Knockdown of Hyaluronan synthase 2 suppresses liver fibrosis in mice via induction of transcriptomic changes similar to 4MU treatment

Hepatic fibrosis remains a significant clinical challenge due to ineffective treatments. 4-methylumbelliferone (4MU), a hyaluronic acid (HA) synthesis inhibitor, has proven safe in phase one clinical trials. In this study, we aimed to ameliorate liver fibrosis by inhibiting HA synthesis. We compared two groups of mice with CCl4-induced fibrosis, treated with 4-methylumbelliferone (4MU) and hyaluronan synthase 2 (HAS2) targeting siRNA (siHAS2). The administration of 4MU and siHAS2 significantly reduced collagen and HA deposition, as well as biochemical markers of hepatic damage induced by repeated CCl4 injections. The transcriptomic analysis revealed converging pathways associated with downstream HA signalling. 4MU- and siHAS2-treated fibrotic livers shared 405 upregulated and 628 downregulated genes. These genes were associated with xenobiotic and cholesterol metabolism, mitosis, endoplasmic reticulum stress, RNA processing, and myeloid cell migration. The functional annotation of differentially expressed genes (DEGs) in siHAS2-treated mice revealed attenuation of extracellular matrix-associated pathways. In comparison, in the 4MU-treated group, DEGs were related to lipid and bile metabolism pathways and cell cycle. These findings confirm that HAS2 is an important pharmacological target for suppressing hepatic fibrosis using siRNA. Overall design: The aim of this study was to validate HAS2 as an important pharmacological target in hepatic fibrosis. Furthermore we also wanted to elucidate key biological processes responsible for 4MU's antifibrotic capacity In this study, we utilized a CCL4-induced murine model of fibrosis to execute a parallel investigation wherein HAS2 was pharmacologically inhibited by 4MU or its mRNA expression was repressed by siRNA. Using Next generation RNAseq we compared the transcriptomic signatures of 4MU or siRNA mediated inhibition of HAS2.This experimental design allowed us to delineate common and unique pathways downstream of HA signalling transduction. Mice were divided into 2 main groups based whether they received CCL4 IP injection. Mice which received CCL4 were assigned fibrotic whilst mice wich did not receive CCL4 were assigned healthy (fibrotic or healthy). Each group comprised 4 subgroups based on the treatment received. In the fibrotic mice group we had the model (CCl4 only), 4MU adminstered (4MU.CCL4), siHAS adminstered (siHAS2.CCL4) siLuc adminstered (siLuc.CCL4/ non-targeting control). For the healthy group we also had 4 groups. Instead of CCL4, all mice in this group were injected with olive oil into the IP namely: control(oil+ tail vein injection of PBS), 4MU administered, siHAS2 adminstered and siLuc adminstered.all siRNA and PBS (for control mice ) was injected intravenously via the tail vein