Inducible nitric oxide synthase accelerates nonalcoholic fatty liver disease progression by regulating macrophage autophagy.

BACKGROUND: Cells and tissues, such as macrophages, express inducible nitric oxide synthase (INOS) after stimulation by certain factors. INOS helps mediate the macrophage inflammatory reaction, but few studies have explored how INOS affects macrophage function in nonalcoholic fatty liver disease (NAFLD). OBJECTIVE: This study investigated the role of INOS-mediated macrophage activity in NAFLD. METHODS: A high-fat diet was used to establish an NAFLD mouse model. After 12 weeks, blood was collected for immune cell and lipid analyses, and liver tissues were collected for pathological analyses with hematoxylin and eosin and Oil Red O staining. Peritoneal macrophages were extracted in situ, cultured in Dulbecco's modified Eagle's medium, and stimulated with palmitic acid to mimic in vivo conditions for further assays. Real-time polymerase chain reaction, western blot analysis, and immunofluorescence were used to verify the expression of target genes or proteins. RESULTS: In the NAFLD model, INOS expression in macrophages increased, and INOS knockdown significantly decreased the number of macrophages. Pathological examinations confirmed that INOS knockdown slowed NAFLD progression and macrophage infiltration during inflammation. INOS knockdown also enhanced phagocytosis and lipid transport by macrophages, and increased the expression of autophagy-related molecules in macrophages, which improved the autophagy level, promoted apoptotic cell degradation, and maintained intracellular environment homeostasis. CONCLUSIONS: These results indicate a correlation between INOS expression and macrophage function in NAFLD.

A novel xenograft model of human hepatocellular carcinoma in immunocompetent mice based on the microcarrier-6.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors that threaten human health; thus, the establishment of an animal model with clinical features similar to human hepatocellular carcinoma is of important practical significance. METHODS: Taking advantage of the novel microcarrier-6, human HCC cells were injected into immunocompetent mice to establish a novel human HCC patient-derived xenograft (PDX) model. Primary HCC cells were isolated from fresh hepatocellular carcinoma tissues, which were subsequently co-cultured with microcarrier-6 to construct a three-dimensional tumor cell culture model in vitro. The HCC-microcarrier complex was implanted into mice by subcutaneous inoculation, and the tumor formation time, tumor formation rate, and pathological manifestation were recorded. Changes of immune parameters in mice were detected by flow cytometry. RESULTS: The success rate was 60% (6/10) in the establishment of hepatocellular carcinoma PDX mouse model, and the total tumor formation rate of the tumor-forming model is 90-100%. H&E staining and immunohistochemical experiments indicate that the model well retained the characteristics of the primary tumor. Interestingly, M2 macrophages in tumor-bearing mice increased significantly, and the levels of CD4+ T cells were significantly reduced. CONCLUSIONS: Through the application of the microcarrier-6 in immunocompetent mice, we successfully established a novel human HCC PDX model, which can be used to better study and further elucidate the occurrence and pathogenic mechanism of HCC, improve the predictability of toxicity and drug sensitivity in HCC.

Inducible nitric oxide synthase regulates macrophage polarization via the MAPK signals in concanavalin A-induced hepatitis.

INTRODUCTION: Acute liver inflammatory reactions contribute to many health problems; thus, it is critical to understand the underlying pathogenic mechanisms of acute hepatitis. In this study, an experimental in vivo model of concanavalin A (ConA)-induced hepatitis was used. MATERIALS AND METHODS: C57BL/6 (wild-type, WT) or inducible nitric oxide synthase-deficient (iNOS-/- ) mice were injected with PBS or 15 mg/kg ConA via tail vein. Detection of liver injury by histological examination and apoptosis, and flow cytometry to detect the effect of immune cells on liver injury. RESULTS: iNOS-/-  mice had lower levels of the liver enzymes aspartate aminotransferase and alanine aminotransferase, suggesting that they were protected against ConA-induced pathological liver injury and that iNOS participated in the regulation of hepatitis. Furthermore, iNOS deficiency was found to lower CD86 expression and suppressed the messenger RNA levels of inflammatory factors in the liver. In vitro experiments also demonstrated that iNOS deficiency suppressed the sequential phosphorylation of the mitogen-activated protein kinase pathway cascade, thereby inhibiting the M1 polarization of macrophages and consequently suppressing the transcription of inflammation factors. CONCLUSION: iNOS may contribute to ConA-induced inflammation by promoting the activation of proinflammatory macrophages.

Human liver stem cells alleviate Con-A induced liver injury by regulating the balance of Treg/Th17 cells.

BACKGROUND: Liver injury is a serious threat to human health that has become a worldwide problem. To date, there is still no effective treatment strategy. In the present study, we examined the protective effects of Human liver stem cells (HLSCs) against concanavalin A (Con A)-induced acute liver injury. METHODS: Isolated HLSCs were characterized by microscopy, functional assays, and gene expression. HLSCs or HLSCs culture medium were transplanted in mice for 12 h and subsequently challenged with Con A via tail-vein injection. The effects were evaluated through survival rate, histology, blood tests, TUNEL assay, quantitative RT-PCR and flow cytometry. CellTracker™ CM-Dil labled HLSCs were tracked by fluorescence microscope. RESULTS: Transplantation of HLSCs reduced the mortality rate, reduced the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL), narrowed the area of liver necrosis, and inhibited hepatocyte apoptosis induced by Con A. Injection of HLSCs culture medium could also alleviate Con A-induced liver injury. Of note, HLSCs-transplanted mice exhibited lower frequencies of Th17 cells and higher frequencies of Tregs in their liver and spleen following Con A injection. Moreover, transplantation of HLSCs significantly reduced the expression of IL-17A, IL-17F and ROR-γt induced by Con A, while reversed Con A-induced downregulation of Foxp3 expression and IL-10. CONCLUSIONS: HLSCs protect mice from immune-mediated liver injury by regulating the balance of Treg/Th17 cells, suggesting that transplantation of HLSCs is a potential and effective therapeutic method for amelioration of liver injury.

Naringin regulates mitochondrial dynamics to protect against acetaminophen-induced hepatotoxicity by activating the AMPK/Nrf2 signaling pathway in vitro

Naringin (Nar) has been reported to exert potential hepatoprotective effects against acetaminophen (APAP)-induced injury. Mitochondrial dysfunction plays an important role in APAP-induced liver injury. However, the protective mechanism of Nar against mitochondrial damage has not been elucidated. Therefore, the aim of this study was to investigate the hepatoprotective effects of Nar against APAP and the possible mechanisms of actions. Primary rat hepatocytes and HepG2 cells were utilized to establish an in vitro model of APAP-induced hepatotoxicity. The effect of APAP and Nar on cell viability was evaluated by a CCK8 assay and detection of the concentrations of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase. The cellular concentrations of biomarkers of oxidative stress were measured by ELISA. The mRNA expression levels of APAP-related phase II enzymes were determined by real-time PCR. The protein levels of Nrf2, phospho (p)-AMPK/AMPK, and biomarkers of mitochondrial dynamics were determined by western blot analysis. The mitochondrial membrane potential (MMP) was measured by high-content analysis and confocal microscopy. JC-1 staining was performed to evaluate mitochondrial depolarization. Nar pretreatment notably prevented the marked APAP-induced hepatocyte injury, increases in oxidative stress marker expression, reductions in the expression of phase II enzymes, significant loss of MMP, mitochondrial depolarization, and mitochondrial fission in vitro. In conclusion, Nar alleviated APAP-induced hepatocyte and mitochondrial injury by activating the AMPK/Nrf2 pathway to reduce oxidative stress in vitro. Applying Nar for the treatment of APAP-induced liver injury might be promising.

Stem cell transplantation for treating liver diseases: progress and remaining challenges.

With the development of regenerative medicine, various stem cells are increasingly considered for treating liver diseases. Various stem cells have been reported to play an essential role in liver recovery, and studies have verified the preliminary effectiveness and safety of these therapies. Stem cell-based therapies will emerge as an effective treatment strategy for liver diseases. Thus, the research progress and challenges to the related stem cells were reviewed, namely the classification of stem cells, cell culture, transplantation, cell tracing in the body, therapies for various liver diseases.

Establishment of a Human Gastric Cancer Xenograft Model in Immunocompetent Mice Using the Microcarrier-6.

Gastric cancer is among the most common malignant tumors of the digestive tract. Establishing a robust and reliable animal model is the foundation for studying the pathogenesis of cancer. The present study established a mouse model of gastric carcinoma by inoculating immunocompetent mice with MKN45 cells using microcarrier. Sixty male C57BL/6 mice were randomly divided into three groups: a 2D group, an empty carrier group, and a 3D group, according to the coculture system of MKN45 and the microcarrier. The mouse models were established by hypodermic injection. Time to develop tumor, rate of tumor formation, and pathological features were observed in each group. In the 3D group, the tumorigenesis time was short, while the rate of tumor formation was high (75%). There was no detectable tumor formation in either the 2D or the empty carrier group. Both H&E and immunohistochemical staining of the tumor xenograft showed characteristic evidence of human gastric neoplasms. The present study successfully established a human gastric carcinoma model in immunocompetent mice, which provides a novel and valuable animal model for the cancer research and development of anticancer drugs.

Transplanted adult human hepatic stem/progenitor cells prevent histogenesis of advanced hepatic fibrosis in mice induced by carbon tetrachloride.

Transplantation of adult human hepatic stem/progenitor cells (hHSPCs) has been considered as an alternative therapy, replacing donor liver transplantation to treat liver cirrhosis. This study assessed the antifibrotic effects of hHSPCs in mice with fibrosis induced by carbon tetrachloride (CCl4) and examined the actions of hHSPCs on the fibrogenic activity of human hepatic stellate cells (HSCs) in a coculture system. Isolated hHSPCs expressed stem/progenitor cell phenotypic markers. Mice were given CCl4 (twice weekly for 7 weeks) and hHSPC transplantation weekly. CCl4 induced advanced fibrosis (bridging fibrosis and cirrhosis) in mice, which was prevented by hHSPC transplantation. The liver of hHSPC-transplanted mice showed only occasional short septa and focal parenchymal fibrosis, and a 50% reduction in hepatic collagen, assessed by Sirius red stain histomorphometry. Moreover, the proteins for α-smooth muscle actin (α-SMA) and collagen I were decreased. While α-SMA, collagen α1(I), and tissue inhibitor of metalloproproteinase-1 mRNAs were decreased, matrix metalloproteinase (MMP)-1 mRNA was increased, consistent with decreased fibrogenesis. MMP-2 and transforming growth factor-ß were not affected. Alanine aminotransferase and aspartate aminotransferase were lower, suggesting improvement of liver function/damage. In coculture, hHSPCs elicited changes of α-SMA and fibrogenic molecules in HSCs similar to those observed in vivo, providing evidence for a functional link between hHSPCs and HSCs. A decreased HSC proliferation was noted. Thus, transplantation of hHSPCs prevents histogenesis of advanced liver fibrosis caused by CCl4. hHSPCs mediate downregulation of HSC activation coincident with modulation of fibrogenic molecule expression, leading to suppression of fibrogenesis both in vivo and in vitro.

Human liver stem cells attenuate concanavalin A-induced acute liver injury by modulating myeloid-derived suppressor cells and CD4+ T cells in mice.

BACKGROUND: Acute liver failure (ALF) is a serious threat to the life of people all over the world. Finding an effective way to manage ALF is important. Human liver stem cells (HLSCs) are early undifferentiated cells that have been implicated in the regeneration and functional reconstruction of the liver. In this study, we aimed to evaluate the protective effects of the HLSC line HYX1 against concanavalin A (ConA)-induced acute liver injury. METHODS: HYX1 cells were characterized by microscopy, functional assays, gene expression, and western blot analyses. We showed that HYX1 cells can differentiate into hepatocytes. We intraperitoneally injected HYX1 cells in mice and administered ConA via caudal vein injection 3, 6, 12, 24, and 48 h later. The effects of HYX1 cell transplantation were evaluated through blood tests, histology, and flow cytometry. RESULTS: HYX1 cells reduced the levels of alanine transaminase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) in serum and dramatically decreased the severity of liver injuries. Mechanistically, HYX1 cells promoted myeloid-derived suppressor cell (MDSC) migration into the spleen and liver, while reducing CD4+ T cell levels in both tissues. In addition, HYX1 cells suppressed the secretion of proinflammatory cytokines, such as tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), but led to increased interleukin-10 (IL-10) production. CONCLUSIONS: These results confirm the efficacy of HLSCs in the prevention of the ConA-induced acute liver injury through modulation of MDSCs and CD4+ T cell migration and cytokine secretion.

Transplanted mouse liver stem cells at different stages of differentiation ameliorate concanavalin A-induced acute liver injury by modulating Tregs and Th17 cells in mice.

Acute liver failure (ALF) is a disease with a considerably high mortality rate that still lacks a safe and effective treatment. Transplantation of liver stem cells (LSCs) has been considered to be a promising therapeutic alternative for ALF since LSCs have been shown to be involved in immunomodulation and functional reconstruction of the liver. Our present study evaluated and compared the protective effects of the two mouse LSC lines, YE and R5, as well as those of adult mouse hepatocyte (HC), on concanavalin A (ConA)-induced acute liver injury. YE and R5 cells were analyzed by microscopy, functional assays, and gene expression. We confirmed that YE and R5 cells were undifferentiated cells that had partial hepatocytic functions and a potential to differentiate into hepatocytes. YE cells has characteristics of LSCs at the early stage of differentiation, whereas the differentiation stage of R5 cells was later than that of YE cells. Subsequently, YE, R5, and HC cells were intraperitoneally transplanted into three groups of mice, followed by injection of ConA through the tail vein of each mouse at 12 h later. Blood tests, histology, flow cytometry, and quantitative PCR were then used to evaluate the therapeutic effects of the cell transplantations at 24 h after ConA injections. Compared with that of the ConA control group, YE, R5, and HC cells reduced the expression of alanine transaminase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) in serum and alleviated the degree of hepatic necrosis. Moreover, transplantation of these cells induced more regulatory T cells (Tregs) and less T-helper 17 (Th17) cells in the liver and spleen, and also promoted the expression of forkhead box protein 3 (Foxp3) and interleukin (IL)-10; in contrast, these transplantations induced various degrees of inhibition in the expression of retinoic acid-related orphan receptor γt (RORγt), IL-17A, IL-17F, and tumor necrosis factor-α (TNF-α). The protective effects of YE and R5 cells were significantly stronger than those of HC cells, and YE cells at the earlier differentiation stage than that of R5 cells exhibited the strongest protective effects. These results demonstrate that mouse LSCs at different stages of differentiation alleviate ConA-induced acute liver injury in mice by modulating Tregs, Th17 cells, and cytokine secretion.

Human Umbilical Cord Wharton's Jelly Derived Mesenchymal Stromal Cells May Attenuate Sarcopenia in Aged Mice Induced by Hindlimb Suspension.

BACKGROUND Since the use of human umbilical cord Wharton's Jelly derived mesenchymal stromal cells (hWJ-MSCs) to treat sarcopenia has not been explored, we studied the effects of hWJ-MSCs in aged male C57BL/6J mice with sarcopenia induced by hindlimb suspension, and explored the potential mechanism. MATERIAL AND METHODS Hindlimb suspension was used to induce sarcopenia in 24-month-old C57BL/6J mice and green fluorescent protein-tagged hWJ-MSCs and controls were transplanted into mice via tail vein or local intramuscular injection. After hWJ-MSC transplantation, changes in whole body muscle strength and endurance, gastrocnemius muscle weight and myofiber cross-sectional area (CSA) were studied. Proliferation of skeletal muscle stem cell, apoptosis, and chronic inflammation were also investigated. RESULTS We demonstrated that whole body muscle strength and endurance, gastrocnemius muscle mass, and CSA were significantly increased in hWJ-MSC-transplanted mice than in controls (P<0.05). In hWJ-MSC-transplanted mice, apoptotic myonuclei was reduced, and BrdU and Pax-7 expression indices of gastrocnemius muscles were increased (P<0.05). Tumor necrosis factor (TNF)-α and interleukin (IL)-6 were downregulated, and IL-4 and IL-10 were upregulated (P<0.05). CONCLUSIONS hWJ-MSCs may ameliorate sarcopenia in aged male C57BL/6J mice induced by hindlimb suspension, and this may be via activation of resident skeletal muscle satellite cells, reduction of apoptosis, and less chronic inflammation.

Direct conversion of fibroblasts to neurons by reprogramming PTB-regulated microRNA circuits.

The induction of pluripotency or trans-differentiation of one cell type to another can be accomplished with cell-lineage-specific transcription factors. Here, we report that repression of a single RNA binding polypyrimidine-tract-binding (PTB) protein, which occurs during normal brain development via the action of miR-124, is sufficient to induce trans-differentiation of fibroblasts into functional neurons. Besides its traditional role in regulated splicing, we show that PTB has a previously undocumented function in the regulation of microRNA functions, suppressing or enhancing microRNA targeting by competitive binding on target mRNA or altering local RNA secondary structure. A key event during neuronal induction is the relief of PTB-mediated blockage of microRNA action on multiple components of the REST complex, thereby derepressing a large array of neuronal genes, including miR-124 and multiple neuronal-specific transcription factors, in nonneuronal cells. This converts a negative feedback loop to a positive one to elicit cellular reprogramming to the neuronal lineage.

Construction and application of a built-in dual luciferase reporter for microRNA functional analysis

Background: As key gene regulators, microRNAs post-transcriptionally modulate gene expression via binding to partially complementary sequence in the 3' UTR of target mRNA. An accurate, rapid and quantitative tool for sensing and validation of miRNA targets is of crucial significance to decipher the functional implications of miRNAs in cellular pathways. Results: Taking advantage of an improved restriction-free cloning method, we engineered a novel built-in dual luciferase reporter plasmid where Firefly and Renilla luciferase genes were assembled in a single plasmid named pFila. This design eliminates the transfection of a separate control plasmid and thus minimizes the time and labor required for miRNA-target sensing assays. pFila consistently produces Firefly and Renilla luciferase activities when transfected into human-, monkey- and mouse-derived mammalian cell systems. Moreover, pFila is capable of recapitulating the interaction of miR-16 and its known target CCNE1 in Hela cells. Additionally, pFila is shown to be a sensitive miR-biosensor by evaluating the inhibition efficiency of endogenous miRNA. Conclusions: pFila would facilitate miRNA target identification and verification in a rapid and simplified manner. Also, pFila is a sensitive biosensor for active miRNA profiling in vivo.