Targeting BRD4 mitigates hepatocellular lipotoxicity by suppressing the NLRP3 inflammasome activation and GSDMD-mediated hepatocyte pyroptosis.

Nod-like receptor family pyrin-containing protein 3 (NLRP3) inflammasome plays a pathologic role in metabolic dysfunction-associated steatohepatitis (MASH), but the molecular mechanism regulating the NLRP3 inflammasome activation in hepatocellular lipotoxicity remains largely unknown. Bromodomain-containing protein 4 (BRD4) has emerged as a key epigenetic reader of acetylated lysine residues in enhancer regions that control the transcription of key genes. The aim of this study is to investigate if and how BRD4 regulated the NLRP3 inflammasome activation and pyroptosis in MASH. Using the AML12 and primary mouse hepatocytes stimulated by palmitic acid (PA) as an in vitro model of hepatocellular lipotoxicity, we found that targeting BRD4 by genetic knockdown or a selective BRD4 inhibitor MS417 protected against hepatosteatosis; and this protective effect was attributed to inhibiting the activation of NLRP3 inflammasome and reducing the expression of Caspase-1, gasdermin D (GSDMD), interleukin (IL)-1ß and IL-6. Moreover, BRD4 inhibition limited the voltage-dependent anion channel-1 (VDAC1) expression and oligomerization in PA-treated AML12 hepatocytes, thereby suppressing the NLRP3 inflammasome activation. Additionally, the expression of BRD4 enhanced in MASH livers of humans. Mechanistically, BRD4 was upregulated during hepatocellular lipotoxicity that in turn modulated the active epigenetic mark H3K27ac at the promoter regions of the Vdac and Gsdmd genes, thereby enhancing the expression of VDAC and GSDMD. Altogether, our data provide novel insights into epigenetic mechanisms underlying BRD4 activating the NLRP3 inflammasome and promoting GSDMD-mediated pyroptosis in hepatocellular lipotoxicity. Thus, BRD4 might serve as a novel therapeutic target for the treatment of MASH.

Dietary inflammatory index is associated with metabolic dysfunction-associated fatty liver disease among United States adults.

Background: Metabolic dysfunction-associated fatty liver disease (MAFLD) is presently the most prevalent chronic liver disorder globally that is closely linked to obesity, dyslipidemia metabolic syndrome, and type 2 diabetes mellitus (T2DM). Its pathogenesis is strongly associated with inflammation, and diet is a major factor in reducing inflammation. However, current research has focused primarily on exploring the relationship between diet and NAFLD, with less research on its link to MAFLD. Methods: In this research, using dietary inflammatory index (DII) as a measure to assess dietary quality, we analyzed the relationship between diet and MAFLD. Data from the National Health and Nutrition Examination Survey (NHANES) 2017-2018, including 3,633 adults with complete DII and MAFLD, were used to develop cross-sectional analyses. Logistic regression analysis was adapted for investigating the relationship between DII and MAFLD development. Additionally, subgroup analysis and threshold effect analysis were carried out. Results: A positive link between DII and MAFLD was found in the fully adjusted model (OR = 1.05; 95%CI, 1.00-1.11, p < 0.05). Subgroup analysis indicated that there was no significant dependence for the connection between DII and MAFLD except for the subgroup stratified by age. Compared with other age groups, people with MAFLD had 20% higher DII scores than non-MAFLD participants in those aged 20-41 years old (OR = 1.20; 95%CI, 1.08-1.33, p < 0.001). Furthermore, we found a U-shaped curve with an inflection point of 3.06 illustrating the non-linear connection between DII and MAFLD. Conclusion: As a result, our research indicates that pro-inflammatory diet may increase the chance of MAFLD development, thus improved dietary patterns as a lifestyle intervention is an important strategy to decrease the incidence of MAFLD.

Identification and Characterization of a Novel Nanobody Against Human CTGF to Reveal Its Antifibrotic Effect in an in vitro Model of Liver Fibrosis.

Background: No agents are currently available for the treatment or reversal of liver fibrosis. Novel antifibrotic therapies for chronic liver diseases are thus urgently needed. Connective tissue growth factor (CTGF) has been shown to contributes profoundly to liver fibrogenesis, which makes CTGF as a promising target for developing antifibrotic agents. Methods: In this study, we identified a novel nanobody (Nb) against human CTGF (anti-CTGF Nb) by phage display using an immunized camel, which showed high affinity and specificity in vitro. LX-2 cells, the immortalized human hepatic stellate cells, were induced by transforming growth factor beta1 (TGFß1) as an in vitro model of liver fibrosis to verify the antifibrotic activity of the anti-CTGF Nb. Results: Our data demonstrated that anti-CTGF Nb effectively alleviated TGFß1-induced LX-2 cell proliferation, activation, and migration, and promoted the apoptosis of activated LX-2 cells in response to TGFß1. Moreover, the anti-CTGF Nb remarkably reduced the levels of TGFß1, Smad2, and Smad3 expression in LX-2 stellate cells stimulated by TGFß1. Conclusion: Taken together, we successfully identified a novel Nb against human CTGF, which exhibited antifibrotic effects in vitro by regulating the biological functions of human stellate cells LX-2.

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.

Quantitative, noninvasive MRI characterization of disease progression in a mouse model of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is an increasing cause of chronic liver disease characterized by steatosis, inflammation, and fibrosis which can lead to cirrhosis, hepatocellular carcinoma, and mortality. Quantitative, noninvasive methods for characterizing the pathophysiology of NASH at both the preclinical and clinical level are sorely needed. We report here a multiparametric magnetic resonance imaging (MRI) protocol with the fibrogenesis probe Gd-Hyd to characterize fibrotic disease activity and steatosis in a common mouse model of NASH. Mice were fed a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) to induce NASH with advanced fibrosis. Mice fed normal chow and CDAHFD underwent MRI after 2, 6, 10 and 14 weeks to measure liver T1, T2*, fat fraction, and dynamic T1-weighted Gd-Hyd enhanced imaging of the liver. Steatosis, inflammation, and fibrosis were then quantified by histology. NASH and fibrosis developed quickly in CDAHFD fed mice with strong correlation between morphometric steatosis quantification and liver fat estimated by MRI (r = 0.90). Sirius red histology and collagen quantification confirmed increasing fibrosis over time (r = 0.82). Though baseline T1 and T2* measurements did not correlate with fibrosis, Gd-Hyd signal enhancement provided a measure of the extent of active fibrotic disease progression and correlated strongly with lysyl oxidase expression. Gd-Hyd MRI accurately detects fibrogenesis in a mouse model of NASH with advanced fibrosis and can be combined with other MR measures, like fat imaging, to more accurately assess disease burden.

Myricetin Modulates Macrophage Polarization and Mitigates Liver Inflammation and Fibrosis in a Murine Model of Nonalcoholic Steatohepatitis.

This study aimed to investigate the beneficial effects of myricetin in a diet-induced nonalcoholic steatohepatitis (NASH) model and the underlying mechanism. C57BL/6J mice were fed a standard chow or the choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for 8 weeks with the treatment of myricetin (100 mg/kg) or vehicle by daily gavage. Hepatic inflammation, steatosis, fibrosis, and hepatic stellate cells (HSC) activation were assessed. We also analyzed M1 and M2 macrophages and its related markers in livers from NASH mice and in RAW264.7 macrophages stimulated by lipopolysaccharide (LPS) or interleukin 4 (IL-4) in vitro. Furthermore, we determined the effect of myricetin on the triggering receptor expressed on myeloid cells-1 (TREM-1), toll like receptor (TLR) 2 and 4, and myeloid differentiation factor 88 (MyD88) signaling both in livers from mice and in RAW264.7 cells stimulated by LPS. Our results revealed that myricetin remarkably ameliorated hepatic steatosis, inflammation, and inhibited hepatic macrophage infiltration in CDAHFD-fed mice. Myricetin-treated to CDAHFD-fed mice also inhibited liver fibrosis and HSC activation when compared with vehicle-treated to those mice. Moreover, myricetin inhibited M1 macrophage polarization and its relative markers in livers of NASH mice while induced M2 polarization. Similarly, in vitro study, myricetin inhibited the LPS-induced mRNA expression of M1 macrophages marker genes and induced IL-4-induced M2 macrophage marker genes in RAW264.7 macrophages. Mechanically, myricetin inhibited the expression of TREM-1 and TLR2/4-MyD88 signaling molecules in livers from NASH mice and in RAW264.7 macrophages stimulated by LPS in vitro. Additionally, myricetin inhibited the activation of nuclear factor (NF)-κB signaling and the phosphorylation of the signal transducer and activation of transcription 3 (STAT3) in LPS-stimulated RAW264.7 macrophages. Taken together, our data indicated that myricetin modulated the polarization of macrophages via inhibiting the TREM-1-TLR2/4-MyD88 signaling molecules in macrophages and therefore mitigated NASH and hepatic fibrosis in the CDAHFD-diet-induced NASH model in mice.

Morin Hydrate Inhibits TREM-1/TLR4-Mediated Inflammatory Response in Macrophages and Protects Against Carbon Tetrachloride-Induced Acute Liver Injury in Mice.

This study aims to investigate the protective effects of morin hydrate (MH) against acute liver injury induced by carbon tetrachloride (CCl4) in mice and to elucidate the possible molecular mechanism of action. Mice were pretreated with MH (50 mg/kg body weight) or vehicle by oral gavage once daily for 5 days, followed by intraperitoneal injection of a single dose of CCl4 (1 ml/kg in olive oil). Mice were sacrificed 24 h later; the blood and liver samples were harvested for analysis. We also used the model of lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages in vitro and examined the effects of MH and its mechanism of action on the inflammatory response. Our results revealed that MH remarkably attenuated liver histopathological alterations, serum transaminases, hepatocytes death, and inflammatory response induced by CCl4. Importantly, MH reduced expression of the triggering receptor expressed on myeloid cells-1 (TREM-1) and toll-like receptor 4 (TLR4) both in vivo and in vitro experiments. This inhibitory effect MH on expression of the TREM-1 and TLR4 in cell culture was further heightened after TREM-1 knockdown with small interfering RNA (siRNA). Moreover, MH dramatically suppressed the inhibitor of kappa B α (IκBα) degradation and subsequent nuclear factor-kappa B (NF-κB) p65 translocation into the nucleus and NF-κB-mediated cytokines, such as tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, and IL-6. Additionally, MH also ameliorated CCl4-induced oxidative stress by enhancing the nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in the injured livers. Taken together, MH has hepatoprotective activity, and this effect may be elicited by attenuating macrophage-mediated inflammatory responses via inhibition TREM-1/TLR4/NF-κB signaling and by regulating hepatic oxidative stress via enhancement Nrf2/HO-1 antioxidant pathway.

Molecular magnetic resonance imaging accurately measures the antifibrotic effect of EDP-305, a novel farnesoid X receptor agonist.

We examined a novel farnesoid X receptor agonist, EDP-305, for its antifibrotic effect in bile duct ligation (BDL) and choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) models of hepatic injury. We used molecular magnetic resonance imaging with the type 1 collagen-binding probe EP-3533 and the oxidized collagen-specific probe gadolinium hydrazide to noninvasively measure treatment response. BDL rats (n = 8 for each group) were treated with either low or high doses of EDP-305 starting on day 4 after BDL and were imaged on day 18. CDAHFD mice (n = 8 for each group) were treated starting at 6 weeks after the diet and were imaged at 12 weeks. Liver tissue was subjected to pathologic and morphometric scoring of fibrosis, hydroxyproline quantitation, and determination of fibrogenic messenger RNA expression. High-dose EDP-305 (30 mg/kg) reduced liver fibrosis in both the BDL and CDAHFD models as measured by collagen proportional area, hydroxyproline analysis, and fibrogenic gene expression (all P < 0.05). Magnetic resonance signal intensity with both EP-3533 in the BDL model and gadolinium hydrazide in the CDAHFD model was reduced with EDP-305 30 mg/kg treatment (P < 0.01). Histologically, EDP-305 30 mg/kg halted fibrosis progression in the CDAHFD model. Conclusion: EDP-305 reduced fibrosis progression in rat BDL and mouse CDAHFD models. Molecular imaging of collagen and oxidized collagen is sensitive to changes in fibrosis and could be used to noninvasively measure treatment response in clinical trials. (Hepatology Communications 2018;2:821-835).

The Flavonoid Quercetin Ameliorates Liver Inflammation and Fibrosis by Regulating Hepatic Macrophages Activation and Polarization in Mice.

At present, there are no effective antifibrotic drugs for patients with chronic liver disease; hence, the development of antifibrotic therapies is urgently needed. Here, we performed an experimental and translational study to investigate the potential and underlying mechanism of quercetin treatment in liver fibrosis, mainly focusing on the impact of quercetin on macrophages activation and polarization. BALB/c mice were induced liver fibrosis by carbon tetrachloride (CCl4) for 8 weeks and concomitantly treated with quercetin (50 mg/kg) or vehicle by daily gavage. Liver inflammation, fibrosis, and hepatic stellate cells (HSCs) activation were examined. Moreover, massive macrophages accumulation, M1 macrophages and their related markers, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and monocyte chemotactic protein-1 (MCP-1) in livers were analyzed. In vitro, we used Raw 264.7 cells to examine the effect of quercetin on M1-polarized macrophages activation. Our results showed that quercetin dramatically ameliorated liver inflammation, fibrosis, and inhibited HSCs activation. These results were attributed to the reductive recruitment of macrophages (F4/80+ and CD68+) into the liver in quercetin-treated fibrotic mice confirmed by immunostaining and expression levels of marker molecules. Importantly, quercetin strongly inhibited M1 polarization and M1-related inflammatory cytokines in fibrotic livers when compared with vehicle-treated mice. In vitro, studies further revealed that quercetin efficiently inhibited macrophages activation and M1 polarization, as well as decreased the mRNA expression of M1 macrophage markers such as TNF-α, IL-1ß, IL-6, and nitric oxide synthase 2. Mechanistically, the inhibition of M1 macrophages by quercetin was associated with the decreased levels of Notch1 expression on macrophages both in vivo and in vitro. Taken together, our data indicated that quercetin attenuated CCl4-induced liver inflammation and fibrosis in mice through inhibiting macrophages infiltration and modulating M1 macrophages polarization via targeting Notch1 pathway. Hence, quercetin holds promise as potential therapeutic agent for human fibrotic liver disease.

Placental Growth Factor Contributes to Liver Inflammation, Angiogenesis, Fibrosis in Mice by Promoting Hepatic Macrophage Recruitment and Activation.

Placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) family, mediates wound healing and inflammatory responses, exerting an effect on liver fibrosis and angiogenesis; however, the precise mechanism remains unclear. The aims of this study are to identify the role of PlGF in liver inflammation and fibrosis induced by bile duct ligation (BDL) in mice and to reveal the underlying molecular mechanism. PlGF small interfering RNA (siRNA) or non-targeting control siRNA was injected by tail vein starting 2 days after BDL. Liver inflammation, fibrosis, angiogenesis, macrophage infiltration, and hepatic stellate cells (HSCs) activation were examined. Our results showed that PlGF was highly expressed in fibrotic livers and mainly distributed in activated HSCs and macrophages. Furthermore, PlGF silencing strongly reduced the severity of liver inflammation and fibrosis, and inhibited the activation of HSCs. Remarkably, PlGF silencing also attenuated BDL-induced hepatic angiogenesis, as evidenced by attenuated liver endothelial cell markers CD31 and von Willebrand factor immunostaining and genes or protein expression. Interestingly, these pathological ameliorations by PlGF silencing were due to a marked reduction in the numbers of intrahepatic F4/80+, CD68+, and Ly6C+ cell populations, which were reflected by a lower expression of these macrophage marker molecules in fibrotic livers. In addition, knockdown of PlGF by siRNA inhibited macrophages activation and substantially suppressed the expression of pro-inflammatory cytokines and chemokines in fibrotic livers. Mechanistically, evaluation of cultured RAW 264.7 cells revealed that VEGF receptor 1 (VEGFR1) mainly involved in mediating the role of PlGF in macrophages recruitment and activation, since using VEGFR1 neutralizing antibody blocking PlGF/VEGFR1 signaling axis significantly inhibited macrophages migration and inflammatory responses. Together, these findings indicate that PlGF plays an important role in liver inflammation, angiogenesis, and fibrosis by promoting hepatic macrophage recruitment and activation, and suggest that blockage of PlGF could be a promising novel therapy for chronic fibrotic liver diseases.

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.

MicroRNA-146a promote cell migration and invasion in human colorectal cancer via carboxypeptidase M/src-FAK pathway.

Colorectal cancer (CRC) is one of the most common cancers worldwide, and microRNAs play important roles in CRC progression. This study aimed to investigate the roles of miR-146a-5p in human CRC and their molecular mechanisms. First, we found that miR-146a-5p was significantly upregulated in CRC tissues and promoted the migration of CRC cells. Then, we identified carboxypeptidase M (CPM) as a direct target of miR-146a-5p, and found that it inhibited the migration and invasion of CRC cells. Our results also showed that CPM expression was positively correlated with overall survival and negatively correlated with recurrence, lymph node invasion, and N stage. Furthermore, we demonstrated that both miR-146a-5p and CPM regulated Src and FAK expression, while the Src-FAK signaling pathway is widely known to be associated with the migration and invasion of multiple tumor cells. This study is the first to demonstrate the functional and mechanistic relationship of the miR-146a-5p/CPM/Src-FAK axis and its effect on the migration and invasion of CRC cells. Thus, miR-146a-5p represents potential targets for CRC diagnosis and therapy.

Curcumin Protects Against Intestinal Origin Endotoxemia in Rat Liver Cirrhosis by Targeting PCSK9.

Intestinal origin endotoxemia always occurs in severe liver injury. The aim of the current study was to test antiendotoxemia effect of curcumin on tetrachloride (CCl4 )-induced liver cirrhosis rats, and to elucidate the underlying molecular mechanism. Rat cirrhosis models were constructed with CCl4 subcutaneous injections with curcumin (200 mg/kg/d) administered via gavages for 12 wk until the rats were sacrificed. We found that the administration of curcumin improved the physiological condition pertaining to activity index and temperature, and ameliorated the liver injury in CCl4 -induced cirrhosis rats. Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative polymerase chain reaction (qRT-PCR) showed that curcumin could reduce c-reaction protein levels and inflammatory cytokine (TNF-α, IL-1ß, IL-6, and CINC-1/IL-8) concentrations in peripheral serum and liver tissue. Furthermore, curcumin treatment decreased lipopolysaccharide (LPS) levels in peripheral vein, but not in portal vein. As low-density lipoprotein receptor (LDLR) is the important receptor on the surface of hepatocyte during LPS detoxification process, we used qRT-PCR, western blot, and immunohistochemistry (IHC), finding that curcumin significantly increased LDLR protein levels, but not gene levels in the liver tissues. We also tested proprotein convertase subtilisin/kexin type 9 (PCSK9), one negative regulator of LDLR, by qRT-PCR, western blot, and IHC. The results showed that PCSK9 significantly decreased both gene and protein levels in the rat liver tissues of curcumin treatment. Thus, we concluded that curcumin could function to protect against intestinal origin endotoxemia by inhibiting PCSK9 to promote LDLR expression, thereby enhancing LPS detoxification as one pathogen lipid through LDLR in the liver.

Quercetin attenuates the activation of hepatic stellate cells and liver fibrosis in mice through modulation of HMGB1-TLR2/4-NF-κB signaling pathways.

This study aimed to investigate the effects of quercetin on liver fibrogenesis in mice and to elucidate the underlying molecular mechanisms. Mice were administered with carbon tetrachloride (CCl4) for eight weeks to induce liver fibrosis and concomitantly orally treated with quercetin (50mgkg-1day-1). Here, we demonstrated that quercetin dramatically ameliorated liver injury, inflammation, and hepatic fibrogenesis induced by CCl4. Quercetin also inhibited the activation of hepatic stellate cells (HSC) in vivo and in vitro, as evaluated by α-smooth muscle actin (α-SMA) expression, which is a specific marker of HSC activation. Moreover, reduced fibrosis was associated with decreased high-mobility group box 1 (HMGB1), toll like receptor (TLR) 2 and TLR4 genes, and protein expression. Quercetin also inhibited the cytoplasmic translocation of HMGB1 in hepatocytes of fibrotic livers. Further investigation demonstrated that quercetin treatment significantly attenuated CCl4-induced nuclear translocation of the nuclear factor-κB (NF-κB) p65 and inhibited degradation of IκBα (an inhibitor of NF-κB) expression in the liver compared with vehicle-treated fibrotic mice. Considered together, our data indicate that quercetin has hepatoprotective and anti-fibrotic effects in animal models of liver fibrosis, the mechanism of which may be involved in modulating the HMGB1-TLR2/4-NF-κB signaling pathways.

MicroRNA-21 promotes phosphatase gene and protein kinase B/phosphatidylinositol 3-kinase expression in colorectal cancer.

AIM: To explore the regulatory mechanism of the target gene of microRNA-21 (miR-21), phosphatase gene (PTEN), and its downstream proteins, protein kinase B (AKT) and phosphatidylinositol 3-kinase (PI3K), in colorectal cancer (CRC) cells. METHODS: Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression levels of miR-21 and PTEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of PTEN mRNA and its downstream proteins AKT and PI3K in HCT116 cells after downregulating miR-21 were investigated. RESULTS: Comparing the miR-21 expression in CRC cells, the expression levels of miR-21 were highest in HCT116 cells, and the expression levels of miR-21 were lowest in SW480 cells. In comparing miR-21 and PTEN expression in CRC cells, we found that the protein expression levels of miR-21 and PTEN were inversely correlated (P < 0.05); when miR-21 expression was reduced, mRNA expression levels of PTEN did not significantly change (P > 0.05), but the expression levels of its protein significantly increased (P < 0.05). In comparing the levels of PTEN protein and downstream AKT and PI3K in HCT116 cells after downregulation of miR-21 expression, the levels of AKT and PI3K protein expression significantly decreased (P < 0.05). CONCLUSION: PTEN is one of the direct target genes of miR-21. Thus, phosphatase gene and its downstream AKT and PI3K expression levels can be regulated by regulating the expression levels of miR-21, which in turn regulates the development of CRC.

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.

ANGPTL2 expression in gastric cancer tissues and cells and its biological behavior.

AIM: To explore expression of angiopoietin-like protein 2 (ANGPTL2) and its effect on biological behavior such as proliferation and invasiveness in gastric cancer. METHODS: Western blotting was used to detect expression of ANGPTL2 in 60 human normal gastric tissues, 60 human gastric cancer tissues and gastric cell lines including GES-1, N87, SGC7901, BGC823 and PAMC82. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Transwell assay were used to detect the proliferation and invasive ability of gastric cancer cells. RESULTS: Compared to normal tissues, ANGPTL2 protein levels were significantly upregulated in gastric tissues, and this level was closely correlated with gastric tumor grade, clinical stage and lymph node metastasis. Compared to GES-1 cells, ANGPTL2 mRNA and protein levels were significantly increased in gastric cancer cells including N87, SGC7901, BGC823 and PAMC82. The expression of ANGPTL2 in highly malignant gastric cancer cell lines BGC823 and PAMC82 was significantly higher than in low malignancy gastric cancer cell lines N87 and SGC7901. MTT and Transwell experiments indicated that the proliferation rate and invasive ability of stable overexpressed gastric cancer cells was faster than in cells transfected with Lv-NC and blank control cells, and the invasive ability of stable overexpressed gastric cancer cells was higher than that of cells transfected with Lv-NC and blank control cells. CONCLUSION: ANGPTL2 contributed to proliferation and invasion of gastric cancer cells. In clinical treatment, ANGPTL2 may become a new target for treatment of gastric cancer.

Quercetin Protects Mice from ConA-Induced Hepatitis by Inhibiting HMGB1-TLR Expression and Down-Regulating the Nuclear Factor Kappa B Pathway.

The dietary flavonoid quercetin has hepatoprotective effects. We analyzed the effects of quercetin on concanavalin A (ConA)-induced hepatitis in mice and its underlying molecular mechanisms of action. Mice were administered quercetin (50 mg/kg body weight, i.p.) or vehicle 30 min before intravenous administration of ConA. Quercetin pretreatment significantly reduced the ConA-induced elevations in plasma aminotransferase concentrations and liver necrosis, as well as reducing serum concentrations of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interferon-γ, and interleukin-4. Quercetin pretreatment also reduced expression of high-mobility group box 1 protein (HMGB1) and toll-like receptor (TLR)-2 and TLR-4 messenger RNA (mRNA) and protein in liver tissues. Quercetin pretreatment significantly inhibited degradation of inhibitory kappa B alpha and modulated ConA-induced nuclear translocation in the liver of nuclear factor kappa B (NF-κB) p65. These results demonstrate that quercetin protects against ConA-mediated hepatitis in mice by attenuating the HMGB1-TLRs-NF-κB signaling pathway.