4-methylumbelliferone (4MU) is an inhibitor of hyaluronan deposition and an active substance of hymecromone, a choleretic and antispasmodic drug. 4MU reported to be anti-fibrotic in mouse models; however, precise mechanism of action still requires further investigation. Here we describe the cellular and molecular mechanisms of 4MU action on CCl₄-induced liver fibrosis in mice using NGS transcriptome, Q-PCR and immunohistochemical analysis. Collagen and hyaluronan deposition were prevented by 4MU. The CCl₄ stimulated expression of Col1a and αSMA were reduced, while the expression of the ECM catabolic gene Hyal1 was increased in the presence of 4MU. Bioinformatic analysis identified an activation of TGF-beta and Wnt/beta-catenin signaling pathways, and inhibition of the genes associated with lipid metabolism by CCL₄ treatment, while 4MU restored key markers of these pathways to the control level. Immunohistochemical analysis reveals the suppression of hepatic stellate cells (HSCs) transdifferentiation to myofibroblasts by 4MU treatment. The drug affected the localization of HSCs and macrophages in the sites of fibrogenesis. CCl₄ treatment induced the expression of FSTL1, which was downregulated by 4MU. Our results support the hypothesis that 4MU alleviates CCl₄-induced liver fibrosis by reducing hyaluronan deposition and downregulating FSTL1 expression, accompanied by the suppression of HSC trans-differentiation and altered macrophage localization.

4-methylumbelliferone (4MU) is a well-known hyaluronic acid synthesis inhibitor and an approved drug for the treatment of cholestasis. In animal models, 4MU decreases inflammation, reduces fibrosis, and lowers body weight, serum cholesterol, and insulin resistance. It also inhibits tumor progression and metastasis. The broad spectrum of effects suggests multiple and yet unknown targets of 4MU. Aiming at 4MU target deconvolution, we have analyzed publicly available data bases, including: 1. Small molecule library Bio Assay screening (PubChemBioAssay); 2. GO pathway databases screening; 3. Protein Atlas Database. We also performed comparative liver transcriptome analysis of mice on normal diet and mice fed with 4MU for two weeks. Potential targets of 4MU public data base analysis fall into two big groups, enzymes and transcription factors (TFs), including 13 members of the nuclear receptor superfamily regulating lipid and carbohydrate metabolism. Transcriptome analysis revealed changes in the expression of genes involved in bile acid metabolism, gluconeogenesis, and immune response. It was found that 4MU feeding decreased the accumulation of the glycogen granules in the liver. Thus, 4MU has multiple targets and can regulate cell metabolism by modulating signaling via nuclear receptors.

In this study, we investigated the effects of NAD(P)H oxidase (NOX) inhibitor VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine) on the histamine-induced elevation of free cytoplasmic calcium concentration ([Ca²⁺]_i) and the secretion of von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVECs) and on relaxation of rat aorta in response to histamine. At 10 μM concentration, VAS2870 suppressed the [Ca²⁺]_i rise induced by histamine. Inhibition was not competitive, with IC50 3.64 and 3.22 μM at 1 and 100 μM concentrations of histamine, respectively. There was no inhibition of [Ca²⁺]_i elevation by VAS2870 in HUVECs in response to the agonist of type 1 protease-activated receptor SFLLRN. VAS2870 attenuated histamine-induced secretion of vWF and did not inhibit basal secretion. VAS2870 did not change the degree of histamine-induced relaxation of rat aortic rings constricted by norepinephrine. We suggest that NOX inhibitors might be used as a tool for preventing thrombosis induced by histamine release from mast cells without affecting vasorelaxation.

Second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) triggers Ca²⁺ release via two-pore channels (TPCs) localized in endolysosomal vesicles. The aim of the present work is to evaluate the role of TPCs in the action of norepinephrine (NE), angiotensin II (AngII), vasopressin (AVP), and 5-hydroxytriptamine (5-HT) on free cytoplasmic calcium concentration ([Ca²⁺]_i) in smooth muscle cells (SMCs) isolated from rat aorta and on aorta contraction. To address this issue, the NAADP structural analogue and inhibitor of TPCs, NED 19, was applied. We have demonstrated a high degree of colocalization of the fluorescent signals of cis-NED 19 and endolysosmal probe LysoTracker in SMCs. Both cis- or trans-NED 19 inhibited the rise of [Ca²⁺]_i in SMCs induced by 100 μM NE by 50–60%. IC₅₀ for cis- and trans-NED 19 were 2.7 and 8.9 μM, respectively. The inhibition by NED 19 stereoisomers of the effects of AngII, AVP, and 5-HT was much weaker. Both forms of NED 19 caused relaxation of aortic rings preconstricted by NE, with relative potency of cis-NED 19 several times higher than that of trans-NED 19. Inhibition by cis-NED 19 of NE-induced contraction was maintained after intensive washing and slowly reversed within an hour of incubation. Cis- and trans-NED 19 did not cause decrease in the force of aorta contraction in response to Ang II and AVP, and only slightly relaxed aorta preconstricted by 5-HT and by KCl. Suppression of TPC1 in SMCs with siRNA caused a 40% decrease in [Ca²⁺]_i in response to NE, whereas siRNA against TPC2 did not change NE calcium signaling. These data suggest that TPC1 is involved in the NE-stimulated [Ca²⁺]_i rise in SMCs. Inhibition of TPC1 activity by NED 19 could be the reason for partial inhibition of aortic rings contraction in response to NE.

Autologous macrophage transfer is an emerging platform for cell therapy. It is anticipated that conventional macrophage reprogramming based on ex vivo polarization using cytokines and ligands of TLRs may enhance the therapeutic effect. We describe an alternative approach based on small interfering RNA (siRNA) knockdown of selected molecular cues of macrophage polarization, namely EGR2, IRF3, IRF5, and TLR4 in Raw264.7 monocyte/macrophage cell line and mouse-bone-marrow-derived macrophages (BMDMs). The impact of IRF5 knockdown was most pronounced, curtailing the expression of other inflammatory mediators such as IL-6 and NOS2, especially in M1-polarized macrophages. Contrary to IRF5, EGR2 knockdown potentiated M1-associated markers while altogether abolishing M2 marker expression, which is indicative of the principal role of EGR2 in the maintenance of alternative phenotypes. IRF3 knockdown suppressed M1 polarization but upregulated Arg 1, a canonical marker of alternative polarization in M1 macrophages. As anticipated, the knockdown of TLR4 also attenuated the M1 phenotype but, akin to IRF3, significantly induced Arginase 1 in M0 and M1, driving the phenotype towards M2. This study validates RNAi as a viable option for the alteration and maintenance of macrophage phenotypes.