Background: Runt-related transcription factor (RUNX1) regulates inflammation in non-alcoholic steatohepatitis (NASH). Methods: We performed in vivo targeted silencing of the RUNX1 gene in liver sinusoidal endothelial cells (LSECs) by using vegfr3 antibody tagged immunonano-lipocarriers encapsulated RUNX1 siRNA (RUNX1 siRNA) in murine models of methionine choline deficient (MCD) diet-induced NASH. MCD mice given nanolipocarriers-encapsulated negative siRNA were vehicle, and mice with standard diet were controls. Results: Liver RUNX1 expression was increased in the LSECs of MCD mice in comparison to controls. RUNX1 protein expression was decreased by 40% in CD31-positive LSECs of RUNX1 siRNA mice in comparison to vehicle, resulting in the downregulation of adhesion molecules, ICAM1 expression, and VCAM1 expression in LSECs. There was a marked decrease in infiltrated T cells and myeloid cells along with reduced inflammatory cytokines in the liver of RUNX1 siRNA mice as compared to that observed in the vehicle. Conclusions: In vivo LSEC-specific silencing of RUNX1 using immunonano-lipocarriers encapsulated siRNA effectively reduces its expression of adhesion molecules, infiltrate on of immune cells in liver, and inflammation in NASH.

The lymphatic vascular system runs parallel to the blood vascular system, comprising a network of lymphatic vessels and secondary lymphoid organs. The intestinal lymphatic capillaries (lacteals) and the associated collecting vessels in the mesentery form the gut lymphatic system. The gut lymphatic vasculature comprises the longest-studied lymphatic vessel bed and plays a significant role in the uptake and transport of dietary fat, abdominal fluid balance, and gut immunosurveillance. Gut is closely connected to liver through the portal circulation. In several experimental and clinical studies, the 'gut-liver-axis' has been demonstrated to contribute to the pathogenesis of portal hypertension, liver cirrhosis, and its complications. Given a significant impact of gut health on the liver, in the current review, we highlight 'gut-liver axis' in context to the circulatory physiology of gut lymphatic vessels. Despite their paramount importance in maintaining fluid and immune homeostasis in the gut, gut lymphatic vessels remain one of the most understudied physiological systems in liver disease pathology. In the current review, we delineate the connections of gut lymphatics with abdominal fluid homeostasis and bacterial translocation in the pathogenesis of liver cirrhosis and portal hypertension. We describe mechanisms and factors that drive gut lymphangiogenesis and lymphatic vessel dysfunction during inflammation. The review also underscores the role of gut lymphatic endothelial cells in regulating gut and liver immunity. We finally discuss the prognostic and therapeutic prospects of studying gut lymphatic vessels in advanced liver cirrhosis.

Liver cirrhosis is a major health problem with multiple associated complications. The presently available drug delivery systems showed moderate site-specific delivery of antifibrotic molecules to the diseased liver; therefore, research on more effective and selective delivery systems in the context of liver cirrhosis remains a necessity in clinical investigation. The aim of the present study was to develop a peptide-based targeted nanocarrier to deliver an oligonucleotide to the hepatic sinusoidal and perivascular regions of the cirrhotic liver. We have synthesized and characterized a conformationally restricted targeted pentapeptide (RΔFRGD), which contains an unnatural amino acid, α,βdehydrophenylalanine (ΔF). The RΔFRGD self-assembled into spherical nanoparticles (NPs) and was characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Next, we investigated the delivery potential of the pentapeptide-based NPs to make a stable complex with a well-established small interference RNA and studied its site-specific delivery in experimental liver cirrhosis. We used siNR4A1 of the orphan nuclear receptor 4A1 (NR4A1), a well-known regulatory checkpoint for controlling liver fibrosis. Peptide NPs and their complex with siNR4A1 showed high biocompatibility against various mammalian cell lines. Hepatic tissue biodistribution analysis illustrated that targeted NPs predominantly accumulated in the cirrhotic liver compared to normal rats, specifically in sinusoidal and perivascular areas. A significant downregulation of the NR4A1 mRNA expression (−70%) andlower levels of the NR4A1/GAPDH ratio (−55%) were observed in the RΔFRGD-siNR4A1 nanocomplextreated group in comparison to the RΔFRGD-vehicle group (RΔFRGD-Veh) at the gene and protein levels, respectively. In addition, in vivo inhibition of NR4A1 produced a significant aggravation in hepatic fibrosis compared with siRNA-vehicle-treated rats (+41% in the MT stain). The novel pentapeptide-based targeted delivery system can be further evaluated and validated for therapeutic purposes in various pathological conditions.

Imidazo[1,2-b]pyridazine compounds are a new class of promising lead molecules to which we have incorporated polar nitro and amino moieties to increase the scope of their biological activity. Two of these substituted 3-nitro-6-amino-imidazo[1,2-b]pyridazine compounds (5c and 5h) showed potent acetylcholinesterase (AChE) inhibitory activity (IC50 40–50 nM), which we have previously reported. In this study, we wanted to test the biological efficacy of these compounds. Cytotoxicity assays showed that compound 5h mediated greater cell death with over 43% of cells dead at 100 μM and activation of caspase 3-mediated apoptosis. On the other hand, compound 5c mediated a dose-dependent decrease in cell proliferation. Both compounds showed cell cycle arrest in the G0/G1 phase and reduced cellular ATP levels leading to activation of adenosine monophosphate-activated protein kinase (AMPK) and enhanced mitochondrial oxidative stress. It has to be noted that all these effects were observed at doses beyond 10 μM, 200-fold above the IC50 for AChE inhibition. Both compounds also inhibited bacterial lipopolysaccharide-mediated cyclooxygenase-2 and nitric oxide release in primary rat microglial cells. These results suggested that the substituted imidazo (1,2-b) pyridazine compounds, which have potent AChE inhibitory activity, were also capable of antiproliferative, anti-migratory, and anti-inflammatory effects at higher doses.

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