S‐Nitrosylation of <scp>NOTCH1</scp> Regulates Mesenchymal Stem Cells Differentiation Into Hepatocyte‐Like Cells by Inhibiting Notch Signalling Pathway

The differentiation of mesenchymal stem cells (MSCs) into hepatocyte-like cells (HLCs) is considered one of the most promising strategies for alternative hepatocyte transplantation to treat end-stage liver disease. To advance this method, it is crucial to gain a deeper understanding of the mechanisms governing hepatogenic differentiation. The study demonstrated that suppression of the intracellular domain release of the Notch pathway receptor via the γ-secretase inhibitor N-[(3, 5-difluorophenyl)acetyl]-L-alanyl-2-phenylglycine-1, 1-dimethylethyl ester (DAPT) significantly promotes the expression of hepatocyte-related genes and proteins in HLCs. Increased expression of intracellular inducible NO synthase (iNOS) during differentiation led to elevated endogenous NO production. Biotin switch assays revealed a gradual increase in S-nitrosylation (SNO)-NOTCH1 and a decrease in overall NOTCH1 expression during hepatogenic differentiation. The addition of the exogenous NO donor S-nitrosoglutathione (GSNO) and the SNO inhibitor dithiothreitol (DTT) further demonstrated that the elevated expression of SNO-NOTCH1 promotes the differentiation of MSCs into mature hepatocytes. Briefly, our results fully demonstrated that the modification of the extracellular domain of NOTCH1 by NO, leading to the formation of SNO-NOTCH1, significantly promotes hepatogenic differentiation by inhibiting the Notch signalling pathway. Our study highlights the critical role of SNO-NOTCH1 in regulating the Notch signalling pathway and offers new insights into the mechanisms driving this differentiation process.