Chuantao Tu

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.

Curcumin ameliorates intrahepatic angiogenesis and capillarization of the sinusoids in carbon tetrachloride-induced rat liver fibrosis

Neoangiogenesis and the development of an abnormal angio-architecture in the liver are strongly linked with progressive fibrogenesis. This study aimed to evaluate the ability of curcumin to protect liver fibrosis-associated angiogenesis and capillarization of the sinusoids in experimental rats. Liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl₄) with or without curcumin for 6 weeks. The results suggest that curcumin treatment markedly attenuated CCl₄-induced liver fibrosis, as assessed by histology and hydroxyproline content, and inhibited hepatic stellate cell activation. Curcumin ameliorated hepatic angiogenesis, as assessed by measuring microvessel density using Von Willebrand factor staining and by examining the expression of the endothelial cell markers CD31 and vascular endothelial growth factor receptor (VEGFR)-2 in the livers. Pathologic remodeling of liver sinusoidal capillarization, as assessed by electron-microscopic analysis of Disses space and by evaluation of the levels of basement membrane protein expression, was also attenuated by curcumin administration. The intrahepatic gene or protein expression of hypoxia-inducible factor-1α, VEGFR-1, placental growth factor, and cyclooxygenase-2 decreased with treatment with curcumin in fibrotic rats. In conclusion, curcumin ameliorates hepatic angiogenesis and sinusoidal capillarization in CCl₄-induced rat liver fibrosis through suppressing multiple proangiogenic factors.

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.

Enhanced high-mobility group box 1 (HMGB1) modulates regulatory T cells (Treg)/T helper 17 (Th17) balance via toll-like receptor (TLR)-4-interleukin (IL)-6 pathway in patients with chronic hepatitis B.

High‐mobility group box 1 (HMGB1) proteins are substantially up‐regulated in acute and chronic hepatitis. However, the immunopathogenic role of HMGB1 in patients with chronic hepatitis B (CHB) has not been elucidated. In this study, using a cohort of 36 CHB patients, we demonstrated a crucial role for HMGB1 to modulate balance between regulatory T (Treg) and T helper 17 (Th17) cells via the toll‐like receptor (TLR)‐4‐interleukin (IL)‐6 pathway. Serum HMGB1 levels were dramatically higher in CHB patients and increased along with liver injury, inflammation and fibrosis. Notably, HMGB1 increased along with decreased Treg/Th17 cells ratios in the periphery or intrahepatic microenvironment, which provides a clue for HMGB1 to favour Th17 responses whereas inhibit Treg responses. For in vitro studies, serum pools were constructed with serum from CHB patients at an advanced stage, whereas peripheral blood mononuclear cells (PBMC) pools were constructed with cells from those at an early stage. CHB‐serum significantly enhanced retinoic acid‐related orphan receptor‐γt (RORγt), whereas they inhibited forkhead box P3 (Foxp3) expression in CHB‐PBMC, which could be reversed by blocking of HMGB1, TLR4, or IL‐6. Besides, recombinant HMGB1 (rHMGB1) dose‐dependently up‐regulated RORγt whereas down‐regulated Foxp3 expression in CHB‐PBMC, and meanwhile, rHMGB1 enhanced TLR4 and IL‐6 expression in CHB‐PBMC. Moreover, the axis of HMGB1–TLR4‐IL‐6–Treg/Th17 required noncontact interactions between CD4 and non‐CD4 cells. In addition, rHMGB1 down‐regulated anti‐inflammatory proteins on CD4+CD25+ cells whereas up‐regulated pro‐inflammatory cytokines in CD4+CD25− cells. In summary, enriched HMGB1 in CHB patients shifts Treg/Th17 balance to Th17 dominance via the TLR4‐IL‐6 pathway, which exacerbates liver injury and inflammation.

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.

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.

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.

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.

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.

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.