Unexpected detection of a submillimeter early hepatocellular carcinoma focus by intraoperative near-infrared fluorescence imaging-a case report.

Background: Prognosis of hepatocellular carcinoma (HCC) is closely related to residual tumor cells and tissues after tumor resection. Thus, close monitoring to ensure complete removal of residual tumor is fundamental. In this regard, intraoperative near-infrared fluorescence (NIRF) imaging has been of great assistance to surgeons for precision cancer surgeries. However, up to now, the identification of tiny lesions has not been reported. Herein, we report our findings on the case of an ultra-small HCC focus of about 430 µm that was successfully detected using NIRF during real-time monitored liver cancer surgery. The patient had a background of hepatitis B cirrhosis, which is the most phenomenon in China. Surgeons usually unable to distinguish sclerotic nodules from small tumor tissue with the naked eyes. Case Description: A 55-year-old man with chronic hepatitis B infection was preoperatively diagnosed with a space-occupying liver lesion. A fluorescence signal was detected on the surface of the liver through the NIRF imaging system which had not been found by preoperative computed tomography (CT) and ultrasound examination. We subsequently tested the residual liver surface and observed a high signal point, less than 1 mm in the right anterior lobe of the liver. Histopathological examination revealed that the tiny fluorescent spot belong to an early HCC focus. Conclusions: Based on these results, we think indocyanine green (ICG)-NIRF imaging may be used as a routine intraoperative detection method for liver cancer surgery in order to remove any residual tumor cells and tissue, hence minimizing further risk of remnant tumor regrowth.

The Impact of Surgical Boot Camp and Subsequent Repetitive Practice on the Surgical Skills and Confidence of Residents.

BACKGROUND: Boot camp can enable residents to acquire surgical skills and confidence, but they can lose these skills over time if they do not use them. The purpose of this study was to explore whether boot camp and subsequent repetitive practice could strengthen residents' clinical skills and self-confidence. METHODS: This is a comparative study of surgical residents who were enrolled in our institution from 2016 to 2017. The residents in the experimental group (enrolled in 2017) received boot camp training and a year of repetitive practice. The control group (enrolled in 2016) only received routine residency training. The rotation assessment pass rates of the two groups during the first year of the residency training were compared. A survey was conducted at different points in time to investigate the influence of boot camp and repetitive practice on the confidence of the residents. RESULTS: The assessment pass rate of the experimental group was significantly higher than that of the control group (p < 0.05). The residents' confidence in themselves improved significantly after the boot camp, and it was comparable to that of the residents in the control group after their first year of residency. The level of self-confidence of the experimental group was further improved after repetitive practice. Finally, residents in the experimental group received better evaluations by their colleagues than the control group received. CONCLUSIONS: This study showed that boot camp can improve the surgical skills and confidence of residents and that repetitive practice can further strengthen them. Residents in the experimental group developed their self-confidence in boot camp, and it increased after repetitive practice.

Mesenchymal stem cells increase heme oxygenase 1-activated autophagy in treatment of acute liver failure.

In recent years, transplantation of mesenchymal stem cells (MSCs) has attracted much attention as a potential cell-based therapy for acute liver failure (ALF). As an inducible enzyme, heme oxygenase 1 (HO-1) has been reported to have cytoprotective, anti-apoptotic and immunoregulatory effects. Autophagy, a conserved catabolic process in cells, may be an important pathway for MSCs to treat ALF. In this study, we aimed to explore whether MSCs treat ALF by regulating autophagy and whether HO-1 was involved in the same pathway. Bone marrow-derived MSCs were isolated from Sprague-Dawley rats and cultured according to an established protocol. Co-culture systems of MSCs and hepatocytes were used to assess autophagy in the treatment of ALF. Meanwhile, MSCs were transplanted into rats with d-galactosamine (Gal)-induced ALF. Autophagy inhibitor (3-methyladenine, 3-MA), HO-1 inhibitor (zinc protoporphyrin, ZnPP) and PI3K specific inhibitor (LY294002) were employed in the study. Blood samples and liver tissues were collected before euthanasia. Survival rate, liver function, inflammatory factors, histology, Ki67 and TUNEL staining were determined. MSCs transplantation alleviated ALF both in vivo and in vitro. Autophagy and autophagy-related proteins were significantly up-regulated during MSCs treatment. 3-MA attenuated the therapeutic effect of MSCs. Administration of LY294002 before ALF induction inhibited hepatocyte autophagy. During the MSCs treatment, the HO-1 expression was increased, while inhibiting HO-1 attenuated the therapeutic effect of MSCs as well as hepatocyte autophagy. These findings suggested MSCs could alleviate ALF by increasing the HO-1 expression, which played an important role in activating autophagy through PI3K/AKT signaling pathway.

Interleukin-10 secreted by mesenchymal stem cells attenuates acute liver failure through inhibiting pyroptosis.

AIM: Recently, the benefit of mesenchymal stem cells (MSCs) as a cell-based therapy for acute liver failure (ALF) has gained much attention, although the mechanism of action of MSCs in the treatment of ALF remains elusive. Pyroptosis is a novel form of programmed cell death with an intense inflammatory response. The aim of the present study was to explore the soluble cytokines secreted by MSCs and their therapeutic effects through inhibiting pyroptosis in ALF. METHODS: Mesenchymal stem cells obtained from C57BL/6 mice were isolated and cultured according to an established protocol. The MSCs were transplanted into mice with D-galactosamine (D-Gal)-induced ALF. Liver function, survival rate, histology, and inflammatory factors were determined. Exogenous recombinant rat interleukin (IL)-10, ShIL-RNA, and MCC950 (NLRP3 inhibitor) were given to the mice to explore the therapeutic mechanism of MSCs. Statistical analyses were carried out with spss version 19.0, and all data were analyzed by independent-samples t-test. RESULTS: Injection of IL-10 or MSC transplantation ameliorated D-Gal-induced increase in alanine aminotransferase, aspartate aminotransferase, total bilirubin, NH3, and inflammatory cytokines. Blockage of IL-10 confirmed the therapeutic significance of this cytokine. CONCLUSION: Pyroptosis was inhibited after IL-10 infusion and inhibition of NLRP3 by MCC950 reversed liver dysfunction.

PARP-1 promotes tumor recurrence after warm ischemic liver graft transplantation via neutrophil recruitment and polarization.

Poly (ADP-ribose) polymerase 1 (PARP-1) is a crucial contributor to exacerbate ischemia and reperfusion (IR) injury and cancer process. However, there is little research into whether PARP-1 affects the hepatocellular carcinoma (HCC) recurrence after liver transplantation. In this study, we investigated the influence of PARP-1 on hepatic neutrophil mobilizing and phenotype shifting which may lead to HCC recurrence after liver transplantation. We found that rats received the grafts with warm ischemic injury had higher risk of HCC recurrence, which was markedly prevented by pharmacological inhibition of PARP-1 after liver transplantation. In mouse models, the up-regulation of PARP-1 was closely related to the greater tumor burden and increased hepatic susceptibility to recurrence after IR injury. The reason was that high hepatic PARP-1 led to increased liver CXCL1 levels, which in turn promoted recruitment of neutrophils. Both blocking CXCL1/CXCR2 signaling pathway and depleting neutrophils decreased tumor burden. Moreover, these infiltrating neutrophils were programmed to a proangiogenic phenotype under the influence of PARP-1 in vivo after hepatic IR injury. In conclusion, IR-induced PARP-1 up-regulation increased the hepatic recruitment of neutrophils through regulation of CXCL1/CXCR2 signaling and polarized hepatic neutrophils to proangiogenic phenotype, which further promoted HCC recurrence after transplantation.

Mesenchymal stem cells increase expression of heme oxygenase-1 leading to anti-inflammatory activity in treatment of acute liver failure.

BACKGROUND: Mesenchymal stem cells (MSCs) have been studied for the treatment of acute liver failure (ALF) for several years. MSCs may exert their effect via complex paracrine mechanisms. Heme oxygenase (HO) 1, a rate-limiting enzyme in heme metabolism, exerts a wide range of anti-inflammatory, anti-apoptotic and immunoregulatory effects in a variety of diseases. However, the relationship between MSCs and HO-1 in the treatment of ALF is still unclear. We investigated the preventive and therapeutic potential of intravenously administered BMSCs. METHODS: Bone marrow-derived mesenchymal stem cells (BMSCs) obtained from Sprague-Dawley rats were isolated and cultured. We employed BMSCs, hemin (a HO-1 inducer) and zinc protoporphyrin (ZnPP, the HO-1 activity inhibitor) in D-galactosamine (D-Gal)/lipopolysaccharides (LPS)-induced ALF rats. Rats were sacrificed at days 1, 3, 5, and 7 post-transfusion, respectively. Blood samples and liver tissues were collected. Hepatic injury, HO-1 activity, chemokines, inflammatory cytokines, the number and oxidative activity of neutrophils, ki67, and TUNEL-positive cells were evaluated. RESULTS: HO-1 induction or BMSCs transplantation attenuated D-galactosamine/lipopolysaccharide-induced increases in alanine aminotransferase, aspartate aminotransferase, total bilirubin (TBIL), ammonia, and inflammatory cytokines. Treatment with hemin or BMSCs also inhibited neutrophil infiltration, oxidative activity, and hepatocyte apoptosis. The protective effect of BMSCs was partially neutralized by ZnPP, suggesting the key role of HO-1 in the process. CONCLUSIONS: These findings may correlate with inhibition of nuclear factor-κ B activation. BMSCs ameliorated ALF by increasing the HO-1 expression, which reduced PMN infiltration and function, and played an important anti-inflammatory and anti-apoptotic role. Proposed mechanism by which BMSCs reduce inflammation, neutrophil activation, and hepatocyte apoptosis and promote hepatocyte proliferation via HO-1. BMSCs increase HO-1 expression in liver via Nrf2. HO-1 protects against LPS/D-Gal-induced ALF by inhibiting neutrophil infiltration and inflammatory burst, and hepatocyte apoptosis and necrosis. HO-1 also promotes hepatocyte proliferation.

Combined treatment with MSC transplantation and neutrophil depletion ameliorates D-GalN/LPS-induced acute liver failure in rats.

BACKGROUND: The imbalance of immunity is an important pathogenesis of acute liver failure (ALF). Neutrophils are the hallmark of acute inflammation, which have an essential role in immune regulation. Mesenchymal stem cell (MSC) transplantation is a promising therapy in ALF treatment. Recent studies indicated a considerable connection between MSCs and neutrophils in immune regulation. AIM: To investigate changes in neutrophils in ALF rats after MSC transplantation, and to explore the therapeutic effect and mechanism of the combined treatment with MSC transplantation and neutrophil depletion in ALF. METHODS: We employed monotherapy and the combination therapy with MSCs and anti-PMN serum in D-galactosamine (D-GalN)/lipopolysaccharides (LPS)-induced ALF rats. Rats were sacrificed at 6, 12 and 24h, respectively. Blood samples and liver tissues were collected. Hepatic injury, inflammatory cytokines (TNF-α, IL-1ß and IL-10), chemokines (CXCL1 and CXCL2), the number and activity of neutrophils and animal survival were assessed at fixed times. RESULTS: MSC transplantation can effectively improve the liver function of ALF rats and reduce the number and activity of neutrophils in both peripheral blood and liver. Compared with MSC transplantation alone, anti-PMN treatment and co-treatment had a better result in diminishing neutrophils. The co-treatment also exhibited a better therapeutical effect in ALF rats compared with monotherapy. In this process, the expressions of inflammatory cytokines in the liver were consistent with liver function. CONCLUSIONS: The regulation of the neutrophil-related microenvironment is affected in D-GalN/LPS-induced ALF rats after MSC transplantation. The combined treatment with MSC transplantation and neutrophil depletion may have a better therapeutic effect in ALF rats.

Puerarin protects against CCl4-induced liver fibrosis in mice: possible role of PARP-1 inhibition.

Liver fibrosis, which is the pathophysiologic process of the liver due to sustained wound healing in response to chronic liver injury, will eventually progress to cirrhosis. Puerarin, a bioactive isoflavone glucoside derived from the traditional Chinese medicine pueraria, has been reported to have many anti-inflammatory and anti-fibrosis properties. However, the detailed mechanisms are not well studied yet. This study aimed to investigate the effects of puerarin on liver function and fibrosis process in mice induced by CCl4. C57BL/6J mice were intraperitoneally injected with 10% CCl4 in olive oil(2mL/kg) with or without puerarin co-administration (100 and 200mg/kg intraperitoneally once daily) for four consecutive weeks. As indicated by the ameliorative serum hepatic enzymes and the reduced histopathologic abnormalities, the data collected showed that puerarin can protect against CCl4-induced chronic liver injury. Moreover, CCl4-induced development of fibrosis, as evidenced by increasing expression of alpha smooth muscle actin(α-SMA), collagen-1, transforming growth factor (TGF)-ß and connective tissue growth factor(CTGF) in liver, were suppressed by puerarin. Possible mechanisms related to these suppressive effects were realized by inhibition on NF-κB signaling pathway, reactive oxygen species(ROS) production and mitochondrial dysfunction in vivo. In addition, these protective inhibition mentioned above were driven by down-regulation of PARP-1 due to puerarin because puerarin can attenuate the PARP-1 expression in CCl4-damaged liver and PJ34, a kind of PARP-1 inhibitor, mimicked puerarin's protection. In conclusion, puerarin played a protective role in CCl4-induced liver fibrosis probably through inhibition of PARP-1 and subsequent attenuation of NF-κB, ROS production and mitochondrial dysfunction.

Interleukin-10 Contributes to Therapeutic Effect of Mesenchymal Stem Cells for Acute Liver Failure via Signal Transducer and Activator of Transcription 3 Signaling Pathway.

BACKGROUND: Mesenchymal stem cells (MSCs) transplantation has been proven to have therapeutic potential for acute liver failure (ALF). However, the mechanism remains controversial. Recently, modulation of inflammation by MSCs has been regarded as a crucial mechanism. The aim of the present study was to explore the soluble cytokines secreted by MSCs and their therapeutic effects in ALF. METHODS: MSCs isolated from Sprague-Dawley rats were identified by fluorescence-activated cell sorting analysis. Conditioned medium derived from MSCs (MSCs-CM) was collected and analyzed by a cytokine microarray. MSCs and MSCs-CM were transplanted into rats with D-galactosamine-induced ALF. Liver function, survival rate, histology, and inflammatory factors were determined. Exogenous recombinant rat interleukin (IL)-10, anti-rat IL-10 antibody, and AG490 (signal transducer and activator of transcription 3 [STAT3] signaling pathway inhibitor) were administered to explore the therapeutic mechanism of MSCs-CM. Statistical analysis was performed with SPSS version 19.0, and all data were analyzed by the independent-sample t-test. RESULTS: There are statistical differences of the survival curve between ALF+MSCs group and ALF+Dulbecco's modified Eagle's medium (DMEM) group, as well as ALF+MSCs-CM group and ALF+DMEM group (all P < 0.05). Serum alanine aminotransferase (ALT) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (865.53±52.80 vs. 1709.75±372.12 U/L and 964.72±414.59 vs. 1709.75±372.12 U/L, respectively, all P < 0.05); meanwhile, serum aspartate aminotransferase (AST) level in the ALF+MSCs and ALF+MSCs-CM groups was lower than that in the ALF+DMEM group (2440.83±511.94 vs. 4234.35±807.30 U/L and 2739.83±587.33 vs. 4234.35±807.30 U/L, respectively, all P < 0.05). Furthermore, MSCs or MSCs-CM treatment significantly reduced serum interferon-γ (IFN-γ), IL-1ß, IL-6 levels and increased serum IL-10 level compared with DMEM (all P < 0.05). Proteome profile analysis of MSCs-CM indicated the presence of anti-inflammatory factors and IL-10 was the most distinct. Blocking of IL-10 confirmed the therapeutic significance of this cytokine. Phosphorylated STAT3 was upregulated after IL-10 infusion and inhibition of STAT3 by AG490 reversed the therapeutic effect of IL-10. CONCLUSIONS: The factors released by MSCs, especially IL-10, have the potential for therapeutic recovery of ALF, and the STAT3 signaling pathway may mediate the anti-inflammatory effect of IL-10.

IL-1ß siRNA adenovirus benefits liver regeneration by improving mesenchymal stem cells survival after acute liver failure.

UNLABELLED:   BACKGROUND: Uncontrolled hapatic inflammatory response is regarded as the primary pathological mechanism of acute liver failure and impairs the regeneration of hepatocytes and stem cell grafts. Interleukin-1 plays a key role for activating immune and inflammatory response. Recently, siRNA has made quite a few progresses in treating inflammatory response. AIM: To assess the effect of IL-1? siRNA adenovirus on MSC and the therapeutic effect of MSC combined with IL-1? siRNA adenovirus in ALF. MATERIAL AND METHODS: We implanted MSC or/and IL-1? siRNA adenovirus via the tail vein, using CCl4-induced ALF in a mice model. Mice were sacrificed at different time points. Blood samples and liver tissues were collected. Hepatic injury, liver regeneration, cytokines (CXCL1, IL-1?, IL-10, IL-6, VEGF and HGF), animal survival and vital MSC were assessed after cell transplantation. RESULTS: MSC combined with IL-1? siRNA reduced the inflammatory levels and prevented liver failure. These animals administrated with MSC and IL-1? siRNA also exhibited improved liver regeneration and increased survival rates. Immunohistochemistry and fluorescence microscopy revealed the number of vital MSC in ALF + MSC + IL-1? siRNA group were significantly more than that in ALF + MSC group. CONCLUSION: IL-1? siRNA adenovirus could enhance MSC ability of tissue regeneration through increasing its survival rate. Accordingly, combination of IL-1? siRNA adenovirus and MSC had a synergistic effect on acute liver failure.

Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes.

Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system.

Targeted migration of mesenchymal stem cells modified with CXCR4 to acute failing liver improves liver regeneration.

AIM: To improve the colonization rate of transplanted mesenchymal stem cells (MSCs) in the liver and effect of MSC transplantation for acute liver failure (ALF). METHODS: MSC was modified with the chemokine CXC receptor 4 (CXCR4) gene (CXCR4-MSC) or not (Null-MSC) through lentiviral transduction. The characteristics of CXCR4-MSCs and Null-MSCs were determined by real-time quantitative polymerase chain reaction, Western blotting and flow cytometry. CXCR4-MSCs and Null-MSCs were infused intravenously 24 h after administration of CCl4 in nude mice. The distribution of the MSCs, survival rates, liver function, hepatocyte regeneration and growth factors of the recipient mice were analyzed. RESULTS: In vitro, CXCR4-MSCs showed better migration capability toward stromal cell-derived factor-1α and a protective effect against thioacetamide in hepatocytes. In vivo imaging showed that CXCR4-MSCs migrated to the liver in larger numbers than Null-MSCs 1 and 5 d after ALF. Higher colonization led to a longer lifetime and better liver function. Either CXCR4-MSCs or Null-MSCs exhibited a paracrine effect through secreting hepatocyte growth factor and vascular endothelial growth factor. Immunohistochemical analysis of Ki-67 showed increased cell proliferation in the damaged liver of CXCR4-MSC-treated animals. CONCLUSION: Genetically modified MSCs expressing CXCR4 showed greater colonization and conferred better functional recovery in damaged liver.

Administration of IL-1Ra chitosan nanoparticles enhances the therapeutic efficacy of mesenchymal stem cell transplantation in acute liver failure.

BACKGROUND AND AIMS: To investigate the synergistic effect of IL-1Ra administration and stem cell transplantation in swine suffering from acute liver failure (ALF), to elucidate the mechanism of IL-1Ra activity and to demonstrate mesenchymal stem cell (MSC) transplantation as a potential treatment for ALF. METHODS: Thirty-five Chinese experimental mini-swine were divided into five groups randomly. Group A (n = 7) is the control group and all swine were injected with saline via portal veins. Group B (n = 7) received IL-1Ra via ear veins 6 h before receiving saline. Group C (n = 7) received MSC transplantation and all swine were injected with 8 × 107 MSCs via portal veins. Group D (n = 7) swine were treated with a combination of IL-1Ra and MSC transplantation E (n = 7) also received a combined treatment of both IL-1Ra and bone marrow (BM-MSC) transplantation, except that the IL-1Ra was in the form of chitosan nanoparticles. Liver function, level of cytokines and liver pathological changes were measured in the following 4 weeks. RESULTS: IL-1Ra chitosan nanoparticles exhibited controlled-release ability in PBS. Swine in Group E showed a significant improvement in inflammation environment, liver function and hepatocyte proliferation. Levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) in Group E were elevated compared to other groups. CONCLUSIONS: IL-1Ra chitosan nanoparticles showed significant liver targeting ability and controlled-release characteristics. Combined therapy with IL-1Ra chitosan nanoparticles and MCS transplantation exhibits great synergistic effects through paracrine function and suppression of inflammation.