Liver B Cells Promotes MASLD Progression via the Apelin/APLNR System

Abstract

Aims: Investigate the role of the apelin/APLNR axis in metabolic dysfunction-associated steatotic liver disease (MASLD), focusing on the progression from metabolic dysfunction-associated simple steatotic liver (MASS) to metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis, with emphasis on liver B cells. Methods: Serum samples from MASLD patients and liver tissues from hepatocellular carcinoma patients were collected to measure apelin and APLNR protein expression. C57BL/6J mouse models of varying MASLD stages were developed using a high-fat diet and CCl₄. RNA sequencing was used to study the apelin/APLNR axis's regulatory functions in the Raji B cell line. Results: Bioinformatic and clinical analyses show that apelin and APLNR are up-regulated in MASLD, correlating with disease severity. Animal models demonstrate that apelin and ML221 injections affect liver steatosis, inflammation, and fibrosis. Sequencing and RT-PCR in Raji cells indicate that the apelin/APLNR axis promotes the expression of inflammatory cytokines and extracellular matrix molecules. Conclusion: The apelin/APLNR axis is crucial in MASLD progression. Targeting this axis may offer therapeutic potential to modulate B cell function and mitigate MASLD advancement.

Keywords: B cells; apelin/APLNR; metabolic dysfunction-associated steatotic liver disease.

Figure 1

Apelin and APLNR expression were up-regulated in livers of MASLD and HCC patients. (A) Box plots showing APLNR expression in the GSE89632 and GSE185051 datasets. (B) NAS score and expression of APLNR in GSE225740 dataset. (C) Expression of APLN and APLNR in the GSE240729 dataset among the low liver fibrosis (fibrosis score<2) and high liver fibrosis groups. (D) Unpaired and paired expression of APLN in the TCGA-LIHC dataset. (E) The concentration of apelin in the serum of MASLD patients (n=74) and healthy individuals (n=14) measured by ELISA. (F) Correlation analysis between serum apelin concentration and serum TG, TC, and LDL-C in MASLD patients (n=74). (G) Representative IHC staining results of apelin and APLNR in human HCC and peritumor tissues. The statistical analysis of the staining scores is shown on the right (n=30). The P values are calculated by two-tailed unpaired Welch's t-test (A-C, E), Mann-Whitney U test (D, G), two-tailed paired Student's t-test (D), or Spearman's correlation (B, F). *P < 0.05; **P < 0.01; ***P < 0.001

Figure 2

Apelin and APLNR expression were up-regulated in livers of HFD mice. (A) Body weight of C57BL/6J mice on ND diet for 15 weeks and HFD diet for 15 weeks, recorded every 3 weeks (n=6). (B) After 15 weeks of feeding, the bilateral epididymal white adipose tissue (EWAT) of the mice was isolated and weighed (n=6). (C-D) Blood was collected from the mouse eye, and serum was separated for the measurement of glucose, TC, and TG using assay kits (n=6). (E) Mouse livers were isolated and photographed. (F) Liver tissues were cryosectioned and stained with an Oil Red O staining kit to visualize and image lipid content under a microscope. (G) Paraffin-embedded sections of the liver tissue were prepared and stained using a Masson's trichrome staining kit to assess the level of fibrosis. (H) RNA was extracted from mouse liver tissues using the Trizol method, and RT-PCR was conducted to determine mRNA levels of Apln and Aplnr (n=3). (I) Total protein was extracted from mouse liver tissues, and Western blot was used to determine the expression of apelin and APLNR (n=6). (J) Immunohistochemistry was performed on liver cryosections to detect apelin and APLNR. The statistical analysis of the staining scores is shown on the right (n=6). The P values are calculated by two-tailed unpaired Student's t-test (A, C, D), two-tailed unpaired Welch's t-test (B, H), Mann-Whitney U test (J). *P < 0.05; **P < 0.01; ***P < 0.001

Figure 3

The expression of apelin and APLNR increased with the progression of MASLD. (A) Schematic diagram of the establishment of mouse models for different stages of MASLD (n=4). (B) Body weight of ND, HFD, HFMCD, and HFMCD+CCl₄ groups of mice after 9 weeks of feeding (n=4). (C) After 9 weeks of feeding, the liver and EWAT were isolated from the mice and weighed (n=4). (D) Images of the liver from each group of mice. (E) RNA was extracted from livers of each group, and RT-PCR was used to determine the mRNA expression of Apln and Aplnr (n=4). (F) Total protein was extracted from each group's mouse liver, and Western blot was used to determine the expression of apelin protein (n=4). (G) Paraffin-embedded liver sections were used for IHC detection of apelin and APLNR. The statistical analysis of the staining scores is shown on the right (n=4). The P values are calculated by one-way ANOVA followed by Tukey's multiple comparisons tests (B-C, E, G). *P < 0.05; **P < 0.01; ***P < 0.001; ns represents no significance

Figure 4

Apelin/APLNR exacerbated hepatic lipid accumulation and fibrosis in MASLD mice. (A) HE staining of liver paraffin sections shows pathological changes in the liver after intraperitoneal injection of apelin or ML221. (B) Oil Red O staining of mouse liver frozen sections. (C) Masson's trichrome staining of mouse liver paraffin sections. (D-F) Blood was collected from each group of mice after 9 weeks of feeding, and serum was separated for the measurement of TC, TG, and glucose using assay kits (n=4). The P values are calculated by one-way ANOVA followed by Tukey's multiple comparisons tests (D-F). *P < 0.05; **P < 0.01; ***P < 0.001

Figure 5

Apelin/APLNR affected the progression of MASLD by regulating liver B cells. (A) Immunofluorescence detection of CD19⁺ B cells in liver paraffin sections of C57BL/6J mice fed with HFD for 15 weeks and controls. (B) Flow cytometry analysis of CD19⁺ and APLNR⁺ cells in human peripheral blood PBMCs. (C-D) Immunofluorescence detection of CD19 and APLNR in livers of mice fed with HFD and ND for 15 weeks. (E) After intraperitoneal injection of apelin, ML221, or saline, single-cell suspensions of livers from different model mice were prepared, and immune cells were isolated and analyzed by flow cytometry for CD45, CD19, and APLNR. The P values are calculated by one-way ANOVA followed by Tukey's multi comparison test (E). *P < 0.05; **P < 0.01; ***P < 0.001

Figure 6

Apelin/APLNR could promote B cell migration and activation. (A-B) Cultured Raji cells were transfected with a plasmid to overexpress APLNR and were treated with apelin recombinant protein in the culture medium. Transcriptome sequencing was conducted along with the control group (n=3). The heatmap and volcano plot of differentially expressed genes are shown. (C) Dot plot of GSEA pathway analysis. (D) GSEA pathway analysis showing extracellular matrix organization pathway and degradation of the extracellular matrix pathway. (E) After overexpressing APLNR in Raji cells through plasmid transfection and adding LPS or apelin to the culture medium, cells were collected for RNA extraction, and RT-PCR was performed to measure the mRNA levels of AHNAK, COL6A3, IL10, CD86, IRF9, and RFX2. The P values are calculated by one-way ANOVA followed by Tukey's multi comparison test (E). *P < 0.05; **P < 0.01; ***P < 0.001