Establishment of Functional Liver Spheroids From Human Hepatocyte-Derived Liver Progenitor-Like Cells for Cell Therapy.

Correction

BackgroundAngiogenesis plays an important role in the progression of glioblastoma, with a high degree of malignancy. Previous studies have proved that glial cell line-derived neurotrophic factor (GDNF) and fibromodulin (FMOD) are strongly expressed in human glioblastoma. The purpose of this study was to explore the roles of GDNF and FMOD in angiogenesis and the molecular mechanisms underlying these roles in human glioblastoma.Material/MethodsThe effects of GDNF on the expression and secretion of vascular endothelial growth factor (VEGF) in human glioblastoma cell line U251 and angiogenesis in human umbilical vein endothelial cells (HUVECs) were investigated. The molecular mechanism of GDNF-induced expression of FMOD was explored. The roles of FMOD in GDNF-induced expression and secretion of VEGF and angiogenesis were also examined.ResultsIn the present study, we showed that GDNF promoted the expression and secretion of VEGF in U251 cells. VEGF mediated GDNF-induced angiogenesis in human glioblastoma. In addition, GDNF significantly upregulated the expression of FMOD in U251 cells. Mechanistically, the results of luciferase reporter assay and methylation-specific PCR (MSP) demonstrated that GDNF facilitated the demethylation of the FMOD promoter. More importantly, we found that FMOD acted as an important mediator in VEGF expression and angiogenesis induced by GDNF in human glioblastoma.ConclusionsCollectively, our data show that GDNF promotes angiogenesis through demethylation of the FMOD promoter in human glioblastoma, indicating that GDNF and FMOD may be potential therapeutic targets for glioblastoma.

Transforming growth factor-beta1 (TGF-β1) is a major factor in pathogenesis of chronic hepatic injury. Carbon tetrachloride (CCl4) is a liver toxicant, and CCl4-induced liver injury in mouse is a classical animal model of chemical liver injury. However, it is still unclear whether TGF-β1 is involved in the process of CCl4-induced acute chemical liver injury. The present study aimed to evaluate the role of TGF-β1 and its signaling molecule Smad3 in the acute liver injury induce by CCl4. The results showed that CCl4 induced acute liver injury in mice effectively confirmed by H&E staining of liver tissues, and levels of not only liver injury markers serum ALT and AST, but also serum TGF-β1 were elevated significantly in CCl4-treated mice, compared with the control mice treated with olive oil. Our data further revealed that TGF-β1 levels in hepatic tissue homogenate increased significantly, and type II receptor of TGF-β (TβRII) and signaling molecules Smad2, 3, mRNA expressions and Smad3 and phospho-Smad3 protein levels also increased obviously in livers of CCl4-treated mice. To clarify the effect of the elevated TGF-β1/Smad3 signaling on CCl4-induced acute liver injury, Smad3 in mouse liver was overexpressed in vivo by tail vein injection of Smad3-expressing plasmids. Upon CCl4 treatment, Smad3-overexpressing mice showed more severe liver injury identified by H&E staining of liver tissues and higher serum ALT and AST levels. Simultaneously, we found that Smad3-overexpressing mice treated with CCl4 showed more macrophages and neutrophils infiltration in liver and inflammatory cytokines IL-1β and IL-6 levels increment in serum when compared with those in control mice treated with CCl4. Moreover, the results showed that the apoptosis of hepatocytes increased significantly, and apoptosis-associated proteins Bax, cytochrome C and the cleaved caspase 3 expressions were up-regulated in CCl4-treated Smad3-overexpressing mice as well. These results suggested that TGF-β1/Smad3 signaling was activated during CCl4-induced acute liver injury in mice, and Smad3 overexpression aggravated acute liver injury by promoting inflammatory cells infiltration, inflammatory cytokines release and hepatocytes apoptosis. In conclusion, the activation of TGF-β signaling contributes to the CCl4-induced acute liver injury. Thus, TGF-β1/Smad3 may serve as a potential target for acute liver injury therapy.

Tubulogenesis, the organization of epithelial cells into tubular structures, is an essential step during renal organogenesis as well as during the regeneration process of renal tubules after injury. In the present study, endothelial cell-derived factors that modulate tubule formation were examined using an in vitro human tubulogenesis system. When human renal proximal tubular epithelial cells (RPTECs) were cultured in gels, tubular structures with lumens were induced in the presence of hepatocyte growth factor (HGF). Aquaporin 1 was localized in the apical membrane of these tubular structures, suggesting that these structures are morphologically equivalent to renal tubules in vivo. HGF-induced tubule formation was significantly enhanced when co-cultured with human umbilical vein endothelial cells (HUVECs) or in the presence of HUVEC-conditioned medium (HUVEC-CM). Co-culture with HUVECs did not induce tubular structures in the absence of HGF. A phospho-receptor tyrosine kinase array revealed that HUVEC-CM markedly enhanced phosphorylation of Ret, glial cell-derived neurotrophic factor (GDNF) receptor, in HGF-induced tubular structures compared to those without HUVEC-CM. HUVECs produced GDNF, and RPTECs expressed both Ret and GDNF family receptor alpha1 (co-receptor). HGF-induced tubule formation was significantly enhanced by addition of GDNF. Interestingly, not only HGF but also GDNF significantly induced phosphorylation of the HGF receptor, Met. These data indicate that endothelial cell-derived GDNF potentiates the tubulogenic properties of HGF and may play a critical role in the epithelial-endothelial crosstalk during renal tubulogenesis as well as tubular regeneration after injury.

Food-borne bioactive peptides have shown promise in preventing and mitigating alcohol-induced liver injury. This study was the first to assess the novel properties of Mactra chinenesis peptides (MCPs) in mitigating acute alcoholic liver injury in mice, and further elucidated the underlying mechanisms associated with this effect. The results showed that MCPs can improve lipid metabolism by modulating the AMPK signaling pathway, decreasing fatty acid synthase activity, and increasing carnitine palmitoyltransferase 1a activity. Meanwhile, MCPs ameliorate inflammation by inhibiting the NF-κB activation, leading to reduced levels of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β). Additionally, a 16S rDNA sequencing analysis revealed that MCPs can restore the balance of gut microbiota and increase the relative abundance of beneficial bacteria. These findings suggest that supplementation of MCPs could attenuate alcohol intake-induced acute liver injury, and, thus, may be utilized as a functional dietary supplement for the successful treatment and prevention of acute liver injury.

The trigeminal root entry zone is the zone at which the myelination switches from peripheral Schwann cells to central oligodendrocytes. Its special anatomical and physiological structure renders it susceptible to nerve injury. The etiology of most primary trigeminal neuralgia is closely related to microvascular compression of the trigeminal root entry zone. This study aimed to develop an efficient in vitro model mimicking the glial environment of trigeminal root entry zone as a tool to investigate the effects of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor on the structural and functional integrity of trigeminal root entry zone and modulation of cellular interactions. Primary astrocytes and Schwann cells isolated from trigeminal root entry zone of postnatal rats were inoculated into a two-well silicon culture insert to mimic the trigeminal root entry zone microenvironment and treated with glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor. In monoculture, glial cell line-derived neurotrophic factor promoted the migration of Schwann cells, but it did not have effects on the migration of astrocytes. In the co-culture system, glial cell line-derived neurotrophic factor promoted the bidirectional migration of astrocytes and Schwann cells. Brain-derived neurotrophic factor markedly promoted the activation and migration of astrocytes. However, in the co-culture system, brain-derived neurotrophic factor inhibited the migration of astrocytes and Schwann cells to a certain degree. These findings suggest that glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor are involved in the regulation of the astrocyte-Schwann cell interaction in the co-culture system derived from the trigeminal root entry zone. This system can be used as a cell model to study the mechanism of glial dysregulation associated with trigeminal nerve injury and possible therapeutic interventions.

Mesenchymal stem cells (MSCs) have emerged as ideal cell-based therapeutic candidates for the structural and functional restoration of the diseased kidney. Glial cell line-derived neurotrophic factor (GDNF) has been demonstrated to promote the therapeutic effect of MSCs on ameliorating renal injury. The mechanism may involve the transfer of endogenous molecules via paracrine factors to salvage injured cells, but these factors remain unknown.Methods: GDNF was transfected into human adipose mesenchymal stem cells via a lentiviral transfection system, and exosomes were isolated (GDNF-AMSC-exos). Using the unilateral ureteral obstruction (UUO) mouse model and human umbilical vein endothelial cells (HUVECs) against hypoxia/serum deprivation (H/SD) injury models, we investigated whether GDNF-AMSC-exos ameliorate peritubular capillary (PTC) loss in tubulointerstitial fibrosis and whether this effect is mediated by the Sirtuin 1 (SIRT1) signaling pathway. Additionally, by using SIRT1 activators or siRNAs, the roles of the candidate mRNA and its downstream gene in GDNF-AMSC-exo-induced regulation of endothelial cell function were assessed. PTC characteristics were detected by fluorescent microangiography (FMA) and analyzed by the MATLAB software.Results: The green fluorescent PKH67-labeled exosomes were visualized in the UUO kidneys and colocalized with CD81. GDNF-AMSC-exos significantly decreased PTC rarefaction and renal fibrosis scores in mice with UUO. In vitro studies revealed that GDNF-AMSC-exos exerted cytoprotective effects on HUVECs against H/SD injury by stimulating migration and angiogenesis as well as conferring apoptosis resistance. Mechanistically, GDNF-AMSC-exos enhanced SIRT1 signaling, which was accompanied by increased levels of phosphorylated endothelial nitric oxide synthase (p-eNOS). We also confirmed the SIRT1-eNOS interaction in HUVECs by immunoprecipitation. Furthermore, we observed a correlation of the PTC number with the SIRT1 expression level in the kidney in vivo.Conclusion: Our study unveiled a mechanism by which exosomes ameliorate renal fibrosis: GDNF-AMSC-exos may activate an angiogenesis program in surviving PTCs after injury by activating the SIRT1/eNOS signaling pathway.

The aim of this study is to delineate the expression patterns of prolyl cis-trans isomerase NIMA-interacting protein 1 (Pin1), Glial cell-derived neurotrophic factor (GDNF), and Angiotensin II (ANG II) during the process of wound repair, and to ascertain the effects of Pin1, GDNF, and ANG II on the healing of wounds in a rat model. A total of 18 rats were allocated into three groups-sham (control), DMSO (vehicle control), and Pin1 inhibitor (treatment with juglone)-with six animals in each group. An animal model of wound healing was established, followed by the intraperitoneal administration of juglone. Tissue samples from the wounds were subsequently collected for histopathological evaluation. Expression levels of Pin1, GDNF, and Ang II were quantified. In addition, an in vitro model of wound healing was created using human umbilical vein endothelial cells (HUVEC), to assess cell proliferation, migration, and tube formation under conditions of juglone pre-treatment. The expression levels of Pin1, GDNF, and ANG II were notably elevated on 7-, and 10- days post-wound compared to those measured on 3-day. Contrastingly, pre-treatment with juglone significantly inhibited the expression of these molecules. Histological analyses, including HE (Hematoxylin and Eosin), Masson's trichrome, and EVG (Elastic van Gieson) staining, demonstrated that vascular angiogenesis, as well as collagen and elastin deposition, were substantially reduced in the juglone pre-treated group when compared to the normal group. Further, immunohistochemical analysis revealed a considerable decrease in CD31 expression in the juglone pre-treatment group relative to the normal control group. Pin1 serves as a pivotal facilitator of wound repair. The findings indicate that the modulation of Pin1, GDNF, and ANG II expression impacts the wound healing process in rats, suggesting potential targets for therapeutic intervention in human wound repair.

Ethanol binge and obesity are the key risk factors for alcohol-related liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD), respectively. The human beings have a habit of drinking alcohol and consuming high calorie foods, these two factors often coexist, and thus contributing to the liver injury. However, the mechanisms of a short-term consumption of high-fat diet (HFD) plus alcohol binge-induced acute liver injury are unclear. Male C57BL/6 mice (aged 8-10weeks) were fed a HFD or HFD Control diet for 3days. Then, they received a single dose of ethanol or the same volume of distilled water by oral gavage. The liver damage was evaluated after 9h of ethanol gavage. Short-term (3 days) HFD feeding plus ethanol binge significantly aggravated liver injury and steatosis in mice as indicated by the increased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglyceride (TG) levels, the upregulated hepatic TG levels, and Oil Red O staining and H&E staining. Mechanistically, short-term HFD feeding plus ethanol binge disturbed hepatic redox homeostasis by increasing 3-nitrotyrosine (3-NT), malondialdehyde (MDA) and myeloperoxidase (MPO) levels, while decreasing glutathione (GSH) levels. HFD and alcohol co-consumption also increased hepatic TNF-α, IL-1β and IL-18 via enhancing the phosphorylation of MAPK (ERK1/2, p38 and JNK) and NF-κB. The canonical (Caspase-1 to GSDMD) and non-canonical pyroptosis signaling (Caspase-8/11 to GSDMD, and Caspase-3 to GSDME) further contributed to the acute liver injury. Short-term HFD feeding plus a single dose of ethanol gavage can significantly exacerbate acute liver injury and hepatic fat deposition in mice by enhancing oxidative stress, MAPK and NF-κB signaling, and Caspase-1/8/11-GSDMD and Caspase-3-GSDME pyroptosis signaling.

The root bark of Illicium henryi has been used in traditional Chinese medicine to treat lumbar muscle strain and rheumatic pain. Its ethanol extract (EEIH) has been previously reported to attenuate lipopolysaccharide (LPS)-induced acute kidney injury in mice. The present study aimed to evaluate the in vitro antioxidant activities and in vivo protective effects of EEIH against LPS-induced acute liver injury (ALI) in mice as well as explore its molecular mechanisms. The mice were injected intraperitoneally (i.p.) with EEIH at the doses of 1.25, 2.5, and 5.0 mg/kg every day for 5 days. One hour after the last administration, the mice were administered i.p. with LPS (8 mg/kg). After fasting for 12 h, blood and liver tissues were collected to histopathological observation, biochemical assay, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analyses. EEIH possessed 2,2-diphenyl-1-picrylhydrazil (DPPH) and 2,2′-azino-bis-(3-ethylbenzothiozoline-6-sulfonic acid) disodium salt (ABTS) radical scavenging activities and ferric-reducing antioxidant capacity in vitro. The histopathological examination, serum biochemical analysis, and liver myeloperoxidase (MPO) activity showed that EEIH pretreatment alleviated LPS-induced liver injury in mice. EEIH significantly dose-dependently decreased the mRNA and protein expression levels of inflammatory factors TNF-α, IL-1β, IL-6, and COX-2 in liver tissue of LPS-induced ALI mice via downregulating the mRNA and protein expressions of toll-like receptor 4 (TLR4) and inhibiting the phosphorylation of nuclear factor-κB (NF-κB) p65. Furthermore, EEIH markedly ameliorated liver oxidative and nitrosative stress burden in LPS-treated mice through reducing the content of thiobarbituric acid reactive substances (TBARS), inducible nitric oxide synthase (iNOS), and nitric oxide (NO) levels, restoring the decreased superoxide dismutase (SOD) and reduced glutathione (GSH) levels, and up-regulating nuclear factor erythroid 2 related factor 2 (Nrf2). These results demonstrate that EEIH has protective effects against ALI in mice via alleviating inflammatory response, oxidative and nitrosative stress burden through activating the Nrf2 and suppressing the TLR4/NF-κB signaling pathways. The hepatoprotective activity of EEIH might be attributed to the flavonoid compounds such as catechin (1), 3′,4′,7-trihydroxyflavone (2), and taxifolin (7) that most possibly act synergistically.

Background: Glial-derived neurotrophic factor (GDNF) has been demonstrated to promote the therapeutic effect of mesenchymal stem cells on ameliorating renal injury. The mechanism may involve the transfer of endogenous molecules, but these factors remain unknown. Methods: GDNF was transfected into human adipose mesenchymal stem cells via a lentiviral transfection system and exosomes were isolated (GDNF-AMSC-exos). Using unilateral ureteral obstruction (UUO) mouse models and human umbilical vein endothelial cells (HUVECs) against hypoxia/serum deprivation (H/SD) injury models we investigated whether GDNF-AMSC-exos ameliorate peritubular capillary (PTC) loss in tubulointerstitial fibrosis, and whether this effect is meditated by SIRT1 signaling pathway. Additionally, by using SIRT1 activators or siRNA, the roles of the candidate mRNA and its downstream gene in GDNF-AMSC-exo-induced regulation of endothelial cell function were assessed. Findings: GDNF-AMSC-exos resulted in significantly decreased PTC rarefaction and renal fibrosis scores in mice with UUO. In vitro studies revealed that GDNF-AMSC-exos exert cytoprotective effects on HUVECs against H/SD injury by stimulating migration, angiogenesis and conferring apoptosis resistance. Mechanistically, GDNF-AMSC-exos enhanced SIRT1 signaling, which accompanied by increased protein levels of phosphorylated eNOS (p-eNOS). In addition, we have confirmed the SIRT1-eNOS interaction in HUVECs by immunoprecipitation. Interpretation: Our study unveils a mechanism by which exosomes ameliorate renal fibrosis: GDNF-AMSC-exos may activate an angiogenesis program in surviving PTCs after injury by activating the SIRT1/eNOS signaling pathway. This finding suggests that inhibition of chronic kidney disease (CKD) progression requires long-term suppression of fibrosis via renal microvascular repair and angiogenesis and that GDNF-AMSC-exos therapy is a promising strategy for achieving this goal. Funding Statement: This work were supported by projects of the National Natural Science Foundation of China (81270769 and 81803473); a project of the Jiangsu Provincial Natural Science Foundation (BK20161172); a project of the Jiangsu Provincial Commission of Health and Family Planning (H201628); projects of the Jiangsu Provincial Post Graduate Innovation Plan (KYCX18-2178, KYCX17-1708, SJCX17-0560, SJCX18-0715); the Municipal Key Research and Development Project of Xuzhou (KC18212); the “Liu ge yi Gong Cheng” project of Jiangsu High-Level Personnel (LGY2016043); a school class project of Xuzhou Medical University (2017KJ13); and the Xuzhou Science and Technology Bureau Applied Basic Research Program( KC18041 and KC 19069). Declaration of Interests: All the authors declared no competing interests. Ethics Approval Statement: Approval of all research involving human participants was obtained from the Institutional Review Board of the Affiliated Hospital of Xuzhou Medical University (permit number: xyfylw2013032). All experiments involving animals followed the animal use protocol enacted by the Institutional Animal Care and Use Committee of Xuzhou Medical University.

Acute liver injury (ALI) is a life-threatening clinical syndrome. Long-lasting liver injury can lead to chronic hepatic inflammation and fibrogenic responses. Zerumbone (ZER), the main constituent of rhizomes of Zingiber zerumbet Smith, has a variety of functions including anticancer activity. We investigated the role of ZER on the progression of hepatotoxin-induced liver injury. Single or repeated injection of CCl4 was used to induce acute or chronic liver injury, respectively. Mice were orally administered with ZER (10, 50mg/kg) during the experimental period. Histopathologic analysis and serum biochemical levels revealed that ZER had hepatoprotective activities against ALI. Similar effects of ZER on injured livers were confirmed by analyses of inflammation and apoptosis-related genes. Western blot analysis showed that protein levels of apoptotic molecules were decreased, whereas antiapoptotic protein levels were conversely increased in injured livers treated with ZER. Furthermore, chronic liver injury and its associated fibrogenesis in mice were reduced by ZER treatment. These findings from our in vivo experiments further indicate that ZER could alleviate hepatocellular toxicity and inhibit activation of primary hepatic stellate cells. Our results suggest that ZER might have potential as a safe and prophylactic alternative to prevent acute and chronic liver injury.

Adhesion of platelets through thromboxane A₂ receptor signaling facilitates liver repair during acute chemical-induced hepatotoxicity.

EuHD1 protects against inflammatory injury driven by NLRP3 inflammasome.

Protective effect of ISO-1 with inhibition of RIPK3 up-regulation and neutrophilic accumulation on acetaminophen-induced liver injury in mice