Zoledronic acid inhibits TSC2-null cell tumor growth via RhoA/YAP signaling pathway in mouse models of lymphangioleiomyomatosis.

BACKGROUND: This study is to investigate the effects of zoledronic acid (ZA) on TSC2-null cell proliferation and on the tumor progression and recurrence in mouse models of lymphangioleiomyomatosis (LAM). METHODS: Subcutaneous mouse models and LAM mouse models were established. Immunohistochemistry and immunofluorescence were performed to detect the protein expression levels. TUNEL assay was conducted to detect cell apoptosis. Immunoprecipitation was carried out to determine the interaction between proteins. RESULTS: ZA prevented the growth of TSC2-null cells both in culture and in LAM mouse models. Compared with rapamycin, ZA more effectively promoted the apoptosis of TSC2-null cells. Moreover, combined with the rapamycin, ZA effectively suppressed the tumor recurrence after drug withdrawal and ZA inhibited the activity of GTPase RhoA by decreasing protein geranylgeranylation, resulting in changes of Yap nucleus translocation. CONCLUSION: ZA promotes cell apoptosis in TSC2-null cells through the RhoA/YAP signaling pathway. ZA may be used for the clinical treatment of LAM.

Prostaglandin E2 secreted by mesenchymal stem cells protects against acute liver failure via enhancing hepatocyte proliferation.

Bone marrow-derived mesenchymal stem cells (MSCs) have been recently used in clinical trials as treatment for liver diseases. However, the underlying mechanism of their effectiveness remains largely unexplored. In the present study, we confirmed that the protective effects of MSCs on mouse model of acute liver failure (ALF) were based on MSC-secreted prostaglandin (PG)E2. Our data confirmed that MSC-secreted PGE2 not only inhibited apoptosis but also enhanced hepatocyte proliferation, thus attenuating ALF. Moreover, Yes-associated protein (YAP) played a major role in PGE2-triggered hepatocyte proliferation. In vitro studies showed that PGE2 increased the expression of PGE4 and enhanced the phosphorylation of cAMP response element binding protein, resulting in YAP activation and increased expression of YAP-related genes. Furthermore, the mammalian target of rapamycin, another major regulator of cell proliferation, was activated by YAP via suppressing phosphatase and tensin homolog through miR-29a-3p. These pathways coordinated to control cell proliferation. Collectively, MSCs could promote the recovery of ALF through PGE2-induced hepatocyte proliferation.-Liu, Y., Ren, H., Wang, J., Yang, F., Li, J., Zhou, Y., Yuan, X., Zhu, W., Shi, X. Prostaglandin E2 secreted by mesenchymal stem cells protects against acute liver failure via enhancing hepatocyte proliferation.

ER-stress regulates macrophage polarization through pancreatic EIF-2alpha kinase.

During the process of NAFLD progression, ER-stress is activated in macrophages and induces the pro-inflammatory polarization of macrophage. As one of the three ER membrane resident proteins, pancreatic eIF-2alpha kinase (PERK) plays an important role in ER stress, but its participation in macrophage polarization is largely unknown. In this study, we found that the PA mediated ER-stress activation could induce M1-type polarization in macrophages, and this phenotype polarization could be inhibited by ER-stress inhibitor 4-PBA as well as GSK2656157, an inhibitor of PERK. Moreover, the knockdown of PERK altered the STAT1 and STAT6 pathways in macrophages, which then led to the M1-to-M2 phenotypic shift. In summary, we found that PERK could regulate the phenotypic polarization of macrophages. This finding may provide new insight into the suppression of pathological progression of fatty liver or liver ischemia reperfusion injury induced by M1-type macrophages.

Hepatic expression of Yin Yang 1 (YY1) is associated with the non-alcoholic fatty liver disease (NAFLD) progression in patients undergoing bariatric surgery.

BACKGROUND: This study is to investigate the association between the hepatic expression of Yin Yang 1 (YY1) and the progression of non-alcoholic fatty liver disease (NAFLD) in patients undergoing bariatric surgery. METHODS: Obese patients undergoing bariatric surgery were included. Liver tissues were subjected to the quantitative real-time PCR, Western blot analysis, and immunohistochemical assay, to determine the expression levels of YY1. RESULTS: Totally 88 patients were included. According to the NAFLD activity score (NAS), these patients were divided into the control (n = 12), steatosis (n = 20), non-defining NASH (n = 38), and NASH (n = 18) groups. Significant differences in the serum glucose, insulin, ALT, AST, and HOMA-IR levels were observed among these different NAFLD groups. Hepatic YY1 expression had correlation with serum glucose, insulin, HOMA-IR, ALT, AST, triglycerides, HDL, and GGT. Immunohistochemical analysis showed that, compared with the control group, the expression levels of YY1 were significantly higher in the non-defining NASH and NASH groups. In addition, multivariate regression model showed that the serum ALT and YY1 levels were strongly associated with the NAFLD activity. CONCLUSIONS: Several factors are associated with NAFLD progression, including the expression of YY1. Our findings contribute to understanding of the pathogenesis of NAFLD. TRIAL REGISTRATION: NCT03296605 , registered on September 28, 2017.

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.

MicroRNA-122 Inhibits Lipid Droplet Formation and Hepatic Triglyceride Accumulation via Yin Yang 1.

BACKGROUND/AIMS: An increase in intracellular lipid droplet formation and hepatic triglyceride (TG) content usually results in nonalcoholic fatty liver disease. However, the mechanisms underlying the regulation of hepatic TG homeostasis remain unclear. METHODS: Oil red O staining and TG measurement were performed to determine the lipid content. miRNA expression was evaluated by quantitative PCR. A luciferase assay was performed to validate the regulation of Yin Yang 1 (YY1) by microRNA (miR)-122. The effects of miR-122 expression on YY1 and its mechanisms involving the farnesoid X receptor and small heterodimer partner (FXR-SHP) pathway were evaluated by quantitative PCR and Western blot analyses. RESULTS: miR-122 was downregulated in free fatty acid (FFA)-induced steatotic hepatocytes, and streptozotocin and high-fat diet (STZ-HFD) induced nonalcoholic steatohepatitis (NASH) in mice. Transfection of hepatocytes with miR-122 mimics before FFA induction inhibited lipid droplet formation and TG accumulation in vitro. These results were verified by overexpressing miR-122 in the livers of STZ-HFD-induced NASH mice. The 3'-untranslated region (3'UTR) of YY1 mRNA is predicted to contain an evolutionarily conserved miR-122 binding site. In silico searches, a luciferase reporter assay and quantitative PCR analysis confirmed that miR-122 directly bound to the YY1 3'UTR to negatively regulate YY1 mRNA in HepG2 and Huh7 cells. The (FXR-SHP) signaling axis, which is downstream of YY1, may play a key role in the mechanism of miR-122-regulated lipid homeostasis. YY1-FXR-SHP signaling, which is negatively regulated by FFA, was enhanced by miR-122 overexpression. This finding was also confirmed by overexpression of miR-122 in the livers of NASH mice. CONCLUSIONS: The present results indicate that miR-122 plays an important role in lipid (particularly TG) accumulation in the liver by reducing YY1 mRNA stability to upregulate FXR-SHP signaling.

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.

No transmission of porcine endogenous retrovirus in an acute liver failure model treated by a novel hybrid bioartificial liver containing porcine hepatocytes.

BACKGROUND: A novel hybrid bioartificial liver (HBAL) was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells (MSCs). This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses (PERVs) into canines with acute liver failure (ALF) undergoing HBAL. METHODS: Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity. RESULTS: PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative. CONCLUSION: No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.

Evaluation of two decellularization methods in the development of a whole-organ decellularized rat liver scaffold.

AIM: Hepatic tissue engineering is considered as a possible alternative to liver transplantation for end-stage liver disease. Several methods of decellularization of xenogeneic liver are available to produce three-dimensional organ scaffolds for engineering liver tissues. However, rare studies have examined and compared the effectiveness of different methods on the structure and composition of intact decellularized liver extracellular matrix. METHODS: Two decellularization methods were adopted herein. Their effects on collagen, elastin, glycosaminoglycans (GAGs), hepatocyte growth factor (HGF) content and influence to the function of hepatocytes cultured in scaffolds were examined and compared. RESULTS: The complete tissue decellularization was successfully achieved after treatment with sodium dodecyl sulphate (SDS) and Triton X-100. The total absence of nuclear structures and removal of viable cells were confirmed by haematoxylin-eosin staining and scanning electron microscopy. Collagen was preserved after both treatments. However, the elastin content decreased to about 20% and 60%, the GAGs content decreased to about 10% and 50% and the HGF content decreased to about 20% and 60% of the native liver level after SDS and Triton X-100 treatment respectively. The Triton X-100-treated scaffolds were much superior than SDS-treated scaffolds in supporting liver-specific function, including albumin secretion (P = 0.001), urea synthesis (P = 0.002), ammonia elimination (P = 0.007) and mRNA expression levels of drug metabolism enzymes. CONCLUSION: This study suggested that liver extracellular matrix scaffolds constructed using perfusion of Triton X-100 as described herein might provide a more effective and ideal material for the usage in tissue engineering and regenerative medicine approaches.

Alleviation of Hepatic Steatosis by 4-azidophlorizin via the Degradation of Geranylgeranyl Diphosphate Synthase by the Ubiquitin-Proteasome Pathway in Vivo and in Vitro.

There are no known approved pharmacotherapies for non-alcoholic fatty liver disease (NAFLD) in the clinical setting. Although studies have provided substantial evidence that geranylgeranyl diphosphate synthase (GGPPS) is a potential therapeutic target for the treatment of NAFLD corresponding drug screening is rare. A GGPPS-targeted inhibitor is identified using a structure-based virtual small molecule screening method. The interaction of 4-AZ and GGPPS is detected by microscale thermophoresis. 4-AZ degradation of GGPPS by the ubiquitin-proteasome pathway is detected by western blotting. The anti-steatotic effect of 4-AZ in vivo is detected by CT. Lipid-related gene detection is detected by real-time PCR both in primary hepatocytes and mice. The compound inhibits the accumulation of lipids in primary hepatocytes and decreases lipogenic gene expression through GGPPS. Pharmacological studies show that 4-AZ can attenuate hepatic steatosis and improve liver injury in high-fat diet-induced mice. This data provides a novel application of 4-AZ NAFLD therapy, proving that the inhibition of GGPPS is a novel strategy for the treatment of NAFLD.

Bone marrow cells: Important role on neovascularization of hepatocellular carcinoma.

BACKGROUND AND AIM: Present antivascular therapies including embolization to hepatocellular carcinoma (HCC) were not as satisfying as expected. The aim was to explore whether or not bone marrow cells (BMCs) played an important role on neovascularization in HCC. METHODS: Bone marrow-GFP(+) orthotropic HCC mice model was used. In controls and HCC mice, the dynamic change of circulating BMCs and serum vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF) were measured by flow cytometry and enzyme linked immunosorbent assay, respectively. Intrahepatic distribution of BMCs was evaluated using immunofluorescent and realtime polymerase chain reaction protocols. BMCs' intrahepatic differentiation and proportion in vessels was investigated by immunofluorescent methods. Immunohistochemistry and western blotting were performed to examine the expression of adhesion molecule in tumor tissues and tumor free tissues. RESULTS: Compared with controls, the frequency of circulating BMCs and serum VEGF, PDGF were much higher in HCC mice. The number of BMCs and the level of CD133 gene in tumor increased significantly relative to the tumor free zone. Since the early stage of HCC, BMCs have been mobilized, recruited into tumor and incorporated into different types of vessels of the liver. Besides into endothelial cells, BMCs also differentiated into vascular fibroblast and hepatic stellate cells. Moreover with tumor growth, the proportion of BMCs in vessels increased gradually. CONCLUSION: Mobilized BMCs played an important role in tumor vasculogenesis of HCC. Combined blockading of bone marrow-mediated vasculogenesis may improve the efficacy of current therapy to HCC patients.

Serum proteome profiling reveals differentially expressed proteins between subjects with metabolically healthy obesity and nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the leading risk factor for common chronic liver disease and is often regarded as a prevalent metabolic disorder tightly associated with obesity. However, the existence of metabolically healthy obesity (MHO) indicates that some important factors may participate in protecting individuals with MHO free of NAFLD, even with excessive adiposity. To explore factors independent of obesity that may be involved in the occurrence of NAFLD, we performed an iTRAQ-based proteomic study to identify proteins differentially expressed in serum between NAFLD and MHO subjects. Compared with the MHO group, ten proteins were upregulated and five were downregulated significantly in the NAFLD group. Gene Ontology analysis indicated significant changes in the immune response and triglyceride metabolism-related pathways between MHO and NAFLD. We further validated three candidates markedly dysregulated in NAFLD by Western blotting and ELISA, including two upregulated proteins (afamin and apolipoprotein H) and one downregulated protein (apolipoprotein C-1). Detection of serum apolipoprotein H levels in a large-scale cohort with MHO and different stages of NAFLD indicated that apolipoprotein H may be a potential blood biomarker for distinguishing NAFLD from MHO and an independent risk factor for predicting NAFLD.

Immunization with DNA vaccine expressing herpes simplex virus type 1 gD and IL-21 protects against mouse herpes keratitis.

The development of novel vaccines to eradicate herpes simplex virus (HSV) is a global public health priority. In this study, we developed a DNA vaccine expressing HSV-1 glycoprotein D (gD) and mouse interleukin-21(IL-21) and intramuscularly inoculated mice 3 times at 2-week intervals with a total of 300 ?g/mouse. Two weeks after the last immunization the specific antibody, splenocyte proliferative response to gD, IFN-? and IL-4 as well as the cytotoxic activities of splenocytes and natural killer (NK) cells were assayed. Immune protection against herpes keratitis was concurrently evaluated in the immunized mice after HSV-1 challenge of the mouse cornea. The results showed that the DNA vaccine pRSC-gD-IL-21 generated higher levels of antibody, IFN-? and IL-4, and enhanced the splenocyte proliferative response to gD as well as the cytotoxic activity of splenocytes and NK cells to target cells compared with the response in either the pRSC-gD or mock plasmid pRSC immunized mice. Importantly, the pRSC-gD-IL-21 ameliorated herpes keratitis severity and time course after corneal infection with HSV-1. The findings suggest that the DNA vaccine pRSC-gD-IL-21 may induce an immune response that can limit HSV-1 infection and development of herpes keratitis in the immunized mice.

WIPI2 depletion inhibits the growth of hepatocellular carcinoma cells through the AMPK signaling pathway.

WD­repeat domain phosphoinositide­interacting protein 2 (WIPI2) is a protein that regulates the assembly of multiprotein complexes by presenting a beta­propeller platform for simultaneous and reversible protein­protein interactions. This study was designed to investigate the association between the expression of WIPI2 and the growth of hepatocellular carcinoma (HCC). Publicly­available data from the UALCAN platform revealed that WIPI2 is upregulated in tumor tissues compared with that noted in normal tissues in many types of tumors especially in HCC, and high WIPI2 expression predicts a poor patient prognosis. WIPI2 expression was significantly higher in tumor tissues compared with that in the corresponding adjacent normal tissues. Depletion of WIPI2 inhibited the proliferation and promoted the apoptosis both in HCC Huh7 and Hep3B cells. In order to explore the mechanisms of WIPI2 in HCC, WIPI2 was depleted in HCC cell lines and a gene microarray was constructed. The bioinformatic analysis showed that WIPI2 regulated the proliferation of HCC cells mainly through the AMPK signaling pathway. Further analysis indicated that the downstream factors of the AMPK signaling pathway were downregulated after WIPI2 depletion. Collectively, our study revealed that WIPI2 plays an important role in the pathogenesis of HCC mainly through the AMPK signaling pathway.

Proteomic analysis to identify differentially expressed proteins between subjects with metabolic healthy obesity and non-alcoholic fatty liver disease.

Obese subjects with non-alcoholic fatty liver disease (NAFLD) and considered metabolically healthy have not been well differentiated. In this study, obese subjects were divided into metabolic healthy obesity (MHO) and NAFLD groups. Liver tissues were sampled from these two types of subjects undergoing bariatric surgery, and proteins in the liver tissues that expressed differently between the two groups of subjects were identified by Tandem Mass Tags (TMT) assay. Compared with the MHO group, 132 proteins were found to be upregulated and 84 proteins were found to be downregulated (mainly localized in mitochondria) in NAFLD group. The KEGG pathway analysis showed that significantly upregulated metabolic pathways include PPAR signaling, ECM-receptor interaction and oxidative phosphorylation was significantly downregulated. The GO analysis revealed that upregulated proteins were involved in extracellular structure organization, extracellular matrix organization and downregulated proteins took part in the oxidation-reduction process and so on. FBLN5 and DHRS2 were further validated by Western blot, immunohistochemistry and ELISA. All results demonstrate that FBLN5 expression was significantly upregulated but DHRS2 was significantly downregulated. SIGNIFICANCE: The variation between MHO and NAFLD was studied by mass spectroscopy to evaluate the mechanism with which MHO subjects resist the harmful effects induced by obesity.

Liver governs adipose remodelling via extracellular vesicles in response to lipid overload.

Lipid overload results in lipid redistribution among metabolic organs such as liver, adipose, and muscle; therefore, the interplay between liver and other organs is important to maintain lipid homeostasis. Here, we show that liver responds to lipid overload first and sends hepatocyte-derived extracellular vesicles (EVs) targeting adipocytes to regulate adipogenesis and lipogenesis. Geranylgeranyl diphosphate synthase (Ggpps) expression in liver is enhanced by lipid overload and regulates EV secretion through Rab27A geranylgeranylation. Consistently, liver-specific Ggpps deficient mice have reduced fat adipose deposition. The levels of several EV-derived miRNAs in the plasma of non-alcoholic fatty liver disease (NAFLD) patients are positively correlated with body mass index (BMI), and these miRNAs enhance adipocyte lipid accumulation. Thus, we highlight an inter-organ mechanism whereby the liver senses different metabolic states and sends corresponding signals to remodel adipose tissue to adapt to metabolic changes in response to lipid overload.

Protective Properties of FOXO1 Inhibition in a Murine Model of Non-alcoholic Fatty Liver Disease Are Associated With Attenuation of ER Stress and Necroptosis.

AIM: The pathogenesis of non-alcoholic fatty liver disease is currently unclear, however, lipid accumulation leading to endoplasmic reticulum stress appears to be pivotal in the process. At present, FOXO1 is known to be involved in NAFLD progression. The relationship between necroptosis and non-alcoholic steatohepatitis has been of great research interest more recently. However, whether FOXO1 regulates ER stress and necroptosis in mice fed with a high fat diet is not clear. Therefore, in this study we analyzed the relationship between non-alcoholic steatohepatitis, ER stress, and necroptosis. MAIN METHODS: Male C57BL/6J mice were fed with an HFD for 14 weeks to induce non-alcoholic steatohepatitis. ER stress and activation of necroptosis in AML12 cells were evaluated after inhibition of FOXO1 in AML12 cells. In addition, mice were fed with AS1842856 for 14 weeks. Liver function and lipid accumulation were measured, and further, ER stress and necroptosis were evaluated by Western Blot and Transmission Electron Microscopy. KEY FINDINGS: Mice fed with a high fat diet showed high levels of FOXO1, accompanying activation of endoplasmic reticulum stress and necroptosis. Further, sustained PA stimulation caused ER stress and necroptosis in AML12 cells. At the same time, protein levels of FOXO1 increased significantly. Inhibition of FOXO1 with AS1842856 alleviated ER stress and necroptosis. Additionally, treatment of mice with a FOXO1 inhibitor ameliorated liver function after they were fed with a high fat diet, displaying better liver condition and lighter necroptosis. SIGNIFICANCE: Inhibition of FOXO1 attenuates ER stress and necroptosis in a mouse model of non-alcoholic steatohepatitis.

IRE1α aggravates ischemia reperfusion injury of fatty liver by regulating phenotypic transformation of kupffer cells.

Fatty liver is one of the widely accepted marginal donor for liver transplantation, but is also more sensitive to ischemia and reperfusion injury (IRI) and produces more reactive oxygen species (ROS). Moreover, so far, no effective method has been developed to alleviate it. Endoplasmic reticulum stress (ER-stress) of hepatocyte is associated with the occurrence of fatty liver disease, but ER-stress of kupffer cells (KCs) in fatty liver is not clear at all. This study evaluates whether ER-stress of KCs is activated in fatty liver and accelerate IRI of fatty livers. ER-stress of KCs was activated in fatty liver, especially the IRE1α signal pathway. KCs with activated ER-stress secreted more proinflammatory cytokine to induce its M1-phenotypic shift in fatty liver, resulting in more severe IRI. Also, activated ER-stress of BMDMs in vitro by tunicamycin can induce its pro-inflammatory shift and can be reduced by 4-PBA, an ER-stress inhibitor. Knockdown of IRE1α could regulate the STAT1 and STAT6 pathway of macrophage to inhibit the M1-type polarization and promote M2-phenotypic shift. Furthermore, transfusion of IRE1α-knockdown KCs significantly reduced the liver IRI as well as the ROS of HFD feeding mice. Altogether, these data demonstrated that IRE1α of KCs may be a potential target to reduce the fatty liver associated IRI in liver transplantation.

Mesenchymal Stem Cells Enhance Liver Regeneration via Improving Lipid Accumulation and Hippo Signaling.

The liver has the potential to regenerate after injury. It is a challenge to improve liver regeneration (LR) after liver resection in clinical practice. Bone morrow-derived mesenchymal stem cells (MSCs) have shown to have a role in various liver diseases. To explore the effects of MSCs on LR, we established a model of 70% partial hepatectomy (PHx). Results revealed that infusion of MSCs could improve LR through enhancing cell proliferation and cell growth during the first 2 days after PHx, and MSCs could also restore liver synthesis function. Infusion of MSCs also improved liver lipid accumulation partly via mechanistic target of rapamycin (mTOR) signaling and enhanced lipid ß-oxidation support energy for LR. Rapamycin-induced inhibition of mTOR decreased liver lipid accumulation at 24 h after PHx, leading to impaired LR. And after infusion of MSCs, a proinflammatory environment formed in the liver, evidenced by increased expression of IL-6 and IL-1ß, and thus the STAT3 and Hippo-YAP pathways were activated to improve cell proliferation. Our results demonstrated the function of MSCs on LR after PHx and provided new evidence for stem cell therapy of liver diseases.

USP39 regulates the growth of SMMC-7721 cells via FoxM1.

The present study investigated ubiquitin specific peptidase 39 (USP39) gene knockdown on SMMC-7721 cells in vitro and in vivo, and the role of USP39 in regulating the growth of hepatocellular carcinoma (HCC). Two small interfering RNAs (siRNA) were constructed, which targeted the USP39 gene and control sequences were synthesized and inserted into a pGCSIL-GFP lentiviral vector. The full length of USP39 cDNA was amplified by polymerase chain reaction (PCR) and cloned into pEGFP-N2, and the recombinant plasmids were transfected into cells. Knockdown efficiency and upregulation of USP39 was determined by reverse transcription-quantitative PCR and western blotting. The impact of USP39 on the growth of SMMC-7721 cells in vitro was examined using an MTT assay, colony formation, flow cytometry (FCM) and immunohistochemical staining. The impact of USP39 on the growth of SMMC-7721 cells in vivo was examined by assessing tumorigenicity in nude mice. Western blotting was performed to examine the mechanism of USP39 regulation on SMMC-7721 cell growth. Recombinant vectors containing specific and scrambled USP39 siRNA sequences were constructed and transfected into SMMC-7721 cells. USP39 knockdown inhibited cell proliferation and colony formation in SMMC-7721 cells, while upregulation of USP39 promoted the growth of tumor cells. FCM indicated that USP39 knockdown led to G2/M arrest and induced apoptosis in SMMC-7721 cells. USP39 knockdown inhibited xenograft tumor growth in nude mice and led to the downregulation of the transcription factor Forkhead Box M1 (FoxM1). Gene expression of FoxM1 targets, including polo-like kinase 1, cyclin B1 and centromere protein A also decreased following USP39 knockdown. The results suggest that knockdown of USP39 inhibits the growth of HCC in vitro and in vivo, potentially through the induction of G2/M arrest by regulating the pre-mRNA splicing of FoxM1.