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.

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.

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.

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.

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.

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.

[Epidemiological study of Helicobacter pylori infection and its risk factors in Shanghai].

OBJECTIVE: To explore the Helicobacter pylori (Hp) infection status in Shanghai and its risk factors. METHODS: A questionnaire survey including 60 items related to Hp was conducted among 1925 persons, 976 males (50.7%) and 949 females (49.3%), aged 37 +/- 11 (15 approximately 72), selected by cluster sampling in 12 rural and urban areas in Shanghai. Peripheral blood was collected to detect Hp IgG antibody so as to evaluate the past infection status and (14)C urea breath test (UBT) was performed so as to evaluate the present infection status. Fecal Hp antigen test was done in the subjects under 18 years of age. RESULTS: 1822 of the 1925 subjects (94.6%) underwent the 3 tests. were followed antibody detection and (14)C UBT and response with questionnaires. The Hp infection rate was 66.4% in general, and 68.4% in the females, somehow higher than in the males (64.3%, P = 0.067). The infection rate was different in different age groups, was the lowest in the age group of 15 approximately 19 (44.4%) and the highest 70% in age group of 20 approximately 30 (70%), then somewhat lower and gradually became higher. Socio-economic status was related to Hp infection. Hp infection was related with such factors as eating raw vegetables and fruits, and never washing raw vegetables and fruits before eating. The knowledge rate about Hp infection was only 33.2% among the respondents. Only 23.8% of the respondents knew well the transmission route of Hp. CONCLUSION: Hp infection rate is rather high in Shanghai. Hp infection is related to socio-economic status, and family dietary and sanitary habits.

Curcumin protects against concanavalin A-induced hepatitis in mice through inhibiting the cytoplasmic translocation and expression of high mobility group box 1.

The aims of this study were to examine the anti-inflammatory effect of curcumin on concanavalin A (ConA) induced hepatitis in mice, and to elucidate its underlying molecular mechanisms. Mice received curcumin by gavage before ConA intravenous administration. The results showed that curcumin pretreatment attenuated ConA-induced hepatitis. Enzyme linked immunosorbent assay (ELISA) results showed that serum levels of high mobility group box 1 (HMGB1) increased at 4 h and reached its peak value at 12 h after challenge with ConA; but this increase was significantly inhibited by curcumin. Furthermore, curcumin significantly decreased the HMGB1 translocation from nucleus to cytoplasm of hepatocytes in ConA-induced mice. The levels of HMGB1 mRNA and protein expression in the liver were also significantly lowered in curcumin-treated mice. In addition, curcumin inhibited intrahepatic expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6 protein. In conclusion, the results indicated that curcumin protected against ConA-induced hepatitis in mice; and the beneficial effects may be partly through inhibition of HMGB1 translocation in hepatocytes, release into the plasma and expression in livers.

Glycyrrhizin regulates CD4+T cell response during liver fibrogenesis via JNK, ERK and PI3K/AKT pathway.

The aims of this study were to elucidate the immunomodulatory effects of glycyrrhizin (GL) on CD4(+)T cell responses during liver fibrogenesis. To obtain in vivo evidence about the effects of GL on CD4(+)T cells in livers and spleens of concanavalin A (ConA)-induced mouse model, mice were administrated with ConA together with or without GL for 8 weeks. Mice treated with GL dramatically prevented liver inflammation and fibrosis. Besides, GL inhibited the infiltration of T helper (Th) cell type 1, Th2, Th17 and regulatory T cells (Treg) in livers and spleens of mouse fibrosis models, and regulated the Th1/Th2 and Treg/Th17 balances respectively to a relative dominance of Th1 and Treg lineages in livers. Moreover, GL dramatically enhanced the antifibrotic cytokine interferon (IFN)-γ and interleukin (IL)-10. GL at a concentration of 10 or 100 µg/mL was respectively incubated with ConA-stimulated splenic CD4(+)T cells in vitro, and JNK inhibitor (SP600125), ERK inhibitor (U0126), p38 inhibitor (SB203580) or PI3K/AKT inhibitor (LY29400225) was added during the incubation. Notably, GL not only inhibited ConA-induced proliferation of splenic CD4(+)T cells but also enhanced the mRNAs of IFN-γ and IL-10 in these cells. Be similar to the effects of GL, SP600125, U0126 and LY29400225, however not SB203580, also inhibited ConA-induced CD4(+)T cell proliferation, indicating the involvement of JNK, ERK and PI3K/AKT in this process. Moreover, GL significantly inhibited ConA-induced phosphorylation of JNK, ERK and PI3K/AKT in vitro. Collectively, GL might alleviate liver injury and fibrosis progression via regulation of CD4(+)T cell response in JNK, ERK and PI3K/AKT-dependent pathways.

Curcumin attenuates Concanavalin A-induced liver injury in mice by inhibition of Toll-like receptor (TLR) 2, TLR4 and TLR9 expression.

Curcumin has antiviral, antioxidant, and anti-inflammatory properties. However, the hepatoprotective effects and molecular mechanisms of curcumin on acute liver injury have not been carefully examined. The aims of this study were to examine the anti-inflammatory effect of curcumin on Concanavalin A (Con A) induced hepatitis, and to elucidate its underlying molecular mechanisms in mice. Mice received curcumin (200 mg/kg body weight) by gavage before Con A intravenous administration. We found that curcumin pretreatment was able to significantly reduce the elevated plasma aminotransferase levels and liver necrosis in Con A-induced hepatitis. Also, curcumin pretreatment reduced intrahepatic expression of genes encoding pro-inflammatory molecules such as tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) as compared with the vehicle controls, but augmented anti-inflammatory cytokine interleukin 10 (IL-10) by enzyme linked immunosorbent assay (ELISA). Furthermore, the expression levels of Toll-like receptor (TLR) 2, TLR4 and TLR9 mRNA or protein in liver tissues were significantly lowered by curcumin treatment. Curcumin pretreatment did not affect hepatic Kupffer cell numbers after Con A injection. These results suggest that curcumin pretreatment protects against T cell-mediated hepatitis in mice. The beneficial effect of curcumin may be partly mediated by inhibiting the expression levels of TLR2, TLR4 and TLR9 in the liver.

Protective effects of curcumin against hepatic fibrosis induced by carbon tetrachloride: modulation of high-mobility group box 1, Toll-like receptor 4 and 2 expression.

The aim of the study was to investigate the effect of curcumin on the liver fibrosis induced by carbon tetrachloride (CCl(4)) in rats, and to elucidate its underlying molecular mechanisms. Rats were administered with CCl(4) together with or without curcumin for 6 weeks. Hepatic damage was evaluated by analysis of liver function tests in serum. Hepatic histopathology and collagen content were employed to quantify liver fibrosis; and activated hepatic stellate cells were assessed. Moreover, the mRNA and protein expression levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, high-mobility group box 1 (HMGB1), Toll like receptor (TLR) 2 and TLR4 were determined by quantitative real time PCR, Western blot or immunohistochemistry. Treatment with curcumin significantly attenuated CCl(4)-induce liver injury, hepatic inflammation and reduced the levels of proinflammatory mediators (TNF-α, IL-6 and MCP-1). Moreover, curcumin significantly inhibited extracellular matrix deposition, reduced the number of activated stellate cells, and decreased the levels of HMGB1, TLR4 and TLR2 expression in the rat model of fibrogenesis. These results suggest that curcumin could be an effective agent for preventing liver fibrosis and its mechanism may in part be a consequence of the reduction TLR2, TLR4 and HMGB1 expression.

Significance of the balance between regulatory T (Treg) and T helper 17 (Th17) cells during hepatitis B virus related liver fibrosis.

BACKGROUND: Hepatitis B virus-related liver fibrosis (HBV-LF) always progresses from inflammation to fibrosis. However, the relationship between these two pathological conditions is not fully understood. Here, it is postulated that the balance between regulatory T (Treg) cells and T helper 17 (Th17) cells as an indicator of inflammation may predict fibrosis progression of HBV-LF. METHODOLOGY/PRINCIPAL FINDINGS: The frequencies and phenotypes of peripheral Treg and Th17 cells of seventy-seven HBeAg-positive chronic hepatitis B (CHB) patients who underwent liver biopsies and thirty healthy controls were determined by flow cytometry. In the periphery of CHB patients, both Treg and Th17 frequencies were significantly increased and correlated, and a lower Treg/Th17 ratio always indicated more liver injury and fibrosis progression. To investigate exact effects of Treg and Th17 cells during HBV-LF, a series of in vitro experiments were performed using purified CD4(+), CD4(+)CD25(+), or CD4(+)CD25(-) cells from the periphery, primary human hepatic stellate cells (HSCs) isolated from healthy liver specimens, human recombinant interleukin (IL)-17 cytokine, anti-IL-17 antibody and HBcAg. In response to HBcAg, CD4(+)CD25(+) cells significantly inhibited cell proliferation and cytokine production (especially IL-17 and IL-22) by CD4(+)CD25(-) cells in cell-contact and dose-dependent manners. In addition, CD4(+) cells from CHB patients, compared to those from HC subjects, dramatically promoted proliferation and activation of human HSCs. Moreover, in a dramatically dose-dependent manner, CD4(+)CD25(+) cells from CHB patients inhibited, whereas recombinant IL-17 response promoted the proliferation and activation of HSCs. Finally, in vivo evidence about effects of Treg/Th17 balance during liver fibrosis was obtained in concanavalin A-induced mouse fibrosis models via depletion of CD25(+) or IL-17(+) cells, and it's observed that CD25 depletion promoted, whereas IL-17 depletion, alleviated liver injury and fibrosis progression. CONCLUSIONS/SIGNIFICANCE: The Treg/Th17 balance might influence fibrosis progression in HBV-LF via increase of liver injury and promotion of HSCs activation.

Early diagnosis and therapeutic choice of Klebsiella pneumoniae liver abscess.

Nowadays, pyogenic liver abscess (PLA) is still a common and severe intra-abdominal infection, and Klebsiella pneumoniae had emerged as the most common pathogenic bacteria worldwide in the past ten years. Our study aims to achieve an early pathogenic diagnosis and rational therapy modality for Klebsiella pneumoniae liver abscess (KLA) through clinical data analysis. A total of 197 inpatients in Zhongshan Hospital, Shanghai, diagnosed as having liver abscess between March 2001 and September 2009 were enrolled. Patients with monomicrobial infection were divided into two groups: patients with K. pneumoniae liver abscess (KLA group, n=106) and those with non-Klebsiella pneumoniae liver abscess (NKLA group, n=56). A retrospective analysis was made between these two groups on the aspects of underlying diseases, clinical characteristics, laboratory data, culture results, and imaging findings. To evaluate the effects of different medical interventions, monomicrobial KLA patients were further divided into four subgroups (percutaneous liver aspiration, aspiration plus antibiotics flushing, aspiration plus retained catheter, and aspiration plus antibiotics flushing and retained catheter), and corresponding therapeutic effects were analyzed. KLA was more likely to occur in patients with coexisting diseases such as diabetes mellitus (53.77% vs 25.00%, P=0.001) and hepatic adipose infiltration (16.04% vs 5.36%, P=0.029). Compared to NKLA group, clinical characteristics including abdominal pain (40.57% vs 57.14%, P=0.044), hypodynamia (19.81% vs 46.43%, P=0.001), and hepatomegaly (4.72% vs 14.29%, P=0.033) were much milder, but with a higher fasting blood glucose level (7.84±0.36 vs 5.76±0.30, P=0.001) on admission in KLA group. In addition, KLA abscess often appeared singly in the right lobe of the liver with gas forming nature (32.88% vs 13.51%, P=0.039), unsmooth rim (71.23% vs 40.54%, P=0.002), and dynamic septum enhancement (41.10% vs 16.22%, P=0.009). Compared to mono aspiration subgroup, additional antibiotic flushing could not further improve clinical outcomes of KLA patients (P>0.05); however, the retained catheter showed obvious advantage in reducing abscess diameter (34.38±3.25 mm vs 22.67±2.37 mm, P=0.017). It can be concluded that the strong association with diabetes, milder clinical symptoms, and gas-forming nature in CT images makes early pathogenic diagnosis of KLA possible. Comparatively, ultrasonography-guided percutaneous liver aspiration with retained catheter may be the most rational intervention modality of KLA.

Antifibrotic activity of rofecoxib in vivo is associated with reduced portal hypertension in rats with carbon tetrachloride-induced liver injury.

BACKGROUND AND AIM: Upregulation of cyclooxygenase-2 (COX-2), an inducible enzyme that is actively involved in inflammation and wound healing, has been found in cirrhotic livers. The aim of this study was to investigate the effects of selective inhibition of COX-2 on the development of liver cirrhosis and portal hypertension in rats. METHODS: Liver cirrhosis was induced by carbon tetrachloride (CCl(4)) in Sprague-Dawley rats. Rofecoxib, a highly selective COX-2 inhibitor, was orally administered to rats at a dose of 10 mg/kg/day. Portal pressure was measured at 8 weeks post CCl(4) administration with the catheterization method followed by the harvesting of liver samples. Liver histopathology was analyzed with hematoxylin and eosin and Masson's trichrome staining. The activated, alpha smooth muscle actin (alpha-SMA) positive hepatic stellate cells (HSCs) and the protein levels of collagen types I, III, IV, as well as laminin and two fibrogenic mediators, vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in the livers, were detected with immunohistochemical staining and western blot methods, respectively. The level of hepatic thromboxane B(2) (TXB(2)), a potent vasoconstrictive substance derived from COX, was measured with enzyme immunoassay. RESULTS: Oral administration of rofecoxib decreased portal pressure in rats that were treated with CCl(4) for 8 weeks. This was associated with a marked reduction in collagen accumulation and TXB(2) level in the rat livers. In addition, rofecoxib administration was found to reduce the number of activated HSCs and to downregulate hepatic protein levels of three detected types of collagen, laminin, VEGF and CTGF in CCl(4)-treated rats. CONCLUSIONS: COX-2 is involved in the fibrogenesis of livers and the formation of portal hypertension in CCl(4)-treated rats. Selective inhibition of COX-2 by rofecoxib reduces portal hypertension and this is associated with antifibrotic activity as well as a reduction of COX-2-derived vasoactive substance.