Tyrosine kinase receptor B attenuates liver fibrosis by inhibiting TGF-ß/SMAD signaling.

BACKGROUND AND AIMS: Liver fibrosis is a leading indicator for increased mortality and long-term comorbidity in NASH. Activation of HSCs and excessive extracellular matrix production are the hallmarks of liver fibrogenesis. Tyrosine kinase receptor (TrkB) is a multifunctional receptor that participates in neurodegenerative disorders. However, paucity of literature is available about TrkB function in liver fibrosis. Herein, the regulatory network and therapeutic potential of TrkB were explored in the progression of hepatic fibrosis. METHODS AND RESULTS: The protein level of TrkB was decreased in mouse models of CDAHFD feeding or carbon tetrachloride-induced hepatic fibrosis. TrkB suppressed TGF-ß-stimulated proliferation and activation of HSCs in 3-dimensional liver spheroids and significantly repressed TGF-ß/SMAD signaling pathway either in HSCs or in hepatocytes. The cytokine, TGF-ß, boosted Nedd4 family interacting protein-1 (Ndfip1) expression, promoting the ubiquitination and degradation of TrkB through E3 ligase Nedd4-2. Moreover, carbon tetrachloride intoxication-induced hepatic fibrosis in mouse models was reduced by adeno-associated virus vector serotype 6 (AAV6)-mediated TrkB overexpression in HSCs. In addition, in murine models of CDAHFD feeding and Gubra-Amylin NASH (GAN), fibrogenesis was reduced by adeno-associated virus vector serotype 8 (AAV8)-mediated TrkB overexpression in hepatocytes. CONCLUSION: TGF-ß stimulated TrkB degradation through E3 ligase Nedd4-2 in HSCs. TrkB overexpression inhibited the activation of TGF-ß/SMAD signaling and alleviated the hepatic fibrosis both in vitro and in vivo . These findings demonstrate that TrkB could be a significant suppressor of hepatic fibrosis and confer a potential therapeutic target in hepatic fibrosis.

ANGPTL2 Deletion Attenuates Neuroinflammation and Cognitive Dysfunction Induced by Isoflurane in Aged Mice through Modulating MAPK Pathway.

Postoperative cognitive dysfunction (POCD) is a well-known complication after surgery with cognitive impairments. Angiopoietin-like protein 2 (ANGPTL2) has been found to be associated with inflammation. However, the role of ANGPTL2 in inflammation of POCD is unclear. Here, mice were subjected into isoflurane anesthesia. It was demonstrated that isoflurane increased ANGPTL2 expression and promoted pathological change in brain tissues. However, downregulation of ANGPTL2 alleviated the pathological change and elevated learning and memory abilities, improving isoflurane-induced cognitive dysfunction in mice. In addition, isoflurane-induced cell apoptosis and inflammation were repressed via ANGPTL2 knockdown in mice. Downregulation of ANGPTL2 was also verified to suppress isoflurane-induced microglial activation, evidenced by a decrease of Iba1 and CD86 expressions and an increase of CD206 expression. Further, the isoflurane-induced MAPK signaling pathway was repressed through downregulation of ANGPTL2 in mice. In conclusion, this study proved that downregulation of ANGPTL2 attenuated isoflurane-induced neuroinflammation and cognitive dysfunction in mice via modulating the MAPK pathway, which provided a new therapeutic target for POCD.

M2-polarized tumor-associated macrophages promote epithelial-mesenchymal transition via activation of the AKT3/PRAS40 signaling pathway in intrahepatic cholangiocarcinoma.

Tumor-associated macrophages (TAMs) have been considered as a major component of the tumor microenvironment. However, the crosstalk between M2-polarized tumor-associated macrophages (M2-TAMs) and intrahepatic cholangiocarcinoma (ICC) remains undetermined. In the present study, we aimed to clarify the role of M2-TAMs in ICC and the underlying mechanism. The in vitro assay demonstrated M2-TAMs promoted epithelial-mesenchymal transition (EMT) of ICC cells, resulting in enhanced cell invasion and metastasis ability. Moreover, M2-TAMs modulated the microenvironment of ICC by increasing the secretion of cytokines (GM-CSF, tumor necrosis factor-α [TNF-α], ICAM-1, interleukin-6 [IL-6], etc) and chemokines (CCL1, CCL3, etc). In addition, p-AKT (Ser473) and p-PRAS40 (Thr246) were upregulated in ICC cells when cocultured with M2-TAMs or treated with M2-TAMs secreted core cytokines (GM-CSF, TNF-α, ICAM-1, and IL-6). Consistently, AKT3 silencing (but not AKT1 silencing and AKT2 silencing) markedly inhibited phosphorylation of AKT and PRAS40 of ICC cells and inhibited the EMT process when cocultured with M2-TAMs. Taken together, the current data indicated that M2-TAMs promoted ICC cells EMT, partially through increasing secretion of cytokines and chemokines, thus modulating the microenvironment and activating the AKT3/PRAS40 signaling pathway.

Smurf2, an E3 ubiquitin ligase, interacts with PDE4B and attenuates liver fibrosis through miR-132 mediated CTGF inhibition.

We previously reported that Smad ubiquitin regulatory factor 2 (Smurf2) activity was decreased in human fibrotic livers. Here, we overexpressed Smurf2 in livers of transgenic mice and observed inhibited collagen deposition and hepatic stellate cell activation in fibrotic model induced by carbon tetrachloride treatment or bile duct ligation. Hepatic Smurf2 overexpression also inhibited the production of connective tissue growth factor (CTGF), a central mediator of liver fibrosis. Using miRNA array and bioinformatics analyses, we identified miR-132 as a mediator of this inhibitory effect. miR-132 directly targets the 3'-untranslated region of CTGF and was transcriptionally upregulated by cAMP-PKA-CREB signaling. In addition, Smurf2 activated cAMP-PKA-CREB pathway by interacting with phosphodiesterase 4B (PDE4B) and facilitating its degradation. Thus, we have demonstrated a previously unrecognized anti-fibrotic pathway controlled by Smurf2.

BH3 Mimetic ABT-199 Enhances the Sensitivity of Gemcitabine in Pancreatic Cancer in vitro and in vivo.

BACKGROUND AND AIMS: Pancreatic cancer is an aggressive malignancy with poor prognosis. Gemcitabine is the standard chemotherapeutic drug used to treat the disease; however, it has a low response rate. Therefore, there is an urgent need to develop new and safe therapies to enhance sensitivity to gemcitabine in treating pancreatic cancer. METHODS: The synergistic effect of gemcitabine combined with specific B cell CLL/lymphoma 2 (Bcl-2) inhibitor ABT-199 against pancreatic cancer was tested using cell viability, cell cycle, and apoptosis assays in vitro and in an MIA Paca-2 xenograft model in vivo. Its underlying mechanism was explored using western blotting analysis of Bcl-2 family proteins. RESULTS: ABT-199 not only enhanced the effect of gemcitabine on cell growth inhibition but also promoted gemcitabine-induced apoptosis in pancreatic cancer cell lines. Gemcitabine decreased the expression of anti-apoptotic protein Mcl-1 but increased the expression of anti-apoptotic protein Bcl-2. ABT-199 downregulated the gemcitabine-induced production of Bcl-2 and increased the expression of pro-apoptotic protein Bcl-2 interacting protein (BIM). Mouse xenograft experiments also confirmed the synergistic effect of gemcitabine and ABT-199 on tumor growth inhibition and the induction of tumor cell apoptosis. CONCLUSION: Our results indicated that ABT-199 improved the anti-tumor effect of gemcitabine on pancreatic cancer by downregulating gemcitabine-induced overexpression of Bcl-2. ABT-199 has already been investigated in phase 3 clinical trials for chronic lymphocytic leukemia; therefore, it may serve as a potential drug to improve the sensitivity of pancreatic cancer to gemcitabine.

Placental growth factor silencing ameliorates liver fibrosis and angiogenesis and inhibits activation of hepatic stellate cells in a murine model of chronic liver disease.

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family and is involved in pathological angiogenesis associated with chronic liver diseases. However, the precise mechanisms underlying PlGF signalling contributing to liver fibrosis and angiogenesis remain largely unexplored. This study aimed to assess the effect of reducing PlGF expression using small interfering RNA (siRNA) on experimental liver fibrosis and angiogenesis, and to elucidate the underlying molecular mechanisms. Fibrosis was induced in mice by carbon tetrachloride (CCl4 ) for 8 weeks, and mice were treated with PlGF siRNA or non-targeting control siRNA starting two weeks after initiating CCl4 injections. The results showed that PlGF was highly expressed in cirrhotic human and mice livers; which mainly distributed in activated hepatic stellate cells (HSCs). PlGF silencing robustly reduced liver inflammation, fibrosis, intrahepatic macrophage recruitment, and inhibited the activation of HSCs in vivo. Moreover, PlGF siRNA-treated fibrotic mice showed diminished hepatic microvessel density and angiogenic factors, such as hypoxia-inducible factor-1α (HIF-1α), VEGF and VEGF receptor-1. Moreover, down-regulation of PlGF with siRNA in HSCs inhibited the activation and proliferation of HSCs. Mechanistically, overexpression of PlGF in activated HSCs was induced by hypoxia dependent on HIF-1α, and PlGF induces HSC activation and proliferation via activation the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathways. These findings indicate that PlGF plays an important role in liver fibrosis-associated angiogenesis and that blockage of PlGF could be an effective strategy for chronic liver disease.

Clinicopathological significance of the microRNA-146a/WASP-family verprolin-homologous protein-2 axis in gastric cancer.

Gastric cancer (GC) is one of the most common malignancies, and cancer invasion and metastasis are the leading causes of cancer-induced death in GC patients. WASP-family verprolin-homologous protein-2 (WASF2), with a role controlling actin polymerization which is critical in the formation of membrane protrusions involved in cell migration and invasion, has been reported to possess cancer-promoting effects in several cancers. However, data of WASF2's role in GC are relatively few and even contradictory. In this study, we analyzed WASF2 expression in GC tissues and their corresponding adjacent normal tissues. We found that WASF2 was upregulated in GC tissues and high level of WASF2 was associated with lymph node metastasis of GC. Through gain- and loss-of-function studies, WASF2 was shown to significantly increase GC cells migration and invasion, but had no effect on proliferation in vitro. Importantly, WASF2 was also found to enhance GC metastasis in vivo. Our previous research suggested that WASF2 was a direct target of microRNA-146a (miR-146a). Furthermore, we analyzed miR-146a's level in GC tissues and their corresponding adjacent normal tissues. We found that miR-146a was downregulated in GC tissues and low miR-146a level was associated with advanced TNM stage and lymph node metastasis. The level of WASF2 in GC tissues was negatively correlated with miR-146a expression and had inverse clinicopathologic features. The newly identified miR-146a/WASF2 axis may provide a novel therapeutic target for GC.

Placental growth factor enhances angiogenesis in human intestinal microvascular endothelial cells via PI3K/Akt pathway: Potential implications of inflammation bowel disease.

BACKGROUND: Angiogenesis plays a major role in the pathogenesis of inflammatory bowel disease (IBD). Placental growth factor (PlGF) is a specific regulator of pathological angiogenesis and is upregulated in the sera of IBD patients. Therefore, the role of PlGF in IBD angiogenesis was investigated here using HIMECs. METHODS: The expression of PlGF and its receptors in human intestinal microvascular endothelial cells (HIMECs) and inflamed mucosa of IBD patients were examined using quantitative PCR and western blot analysis and the role of PlGF in IBD HIMECs was further explored using small interfering RNA (siRNA). The induction of pro-inflammatory cytokine by PlGF in HIMECs was confirmed by ELISA. The capacity of PlGF to induce angiogenesis in HIMECs was tested through proliferation, cell-migration, matrigel tubule-formation assays and its underlying signaling pathway were explored by western blot analysis of ERK1/2 and PI3K/Akt phosphorylation. RESULTS: mRNA and protein expression of PlGF and its receptor NRP-1 were significantly increased in IBD HIMECs. Inflamed mucosa of IBD patients also displayed higher expression of PIGF. The production of IL-6 and TNF-α in culture supernatant of HIMECs treated with exogenous recombinant human PlGF-1 (rhPlGF-1) were increased. Furthermore, rhPlGF-1 significantly induced HIMECs migration and tube formation in a dose-dependent manner and knockdown of endogenous PlGF in IBD HIMECs using siRNA substantially reduced these angiogenesis activities. PlGF induced PI3K/Akt phosphorylation in HIMECs and pretreatment of PlGF-stimulated HIMECs with PI3K inhibitor (LY294002) significantly inhibited the PlGF-induced cell migration and tube formation. CONCLUSION: Our results demonstrated the pro-inflammatory and angiogenic effects of PlGF on HIMECs in IBD through activation of PI3K/Akt signaling pathway. PlGF/PI3K/Akt signaling may serve as a potential therapeutic target for IBD.

Age-related changes in small intestinal mucosa epithelium architecture and epithelial tight junction in rat models.

BACKGROUND AND AIMS: Functions of the intestinal mucosal barrier are often impaired in the elderly and are closely associated with many age-related diseases. However, mechanisms by which aging influences intestinal barrier function still remain unclear. The aim of this study was to investigate age-related changes in small intestinal morphology, bacteria contents and expression of epithelial tight junction (TJ) proteins. METHODS: Thirty Sprague-Dawley rats were divided into groups: young (3 months), adult (12 months), and old (24 months). The small intestinal mucosal architecture and TJ of intestinal epithelial cells were examined by light microscopy and transmission electron microscopy. Jejunum and cecum contents were cultured to identify and measure bacterial species. mRNA expression of Zonula occludens-1 (ZO-1) and occludin were measured by semi-quantitative RT-PCR. Protein expression of ZO-1 and occludin were detected by immunohistochemistry and Western blot. RESULTS: Normal ileum villi, which were thick and regularly arranged, though increasingly scattered and atrophic in character with shorter and narrower dimensions (P < 0.01), were observed in old rats, along with an elevated number of Gram-positive and Gram-negative bacteria in the jejunum. The TJs of intestinal epithelial cells, as detected by transmission electron microscopy, were wider and discontinuous in old rats. Age-induced down-regulation of mRNA expression and decreased protein expression of ZO-1 and occludin were observed in the ileum (P < 0.01). CONCLUSIONS: Our study indicated that age-related intestinal barrier dysfunction may be associated with mucosal atrophy, damages to TJ structure, increased small intestine bacteria counts, and decreased epithelial TJ protein.

Anti-PCSK9 Treatment Attenuates Liver Fibrosis via Inhibiting Hypoxia-Induced Autophagy in Hepatocytes.

Hypoxia and its induced autophagy are involved in the initiation and progression of liver fibrosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been recognized as a potential regulator of autophagy. Our previously reported study found that PCSK9 expression increased in liver fibrosis and that anti-PCSK9 treatment alleviated liver injury. This study aimed to investigate the mechanism of anti-PCSK9 treatment on liver fibrosis by inhibiting hypoxia-induced autophagy. Carbon tetrachloride-induced mouse liver fibrosis and mouse hepatocyte line AML12, cultured under the hypoxic condition, were established to undergo PCSK9 inhibition. The degree of liver fibrosis was shown with histological staining. The reactive oxygen species (ROS) generation was detected by flow cytometry. The expression of PCSK9, hypoxia-inducible factor-1α (HIF-1α), and autophagy-related proteins was examined using Western blot. The autophagic flux was assessed under immunofluorescence and transmission electron microscope. The mouse liver samples were investigated via RNA-sequencing to explore the underlying signaling pathway. The results showed that PCSK9 expression was upregulated with the development of liver fibrosis, which was accompanied by enhanced autophagy. In vitro data verified that PCSK9 increased via hypoxia and inflammation, accompanied by the hypoxia-induced autophagy increased. Then, the validation was acquired of the bidirectional interaction of hypoxia-ROS and PCSK9. The hypoxia reversal attenuated PCSK9 expression and autophagy. Additionally, anti-PCSK9 treatment alleviated liver inflammation and fibrosis, reducing hypoxia and autophagy in vivo. In mechanism, the AMPK/mTOR/ULK1 signaling pathway was identified as a target for anti-PCSK9 therapy. In conclusion, anti-PCSK9 treatment could alleviate liver inflammation and fibrosis by regulating AMPK/mTOR/ULK1 signaling pathway to reduce hypoxia-induced autophagy in hepatocytes.

Inhibition by curcumin of multiple sites of the transforming growth factor-beta1 signalling pathway ameliorates the progression of liver fibrosis induced by carbon tetrachloride in rats.

BACKGROUND: At present there is no effective and accepted therapy for hepatic fibrosis. Transforming growth factor (TGF)-ß1 signaling pathway contributes greatly to hepatic fibrosis. Reducing TGF-ß synthesis or inhibiting components of its complex signaling pathway represent important therapeutic targets. The aim of the study was to investigate the effect of curcumin on liver fibrosis and whether curcumin attenuates the TGF-ß1 signaling pathway. METHODS: Sprague-Dawley rat was induced liver fibrosis by carbon tetrachloride (CCl4) for six weeks together with or without curcumin, and hepatic histopathology and collagen content were employed to quantify liver necro-inflammation and fibrosis. Moreover, the mRNA and protein expression levels of TGF-ß1, Smad2, phosphorylated Smad2, Smad3, Smad7 and connective tissue growth factor (CTGF) were determined by quantitative real time-PCR, Western blot, or immunohistochemistry. RESULTS: Rats treated with curcumin improved liver necro-inflammation, and reduced liver fibrosis in association with decreased α-smooth muscle actin expression, and decreased collagen deposition. Furthermore, curcumin significantly attenuated expressions of TGFß1, Smad2, phosphorylated Smad2, Smad3, and CTGF and induced expression of the Smad7. CONCLUSIONS: Curcumin significantly attenuated the severity of CCl4-induced liver inflammation and fibrosis through inhibition of TGF-ß1/Smad signalling pathway and CTGF expression. These data suggest that curcumin might be an effective antifibrotic drug in the prevention of liver disease progression.

Smurf2 suppresses the metastasis of hepatocellular carcinoma via ubiquitin degradation of Smad2.

Purpose: Smurf2, one of C2-WW-HECT domain E3 ubiquitin ligases, is closely related to the development and progression in different cancer types, including hepatocellular carcinoma (HCC). This study aims to illustrate the expression and molecular mechanism of Smurf2 in regulating the progression of HCC. Methods: The expression of Smurf2 in human HCC and adjacent non-tumor liver specimens was detected using tissue microarray studies from 220 HCC patients who underwent curative resection. The relationships of Smurf2 and HCC progression and survival were analyzed using the chi-square test, Kaplan-Meier analysis, and Cox proportional hazards model. For Smurf2 was low expression in HCC cell lines, Smurf2 overexpression cell lines were established. The effect of Smurf2 on cell proliferation and migration was detected by Cell Counting Kit-8 and colony formation assay, and the epithelial-mesenchymal transition (EMT) markers and its transcription factors were tested by immunoblotting. The interaction and ubiquitination of Smad2 by Smurf2 were detected by co-immunoprecipitation and immunoprecipitation assay. Finally, the effect of Smurf2 on HCC was verified using the mouse lung metastasis model. Results: Smurf2 was downregulated in HCC tissues compared to that of corresponding non-tumor liver specimens. The low expression of Smurf2 in HCC was significantly associated with macrovascular or microvascular tumor thrombus and the impairment of overall survival and disease-free survival. In vitro and in vivo analysis showed that Smurf2 overexpression decreased the EMT potential of HCC cells by promoting the ubiquitination of Smad2 via the proteasome-dependent degradation pathway. Conclusion: The expression of Smurf2 was downregulated in HCC specimens and affected the survival of patients. Smurf2 inhibited the EMT of HCC by enhancing Smad2 ubiquitin-dependent proteasome degradation.

Yinzhihuang oral liquid protects against non-alcoholic steatohepatitis via modulation of the gut-liver axis in mice.

Background: Yinzhihuang (YZH) oral liquid is a traditional Chinese medicine compound that has emerged as a promising therapeutic agent for non-alcoholic fatty liver disease (NAFLD). Here, we aimed to investigate the therapeutic effects of YZH on non-alcoholic steatohepatitis (NASH) and elucidate its underlying molecular mechanisms. Methods: Mice fed on a high-fat diet plus fructose/glucose drinking water (HFGD) were treated with YZH (30 mL/kg/d). The effects of YZH on mice with NASH were assessed through serological analysis and histological examination. Microbiota analysis based on 16S ribosomal ribonucleic acid (16S rRNA) and intestinal mucosal barrier function, serum inflammatory factors, hepatic macrophage infiltration, as well as hepatic toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), nuclear factor kappa B (NFκB) pathway were carried out to explore the mechanism of YZH for treatment of NASH. Results: Results of the current study found that YZH effectively reduced body weight gain and adiposity and alleviated hepatocyte steatosis, hepatocyte ballooning, liver tissue lobular inflammation, as well as fibrosis. It also reduced the accumulation of triglycerides, cholesterol, and free fatty acids in the liver of the treated mice and normalized serum aspartate transaminase, alanine transaminase, and glucose levels as well as lipid metabolism. Meanwhile, YZH treatment significantly decreased the abundance of harmful bacteria, such as Mucispirillum, Helicobacter, and Desulfovibrionaceae. Mechanistically, the present study found that YZH upregulated the expression of tight junction proteins, decreased serum lipopolysaccharide, interleukin 6, and tumor necrosis factor α levels, and increased interleukin 10 levels in serum. In the liver, YZH alleviated macrophage infiltration, especially that of pro-inflammatory macrophages. Moreover, it was found that YZH inhibited the canonical TLR4, MyD88, NFκB signaling pathway. Conclusions: In conclusion, YZH may be a new agent for the prevention of NASH. Further, YZH alleviates gut microbiota dysbiosis, restores the intestinal mucosal barrier, and inhibits the canonical TLR4, MyD88, NFκB signaling pathway.

Nobiletin mitigates hepatocytes death, liver inflammation, and fibrosis in a murine model of NASH through modulating hepatic oxidative stress and mitochondrial dysfunction.

This study aimed to investigate the therapeutic effects of nobiletin (NOB) on nonalcoholic steatohepatitis (NASH) and liver fibrosis in mice and to elucidate its underlying molecular mechanisms. BALB/c mice were fed a normal chow diet or a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 8 wks and treated with NOB (50 mg/kg) or vehicle by daily intraperitoneally injection for the last 4 wks. In vitro, we used palmitate (PA) stimulated AML12 cells as the model of hepatocyte lipotoxicity to dissect the effect and molecular mechanisms of NOB' action. Our results exhibited that NOB dramatically reduced hepatic steatosis, lipid accumulation and hepatocyte apoptosis, and inhibited the infiltration of F4/80+ macrophages into the NASH livers. Furthermore, NOB limited liver fibrosis and hepatic stellate cells activation in NASH mice. In parallel, NOB alleviated hepatocytes apoptosis and lipid accumulation in PA-treated AML12 cells. Most importantly, these histological ameliorations in NASH and fibrosis in NOB-treated NASH mice were associated with improvement hepatic oxidative stress, lipid peroxidation product, mitochondrial respiratory chain complexes I and restored ATP production. Similarly, NOB attenuated PA-induced reactive oxygen species (ROS) generation and mitochondrial disfunction in cultured AML12 cells. Additionally, NOB diminished the expression of mitochondrial Ca2+ uniporter (MCU) both in NASH livers and in PA-treated AML12. Taken together, our results indicate that NOB mitigated NASH development and fibrosis through modulating hepatic oxidative stress and attenuating mitochondrial dysfunction. Therefore, NOB might be a novel and promising agent for treatment of NASH and liver fibrosis.

Validity of plasma macrophage migration inhibitory factor for diagnosis and prognosis of hepatocellular carcinoma.

We performed our study to determine whether plasma macrophage migration inhibitory factor (MIF) levels have diagnostic and prognostic value in hepatocellular carcinoma (HCC) patients. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were used to measure the expression of MIF in plasma and tissues, respectively. Plasma MIF levels were compared to HCC occurrence, clinicopathological features and outcomes. Cutpoints of plasma MIF levels for diagnosis and prognosis were, respectively, determined by receiver operating characteristic analysis and X-tile in corresponding training cohort, and then were confirmed in the validation cohort. The postoperative plasma MIF levels of HCC patients were detected in an independent cohort (80 HCC patients). As a result, MIF expression in situ was mainly observed in the cytoplasm of HCC cells. Intratumoral MIF expression was positively correlated with plasma MIF levels (r = 0.759, p < 0.001). Compared to serum α-fetoprotein (AFP), plasma MIF had a higher diagnostic value for discrimination of HCC from controls at 35.3 ng/ml. With determined cutpoints, plasma MIF levels demonstrated a significant association with overall survival (OS) and disease-free survival (DFS) of HCC patients even in patients with normal serum AFP levels and Tumor Node Metastasis (TNM) stage I. In addition, the plasma MIF levels were identified as an independent factor for OS [hazard ratio (HR) = 1.754; p = 0.012] and DFS (HR = 2.121; p < 0.001). Plasma MIF levels decreased markedly within 30 days after tumor resection (p < 0.001). Therefore, plasma MIF levels have potential as a diagnostic and prognostic factor for HCC.

Validation of the simplified criteria for diagnosis of autoimmune hepatitis in Chinese patients.

BACKGROUND & AIMS: In 1999, the International Autoimmune Hepatitis Group (IAIHG) revised the diagnostic criteria for autoimmune hepatitis (AIH). It subsequently developed the simplified criteria in 2008 to enhance clinical applicability and practicability. In this study, we validated the simplified diagnostic criteria in Chinese patients with AIH or other chronic liver diseases in comparison with the revised original criteria. METHODS: Diagnostic scores were determined using the revised original criteria and the simplified criteria in 405 patients with diverse liver diseases. The sample included 127 patients with AIH type I diagnosed by the descriptive criteria, 77 patients with primary biliary cirrhosis (PBC), 6 patients with AIH-PBC overlap syndrome, 47 patients with drug-induced liver injury (DILI), 36 patients with non-alcoholic steatohepatitis (NASH), 82 patients with chronic hepatitis B (CHB), and 30 patients with chronic hepatitis C (CHC). The simplified criteria were compared to the revised original criteria based on sensitivity, specificity, and predictability for the pre-treatment diagnosis of AIH. RESULTS: The simplified criteria had sensitivity and specificity of 90% and 95%, respectively, for the diagnosis of probable AIH in the Chinese patients. This compares well with the more rigorous revised original criteria, which had sensitivity and specificity of 100% and 93%, respectively, for probable AIH. On definite AIH, the simplified criteria had sensitivity and specificity of 62% and 99%, respectively, compared to 64% and 100% for definite AIH by the revised original criteria. In addition, the predictabilities of the revised original criteria and simplified criteria were 96% and 94% for probable AIH, and 88% and 87% for definite AIH, respectively, in our groups. Using the revised original criteria, 84 patients were diagnosed with definite AIH. On the other hand, among these 84 patients, the simplified criteria diagnosed only 61 patients with definite AIH (accordant diagnosis) and provided the 23 other patients with downgraded diagnosis. Comparison of the clinical and laboratory features of these two groups (accordant diagnosis vs. downgraded/excluded diagnosis) showed that the patients with downgraded diagnosis had significantly higher histological scores than the patients with accordant diagnosis. CONCLUSIONS: The simplified criteria are comparable to the revised original criteria and have high sensitivity and specificity for the diagnosis of AIH in Chinese patients. Liver histology is critical for the diagnosis of AIH especially when using the simplified criteria. Further study or prospective evaluation is needed to confirm these observations, however, due to the small group of CHC patients as well as the absence of primary sclerosing cholangitis (PSC) patients in our study.

Curcumin protects mice against concanavalin A-induced hepatitis by inhibiting intrahepatic intercellular adhesion molecule-1 (ICAM-1) and CXCL10 expression.

The effect of curcumin on liver injury caused by Concanavalin A (Con A) has not been carefully examined. This study was designed to evaluate the protective effect of curcumin on Con A-induced hepatitis in mice. Liver injured mice received curcumin by gavage at a dose of 200 mg/kg body weight before Con A intravenous administration. Curcumin was effective in reducing the elevated plasma levels of aminotransferases and the incidence of liver necrosis compared with Con A-injected control group. Enzyme-linked immunosorbent assay (ELISA) showed that curcumin suppressed proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-4 production in Con A-injected mice. The reduced severity of hepatitis in curcumin pretreated mice correlated with decrease in numbers of liver CD4(+) T cells but not CD8(+) T cells by immunohistochemical analysis. Furthermore, the expression levels of intercellular adhesion molecule-1 (ICAM-1) and the interferon-inducible chemokine CXCL10 in hepatic tissue were significantly decreased by curcumin pretreatment. In conclusion, curcumin pretreatment protects against T cell-mediated hepatitis in mice.

Hepatic NOD2 promotes hepatocarcinogenesis via a RIP2-mediated proinflammatory response and a novel nuclear autophagy-mediated DNA damage mechanism.

BACKGROUND: Key hepatic molecules linking gut dysbiosis and hepatocarcinogenesis remain largely unknown. Gut-derived gut microbiota contains pathogen-associated molecular patterns (PAMPs) that may circulate into the liver and, consequently, be recognized by hepatic pattern recognition receptors (PRRs). NOD2, a general intracellular PRR, recognizes muramyl dipeptide (MDP), present in both gram (+) and gram (-) bacteria. Here, we investigated the role of NOD2 as a molecular sensor translating gut dysbiosis signaling into hepatocarcinogenesis. METHODS: NOD2 expression was measured in clinical hepatocellular carcinoma (HCC) samples using qPCR (80 pairs), western blotting (30 pairs) and immunostaining (141 pairs). The role of NOD2 in hepatocarcinogenesis was examined in the hepatocyte-specific Nod2-knockout (Nod2△hep), Rip2-knockout (Rip2△hep), Lamin A/C-knockout (Lamn△hep) and Rip2/Lamin A/C double-knockout (Rip2/Lamn△hep) mice models of diethylnitrosamine (DEN)/CCl4-induced HCC. RESULTS: NOD2 was upregulated and activated in HCC samples, and high NOD2 expression correlated with poor prognosis in HCC patients. Hepatic NOD2 deletion in vivo decreased DEN/CCl4-induced HCC by reducing the inflammatory response, DNA damage and genomic instability. NOD2 activation increased liver inflammation via RIP2-dependent activation of the MAPK, NF-κB and STAT3 pathways. Notably, a novel RIP2-independent mechanism was discovered, whereby NOD2 activation induces the nuclear autophagy pathway. We showed that NOD2 undergoes nuclear transport and directly binds to a component of nuclear laminae, lamin A/C, to promote its protein degradation, leading to impaired DNA damage repair and increased genomic instability. CONCLUSIONS: We reveal a novel bridge, bacterial sensor NOD2, linking gut-derived microbial metabolites to hepatocarcinogenesis via induction of the inflammatory response and nuclear autophagy. Thus, we propose hepatic NOD2 as a promising therapeutic target against HCC.

[Application of serum protein profiling in diagnosis, prognosis and evaluation of curative effect of pancreatic adenocarcinoma].

OBJECTIVE: To establish decision tree and logistic regression classification models for diagnosing pancreatic adenocarcinoma (PaCa) and for screening serum biomarkers related to evaluation of different stages and curative effects. METHODS: Serum samples obtained from subjects with pancreatic adenocarcinoma (n = 58) and normal pancreas (n = 51) were applied to strong anion exchange chromatography (SAX2) chips for protein profiling by SELDI-TOF-MS to screen multiple serum biomarkers. Biomarker Wizard software and several statistical methods including algorithm of decision tree, logistic regression and ROC curves were used to construct the decision tree or logistic regression classification models. RESULTS: Average of 61 mass peaks were detected at the molecular range of 2000-30,000, ten decision trees with the highest cross validation rate were chosen to construct the classification models, which can differentiate PaCa from normal pancreas with a sensitivity of 83.3% and a specificity of 100%. Logistic regression was used to achieve the AUC (0.976 +/- 0.011, P < 0.001) with a sensitivity of 77.6% - 91.4% and a specificity of 92.2% - 100%. Six mass peaks were combined by logistic regression to achieve the AUC 0.897 +/- 0.054, 0.978 +/- 0.021 and 0.792 +/- 0.107 (P < 0.05) in the three groups (patients at stage I and II, stage II and III, stage III and IV). One mass peak (M/Z 4,016) was screened (P < 0.05) significantly between the preoperative and postoperative PaCa samples and the intensity decreased weeks after operation. CONCLUSION: Decision tree and logistic regression classification models of the mass peaks screened by SELDI-TOF-MS serum profiling can be used to differentiate pancreatic adenocarcinoma from normal pancreas, and is superior to CA 199. The detected mass peaks are helpful for the evaluation of curative effect and prognosis of pancreatic adenocarcinoma.

Proprotein Convertase Subtilisin/Kexin Type 9 Promotes Gastric Cancer Metastasis and Suppresses Apoptosis by Facilitating MAPK Signaling Pathway Through HSP70 Up-Regulation.

OBJECTIVE: To examine the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on gastric cancer (GC) progression and prognosis, and to explore the underlying mechanism. METHODS: PCSK9 expression levels in human GC tissues were determined by quantitative real-time PCR, western blotting, and immunohistochemical assay. PCSK9 serum levels were detected by enzyme-linked immunosorbent assay. The relationships of PCSK9 and GC progression and survival were analyzed using the Chi-square test, Kaplan-Meier analysis, and Cox proportional hazards model. The effect of PCSK9 on cell invasion, migration, and apoptosis were determined in human GC cell lines and mouse xenograft model separately using PCSK9 knockdown and overexpression strategies. The PCSK9 interacting molecules, screened by co-immunoprecipitation combined with LC-MS/MS, were identified by immunofluorescence localization and western blotting. Additionally, the mitogen-activated protein kinase (MAPK) pathway was assessed by western blotting. RESULTS: PCSK9 mRNA and protein levels were significantly elevated in GC tissues compared with the paired normal tissues at our medical center (P < 0.001). Notably, the up-regulation of PCSK9 expression in GC tissues was related to tumor progression and poor survival. GC patients had higher serum levels of PCSK9 than the age-matched healthy controls (P < 0.001); PCSK9 promoted invasive and migratory ability and inhibited apoptosis in GC cells with no apparent affection in cell proliferation. The silencing of PCSK9 reversed these effects, suppressing tumor metastasis in vitro and in vivo. Furthermore, PCSK9 maintained these functions through up-regulating heat shock protein 70 (HSP70), ultimately facilitating the mitogen-activated protein kinase (MAPK) pathway. CONCLUSION: Collectively, our data revealed that high PCSK9 expression levels in GC tissue were correlated with GC progression and poor prognosis and that PCSK9 could promote GC metastasis and suppress apoptosis by facilitating MAPK signaling pathway through HSP70 up-regulation. PCSK9 may represent a novel potential therapeutic target in GC.