Fig. 4: Metrnl regulates HSCs activation in vitro.

A The level of Metrnl in the cultured medium, secreted by LX-2 cells overexpressing Metrnl (OE-Met) and knockdown Metrnl (sh-Met) induced by adenovirus, was measured using ELISA (n = 3 per group). Representative Western blot images and quantification analysis of FN, Col4, and α-SMA expression in LX-2 cells transfected with adenovirus-mediated Metrnl knockdown (B), rMetrnl treatment (200 ng/mL) (C), and adenovirus-mediated Metrnl overexpression (D) combined with TGF-β (2 ng/mL) treatment for 48 h (n = 3 blots). E Volcano plot analysis was performed on the RNA sequencing results of the indicated samples. A stringent set of genes (p < 0.05) was identified in Metrnl overexpressing LX-2 cells compared to vector cells treated with TGF-β for 48 h (n = 3 per group). F Heat map of representative, differentially expressed genes associated with matrix remodeling, inflammation, and fibrosis is shown. The analysis includes genes involved in fibrosis-related signaling pathways (vector and OE-Met, both with TGF-β, n = 3 per group). G Changes in relative mRNA levels of genes related to matrix remodeling, inflammation, and fibrosis were observed in Metrnl overexpressing LX-2 cells. These mRNA levels were normalized to that of GAPDH (n = 3 per group). H Relative mRNA levels of genes related to fibrosis in primary HSCs isolated from C57BL/6 mice and treated with TGF-β (2 ng/mL) for 48 h. These mRNA levels were normalized to that of GAPDH (n = 3 per group). All data are mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.