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Circulating microRNAs (miRNAs) can be employed as biomarkers to diagnose liver and other diseases. Noninvasive approaches are needed to complement and improve the current strategies for screening for biomarkers liver cirrhosis. We determined whether the serum levels of miRNAs can distinguish between chronic hepatitis B (CHB) and CHB-induced cirrhosis (HBC) and investigated the potential mechanisms involved. We found that serum miR-27a was significantly up-regulated in HBC, distinguishing HBC from CHB and healthy controls (Ctrl) (P

// Guiju Tang 1 , Zhihong Weng 2 , Jun Song 3 and Yixiong Chen 3 1 Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 2 Department of Infectious Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China 3 Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China Correspondence to: Yixiong Chen, email: // Keywords : recombinant adeno-associated virus, Jagged1, hepatic fibrosis, hepatic stellate cells, epithelial-mesenchymal transition, Pathology Section Received : November 30, 2016 Accepted : June 02, 2017 Published : June 15, 2017 Abstract The role of the Notch ligand Jagged1 in hepatic fibrosis remains to be elucidated. In the current study, we investigated the role of Jagged1 in the activation of hepatic stellate cells (HSCs) and development of hepatic fibrosis in rats. In vitro , Jagged1 in HSCs was downregulated and upregulated by Jagged1 siRNA and pcDNA3.1 Jagged1, respectively. The levels of epithelial-mesenchymal transition (EMT) markers and HSC activation markers were assessed using western blot analysis. The proliferation and migration capacity of HSCs were assessed using 5-ethynyl-2′-deoxyuridine (EdU) incorporation and Transwell migration assays. In vivo , a recombinant adeno-associated virus type 1 (rAAV1) vector carrying Jagged1 shRNA (rAAV1-Jagged1-shRNA) was constructed and transferred to rat livers via the tail vein. Reversion of liver fibrosis and the effect of Jagged1 signaling on EMT were studied using pathological, immunohistochemical and immunofluorescence methods. Our findings revealed that downregulation and upregulation of Jagged1 inhibited and promoted, respectively, HSC activation. The migratory capacity of HSCs was markedly restrained by Jagged1 siRNA. Furthermore, downregulation of Jagged1 suppressed EMT in HSCs. rAAV1-Jagged1-shRNA was generated to treat CCl4-induced hepatic fibrosis in rats. Treatment with rAAV1-Jagged1-shRNA reversed hepatic fibrosis by decreasing EMT. The results of the present study suggest that inhibition of Jagged1 is a potential treatment to ameliorate liver fibrosis.

// Yinong Duan 1, * , Lei Lyu 1, * , Dandan Zhu 1, * , Jianxin Wang 2 , Jinling Chen 1 , Liuting Chen 1 , Chunzhao Yang 1 and Xiaolei Sun 1 1 Department of Pathogen Biology, School of Medicine, Nantong University, Nantong 226001, Jiangsu, People’s Republic of China 2 Laboratory Medicine Center, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, People’s Republic of China * These authors contributed equally to this work Correspondence to: Yinong Duan, email: yinongduan@aliyun.com Keywords: liver fibrosis, P40, p27, E2F1, Schistosoma japonicum Received: December 20, 2016 Accepted: April 06, 2017 Published: April 19, 2017 ABSTRACT The p27 protein plays a critical role in cell cycle arrest. Our previous studies have demonstrated that recombinant P40 protein from Schistosoma japonicum (rSjP40) could induce G1 phase arrest of cell cycle. We, therefore, attempted to observe the effect of rSjP40 on p27 promoter activity in LX-2 cells and to explore its potential mechanisms in this study. Using both Western blot and dual-luciferase reporter assay, we demonstrated that rSjP40 could enhance the expression of p27 in LX-2 cells. Results obtained using truncated fragments of p27 promoter showed that rSjP40 increased p27 promoter activity in LX-2 cells, mainly via some transcription factors that bind to the -1740/-873 region of p27 promoter. Further studies confirmed that the enhancement of p27 promoter activity induced by rSjP40 was related to E2F1 in LX-2 cells. Transfection of siRNA of E2F1 could also restore the effect of rSjP40 on expression of p27 and partially on α-SMA. Therefore, our study provided further insights into the mechanism by which rSjP40 induces LX-2 cell cycle arrest at G1 phase and inhibits HSC activation. Our results provide basis for future study of the blocking effect of rSjP40 in liver fibrosis.

// Xian-Yi Zeng 1, 2, 4, * , Yan-Qiong Zhang 2, 5, * , Xiao-Min He 2, 4, * , Lin-Yan Wan 1, 2, 3, 4, 5, * , Hu Wang 1, 2, 3, 4 , Yi-Ran Ni 2 , Jie Wang 2 , Jiang-Feng Wu 1, 2, 3, 4, 5 , Chang-Bai Liu 1, 2, 3, 4, 5 1 The Institute of Cell Therapy, China Three Gorges University, Yichang, 443000, China 2 Medical College, China Three Gorges University, Yichang, 443002, China 3 The First People’s Hospital of Yichang, Hubei Yichang, 443000, China 4 Hubei Key Laboratory of Tumor Microenvironment and Immunotherapy, China Three Gorges University, Yichang, 443002, China 5 Institute of Liver Diseases, China Three Gorges University, Yichang, 443002, China * These authors contributed equally to this work Correspondence to: Jiang-Feng Wu, email: wujiangfeng@ctgu.edu.cn Chang-Bai Liu, email: cbliu@ctgu.edu.cn Keywords: hepatic fibrosis, TGF-β, BMP-7, miR-23b/27b cluster, siRNA Received: September 24, 2016 Accepted: November 08, 2016 Published: November 15, 2016 ABSTRACT The imbalance between transforming growth factor β and bone morphogenetic protein 7 signaling pathways is a critical step in promoting hepatic stellate cell activation during hepatic fibrogenesis. Gremlin1 may impair the balance. Something remains unclear about the regulatory mechanisms of gremlin1 action on hepatic stellate cell activation and hepatic fibrosis. In the current study, gremlin1 overexpression promotes activation of hepatic stellate cells. Knockdown of gremlin1 with siRNAs suppresses hepatic stellate cell activation and attenuates hepatic fibrosis in rat model. Our results also show that miR-23b/27b cluster members bind to 3′-untranslated region of gremlin1 resulting in reduction of transforming growth factor β, α-smooth muscle actin and collagenI α1/2 gene expression. Our findings suggest that gremlin1 promotes hepatic stellate cell activation and hepatic fibrogenesis through impairment of the balance between transforming growth factor β and bone morphogenetic protein 7 signaling pathways. The miR-23b/27b cluster suppresses activation of hepatic stellate cells through binding gremlin1 to rectify the imbalance.

// Lei Liang 1, 2, * , Xue Yang 1, * , Yang Yu 1, 3, * , Xiaoyong Li 1, 2 , Yechen Wu 1, 2 , Rongyu Shi 1 , Jinghua Jiang 1 , Lu Gao 1 , Fei Ye 1 , Qiudong Zhao 1 , Rong Li 1 , Lixin Wei 1 , Zhipeng Han 1 1 Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, the Second Military Medical University, Shanghai, China 2 Medical College of Soochow University, Suzhou, China 3 Department of General Surgery, Yangpu Hospital, Tongji University School of Medicine, Shanghai, 200065, China * These authors contributed equally to this work Correspondence to: Zhipeng Han, email: hanzhipeng0311@126.com Lixin Wei, email: weilixin_smmu@163.com Keywords: hepatic fibrosis, hepatic stellate cells, chinese medicine, Babao Dan, toll-like receptor 4 Received: August 01, 2016 Accepted: October 12, 2016 Published: October 20, 2016 ABSTRACT Babao Dan (BBD), a traditional Chinese medicine, has been widely used as a complementary and alternative medicine to treat chronic liver diseases. In this study, we aimed to observe the protective effect of BBD on rat hepatic fibrosis induced by diethylnitrosamine (DEN) and explore it possible mechanism. BBD was administrated while DEN was given. After eight weeks, values of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicated that BBD significantly protected liver from damaging by DEN and had no obvious side effect on normal rat livers. Meanwhile, BBD attenuated hepatic inflammation and fibrosis in DEN-induced rat livers through histopathological examination and hepatic hydroxyproline content. Furthermore, we found that BBD inhibited hepatic stellate cells activation and proliferation without altering the concentration of lipopolysaccharide (LPS) in portal vein. In vitro study, serum from BBD treated rats (BBD-serum) could also significantly suppress LPS-induced HSCs activation through TLR4/NF-κB pathway. In addition, BBD-serum also inhibited the proliferation of HSCs by regulating TLR4/ERK pathway. Our study demonstrated that BBD may provide a new therapy strategy of hepatic injury and hepatic fibrosis.

// Yu You 1 , Jia-Xin Tan 1 , Hai-Su Dai 1 , Hao-Wei Chen 1 , Xue-Jun Xu 1 , Ai-Gang Yang 1 , Yu-Jun Zhang 1 , Lian-Hua Bai 1 , Ping Bie 1 1 Department of Hepatobiliary Surgery Institute, South Western Hospital, Third Military Medical University, Chongqing 400038, China Correspondence to: Ping Bie, email: bieping@medmail.com.cn Lian-Hua Bai, email: 870426131@qq.com Keywords: hepatocellular carcinoma, hepatic stellate cells, galectin-1, miRNA-22 Received: April 14, 2016 Accepted: July 10, 2016 Published: August 1, 2016 ABSTRACT Hepatic stellate cells (HSCs) induce immune privilege and promote hepatocellular carcinoma (HCC) by suppressing the immune system. On the other hand, galectin-1 and miRNA-22 (miR-22) are dysregulated in HCC and serve as prognostic indicators for patients. In this study, therefore, we measured galectin-1 and miR-22 expression in HSCs isolated from HCC tissues (Ca-HSCs), and in normal liver tissues (N-HSCs) as a control. We also investigated the apoptosis rate among T cells and the production of cytokines (IFN-γ and IL-10) in HSCs co-cultured with T cells. And we used immunohistochemical staining to tested for correlation between galectin-1 expression, CD3 expression and clinicopathological features in 162 HCC patients. Our results showed that galectin-1 expression was much higher in Ca-HSCs than in N-HSCs. Overexpression of galectin-1 promoted HSC-induced T cell apoptosis and cytokine production (IFN-γ and IL-10), while miR-22 expression inhibited it. Galectin-1 expression correlated negatively with miR-22 expression in HSCs. High galectin-1 and low CD3 expression levels were associated with poor prognosis in HCC patients. These results suggest that the immunosuppressive microenvironment promoted by HSC-derived galectin-1 in HCC can be inhibited by miR-22. Galectin-1 and miR-22 could potentially serve as prognostic markers and therapeutic targets in HCC.

// Nan Meng 1 , Min Xia 2 , Ya-Qi Lu 1 , Mi Wang 1 , Krishna M. Boini 2 , Pin-Lan Li 2 and Wang-Xian Tang 1 1 Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 2 Department of Pharmacology and Toxicology, Virginia Commonwealth University, School of Medicine, Richmond, VA, USA Correspondence to: Wang-Xian Tang, email: // Keywords : NLRP3 inflammasome, hepatic stellate cell, cathepsin B, Schistosoma J., Pathology Section Received : February 23, 2016 Accepted : June 01, 2016 Published : June 14, 2016 Abstract The major pathological changes during Schistosoma J. infection are characterized by granulomatous inflammation in the liver, a cellular immune response to schistosomal egg antigens. The molecular mechanisms initiating or promoting this schistosomal granulomatous inflammation remain poorly understood. In the present study, we first demonstrated that in mice infected with Schistosoma J . for 6 weeks exhibited increased levels of IL-1β in liver, a major product of NLRP3 inflammasomes and collagen deposition around the eosinophilic granuloma with Schistosoma J. eggs, which was substantially attenuated by caspase-1 inhibitor, YVAD. This activation of the NLRP3 inflammasome occurred in hepatic stellate cells (HSCs), as shown by a marked increase in co-localization of IL-1β with HSCs marker, desmin. Using isolated, cultured mouse HSCs, we further explored the mechanisms by which soluble egg antigen (SEA) from Schistosoma J. activates NLRP3 inflammasomes. SEA induced the formation and activation of NLRP3 inflammasomes, which was associated with both redox regulation and lysosomal dysfunction, but not with potassium channel activation. These results suggest that NLRP3 inflammasome activation in HSCs may serve as an early mechanism to turn on the inflammatory response and thereby instigate liver fibrosis during Schistosoma J. infection.

// Fujun Yu 1,* , Zhongqiu Lu 2,* , Kate Huang 3 , Xiaodong Wang 1 , Ziqiang Xu 4 , Bicheng Chen 5 , Peihong Dong 1 and Jianjian Zheng 5 1 Department of Infectious Diseases, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China 2 Emergency Department, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China 3 Department of Pathology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China 4 Institute of Organ Transplantation, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China 5 Key Laboratory of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China * These authors have contributed equally to this work Correspondence to: Jianjian Zheng, email: // Peihong Dong, email: // Keywords : microRNA-17-5p, Wnt/β-catenin pathway, hepatic stellate cells, Salvianolic acid B, Wnt inhibitory factor 1 (WIF1), Pathology Section Received : July 09, 2015 Accepted : November 22, 2015 Published : December 02, 2015 Abstract Aberrant Wnt/β-catenin pathway contributes to the development of liver fibrosis. MicroRNAs (MiRNAs) are found to act as regulators of the activation of hepatic stellate cell (HSC) in liver fibrosis. However, whether miRNAs activate Wnt/β-catenin pathway in activated HSCs during liver fibrosis is largely unknown. In this study, we found that Salvianolic acid B (Sal B) treatment significantly inhibited liver fibrosis in CCl 4 -treated rats, HSC-T6 cells and rat primary HSCs, resulting in the suppression of type Ⅰcollagen and alpha-smooth muscle actin. Also, Sal B suppressed HSC activation and cell proliferation in vitro . Interestingly, Sal B treatment induced the inactivation of Wnt/β-catenin pathway, with an increase in P-β-catenin and Wnt inhibitory factor 1 (WIF1). We demonstrated that the anti-fibrotic effects caused by Sal B were, at least in part, via WIF1. Moreover, our study revealed that miR-17-5p was reduced in vivo and in vitro after Sal B treatment. As confirmed by luciferase activity assays, WIF1 was a direct target of miR-17-5p. Notably, the suppression of HSCs induced by Sal B was almost inhibited by miR-17-5p mimics. Collectively, we demonstrated that miR-17-5p activates Wnt/β-catenin pathway to result in HSC activation through inhibiting WIF1 expression.

Lipopolysaccharide (LPS) is currently considered one of the major players in non-alcoholic fatty liver disease (NAFLD) pathogenesis and progression. Here, we aim to investigate the possible role of LPS-induced TNF-α factor (LITAF) in inducing a pro-inflammatory and pro-fibrogenic phenotype of non-alcoholic steatohepatitis (NASH). We found that children with NAFLD displayed, in different liver-resident cells, an increased expression of LITAF which correlated with histological traits of hepatic inflammation and fibrosis. Total and nuclear LITAF expression increased in mouse and human hepatic stellate cells (HSCs). Moreover, LPS induced LITAF-dependent transcription of IL-1β, IL-6 and TNF-α in the clonal myofibroblastic HSC LX-2 cell line, and this effect was hampered by LITAF silencing. We showed, for the first time in HSCs, that LITAF recruitment to these cytokine promoters is LPS dependent. However, preventing LITAF nuclear translocation by p38MAPK inhibitor, the expression of IL-6 and TNF-α was significantly reduced with the aid of p65NF-ĸB, while IL-1β transcription exclusively required LITAF expression/activity. Finally, IL-1β levels in plasma mirrored those in the liver and correlated with LPS levels and LITAF-positive HSCs in children with NASH. In conclusion, a more severe histological profile in paediatric NAFLD is associated with LITAF over-expression in HSCs, which in turn correlates with hepatic and circulating IL-1β levels outlining a panel of potential biomarkers of NASH-related liver damage. The in vitro study highlights the role of LITAF as a key regulator of the LPS-induced pro-inflammatory pattern in HSCs and suggests p38MAPK inhibitors as a possible therapeutic approach against hepatic inflammation in NASH.

// Adil El Taghdouini 1, * , Anita L. Sorensen 2, * , Andrew H. Reiner 2 , Mar Coll 3 , Stefaan Verhulst 1 , Inge Mannaerts 1 , Cristina I. Oie 4 , Bard Smedsrod 4 , Mustapha Najimi 5 , Etienne Sokal 5 , Aernout Luttun 6 , Pau Sancho-Bru 3 , Philippe Collas 2 , Leo A. van Grunsven 1 1 Liver Cell Biology Lab, Vrije Universiteit Brussel (VUB), Brussels, Belgium 2 Department of Molecular medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway 3 Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Barcelona, Spain 4 Department of Medical Biology, Vascular Biology Research Group, UiT, The Arctic University of Norway, Tromso, Norway 5 Universite Catholique de Louvain, Institut de Recherche Experimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy, Brussels, Belgium 6 Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven (KU Leuven), Leuven, Belgium * These authors have contributed equally to this work Correspondence to: Leo A. van Grunsven, e-mail: leo.van.grunsven@vub.ac.be Philippe Collas, e-mail: philippe.collas@medisin.uio.no Keywords: Pathology section, hepatic stellate cells, liver fibrosis, DNA methylation, epigenetics Received: July 08, 2015 Accepted: August 20, 2015 Published: August 31, 2015 ABSTRACT Background & Aims: Liver fibrogenesis – scarring of the liver that can lead to cirrhosis and liver cancer – is characterized by hepatocyte impairment, capillarization of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cell (HSC) activation. To date, the molecular determinants of a healthy human liver cell phenotype remain largely uncharacterized. Here, we assess the transcriptome and the genome-wide promoter methylome specific for purified, non-cultured human hepatocytes, LSECs and HSCs, and investigate the nature of epigenetic changes accompanying transcriptional changes associated with activation of HSCs. Material and methods: Gene expression profile and promoter methylome of purified, uncultured human liver cells and culture-activated HSCs were respectively determined using Affymetrix HG-U219 genechips and by methylated DNA immunoprecipitation coupled to promoter array hybridization. Histone modification patterns were assessed at the single-gene level by chromatin immunoprecipitation and quantitative PCR. Results: We unveil a DNA-methylation-based epigenetic relationship between hepatocytes, LSECs and HSCs despite their distinct ontogeny. We show that liver cell type-specific DNA methylation targets early developmental and differentiation-associated functions. Integrative analysis of promoter methylome and transcriptome reveals partial concordance between DNA methylation and transcriptional changes associated with human HSC activation. Further, we identify concordant histone methylation and acetylation changes in the promoter and putative novel enhancer elements of genes involved in liver fibrosis. Conclusions: Our study provides the first epigenetic blueprint of three distinct freshly isolated, human hepatic cell types and of epigenetic changes elicited upon HSC activation.

// Xinrui Han 1 , Chenzhi Hao 2 , Luyang Li 2 , Jianfei Li 2 , Mingming Fang 2 , Yuanlin Zheng 1 , Jun Lu 1 , Ping Li 3 and Yong Xu 2 1 Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, Xuzhou, China 2 Department of Pathophysiology, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Nanjing Medical University, Nanjing, China 3 Department of Gastroenterology, 2nd Affiliated Hospital to Nanjing Medical University, Nanjing, China Correspondence to: Jun Lu, email: lu-jun75@163.com Ping Li, email: pinglinjmu@gmail.com Yong Xu, email: yjxu@njmu.edu.cn Keywords: HDAC4, hepatic stellate cell, liver fibrosis, MRTF-A, miRNA Received: February 06, 2017 Accepted: April 26, 2017 Published: May 10, 2017 ABSTRACT Activation of hepatic stellate cells (HSCs) is a hallmark event during liver fibrogenesis. We have previously shown that the transcriptional modulator MRTF-A contributes to liver fibrosis by programming epigenetic activation of HSCs. In the present study we investigated the mechanism whereby MRTF-A expression is regulated in this process. We report here that MRTF-A protein levels, but not mRNA levels, were up-regulated in vivo in the livers of mice induced to develop hepatic fibrosis. Pro-fibrogenic stimuli (TGF-β and PDGF-BB) also activated MRTF-A expression post-transcriptionally in vitro in cultured HSCs. miR-206 bound to the 3′-UTR of MRTF-A presumably to inhibit translation. miR-206 levels were down-regulated in response to pro-fibrogenic stimuli in vivo and in vitro allowing MRTF-A proteins to accumulate. Mechanistically, histone deacetylase 4 (HDAC4) was induced by pro-fibrogenic stimuli and recruited to the miR-206 promoter to repress miR-206 transcription. HDAC4 stimulated MRTF-A expression and drove fibrogenesis in HSCs in a miR-206 dependent manner. Therefore, our data reveal an HDAC4-miR-206-MRTF-A axis that can play a potentially important role in HSC activation and liver fibrosis.

// Li Xuanfei 1, * , Chen Hao 1, * , Yi Zhujun 1 , Liu Yanming 2 , Gong Jianping 1 1 Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, P. R. China 2 Department of Gastroenterology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430041, Hubei, P. R. China * These authors contributed equally to this work Correspondence to: Gong Jianping, email: gongjianping11@126.com Keywords: hepatic fibrosis, hepatic stellate cell, imidazoline I 2 receptor, idazoxan, NF-E2-related factor 2 Received: November 10, 2016 Accepted: February 07, 2017 Published: February 18, 2017 ABSTRACT Liver fibrosis is a global health problem and its relationship with imidazoline I 2 receptor has not been reported. This study aimed to investigate the effects and underlying mechanisms of imidazoline I 2 receptor (I 2 R) inhibitor idazoxan (IDA) on carbon tetrachloride (CCl 4 )-induced liver fibrosis. In vivo liver fibrosis in mice was induced by intraperitoneally injections of CCl 4 for eight weeks, and in vitro studies were performed on activated LX2 cells treated with transforming growth factor-β (TGF-β). Our results showed that IDA significantly improved liver inflammation, ameliorated hepatic stellate cells activation and reduced collagen accumulation by suppressing the pro-fibrogenic signaling of TGF-β/Smad. Further investigation showed that IDA significantly balanced oxidative stress through improving the expressions and activities of anti-oxidant and detoxifying enzymes and activating Nrf2-the key defender against oxidative stress with anti-fibrotic potentials. Even more impressively, knock out of Nrf2 or suppression of Akt by perifosine (PE) eliminated the anti-oxidant and anti-fibrotic effects of IDA in vivo and in vitro , suggesting that Akt/Nrf2 constitutes a critical component of IDA's protective functions. Taken together, IDA exhibits potent effects against liver fibrosis via Akt-Nrf2-Smad2/3 signaling pathway, which suggests that specifically targeting I 2 R may be a potentially useful therapeutic strategy for liver fibrosis.

// Huafeng Wang 1, 2, 3, * , Huan Zhang 1, 4, * , Yuqing Zhang 5, * , Dan Wang 1, * , Xixi Cheng 1 , Fengrui Yang 1, 2 , Qi Zhang 1 , Zhenyi Xue 1, 2 , Yan Li 1, 2 , Lijuan Zhang 1, 2 , Luhong Yang 3 , Guolin Miao 1, 2 , Daiqing Li 6 , Zhiyu Guan 7 , Yurong Da 1, 2 , Zhi Yao 2 , Fei Gao 8 , Liang Qiao 9 , Li Kong 10 , Rongxin Zhang 1, 2 1 Laboratory of Immunology and Inflammation, Department of Immunology and Research Center of Basic Medical Sciences, Tianjin Medical University, Tianjin, China 2 Department of Immunology, Tianjin Key Laboratory of Cellular and Molecular Immunology, Key Laboratory of Immune Microenvironment and Diseases, Ministry of Education of China, Tianjin Medical University, Tianjin, China 3 School of Life Science, Shanxi Normal University, Linfen, China 4 Clinical Laboratory, Tianjin Academy of Traditional Chinese Medicine Affiliated Hospital, Tianjin, China 5 Department of Hepatobiliary and Pancreatic Surgery, Tianjin Nankai Hospital, Nankai Clinical School of Medicine, Tianjin Medical University, Tianjin, China 6 Key Laboratory of Hormones and Development (Ministry of Health), Metabolic Diseases Hospital and Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, China 7 Department of Pathogenic Biology, Weifang Medical University, Shandong, China 8 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China 9 Storr Liver Unit, Westmead Millennium Institute, The Western Clinical School of the University of Sydney, Westmead, NSW, Australia 10 Department of Histology and Embryology, Dalian Medical University, Dalian, China * These authors have contributed equally to this work Correspondence to: Rongxin Zhang, email: rxzhang@tmu.edu.cn , rongxinz@yahoo.com Li Kong, email: kongli@dmu.edu.cn Keywords: plumbagin, fulminant hepatic failure, liver fibrosis, hepatic stellate cell, inflammation Received: August 08, 2016 Accepted: October 04, 2016 Published: October 14, 2016 ABSTRACT Plumbagin is a quinonoid constituent extracted from Plumbago genus, and it exhibits diverse pharmacological effects. This study thoroughly investigated the effects of plumbagin on thioacetamide-induced acute and chronic liver injury. Results shown that plumbagin increased survival rate, reduced liver congestion and inflammation, and decreased macrophages and neutrophils in the fulminant hepatic failure model, and remarkably diminished liver fibrosis and inflammation in the chronic liver injury model. Furthermore, plumbagin significantly suppress the HSCs/myofibroblasts activation by reduced expression of markers α-SMA and COL-1/3, and reduced macrophage in liver. In the in vitro study, plumbagin induced apoptosis and suppressed the proliferation of LX-2 cells (human HSCs). Plumbagin treatment increased AMPK phosphorylation and attenuated NF-κB, STAT3, and Akt/mTOR signals in LX-2 cells, while SMAD2 phosphorylation was not changed. Noticeably, plumbagin promoted AMPK binding to p300 which is a cofactor of SMAD complex, this may further competitively decreases the p300/SMAD complex initiated transcription of COL-1/3 and α-SMA. Additionally, plumbagin hampered inflammation related NF-κB signal in RAW 264.7 cells. In conclusion, these findings indicate that plumbagin may be a powerful drug candidate to protect the liver from acute and chronic damage by inhibiting inflammation and collagen production.

// Xinguang Yin 1, 2 , Huixing Yi 3 , Linlin Wang 4 , Wanxin Wu 5 , Xiaojun Wu 2 , Linghua Yu 2 1 Centre for Gastroenterology and Hepatology, The Maternity and Child Health Care Hospital affiliated to Jiaxing College, Jiaxing, 314001, Zhejiang Province, PR China 2 Centre for Gastroenterology and Hepatology, The First Affiliated Hospital of Jiaxing College, Jiaxing, 314001, Zhejiang Province, PR China 3 Intensive Care Unit, The Second Affiliated Hospital of Zhejiang University, Hangzhou, 310009, Zhejiang Province, PR China 4 Department of Basic Medicine Sciences, School of Medicine, Zhejiang University, Hangzhou, 310058, China 5 Deparment of Pathology, The First Affiliated Hospital of Jiaxing College, Jiaxing, 314001, Zhejiang Province, PR China Correspondence to: Linghua Yu, email: yu.lh70s@gmail.com Keywords: RSPO, hepatic fibrosis, Wnt pathway, HSC Received: February 18, 2016 Accepted: August 24, 2016 Published: August 27, 2016 ABSTRACT Roof plate-specific spondin (RSPO) proteins are potent Wnt pathway agonists and involve in a broad range of developmental and physiological processes. This study investigated the activities and mechanisms of RSPOs in liver fibrogenesis, especially in hepatic stellate cell (HSC) activation. HSC activation was assessed by fibrosis biomarker (α-smooth muscle actin and Collagen-I), phenotypic change (accumulation of lipid droplets), and increased proliferation. Similarly, Wnt pathway activity was evaluated by the expression of nuclear β-catenin and T cell-specific transcription factors (TCF) activity. We found RSPOs were overexpressed in human fibrotic liver tissue and the expressions were correlated with liver fibrosis stages. In vitro studies showed RSPOs level increased during HSC activation, and stimuli with RSPOs enhanced Wnt pathway activity and promoted HSC activation subsequently. Furthermore, in vivo experiments demonstrated that the knockdown of RSPOs suppressed both Wnt pathway activity and HSC activation. Interestingly, the inhibitor of the Wnt signaling pathway Dickkopf1 impairs RSPOs effects on HSCs. Taken together, our results revealed that RSPOs facilitated HSC activation and promote liver fibrogenesis by enhancing the Wnt pathway.

Oleoylethanolamide (OEA), an endocannabinoid-like molecule, was revealed to modulate lipid metabolism through a peroxisome proliferator-activated receptor-α (PPAR-α) mediated mechanism. In present study, we further investigated the activities and mechanisms of OEA in ameliorating hepatic fibrosis in Sv/129 mice induced by a methionine choline-deficient (MCD) diet or thioacetamide (TAA) treatment. Liver fibrosis development was assessed by Hematoxylin-eosin and Sirius red staining. Treatment with OEA (5 mg/kg/day, intraperitoneal injection, i.p.) significantly attenuated the progress of liver fibrosis in both two experimental animal models by blocking the activation of hepatic stellate cells (HSCs). Gene expression analysis of hepatic tissues indicated that OEA inhibited the expression of α-smooth muscle action (α-SMA) and collagen matrix, fibrosis markers, and genes involved in inflammation and extracellular matrix remodeling. In vitro studies showed that OEA inhibited transforming growth factor β1-stimulated HSCs activation through suppressing Smad2/3 phosphorylation, α-SMA expression and myofibroblast transformation. These improvements could not be observed in PPAR-α knockout mice models with OEA administration, which suggested all the anti-fibrotic effects of OEA in vivo and in vitro were mediated by PPAR-α activation. Collectively, our results suggested that OEA exerted a pharmacological effect on modulating hepatic fibrosis development through the inhibition of HSCs activation in liver and therefore may be a potential therapeutic agent for liver fibrosis.

Hepatic stellate cells (HSCs) are critical mediators of immunosuppression and the pathogenesis of hepatocellular carcinoma (HCC). Our previous work indicates that HSCs promote HCC progression by enhancing immunosuppressive cell populations including myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). MDSCs are induced by inflammatory cytokines (e.g., prostaglandins) and are important in immune suppression. However, how HSCs mediate expansion of MDSCs is uncertain. Thus, we studied activated HSCs that could induce MDSCs from bone marrow cells and noted that HSC-induced MDSCs up-regulated immunosuppressive activity via iNOS, Arg-1, and IL-4Rα. After treating cells with a COX-2 inhibitor or an EP4 antagonist, we established that HSC-induced MDSC accumulation was mediated by the COX2-PGE2-EP4 signaling. Furthermore, in vivo animal studies confirmed that inhibition of HSC-derived PGE2 could inhibit HSC-induced MDSC accumulation and HCC growth. Thus, our data show that HSCs are required for MDSC accumulation mediated by the COX2-PGE2-EP4 pathway, and these data are the first to link HSC and MDSC subsets in HCC immune microenvironment and provide a rationale for targeting PGE2 signaling for HCC therapy.