m6A Modification Destabilizes Prss8 and Activates Hepatic Stellate Cells via TLR4-Mediated Inflammatory Responses

The 6-methyladenine (m6A) modification plays a major role in various diseases. Serine protease 8 (<i>Prss8</i>) contributes to the initiation and progression of liver fibrosis (LF). However, the mechanism by which the m6A modification of <i>Prss8</i> induces hepatic stellate cells (HSCs) activation in the LF is unclear. This study focused on exploring the contribution of <i>Prss8</i> m6A modification to the pathogenesis of LF. First, primary hepatic parenchymal cells (hepatocytes) and HSCs were isolated from a mouse model of LF, and a coculture of these two types of cells was used as the object of study. Then, real-time fluorescence quantitative PCR, methylated RNA immunoprecipitation, and Western blotting were used to test the expression levels of <i>Prss8</i> mRNA and protein, <i>Prss8</i> m6A modification, <i>Collagen I</i>, <i>α-SMA</i>, and <i>TLR4</i>. Finally, the expression levels of inflammatory markers were measured via an enzyme-linked immunosorbent assay. Compared with the control group, the model group presented significantly lower <i>Prss8</i> mRNA and protein levels in hepatocytes but greater levels of <i>Prss8</i> m6A modification; moreover, the expression of HSC activation markers and the <i>TLR4</i>, <i>IL-1β</i>, and <i>IL-18</i> proteins was significantly elevated. Mutation of the <i>Prss8</i> m6A modification site led to upregulation of <i>Prss8</i> mRNA and protein and decreased levels of m6A modification, <i>TLR4</i>, <i>IL-1β</i>, and <i>IL-18</i>. Furthermore, mutation of the <i>Prss8</i> m6A modification site increased the stability of <i>Prss8</i> mRNA. Rescue experiments confirmed the regulatory link between <i>Prss8</i> m6A modification and <i>TLR4</i>. Overall, <i>Prss8</i> m6A modification decreases the stability of its mRNA, promoting <i>TLR4</i>-mediated inflammatory cascades and leading to excessive activation of HSCs. Targeting <i>Prss8</i> m6A modification is a promising therapeutic strategy for LF.