BACKGROUND:Mesenchymal stem cells (MSCs)-derived exosomes have been previously demonstrated to promote tissue regeneration in various animal disease models. This study investigated the protective effect of exosome treatment in carbon tetrachloride (CCl4)-induced acute liver injury and delineated possible underlying mechanism.METHODS:Exosomes collected from conditioned media of previously characterized human umbilical cord-derived MSCs were intravenously administered into male CD-1 mice with CCl4-induced acute liver injury. Biochemical, histological and molecular parameters were used to evaluate the severity of liver injury. A rat hepatocyte cell line, Clone-9, was used to validate the molecular changes by exosome treatment.RESULTS:Exosome treatment significantly suppressed plasma levels of AST, ALT, and pro-inflammatory cytokines, including IL-6 and TNF-α, in the mice with CCl4-induced acute liver injury. Histological morphometry revealed a significant reduction in the necropoptic area in the injured livers following exosome therapy. Consistently, western blot analysis indicated marked elevations in hepatic expression of PCNA, c-Met, Ets-1, and HO-1 proteins after exosome treatment. Besides, the phosphorylation level of signaling mediator JNK was significantly increased, and that of p38 was restored by exosome therapy. Immunohistochemistry double staining confirmed nuclear Ets-1 expression and cytoplasmic localization of c-Met and HO-1 proteins. In vitro studies demonstrated that exosome treatment increased the proliferation of Clone-9 hepatocytes and protected them from CCl4-induced cytotoxicity. Kinase inhibition experiment indicated that the exosome-driven hepatoprotection might be mediated through the JNK pathway.CONCLUSION:Exosome therapy activates the JNK signaling activation pathway as well as up-regulates Ets-1 and HO-1 expression, thereby protecting hepatocytes against hepatotoxin-induced cell death.

This study investigated whether xenotransplantation of human Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) reduces thioacetamide (TAA)-induced mouse liver fibrosis and the underlying molecular mechanism. Recipient NOD/SCID mice were injected intraperitoneally with TAA twice weekly for 6 weeks before initial administration of WJ-MSCs. Expression of regenerative and pro-fibrogenic markers in mouse fibrotic livers were monitored post cytotherapy. A hepatic stallate cell line HSC-T6 and isolated WJ-MSCs were used for in vitro adhesion, migration and mechanistic studies. WJ-MSCs were isolated from human umbilical cords by an explant method and characterized by flow cytometry. A single infusion of WJ-MSCs to TAA-treated mice significantly reduced collagen deposition and ameliorated liver fibrosis after 2-week therapy. In addition to enhanced expression of hepatic regenerative factor, hepatocyte growth factor, and PCNA proliferative marker, WJ-MSC therapy significantly blunted pro-fibrogenic signals, including Smad2, RhoA, ERK. Intriguingly, reduction of plasma fibronectin (pFN) in fibrotic livers was noted in MSC-treated mice. In vitro studies further demonstrated that suspending MSCs triggered pFN degradation, soluble pFN conversely retarded adhesion of suspending MSCs onto type I collagen-coated surface, whereas pFN coating enhanced WJ-MSC migration across mimicked wound bed. Moreover, pretreatment with soluble pFN and conditioned medium from MSCs with pFN strikingly attenuated the response of HSC-T6 cells to TGF-β1-stimulation in Smad2 phosphorylation and RhoA upregulation. These findings suggest that cytotherapy using WJ-MSCs may modulate hepatic pFN deposition for a better regenerative niche in the fibrotic livers and may constitute a useful anti-fibrogenic intervention in chronic liver diseases.

and Laboratory of Regenerative Medicine, Department of Medical Research, and Department of Surgery, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan. Received: June 24, 2015 Accepted: March 16, 2016 Address reprint request and correspondence to: Ying-Hsien Kao, Laboratory of Regenerative Medicine, Department of Medical Research, and Po-Huang Lee, Department of Surgery, E-Da Hospital, No. 1, Yida Road, Jiaosu Village, Yanchao District, Kaohsiung City 82445, Taiwan. Tel: +886-7-6151100 ext. 5059, Fax: +886-7-6150945, E-mail: danyhkao@gmail.com Tel: +886-2-23123456 ext. 65104, Fax: +886-2-23568810, E-mail: pohuang1115@ntu.edu.tw Original Article

Toll-like receptor-4 (TLR4) is known to influence growth and migration of hepatocellular tumors; however, its role in hepatoblastoma remains poorly understood. This study investigated the regulatory role of TLR4 in proliferation and chemoresistance of HepG2 hepatoblastoma cells. Treatment with lipopolysaccharide (LPS), a TLR4 agonist, was found to significantly upregulate TLR4 expression in HepG2 cells, but not in malignant Huh-7 and Sk-Hep1 hepatocellular carcinoma cells. Additionally, IL-6 enhanced LPS-induced TLR4 upregulation. LPS-stimulated TLR4 activation increased proliferation, nitric oxide synthase (NOS) expression, and NO production in HepG2 cells. Chemotherapeutic agents, cisplatin and doxorubicin, effectively inhibited TLR4 expression in HepG2 cells. Characterization of LPS-induced signaling activation and blockade with kinase inhibitors revealed the involvement of Akt and MAPK pathways in LPS-enhanced NO release from, and proliferation of HepG2 cells. Mechanistically, gene modifications as a result of TLR4 transfection and siRNA-mediated knockdown further demonstrated a crucial role for TLR4 in the regulation of NOS expression, cell proliferation, and chemoresistance in HepG2 cells. These findings suggest that targeting TLR4 expression and its cognate signaling may modulate proliferation and chemosensitivity in hepatoblastoma cells and serve as a potential therapeutic target.

AimsPropofol (PPF), an intravenous anesthetic agent, is previously reported to attenuate oxidative stress- and inflammation-induced endothelial cell dysfunction. This study investigated its effect on endothelial cell biology.Main methodsCultured human umbilical vein endothelial cells (HUVECs) were treated with PPF and subject to measurements for nitric oxide (NO) production, autophagy flux, signal transduction, migration, and in vitro angiogenesis.Key findingsNon-cytotoxic PPF treatment was found to significantly upregulate inducible nitric oxide synthase (NOS2) but downregulate constitutive NOS3 expression. It also potentiated LPS-induced ICAM-1 overexpression and NO overproduction. Mechanistically, the PPF-activated signal transduction in PI3K/Akt, ERK1/2, p38 MAPK, and JNK pathways were involved in the PPF-driven NO overproduction. PPF exhibited a stimulatory effect on autophagy flux by increasing expression of autophagy markers including mTOR, Beclin-1, Atg5, and LC3I/II, as well as a late endosomal indicator, Rab7. However, PPF appeared to antagonize the Rab7 upregulation by LPS. Functionally, PPF enhanced in vitro migratory and angiogenic capacities of HUVECs, but this enhancement was drastically abrogated by the presence of autophagy inhibitors, indicating a pro-angiogenic contribution of PPF-enhanced autophagy in cultured HUVECs.SignificanceOur findings support the notion that PPF enhances motility and angiogenic capacity of cultured HUVECs through an autophagy-involved regulatory mechanism.

This study investigated the pro-fibrogenic role of high mobility group box 1 (HMGB1) peptides in liver fibrogenesis. An animal model of carbon tetrachloride (CCl4)-induced liver fibrosis was used to examine the serum HMGB1 levels and its intrahepatic distribution. The increased serum HMGB1 levels were positively correlated with elevation of transforming growth factor-β1 (TGF-β1) and collagen deposition during fibrogenesis. The cytoplasmic distribution of HMGB1 was noted in the parenchymal hepatocytes of fibrotic livers. In vitro studies confirmed that exposure to hydrogen peroxide and CCl4 induced an intracellular mobilization and extracellular release of nuclear HMGB1 peptides in clone-9 and primary hepatocytes, respectively. An uptake of exogenous HMGB1 by hepatic stellate cells (HSCs) T6 cells indicated a possible paracrine action of hepatocytes on HSCs. Moreover, HMGB1 dose-dependently stimulated HSC proliferation, up-regulated de novo synthesis of collagen type I and α-smooth muscle actin (α-SMA), and triggered Smad2 phosphorylation and its nuclear translocation through a TGF-β1-independent mechanism. Blockade with neutralizing antibodies and gene silencing demonstrated the involvement of the receptor for advanced glycation end-products (RAGE), but not toll-like receptor 4, in cellular uptake of HMGB1 and the HMGB1-mediated Smad2 and ERK1/2 phosphorylation as well as α-SMA up-regulation in HSC-T6 cells. Furthermore, anti-RAGE treatment significantly ameliorated CCl4-induced liver fibrosis. In conclusion, the nuclear HMGB1 peptides released from parenchymal hepatocytes during liver injuries may directly activate HSCs through stimulating HSC proliferation and transformation, eventually leading to the fibrotic changes of livers. Blockade of HMGB1/RAGE signaling cascade may constitute a therapeutic strategy for treatment of liver fibrosis.