Objective: Sodium ⁺ / taurocholate cotransporting polypeptide (NTCP) is a membrane transporter affecting the enterohepatic circulation of bile acids (BAs). We aimed to evaluate NTCP’s roles in humans and animal models of liver fibrosis (LF). Design: Primary hepatic stellate cells (pHSCs) isolated from livers biopsies of patients with LF with different fibrosis grading were stained for NTCP. NTCP gene silencing, taurocholic acid (TCA), obeticholic acid (OCA), epigallocatechin gallate (EGCG) and HA-100 dihydrochloride (HA-100) were used as tools to modulate NTCP expression on human HSC line (LX2). BA trafficking/uptake were assessed extracellularly (LX2 culture medium) and intracellularly following treatment with/without NTCP neutralizing antibody. LF models of C57/BL6 mice of carbon tetrachloride (CCl ₄ ) and leptin-deficient ( Ob/Ob ) fed with high-fat diet ( Ob/Ob ^HFD ) were evaluated for pHSCs-NTCP expressions, metabolic and LF profiles following intraperitoneal injections of NTCP neutralizing antibody. Results: pHSCs from F3/F4-scored patients of LF exhibit threefold increased NTCP expressions compared with F0-scored patients (p<0.0001). Sorted-activated HSCs (LX2 ^αSMA+ ) showed high expressions of NTCP and high TCA uptake in vitro and triggered a further increase in their activations. This phenomenon was inhibited with NTCP small interfering RNA and the NTCP neutralizing antibody. Sorted LX2 ^NTCP+ (high alpha smooth muscle actin (αSMA)/high NTCP) cells showed high phosphorylated pathways of AKT/mTOR and protein kinase C (PKC) accompanied with a decrease in farnesoid X receptor expression. Moreover, LX2 ^NTCP+ cells treated with EGCG, OCA and PKC inhibitor HA-100 significantly decreased NTCP and αSMA. NTCP neutralizing antibody inhibited NTCP (less TCA uptake); it attenuated LF in both CCl ₄ and Ob/Ob ^HFD animal models with ameliorated metabolic profile. Conclusion: NTCP expression is linearly correlated with fibrosis severity. Modulated BA trafficking could be an important step in LF pathogenesis. Antagonising BA uptake may suggest a therapeutic strategy for preventing disease progression.

ˆCCL24 is a pro-fibrotic, pro-inflammatory chemokine expressed in several chronic fibrotic diseases. In the liver, CCL24 plays a role in fibrosis and inflammation, and blocking CCL24 led to reduced liver injury in experimental models. We studied the role of CCL24 in primary sclerosing cholangitis (PSC) and evaluated the potential therapeutic effect of blocking CCL24 in this disease. Multidrug resistance gene 2–knockout (Mdr2 ^–/– ) mice demonstrated CCL24 expression in liver macrophages and were used as a relevant experimental PSC model. CCL24-neutralizing monoclonal antibody, CM-101, significantly improved inflammation, fibrosis, and cholestasis-related markers in the biliary area. Moreover, using spatial transcriptomics, we observed reduced proliferation and senescence of cholangiocytes following CCL24 neutralization. Next, we demonstrated that CCL24 expression was elevated under pro-fibrotic conditions in primary human cholangiocytes and macrophages, and it induced proliferation of primary human hepatic stellate cells and cholangiocytes, which was attenuated following CCL24 inhibition. Correspondingly, CCL24 was found to be highly expressed in liver biopsies of patients with PSC. CCL24 serum levels correlated with Enhanced Liver Fibrosis score, most notably in patients with high alkaline phosphatase levels. These results suggest that blocking CCL24 may have a therapeutic effect in patients with PSC by reducing liver inflammation, fibrosis, and cholestasis.

Insulin resistance is a key risk factor in the progression of nonalcoholic fatty liver disease (NAFLD) and may lead to liver fibrosis. Natural killer (NK) cells are thought to exert an antifibrotic effect through their killing of activated hepatic stellate cells (HSCs). Here, we investigated how the interplay between NK cells and HSCs are modified by insulin resistance in NAFLD. Fresh peripheral blood NK cells (clusters of differentiation [CD]56^dim, CD16⁺) were collected from 22 healthy adults and 72 patients with NAFLD not currently taking any medications and without signs of metabolic syndrome. NK cells were assessed for insulin receptor expressions and cytotoxic activity when cultured in medium with HSCs. Fibrosis severities in patients with NAFLD were correlated linearly with elevated serum proinflammatory cytokine expression and insulin resistance severity. At the same time, fibrosis severities inversely correlated with insulin receptor expressions on NK cells as well as with their cytotoxic activities determined by CD107a by flow cytometry. NK cells from donors exhibiting severe fibrosis and insulin resistance exhibited significant mammalian target of rapamycin and extracellular signal‐regulated kinase depletion (through NK cell western blot quantitation), increased apoptosis, and failure to attenuate HSC activationin vitro. While exposure to insulin stimulated the cytotoxic activity of healthy NK cells, rapamycin prevented this effect and reduced NK insulin receptor expressions.Conclusion: Elevated insulin levels in F1 and F2 fibrosis enhances NK cell cytotoxic activity toward HSCs and prevents fibrosis progression by insulin receptors and downstream mammalian target of rapamycin and extracellular signal‐regulated kinase pathways. At more advanced stages of insulin resistance (F3 and F4 fibrosis), impaired NK cell activity rooted in low insulin receptor expression and or low serum insulin levels could further deteriorate fibrosis and may likely lead to cirrhosis development. (Hepatology Communications2018;2:285‐298)

The toll-like receptor-9 (TLR9) agonist cytosine phosphate guanine (CpG), activates hepatic stellate cells (HSCs) and mediates fibrosis. We investigated the TLR9 effects on lymphocyte/HSCs interactions. Liver fibrosis was induced in wild-type (WT) mice by intra-peritoneal carbon-tetrachloride (CCl₄) induction for 6 weeks. Fibrotic groups were intravenously treated by a vehicle versus CpG along last 2 weeks. Compared to vehicle-treated fibrotic WT, the in-vivo CpG-treatment significantly attenuated hepatic fibrosis and inflammation, associated with decreased CD8 and increased NK liver cells. In-vitro, co-cultures with vehicle-treated fibrotic NK cells increased HSCs proliferation (P<0.001) while their CpG-treated counterparts achieved a significant decrease. To investigate the role of lymphocytes, TLR9^-/- mice induced-hepatic fibrosis were used. Although TLR9^-/- mice manifested lower fibrotic profile as compared to their wild-type (WT) counterparts, senescence (SA-β-Gal activity) in the liver and ALT serum levels were significantly greater. In an adoptive transfer model; irradiated WT and TLR9^-/- recipients were reconstituted with naïve WT or TLR9^-/- lymphocytes. The adoptive transfer of TLR9^-/- versus WT lymphocytes led to increased fibrosis of WT recipients. TLR9^-/- fibrotic recipients reconstituted with TLR9^-/- or WT lymphocytes showed no changes in hepatic fibrosis severity or ALT serum levels. TLR9 activation had inconsistent effects on lymphocytes and HSCs. The net balance of TLR9 activation in WT, displayed significant anti-fibrotic activity, accompanied by CD8 suppression and increased NK-cells, activity and adherence to HSCs. The pro-fibrotic and pro-inflammatory properties of TLR9^-/- lymphocytes fail to activate HSCs with an early senescence in TLR9^-/- mice.

Gundelia (G.) tournefortii has antibacterial, anti-inflammatory, and hypolipemic effects. We evaluated the anticancer effect of G. tournefortii in an hepatocellular carcinoma (HCC) mouse model of an HCC cell line (Hep3B) injected into NOD.CB17-Prkdc-SCID/NCrHsD male mice. Tumorigenicity was assessed by tumor size, histology, serum α-fetoprotein (αFP), and glypican 3 (GPC3). HCC-related gene expression of the cell cycle (Cyclin-dependent kinase inhibitor 2A (CDNK2A)), proliferation (MKI67), and platelet-derived growth factor receptor α (PDGFA) were measured. HCC cell cycle alterations, apoptosis, and antioxidant markers in serum and liver following treatment with G. tournefortii were determined. Signaling pathways of liver p53 and phosphorylated PI3K, AKT, and mTOR were also evaluated. Results indicate a significant increase in tumor size in HCC animals associated with elevated αFP, GPC3, and MKI67. Tumor markers of p53 and phosphorylated AKT/PI3K/mTOR signaling pathway were diminished, with less proliferating cells and reduced PDGFRA gene expression following G. tournefortii infection. H&E staining showed a remarkable reduction in inflammatory lesions in HCC mice treated with G. tournefortii. This result was in line with a significant delay in the G2/M phase of HCC-primary hepatocytes by 1.39- to 2.4-fold and reduced HCC necrosis associated with inhibited CDNK2A gene expression. Antioxidant activity was significantly lower in the HCC mice than in the control group. Moreover, G. tournefortii inhibited the HCC formation of 3D MCTS spheroids. G. tournefortii treatment markedly restored antioxidant levels and displayed anticancer and antiproliferative effects and could be a promising cancer therapy.