Liyong Pu

Antifibrotic Effect of Hepatocyte Growth Factor-Expressing Mesenchymal Stem Cells in Small-for-Size Liver Transplant Rats

Ischemia-reperfusion and chronic injuries associated with small-for-size liver transplantation (SFSLT) impair the regeneration of liver graft and induce liver fibrosis. Mesenchymal stem cells (MSCs) can prevent the development of liver fibrosis, and hepatocyte growth factor (HGF) can also attenuate liver cirrhosis. Our previous studies have demonstrated that higher occurrence of liver fibrosis existed in rats post-SFSLT, and that implantation of HGF/MSCs, the human HGF (hHGF)-expressing MSCs, can improve liver regeneration, reduce mortality of rats, as well as have the potent antifibrotic effect in this SFSLT model. In the present study, we implanted HGF/MSCs into liver grafts via the portal vein and investigated their role in antifibrosis effect, using a 30% SFSLT rat model. Fibrosis indexes, including laminin (LN), hyaluronic acid (HA) levels in serum and hydroxyproline (Hyp) content in the liver grafts, the expression of transforming growth factor-beta1 (TGF-beta(1)), rat HGF (rHGF), alpha-smooth muscle actin (alpha-SMA) in hepatic stellate cells (HSCs), alanine aminotransferase (ALT), total bilirubin (BIL), and albumin (ALB) levels in serum, in rats in different treatment groups were assessed at different time points. We found that HGF/MSCs significantly inhibited the formation of liver fibrosis in rats undergoing SFSLT, while MSCs and HGF had synergistic effects in the process. The antifibrosis effect of HGF/MSCs may have contributed in modulating the activation and apoptosis of HSCs, elevating the rHGF expression level, and decreasing the TGF-beta(1) secretion of activated HSCs. These studies suggest that HGF/MSCs may be a novel therapeutic option for the treatment of liver fibrosis after SFSLT.

N-acetylcysteine attenuates reactive-oxygen-species-mediated endoplasmic reticulum stress during liver ischemia-reperfusion injury

AIM To investigate the effects of N-acetylcysteine (NAC) on endoplasmic reticulum (ER) stress and tissue injury during liver ischemia reperfusion injury (IRI). METHODS Mice were injected with NAC (300 mg/kg) intraperitoneally 2 h before ischemia. Real-time polymerase chain reaction and western blotting determined ER stress molecules (GRP78, ATF4 and CHOP). To analyze the role of NAC in reactive oxygen species (ROS)-mediated ER stress and apoptosis, lactate dehydrogenase (LDH) was examined in cultured hepatocytes treated by H2O2 or thapsigargin (TG). RESULTS NAC treatment significantly reduced the level of ROS and attenuated ROS-induced liver injury after IRI, based on glutathione, malondialdehyde, serum alanine aminotransferase levels, and histopathology. ROS-mediated ER stress was significantly inhibited in NAC-treated mice. In addition, NAC treatment significantly reduced caspase-3 activity and apoptosis after reperfusion, which correlated with the protein expression of Bcl-2 and Bcl-xl. Similarly, NAC treatment significantly inhibited LDH release from hepatocytes treated by H2O2 or TG. CONCLUSION This study provides new evidence for the protective effects of NAC treatment on hepatocytes during IRI. Through inhibition of ROS-mediated ER stress, NAC may be critical to inhibit the ER-stress-related apoptosis pathway.

In vitro induced CD4(+)CD25(+)Foxp3(+) Tregs attenuate hepatic ischemia-reperfusion injury.

Reperfusion injury causes liver dysfunction after warm or cold ischemia. Emerging data suggest a role of T cells as mediators in this ischemia/reperfusion (I/R) injury. In the T cells, a part of CD4(+)CD25(+)FoxP3(+) T regulatory cells (Tregs) were reported to facilitate recovery from I/R injury. These Tregs can be induced by TGF-beta in vitro. Interestingly, rapamycin was reported to selectively expand these Tregs in vitro. In the present study, addition of rapamycin to cultures containing TGF-beta further increased the frequency and absolute number of functional CD4(+) Tregs. Using a partial (70%) hepatic warm ischemia model, we investigated the effects of liver function recovery under the treatment of Tregs induced by rapamycin and TGF-beta. The treatment of Tregs significantly reduced serum alanine aminotransferase and aspartate aminotransferase compared to I/R control animals at 24 h after reperfusion (P<0.05). They also significantly attenuated the up-regulation of IFN-gamma and IL-17 compared to the I/R control animals (P<0.05). In conclusion, Tregs ameliorate the biochemical of hepatic I/R injury by preventing proinflammatory cytokines following a warm I/R insult. These data may pave the way to use Tregs as cell therapy to prevent hepatic I/R injury.

PNPLA3 polymorphisms (rs738409) and non-alcoholic fatty liver disease risk and related phenotypes: a meta-analysis.

One single‐nucleotide polymorphisms (SNPs) rs738409 in the patatin‐like phospholipase domain‐containing 3 gene (PNPLA3) has been implicated in susceptibility to non‐alcoholic fatty liver disease (NAFLD) across different populations. One meta‐analysis confirmed this association, but within it, only two Asian studies were included. This meta‐analysis aimed to investigate the association in Asian population.

Comparative Proteomic Profiling of Human Bile Reveals SSP411 as a Novel Biomarker of Cholangiocarcinoma

Background Cholangiocarcinoma (CC) is an intractable cancer, arising from biliary epithelial cells, which has a poor prognosis and is increasing in incidence. Early diagnosis of CC is essential as surgical resection remains the only effective therapy. The purpose of this study was to identify improved biomarkers to facilitate early diagnosis and prognostication in CC. Methods A comparative expression profile of human bile samples from patients with cholangitis and CC was constructed using a classic 2D/MS/MS strategy and the expression of selected proteins was confirmed by Western blotting. Immunohistochemistry was performed to determine the expression levels of selected candidate biomarkers in CC and matched normal tissues. Finally, spermatogenesis associated 20 (SSP411; also named SPATA20) was quantified in serum samples using an ELISA. Results We identified 97 differentially expressed protein spots, corresponding to 49 different genes, of which 38 were upregulated in bile from CC patients. Western blotting confirmed that phosphoglycerate mutase 1 (brain) (PGAM-1), protein disulfide isomerase family A, member 3 (PDIA3), heat shock 60 kDa protein 1 (chaperonin) (HSPD1) and SSP411 were significantly upregulated in individual bile samples from CC patients. Immunohistochemistry demonstrated these proteins were also overexpressed in CC, relative to normal tissues. SSP411 displayed value as a potential serum diagnostic biomarker for CC, with a sensitivity of 90.0% and specificity of 83.3% at a cutoff value of 0.63. Conclusions We successfully constructed a proteomic profile of CC bile proteins, providing a valuable pool novel of candidate biomarkers. SSP411 has potential as a biomarker for the diagnosis of CC.

Outcomes of Living-Related Liver Transplantation for Wilson's Disease : A Single-Center Experience in China

Background. Although orthotopic liver transplantation provides a therapeutic option for patients with Wilson’s disease (WD) presenting fulminant liver failure or drug resistance, it is still unclear whether the living-related liver transplantation (LRLT) can result in long-term therapeutic effect on WD. Methods. Here, we report a retrospective analysis of LRLT for 36 cases of WD patients. The indications for LRLT were fulminant hepatic failure in two patients and chronic advanced liver disease in 32 patients including 13 patients with Wilsonian neurologic manifestations. Two patients presented with severe Wilsonian neurologic manifestations even though their liver functions were stable. Results. Results revealed that the survival of posttransplant patients or grafts at 1, 3, and 5 years was 91.7%, 83.3%, 75%, or 86.1%, 77.8%, 75%, respectively. Pretransplant intensive care unit-bound and model for end-stage liver disease score were indicated as independent factors predictive of patient survival. Patients with neurologic abnormalities showed significant improvement after liver transplant. Conclusion. Our results indicate LRLT is an excellent therapeutic modality for WD patients with end-stage liver disease. Better pretransplant conditions appeared to be advantageous in gaining better survival outcomes of patients undergoing LRLT.

All-trans retinoic acid preconditioning protects against liver ischemia/reperfusion injury by inhibiting the nuclear factor kappa B signaling pathway

BACKGROUND Inflammatory response plays a pathogenic role in liver ischemia/reperfusion (I/R) injury. All-trans retinoic acid (ATRA) is an active metabolite of vitamin A with anti-inflammatory effects. However, there are few reports on the anti-inflammatory effects of ATRA on liver I/R injury. The purpose of this study was to investigate the effects of ATRA on liver I/R injury and related mechanisms. METHODS A total of 54 male Sprague-Dawley rats were randomly divided into three groups (18 rats each), namely, sham, I/R, and I/R+ATRA groups. ATRA was intraperitoneally administered at a dose of 15mg/kg/d 14d before ischemia surgery. The segmental (70%) hepatic ischemia model was used by clamping the portal vein, hepatic artery, and bile duct of the left and median for 1h. The rats were sacrificed 3, 6, and 24h after reperfusion, and blood and liver tissue samples were obtained. Liver injury was evaluated by biochemical and histopathologic examinations. Myeloperoxidase activity was spectrophotometrically measured. The expression of pro-inflammatory cytokines, such as tumor necrosis factor-α and interleukin-6 was measured by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Liver nuclear factor kappa B (NF-κB) was detected by immunohistochemistry. The expression of NF-κB p65 and inhibitor NF-κB-α (IκBα) was determined by Western blot analysis. RESULTS The serum alanine aminotransferase level, Suzuki scores of hepatic histology, and hepatic myeloperoxidase activity, as indices of hepatic injury, were increased after reperfusion. The increase was attenuated by preadministration with ATRA. Compared with the I/R group, ATRA treatment increased IκBα expression and suppressed NF-κB p65 expression. Subsequently, the levels of tumor necrosis factor-α and interleukin-6 after liver I/R were effectively downregulated. CONCLUSIONS ATRA administration can significantly attenuate I/R injury in rat liver. The protective mechanism is related to its anti-inflammatory function of inhibiting NF-κB activation.

miR-146a Ameliorates Liver Ischemia/Reperfusion Injury by Suppressing IRAK1 and TRAF6

A critical role of the Toll-like receptor(TLR) and its downstream molecules, including IL-1 receptor-associated kinase 1(IRAK1) and tumor necrosis factor receptor– associated factor 6(TRAF6), in the pathogenesis of liver ischemia/reperfusion (I/R) injury has been documented. Recently a microRNA, miR-146a, was identified as a potent negative regulator of the TLR signaling pathway. In this study, we investigated the role of miR-146a to attenuate TLR signaling and liver I/R injury in vivo and in vitro. miR-146a was decreased in mice Kupffer cells following hepatic I/R, whereas IRAK1 and TRAF6 increased. Overexpression of miR-146a directly decreased IRAK1 and TRAF6 expression and attenuated the release of proinflammatory cytokines through the inactivation of NF-κB P65 in hypoxia/reoxygenation (H/R)-induced macrophages, RAW264.7 cells. Knockdown experiments demonstrated that IRAK1 and TRAF6 are two potential targets for reducing the release of proinflammatory cytokines. Moreover, co-culture assays indicated that miR-146a decreases the apoptosis of hepatocytes after H/R. In vivo administration of Ago-miR-146a, a stable version of miR-146a in vivo, protected against liver injury in mice after I/R via inactivation of the TLR signaling pathway. We conclude that miR-146a ameliorates liver ischemia/reperfusion injury in vivo and hypoxia/reoxygenation injury in vitro by directly suppressing IRAK1 and TRAF6.

CD8+CD103+ regulatory T cells in spontaneous tolerance of liver allografts

It has been shown that rat liver allografts between certain inbred major histocompatibility complex (MHC) disparate strains are accepted spontaneously, and regulatory T cells (Tregs) have been suggested to play a role in the spontaneous liver tolerance. CD8(+)CD103(+) T cells bear the phenotypes of effector cells, and they are implicated in allograft destruction. Here we provide evidence that CD8(+)CD103(+) T cells possess regulatory function and may contribute to prevent liver allograft rejection. We show that the expression of CD103 in the CD8(+) T cells was increased in spontaneous liver grafts tolerant recipients. We further show that CD8(+)CD103(-) T cells can also upregulate the expression of CD103 and Foxp3 after stimulation with alloantigen or TGF-beta in vitro, and the CD8(+)CD103(+) T cells acquired regulatory properties. The suppressive function of the alloantigen or TGF-beta conditioned CD8(+)CD103(+) T cells was cell-cell contact dependent. These results imply that liver-specific factor(s) would be involved in the generation of CD8(+)CD103(+) Tregs that contribute to spontaneous liver allografts tolerance.

Adenoviral cardiotrophin‐1 transfer improves survival and early graft function after ischemia and reperfusion in rat small‐for‐size liver transplantation model

This study was to investigate the effect of donor liver adenoviral cardiotrophin‐1 (CT‐1) gene transfer on early graft survival and function in rat small‐for‐size liver transplantation. We constructed a recombinant murine CT‐1 adenoviral vector. Donor rats were transduced in vivo with adenoviruses expressing CT‐1 (AdCT‐1) or control vector (AdEGFP). Livers were harvested 4 days later, reduced to 40% of weight, and transplanted. A syngeneic rat orthotopic liver transplantation model was performed using 40% small‐for‐size grafts. Graft survival, liver function, hepatic architecture change, the degree of necrosis and apoptosis, and cell survival signaling pathways were assessed. AdCT‐1 pretreatment markedly improved liver function and the survival of small‐for‐size grafts. In the CT‐1 treatment group, hepatic architecture was well protected, apoptotic and necrotic cells were reduced; anti‐apoptotic protein bcl‐2 was up‐regulated and pro‐apoptotic cleaved caspase‐3 was down‐regulated, cell survival signaling pathways were activated by phosphorylation of protein kinase B (Akt), extracellular‐regulated kinase (ERK) and Signal transducer and activator of transcription‐3 (Stat‐3) after transplantation. In conclusion, donor liver adenoviral CT‐1 transfer ameliorated ischemia/reperfusion injury by decreasing hepatic necrosis and apoptosis in small‐for‐size liver transplantation, mediated in part by activation of the Akt, ERK, and Stat‐3 survival signaling pathways. These results may provide a potential clinical strategy to improve the outcome of small‐for‐size liver grafts.