assessed with Qupath software (Belfast, Northern Ireland, version 0.5.0) in ten aleatory selected images. **** p < 0.0001, M-W test. Yellow arrows indicate liver structures and CK-18 filaments. (c) Western blot analysis of immunized mice liver tissue lysates revealed a trend toward a decrease in CK-18 levels in AAA-1-immunized mice compared to control mice, p = 0.06, M-W test. The relative protein expression of CK-18, indicated by the red band, is measured in comparison to the constitutive protein GAPDH, represented by the green band. Abbreviations, PT: portal triad; CV: centrilobular vein; CK-18: Cytokeratin 18.

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  Figure 5. Identification of 32 significantly upregulated genes in AAA-1-immunized mice compared to control IgG-immunized mice through differential gene expression analysis. Total RNA was purified from the cryopreserved liver of CDAHFD mice immunized with AAA-1s or Ctl IgG. Quantification of mRNA transcripts was performed by NanoString nCounter hybridization using 20 ng purified RNA and the pre-designed mouse Fibrosis Profiling Panel (NanoString Technologies, Seattle, WA, USA). A |log fold change| > 1.5 and a p-value < 0.05 were defined as differentially expressed mRNAs via R software screening (version 3.56.2, R Foundation for Statistical Computing, Vienna, Austria). The diagram illustrates the gene function even though genes in the inflammation group can indirectly relate to fibrosis, angiogenesis, and extracellular matrix remodeling through their role in immune regulation.

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  Figure 6. Liver fibrosis levels remain unchanged at the microscopic level. Hepatic fibrosis, marked by excess collagen in the liver, is shown by Sirius red fast green staining where collagen fibers stain bright red while cytoplasmic components stain green. The fibrotic zone is magnified in the right figure. Representative Sirius red fast green staining of liver sections from CDAHFD mice immunized with AAA-1s or control IgG. The results are presented as median and interquartile range. The amount of liver fibrosis was unaffected. p = 0.7, M-W test. Non significant values are denoted as “ns”. Arrows indicate liver structures Abbreviations, PT: portal triad; CV: centrilobular vein; CF: collagen fiber.