N-n-Butyl haloperidol iodide ameliorates liver fibrosis and hepatic stellate cell activation in mice.

N-n-Butyl haloperidol iodide (F2) is a novel compound that has antiproliferative and antifibrogenic activities. In this study we investigated the therapeutic potential of F2 against liver fibrosis in mice and the underlying mechanisms. Two widely used mouse models of fibrosis was established in mice by injection of either carbon tetrachloride (CCl4) or thioacetamide (TAA). The mice received F2 (0.75, 1.5 or 3 mg·kg-1·d-1, ip) for 4 weeks of fibrosis induction. We showed that F2 administration dose-dependently ameliorated CCl4- or TAA-induced liver fibrosis, evidenced by significant decreases in collagen deposition and c-Jun, TGF-ß receptor II (TGFBR2), α-smooth muscle actin (α-SMA), and collagen I expression in the liver. In transforming growth factor beta 1 (TGF-ß1)-stimulated LX-2 cells (a human hepatic stellate cell line) and primary mouse hepatic stellate cells, treatment with F2 (0.1, 1, 10 µM) concentration-dependently inhibited the expression of α-SMA, and collagen I. In LX-2 cells, F2 inhibited TGF-ß/Smad signaling through reducing the levels of TGFBR2; pretreatment with LY2109761 (TGF-ß signaling inhibitor) or SP600125 (c-Jun signaling inhibitor) markedly inhibited TGF-ß1-induced induction of α-SMA and collagen I. Knockdown of c-Jun decreased TGF-ß signaling genes, including TGFBR2 levels. We revealed that c-Jun was bound to the TGFBR2 promoter, whereas F2 suppressed the binding of c-Jun to the TGFBR2 promoter to restrain TGF-ß signaling and inhibit α-SMA and collagen I upregulation. In conclusion, the therapeutic benefit of F2 against liver fibrosis results from inhibition of c-Jun expression to reduce TGFBR2 and concomitant reduction of the responsiveness of hepatic stellate cells to TGF-ß1. F2 may thus be a potentially new effective pharmacotherapy for human liver fibrosis.

Hypoxia Suppresses TGF-B1-Induced Cardiac Myocyte Myofibroblast Transformation by Inhibiting Smad2/3 and Rhoa Signaling Pathways.

BACKGROUND/AIMS: Hypoxia modulation of transforming growth factor (TGF)- ß-induced signaling during myofibroblast transformation is dependent on the specific cell type. The purpose of this study was to explore the effects of hypoxia on myofibroblast transformation of TGF-ß1-induced cardiomyocyte H9c2 cells. METHODS: H9c2 cells were cultured for intermittent hypoxia treatment and TGF-ß1 treatment. α-Smooth muscle actin (α-SMA) expression was examined by western blotting and immunofluorescence after treatment. To further explore the possible mechanism for this effect, the effects of hypoxia on three early TGF-ß-dependent signaling pathways, i.e. the Smad2/3, RhoA and mitogen-activated protein kinase (MAPK) pathways, were screened by western blotting. RESULTS: Intermittent hypoxia induced TGF-ß1 expression, but had no effect on α-SMA expression. Exogenous TGF-ß1 alone upregulated α-SMA expression in H9c2 cells in a concentration- and time-dependent manner. α-SMA expression declined with the duration of hypoxia after intermittent hypoxia and exogenous TGF-ß1 co-treatment. Phospho-JNK and phospho-p38 levels were not significantly altered after TGF-ß1 and hypoxia treatment. However, levels of phospho-ERK increased after TGF-ß1 treatment and continued to increase after hypoxia co-treatment. The activation of phospho-Smad2/3 and phospho-RhoA induced by TGFß1 was significantly reduced after hypoxia co-treatment. CONCLUSION: Hypoxia can inhibit TGF-ß1-induced H9c2 myofibroblast transformation, based on inhibition of α-SMA expression by suppressing signaling downstream of TGF-ß1, Smad2/3 and RhoA. It suggested that TGF-ß-mediated cardiomyocyte transformation is not involved in hypoxia-mediated fibrosis.

Phenotypic Switching of Vascular Smooth Muscle Cells in Atherosclerosis.

The medial layer of the arterial wall is composed mainly of vascular smooth muscle cells (VSMCs). Under physiological conditions, VSMCs assume a contractile phenotype, and their primary function is to regulate vascular tone. In contrast with terminally differentiated cells, VSMCs possess phenotypic plasticity, capable of transitioning into other cellular phenotypes in response to changes in the vascular environment. Recent research has shown that VSMC phenotypic switching participates in the pathogenesis of atherosclerosis, where the various types of dedifferentiated VSMCs accumulate in the atherosclerotic lesion and participate in the associated vascular remodeling by secreting extracellular matrix proteins and proteases. This review article discusses the 9 VSMC phenotypes that have been reported in atherosclerotic lesions and classifies them into differentiated VSMCs, intermediately dedifferentiated VSMCs, and dedifferentiated VSMCs. It also provides an overview of several methodologies that have been developed for studying VSMC phenotypic switching and discusses their respective advantages and limitations.

Effects of ghrelin on the intracellular calcium concentration in rat aorta vascular smooth muscle cells.

BACKGROUND/AIMS: Ghrelin has been regarded as a cardioprotective factor with complicated mechanisms. Whether ghrelin is vasodilative or vasoconstrictive in nature is controversial, and the effects of ghrelin on intracellular calcium concentration are still unclear. To explore the mechanisms involved in the vasoactive regulation of ghrelin at the cellular level, we investigated the effects of ghrelin on calcium concentrations in rat aorta vascular smooth muscle cells (VSMCs). METHODS: We obtained VSMCs via cell culture and stained the cells with Furo-2 AM. Western blotting was used to verify growth hormone secretagogue receptor (GHS-R1a) expression in VSMCs. The intracellular calcium variations affected by ghrelin and the interactions of ghrelin with angiotensin II (AngII), Sq22536, and potassium chloride (KCl) were observed using a calcium imaging and analysis system. RESULTS: Western blotting revealed good GHS-R1a expression in VSMCs. The most prominent finding in the present study was that ghrelin inhibited the AngII-induced increase in the calcium concentration. This inhibition was reversed by the adenylate cyclase inhibitor Sq22536 and the GHS-R1a antagonist (D-Lys(3))- GHRP-6. This finding revealed the potential vasodilative effects of ghrelin at the cellular level. We did not observe any effects of ghrelin on intracellular calcium concentrations in resting VSMCs or the increase of calcium concentration induced by KCl. CONCLUSION: Ghrelin inhibited the increase in the intracellular calcium concentration of rat aorta VSMCs induced by AngII, which may depend on the activation of the cAMP/PKA pathway.

Corrigendum: Sevoflurane Alleviates Myocardial Ischemia Reperfusion Injury by Inhibiting P2X7-NLRP3 Mediated Pyroptosis.

[This corrects the article DOI: 10.3389/fmolb.2021.768594.].

Sevoflurane Alleviates Myocardial Ischemia Reperfusion Injury by Inhibiting P2X7-NLRP3 Mediated Pyroptosis.

Myocardial ischemia is common in aging population. This study investigates the protective effect of Sevoflurane on myocardial ischemia reperfusion injury (MIRI) and its underlying mechanism. A total of 87 patients with a history of myocardial ischemia who underwent abdominal surgery with Sevoflurane general anesthesia were recruited in the study. The clinical data, blood pressure, heart rate, pressure-rate quotient (PRQ) and rate-pressure product (RPP) were recorded. Serum samples were collected and heart-type fatty acid binding protein (H-FABP), ischemia modified albumin (IMA), interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) were measured to observe whether Sevoflurane anesthesia had protective effect on myocardium. In addition, MIRI rats and hypoxia/reoxygenation (H/R) injury cell model was established using neonatal rat ventricular myocytes (NRVM). Rats or NRVM were pretreated with sevoflurane for 45min before hypoxia. The mRNA expression of purinergic receptor-7 (P2X7) and NLR family pyrin domain containing 3(NLRP3) were examined. The protein expression of P2X7, NLRP3, apoptosis-associated speck-like protein (ASC), cysteine aspartic acid specific protease-1(Caspase-1), Gasdermin-D (GSDMD), Bcl-2 Associated X Protein (Bax), B-cell lymphoma-2 (Bcl-2) in myocardial tissue and cells were evaluated. The serum contents of IL-1ß, IL-18, Malondialdehyde (MDA), Superoxide dismutase (SOD), Lactate dehydrogenase (LDH), Creatine kinase (CK), and Creatine kinase isoenzymes (CK-MB) were measured. The cellular localization and fluorescence intensity of NLRP3 and ASC in cells were detected. It was found that the secretion of IL-1ß and IL-18 decreased in the patients. After I45 min/R3h in SD rats and H3h/R1h in NRVM, the protein expressions of P2X7, NLRP3, ASC, Caspase-1 and GSDMD were increased, the release of IL-1ß, IL-18, CK, CK-MB, LDH and MDA were increased, and SOD activity was decreased. Sevoflurane treatment inhibited the high expression of P2X7, NLRP3, ASC, Caspase-1 and GSDMD, inhibited the release of LDH, CK,CK-MB and MDA in cells, and improved the activity of SOD, indicating that Sevoflurane alleviated the damage of MIRI of rats and H/R of NRVM, and had myocardial protective effect. Taken together, our study suggests that Sevoflurane inhibited the expression of IL-1ß, IL-18 and GSDMD by inhibiting the P2X7-NLRP3 signaling pathway. It reduced the H/R injury of cardiomyocytes and protected the cardiac function by regulating inflammatory reaction and pyroptosis.

The protective effect of the natural compound hesperetin against fulminant hepatitis in vivo and in vitro.

BACKGROUND AND PURPOSE: Liver diseases are mostly accompanied by inflammation and hepatocyte death. Therapeutic approaches targeting both hepatocyte injury and inflammation are not available. Natural compounds are considered as potential treatment for inflammatory liver diseases. Hesperetin, a flavonoid component of citrus fruits, has been reported to have anti-inflammatory properties. The aim of this study was to evaluate the cytoprotective and anti-inflammatory properties of hesperetin both in vitro and in models of fulminant hepatitis. EXPERIMENTAL APPROACH: Apoptotic cell death and inflammation were induced in primary cultures of rat hepatocytes by bile acids and cytokine mixture respectively. Apoptosis was quantified by caspase-3 activity and necrosis by LDH release. The concanavalin A (ConA) and D-galactosamine/LPS (D-GalN/LPS) were used as models of fulminant hepatitis. Liver injury was assessed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, liver histology and TUNEL assay and inflammation by inducible NOS (iNOS) expression. KEY RESULTS: Hesperetin blocked bile acid-induced apoptosis and cytokine-induced inflammation in rat hepatocytes. Moreover, hesperetin improved liver histology and protected against hepatocyte injury in ConA- and D-GalN/LPS-induced fulminant hepatitis, as assessed by TUNEL assay and serum AST and ALT levels. Hesperetin also reduced expression of the inflammatory marker iNOS and the expression and serum levels of TNFα and IFN-γ, the main mediators of cell toxicity in fulminant hepatitis. CONCLUSION AND IMPLICATIONS: Hesperetin has anti-inflammatory and cytoprotective actions in models of acute liver toxicity. Hesperetin therefore has therapeutic potential for the treatment of inflammatory liver diseases accompanied by extensive hepatocyte injury, such as fulminant hepatitis.