Oleanolic acid alleviating ischemia-reperfusion injury in rat severe steatotic liver via KEAP1/NRF2/ARE.

Severe steatosis in donor livers is contraindicated for transplantation due to the high risk of ischemia-reperfusion injury (IRI). Although Ho-1 gene-modified bone marrow mesenchymal stem cells (HO-1/BMMSCs) can mitigate IRI, the role of gut microbiota and metabolites in this protection remains unclear. This study aimed to explore how gut microbiota and metabolites contribute to HO-1/BMMSCs-mediated protection against IRI in severe steatotic livers. Using rat models and cellular models (IAR20 and THLE-2 cells) of steatotic liver IRI, this study revealed that ischemia-reperfusion led to significant liver and intestinal damage, heightened immune responses, impaired liver function, and altered gut microbiota and metabolite profiles in rats with severe steatosis, which were partially reversed by HO-1/BMMSCs transplantation. Integrated microbiome and metabolome analyses identified gut microbial metabolite oleanolic acid as a potential protective agent against IRI. Experimental validation showed that oleanolic acid administration alone alleviated IRI and inhibited ferroptosis in both rat and cellular models. Network pharmacology and molecular docking implicated KEAP1/NRF2 pathway as a potential target of oleanolic acid. Indeed, OA experimentally upregulated NRF2 activity, which underlies its inhibition of ferroptosis and protection against IRI. The gut microbial metabolite OA protects against IRI in severe steatotic liver by promoting NRF2 expression and activity, thereby inhibiting ferroptosis.

Ferrostatin-1 improves prognosis and regulates gut microbiota of steatotic liver transplantation recipients in rats.

Aims: To investigate the effects of Ferrostatin-1 (Fer-1) on improving the prognosis of liver transplant recipients with steatotic liver grafts and regulating gut microbiota in rats. Methods: We obtained steatotic liver grafts and established a liver transplantation model. Recipients were divided into sham, liver transplantation and Fer-1 treatment groups, which were assessed 1 and 7 days after surgery (n = 6). Results & conclusion: Fer-1 promotes recovery of the histological structure and function of steatotic liver grafts and the intestinal tract, and improves inflammatory responses of recipients following liver transplantation. Fer-1 reduces gut microbiota pathogenicity, and lowers iron absorption and improves fat metabolism of recipients, thereby protecting steatotic liver grafts.

Exosomes derived from bone marrow mesenchymal stem cells alleviate biliary ischemia reperfusion injury in fatty liver transplantation by inhibiting ferroptosis.

Fatty liver grafts are susceptible to ischemia reperfusion injury (IRI), increasing the risk of biliary complications after liver transplantation (LT). Ferroptosis, a newly recognized programmed cell death, is expected to be a novel therapeutic target for IRI. We investigated whether exosomes derived from heme oxygenase 1-modified bone marrow mesenchymal stem cells (HExos) relieve ferroptosis and protect biliary tracts from IRI in a rat fatty liver transplantation model. Rats were fed with a methionine choline deficient (MCD) diet for 2 weeks to induce severe hepatic steatosis. Steatotic grafts were implanted and HExos were administered after liver transplantation. A series of functional assays and pathological analysis were performed to assess ferroptosis and biliary IRI. The HExos attenuated IRI following liver transplantation, as demonstrated by less ferroptosis, improved liver function, less Kupffer and T cell activation, and less long-term biliary fibrosis. MicroRNA (miR)-204-5p delivered by HExos negatively regulated ferroptosis by targeting a key pro-ferroptosis enzyme, ACSL4. Ferroptosis contributes to biliary IRI in fatty liver transplantation. HExos protect steatotic grafts by inhibiting ferroptosis, and may become a promising strategy to prevent biliary IRI and expand the donor pool.

Heme Oxygenase-1-Modified BMMSCs Activate AMPK-Nrf2-FTH1 to Reduce Severe Steatotic Liver Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury (IRI) is an important cause of graft dysfunction post-liver transplantation, where donor liver with severe steatosis is more sensitive to IRI. Liver IRI involves ferroptosis and can be alleviated by heme oxygenase-1-modified bone marrow mesenchymal stem cells (HO-1/BMMSCs). AIMS: To explore the role and mechanism of HO-1/BMMSCs in severe steatotic liver IRI. METHODS: A severe steatotic liver IRI rat model and a hypoxia/reoxygenation (H/R) of severe steatosis hepatocyte model were established. Liver and hepatocyte damage was evaluated via liver histopathology and cell activity. Ferroptosis was evaluated through ferroptosis indexes. Nuclear factor erythroid 2-related factor 2 (Nrf2) was knocked down in severe steatotic hepatocytes. The role of Nrf2 and AMPK in HO-1/BMMSC inhibition of ferroptosis was examined using the AMP-activated protein kinase (AMPK) pathway inhibitor Compound C. RESULTS: The HO-1/BMMSCs alleviated severe steatotic liver IRI and ferroptosis. HO-1/BMMSCs promoted ferritin heavy chain 1(FTH1), Nrf2, and phosphorylated (p)-AMPK expression in the H/R severe steatotic hepatocytes. Nrf2 knockdown decreased FTH1 expression levels but did not significantly affect p-AMPK expression levels. The protective effect of HO-1/BMMSCs against H/R injury in severe steatotic hepatocytes and the inhibitory effect on ferroptosis were reduced. Compound C decreased p-AMPK, Nrf2, and FTH1 expression levels, weakened the HO-1/BMMSC protective effect against severe steatotic liver IRI and H/R-injured severe steatotic hepatocytes, and reduced the inhibition of ferroptosis. CONCLUSIONS: Ferroptosis was involved in HO-1/BMMSC reduction of severe steatotic liver IRI. HO-1/BMMSCs protected against severe steatotic liver IRI by inhibiting ferroptosis through the AMPK-Nrf2-FTH1 pathway. HO-1/BMMSCs activate AMPK, which activates Nrf2, promotes its nuclear transcription, then promotes the expression of its downstream protein FTH1, thereby inhibiting ferroptosis and attenuating severe steatotic liver IRI in rats. Glu: glutamic acid; Cys: cystine; GSH: glutathione; GPX4: glutathione peroxidase 4; HO-1/BMMSCs: HO-1-modified BMMSCs; Fer-1: ferrostatin-1; DFO: deferoxamine; FTH1: ferritin heavy chain1; p-AMPK: phosphorylated AMP-activated protein kinase; Nrf2: nuclear factor erythroid 2-related factor 2; IRI: ischemia-reperfusion injury; MCD: methionine-choline deficiency.

Identification and characterization of colorectal-cancer-associated SNPs on the SMAD7 locus.

PURPOSE: Genome-wide association studies have identified SMAD7 as a colorectal cancer (CRC) susceptibility gene. However, its underlying mechanism has not yet been characterized. This study screened functional SNPs (fSNPs) related to colorectal cancer through Reel-seq and obtained regulatory proteins on functional SNPs. METHODS: The candidate fSNPs on the SMAD7 locus were screened by Reel-seq method. Eight SNPs such as rs8085824 were identified as functional SNPs by luciferase reporter assay and EMSA, SDCP-MS and AIDP-WB revealed that HNRNPK can specifically bind to the rs8085824-C allele. The knockdown of HNRNPK by RNAi proved that HNRNPK could affect cell function by regulating SMAD7. RESULTS: Eight functional SNPs was found on the SMAD7 locus in linkage disequilibrium (LD) with R2 > 0.8, i.e., rs12953717, rs7227023, rs34007497, rs58920878, rs8085824, rs4991143, rs4939826, and rs7227023. We also identified allele-imbalanced binding of HNRNPK to rs8085824, H1-3 to rs12953717, THOC6 to rs7227023, and DDX21 to rs58920878. Further functional analysis revealed that these proteins are the regulatory proteins that modulate the expression of SMAD7 in the human colorectal cancer cell line DLD1. In particular, we discovered that siRNA knockdown of HNRNPK inhibits cell proliferation and cell clonal formation by downregulating SMAD7, as the decreased cell proliferation and cell clonal formation in the siRNA HNRNPK knockdown cells was restored by SMAD7 overexpression. CONCLUSION: Our findings reveal a mechanism which underlies the contribution of the fSNP rs8085824 on the SMD7 locus to CRC susceptibility.

Small extracellular vesicles from HO-1-modified bone marrow-derived mesenchymal stem cells attenuate ischemia-reperfusion injury after steatotic liver transplantation by suppressing ferroptosis via miR-214-3p.

Donor shortage is a major problem that limits liver transplantation availability. Steatotic donor liver presents a feasible strategy to solve this problem. However, severe ischemia-reperfusion injury (IRI) is an obstacle to the adoption of steatotic transplanted livers. Evidence from our prior studies indicated that bone marrow mesenchymal stem cells modified with heme oxygenase-1 (HMSCs) can attenuate non-steatotic liver IRI. However, the contribution of HMSCs in transplanted steatotic liver IRI is unclear. Here, HMSCs and their derived small extracellular vesicles (HM-sEVs) alleviated IRI in transplanted steatotic livers. After liver transplantation, there was significant enrichment of the differentially expressed genes in the glutathione metabolism and ferroptosis pathways, accompanied by ferroptosis marker upregulation. The HMSCs and HM-sEVs suppressed ferroptosis and attenuated IRI in the transplanted steatotic livers. MicroRNA (miRNA) microarray and validation experiments indicated that miR-214-3p, which was abundant in the HM-sEVs, suppressed ferroptosis by targeting cyclooxygenase 2 (COX2). In contrast, COX2 overexpression reversed this effect. Knockdown of miR-214-3p in the HM-sEVs diminished its ability to suppress ferroptosis and protect liver tissues/cells. The findings suggested that HM-sEVs suppressed ferroptosis to attenuate transplanted steatotic liver IRI via the miR-214-3p-COX2 axis.

ISG15 is a potential therapeutic target for osteosarcoma: a comprehensive analysis based on bioinformatics and in vitro experiments.

BACKGROUND: The expression of aberrant interferon-stimulated gene 15 (ISG15) is connected with various human diseases, including cancer. ISG15 is involved in tumor formation and metastasis. However, its role in osteosarcoma is uncertain. METHODS: ISG15 expression in pan-cancer from RNA Sequencing data were obtained from The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression (GTEx) databases. The relationship between ISG15 expression and prognosis was assessed through TCGA clinical survival data. Immunohistochemistry (IHC) images of ISG15 were retrieved using the Human Protein Atlas to analyze the differences in selected normal and tumor tissues. Gene enrichment analysis and signaling pathway analysis were used to assess the potential role of ISG15 in sarcoma, and the correlation between ISG15 expressions and immune cell infiltration levels was estimated by immune infiltration analysis. The expression levels of ISG15 were assessed by qRT-PCR and IHC. Colony formation, wound healing assay and transwell assay were used to detect the effects of ISG15 on the biological behaviors of osteosarcoma cells. The correlation between ISG15 levels and CD8+/CD68+ cells was further examined by double-labeled immunofluorescence. The chemotactic effect of ISG15 on CD8+/CD68+ cells was demonstrated by chemotactic experiments and flow cytometry. RESULTS: ISG15 was highly expressed in most cancers, while high ISG15 expression was significantly correlated with poor overall survival. Gene enrichment analysis in sarcoma suggested that antigen processing and presentation might be involved in the oncogenic mechanism of ISG15. Further immune infiltration analysis showed that high ISG15 expression might reflect the infiltration level of certain immune cells. Additionally, our verification showed that ISG15 was significantly related to the occurrence and metastasis of osteosarcoma, and knockdown of ISG15 significantly altered cell biological behavior, resulting in decreased proliferation, migration and invasion capabilities of osteosarcoma cells. The high expression of ISG15 in osteosarcoma tissue was associated with a high level of CD68+ immune cell infiltration while a low level of CD8+ T cell infiltration. CD68+ immune cells were recruited in vitro by overexpression of ISG15, which on the contrary could weaken the chemotaxis of CD8+ T cells. CONCLUSION: High ISG15 expression is an inherent feature of osteosarcoma and triggers tumorigenesis and metastasis by regulating tumor immunogenicity. ISG15 is expected to be the target of osteosarcoma treatment.

Development of magnetic poly(L-lactic Acid) nanofibrous microspheres for transporting and delivering targeted cells.

To avoid infection and other risks caused by large open-surgery incisions using scaffold transplants, it is very important to study injectable microcarrier-loaded cells for targeted therapy and tissue regeneration. In this study, on the one hand, to simulate the hierarchical structure of the extracellular matrix and carry cells, poly(L-lactic acid) (PLLA) nanofibrous microspheres (large microspheres) were initially fabricated as cell carriers. On the other hand, to precisely deliver cells through a magnetic field and promote stem cell differentiation, drug-loaded mesoporous Fe3O4@SiO2 microspheres (small microspheres) were prepared and coated on the surface PLLA nanofibrous microspheres. The coating conditions were systematically studied and optimized. The results showed that planetary-satellite-like cell carriers were successfully prepared and the carriers were capable of freely translocating under the influence of a magnetic field. It has been demonstrated in vitro experiments that the carriers are biocompatible and are capable of acting as drug carriers. Specifically, they were able to load and release cells in response to magnetic fields. In vivo experiments indicated that the carriers could successfully load and release GFP-labelled cells in nude mice. The study presented in this paper provides a versatile and promising platform for the cell-based therapy in tissue engineering and regenerative medicine.

miR-29a-3p in Exosomes from Heme Oxygenase-1 Modified Bone Marrow Mesenchymal Stem Cells Alleviates Steatotic Liver Ischemia-Reperfusion Injury in Rats by Suppressing Ferroptosis via Iron Responsive Element Binding Protein 2.

Hepatic ischemia-reperfusion injury (IRI) is an inevitable result of liver surgery. Steatotic livers are extremely sensitive to IRI and have worse tolerance. Ferroptosis is considered to be one of the main factors of organ IRI. This study is aimed at exploring the role of ferroptosis in the effect of heme oxygenase-1-modified bone marrow mesenchymal stem cells (HO-1/BMMSCs) on steatotic liver IRI and its mechanism. An IRI model of a steatotic liver and a hypoxia reoxygenation (HR) model of steatotic hepatocytes (SHPs) were established. Rat BMMSCs were extracted and transfected with the Ho1 gene to establish HO-1/BMMSCs, and their exosomes were extracted by ultracentrifugation. Ireb2 was knocked down to verify its role in ferroptosis and cell injury in SHP-HR. Public database screening combined with quantitative real-time reverse transcription PCR identified microRNAs (miRNAs) targeting Ireb2 in HO-1/BMMSCs exosomes. miR-29a-3p mimic and inhibitor were used for functional verification experiments. Liver function, histopathology, terminal deoxynulceotidyl transferase nick-end-labeling staining, cell viability, mitochondrial membrane potential, and cell death were measured to evaluate liver tissue and hepatocyte injury. Ferroptosis was assessed by detecting the levels of IREB2, Fe2+, malondialdehyde, glutathione, lipid reactive oxygen species, glutathione peroxidase 4, prostaglandin-endoperoxide synthase 2 mRNA, and mitochondrial morphology. The results revealed that HO-1/BMMSCs improved liver tissue and hepatocyte injury and suppressed ferroptosis in vivo and in vitro. The expression of IREB2 was increased in steatotic liver IRI and SHP-HR. Knocking down Ireb2 reduced the level of Fe2+ and inhibited ferroptosis. HO-1/BMMSC exosomes reduced the expression of IREB2 and inhibited ferroptosis and cell damage. Furthermore, we confirmed high levels of miR-29a-3p in HO-1/BMMSCs exosomes. Overexpression of miR-29a-3p downregulated the expression of Ireb2 and inhibited ferroptosis. Downregulation of miR-29a-3p blocked the protective effect of HO-1/BMMSC exosomes on SHP-HR cell injury. In conclusion, ferroptosis plays an important role in HO-1/BMMSC-mediated alleviation of steatotic liver IRI. HO-1/BMMSCs could suppress ferroptosis by targeting Ireb2 via the exosomal transfer of miR-29a-3p.

Intestinal Microbiota Participates in the Protective Effect of HO-1/BMMSCs on Liver Transplantation With Steatotic Liver Grafts in Rats.

The present study aimed to explore whether heme oxygenase-1 (HO-1)-modified bone marrow mesenchymal stem cells (BMMSCs) have a protective effect on liver transplantation with steatotic liver grafts in rats, and to determine the role of the intestinal microbiota in such protection. HO-1/BMMSCs were obtained by transduction of Hmox1 gene [encoding heme oxygenase (HO-1)]-encoding adenoviruses into primary rat BMMSCs. Steatotic livers were obtained by feeding rats a high-fat diet, and a model of liver transplantation with steatotic liver grafts was established. The recipients were treated with BMMSCs, HO-1/BMMSCs, or neither, via the portal vein. Two time points were used: postoperative day 1 (POD 1) and POD 7. The results showed that under the effect of HO-1/BMMSCs, the degree of steatosis in the liver grafts was significantly reduced, and the level of liver enzymes and the levels of pro-inflammatory cytokines in plasma were reduced. The effect of HO-1/BMMSCs was better than that of pure BMMSCs in the prolongation of the rats' postoperative time. In addition, HO-1/BMMSCs promoted the recovery of recipients' intestinal structure and function, especially on POD 7. The intestinal villi returned to normal, the expression of tight junction proteins was restored, and intestinal permeability was reduced on POD 7. The intestinal bacterial of the LT group showed significantly weakened energy metabolism and overgrowth. On POD 1, the abundance of Akkermansiaceae was higher. On POD 7, the abundance of Clostridiaceae increased, the level of lipopolysaccharide increased, the intestinal mucosal barrier function was destroyed, and the levels of several invasive bacteria increased. When treated with HO-1/BMMSCs, the energy metabolism of intestinal bacteria was enhanced, and on POD 1, levels bacteria that protect the intestinal mucosa, such as Desulfovibrionaceae, increased significantly. On POD 7, the changed intestinal microbiota improved lipid metabolism and increased the levels of butyrate-producing bacteria, such as Lachnospiraceae. In conclusion, HO-1/BMMSCs have protective effects on steatotic liver grafts and the intestinal barrier function of the recipients. By improving lipid metabolism and increasing the abundance of butyrate-producing bacteria, the changed intestinal microbiota has a protective effect and prolongs the recipients' survival time.

miR-124-3p delivered by exosomes from heme oxygenase-1 modified bone marrow mesenchymal stem cells inhibits ferroptosis to attenuate ischemia-reperfusion injury in steatotic grafts.

BACKGROUND: Steatotic livers tolerate ischemia-reperfusion injury (IRI) poorly, increasing the risk of organ dysfunction. Ferroptosis is considered the initiating factor of organ IRI. Heme oxygenase oxygen-1 (HO-1)-modified bone marrow mesenchymal stem cells (BMMSCs) (HO-1/BMMSCs) can reduce hepatic IRI; however, the role of ferroptosis in IRI of steatotic grafts and the effect of HO-1/BMMSCs-derived exosomes (HM-exos) on ferroptosis remain unknown. METHODS: A model of rat liver transplantation (LT) with a severe steatotic donor liver and a model of hypoxia and reoxygenation (H/R) of steatotic hepatocytes were established. Exosomes were obtained by differential centrifugation, and the differentially expressed genes (DEGs) in liver after HM-exo treatment were detected using RNA sequencing. The expression of ferroptosis markers was analyzed. microRNA (miRNA) sequencing was used to analyze the miRNA profiles in HM-exos. RESULTS: We verified the effect of a candidate miRNA on ferroptosis of H/R treated hepatocytes, and observed the effect of exosomes knockout of the candidate miRNA on hepatocytes ferroptosis. In vitro, HM-exo treatment reduced the IRI in steatotic grafts, and enrichment analysis of DEGs suggested that HM-exos were involved in the regulation of the ferroptosis pathway. In vitro, inhibition of ferroptosis by HM-exos reduced hepatocyte injury. HM-exos contained more abundant miR-124-3p, which reduced ferroptosis of H/R-treated cells by inhibiting prostate six transmembrane epithelial antigen 3 (STEAP3), while overexpression of Steap3 reversed the effect of mir-124-3p. In addition, HM-exos from cell knocked out for miR-124-3p showed a weakened inhibitory effect on ferroptosis. Similarly, HM-exo treatment increased the content of miR-124-3p in grafts, while decreasing the level of STEAP3 and reducing the degree of hepatic ferroptosis. CONCLUSION: Ferroptosis is involved in the IRI during LT with a severe steatotic donor liver. miR-124-3p in HM-exos downregulates Steap3 expression to inhibit ferroptosis, thereby attenuating graft IRI, which might be a promising strategy to treat IRI in steatotic grafts.

Bone marrow mesenchymal stem cells modified with heme oxygenase-1 alleviate rejection of donation after circulatory death liver transplantation by inhibiting dendritic cell maturation in rats.

Immature dendritic cells induce immune tolerance and mature dendritic cells induce acute rejection. We infused bone marrow mesenchymal stem cells (BMMSCs) expressing heme oxygenase 1 (HO-1) (HO-1/BMMSCs) into donation after circulatory death (DCD) livers using normothermic machine perfusion (NMP), and then performed transplantation, with the aim of determining the effects of HO 1/BMMSCs on liver DC maturation and graft rejection. A rat model of acute liver transplantation rejection was established from Lewis to BN rats, in which six experimental groups were set up: Sham operation, static cold storage, NMP, BMMSCs + NMP, HO-1/BMMSCs + NMP (HBP), and NMP + FK506 gavage. Flow cytometry was performed to detect the maturation of DCs and the activation of CD4+ T cells in the liver. In vitro, HO-1/BMMSCs were cocultured with liver DCs, and then the phenotype and ability to stimulate lymphocyte proliferation of DCs were measured. MAPK inhibitors were added to observe the effect of MAPK signaling on DC maturation. The resultsindicatedthatHO-1/BMMSCs could stably colonize the transplanted liver. In the HBP group, rejection was reduced, the maturation of DCs was inhibited, and the infiltration and activation of CD4+ T cells were reduced. In vitro, DCs cocultured with HO-1/BMMSCs showed an immature phenotype and inhibited T cell proliferation. HO-1/BMMSCs inhibited the maturation of DCs by blocking the phosphorylation of p38 and ERK1/2. This study suggested that infusion of HO-1/BMMSCs into DCD livers could reduce acute rejection significantly by inhibiting DC maturation. DC maturation regulation by HO-1/BMMSCs involves ERK1/2/MAPK and p38/MAPK signaling.

HO-1/BMMSC perfusion using a normothermic machine perfusion system reduces the acute rejection of DCD liver transplantation by regulating NKT cell co-inhibitory receptors in rats.

BACKGROUND: Liver transplantation (LT) is required in many end-stage liver diseases. Donation after cardiac death (DCD) livers are often used, and treatment of acute rejection (ACR) requires the use of immunosuppressive drugs that are associated with complications. Bone marrow mesenchymal stem cells (BMMSCs) are used in treatment following LT; however, they have limitations, including low colonization in the liver. An optimized BMMSC application method is required to suppress ACR. METHODS: BMMSCs were isolated and modified with the heme oxygenase 1 (HO-1) gene. HO-1/BMMSCs were perfused into donor liver in vitro using a normothermic machine perfusion (NMP) system, followed by LT into rats. The severity of ACR was evaluated based on liver histopathology. Gene chip technology was used to detect differential gene expression, and flow cytometry to analyze changes in natural killer (NK) T cells. RESULTS: NMP induced BMMSCs to colonize the donor liver during in vitro preservation. The survival of HO-1/BMMSCs in liver grafts was significantly longer than that of unmodified BMMSCs. When the donor liver contained HO-1/BMMSCs, the local immunosuppressive effect was improved and prolonged, ACR was controlled, and survival time was significantly prolonged. The application of HO-1/BMMSCs reduced the number of NKT cells in liver grafts, increased the expression of NKT cell co-inhibitory receptors, and reduced NKT cell expression of interferon-γ. CONCLUSIONS: NK cell and CD8+ T cell activation was inhibited by application of HO-1/BMMSCs, which reduced ACR of transplanted liver. This approach could be developed to enhance the success rate of LT.

Long non-coding RNAs HERH-1 and HERH-4 facilitate cyclin A2 expression and accelerate cell cycle progression in advanced hepatocellular carcinoma.

BACKGROUND: The advanced hepatocellular carcinoma (HCC), such as the recurrent tumor after liver transplantation (LT), is an obstacle of HCC treatment. The aim of this study was to discover the underlying mechanism of HCC progression caused by non-coding RNAs (ncRNAs). METHODS: To this end, we investigated the selected patient cohort of matching primary and recurrent HCC after receiving LT. The recurrent tumors after LT were regarded as clinical models of the advanced HCC. Microarrays were used to profile lncRNA and mRNA expression in HCC recurrent and primary tissue samples. The mRNA profile characteristics were analyzed by bioinformatics. Two cell lines, HepG2 and QGY-7703, were used as HCC cell models. The protein-coding potential, length, and subcellular location of the interested lncRNAs were examined by bioinformatics, Northern blot, fluorescent in situ hybridization (FISH), and quantitative RT-PCR (qRT-PCR) assays. HCC cell proliferation was detected by CCK-8, doubling time and proliferation marker gene quantitation assays. DNA replication during the cell cycle was measured by EdU/PI staining and flow cytometry analyses. Promoter activity was measured using a luciferase reporter assay. Interactions between DNA, RNA, and protein were examined by immunoprecipitation and pull-down assays. The miRNA-target regulation was validated by a fluorescent reporter assay. RESULTS: Both lncRNA and mRNA profiles exhibited characteristic alterations in the recurrent tumor cells compared with the primary HCC. The mRNA profile in the HCC recurrent tissues, which served as model of advanced HCC, showed an aberrant cell cycle regulation. Two lncRNAs, the highly expressed lncRNA in recurrent HCC (HERH)-1 and HERH-4, were upregulated in the advanced HCC cells. HERH-1/4 enhanced proliferation and promoted DNA replication and G1-S transition during the cell cycle in HCC cells. HERH-1 interacted with the transcription factor CREB1. CREB1 enhanced cyclin A2 (CCNA2) transcription, depending on HERH-1-CREB1 interaction. HERH-4 acted as an miR-29b/c sponge to facilitate CCNA2 protein translation through a competing endogenous RNA (ceRNA) pathway. CONCLUSIONS: The oncogenic lncRNA HERH-1/4 promoted CCNA2 expression at the transcriptional and post-transcriptional levels and accelerated cell cycle progression in HCC cells. The HERH-1-CREB1-CCNA2 and HERH-4-miR-29b/c-CCNA2 axes served as molecular stimuli for HCC advance.

Heme Oxygenase-1-Modified Bone Marrow Mesenchymal Stem Cells Combined with Normothermic Machine Perfusion Repairs Bile Duct Injury in a Rat Model of DCD Liver Transplantation via Activation of Peribiliary Glands through the Wnt Pathway.

Livers from donors after circulatory death (DCD) are inevitably exposed to a longer warm ischemic period, which might increase the incidence of postoperative bile duct complications. Bone marrow mesenchymal stem cells (BMMSCs) have tissue repair properties. The present study was aimed at exploring the repair effect of heme oxygenase-1- (HO-1-) modified BMMSCs (HO-1/BMMSCs) combined with normothermic machine perfusion (NMP) on bile duct injury after DCD liver transplantation and at revealing the underlying mechanisms. Rat livers were exposed to in situ warm ischemia for 30 min; then, NMP was performed through the portal vein for 4 h with BMMSCs, HO-1/BMMSCs, or neither before implantation. Obvious bile duct histological damage and liver functional damage were observed postoperatively. In the group treated with HO-1/BMMSCs combined with NMP (HBP group), liver functions and bile duct histology were improved; meanwhile, cell apoptosis was reduced and cell proliferation was active. A large number of regenerative cells appeared at the injured site, and the defective bile duct epithelium was restored. Dilatation of peribiliary glands (PBGs), proliferation of PBG cells, high expression of vascular endothelial growth factor (VEGF), and increased proportion of bile duct progenitor cells with stem/progenitor cells biomarkers were observed. Blocking Wnt signaling significantly inhibited the repair effect of HO-1/BMMSCs on bile duct injury. In conclusion, HO-1/BMMSCs combined with NMP were relevant to the activation of biliary progenitor cells in PBGs which repaired bile duct injury in DCD liver transplantation via the Wnt signaling pathway. Proliferation and differentiation of PBG cells were involved in the renewal of the injured biliary epithelium.

Protective Effects of Bone Marrow Mesenchymal Stem Cells (BMMSCS) Combined with Normothermic Machine Perfusion on Liver Grafts Donated After Circulatory Death via Reducing the Ferroptosis of Hepatocytes.

BACKGROUND To improve the quality of liver grafts from extended-criteria donors donated after circulatory death (DCD), this study explored whether bone marrow mesenchymal stem cells (BMMSCs) combined with normothermic machine perfusion (NMP) have protective effects on DCD donor livers and the effects of ferroptosis in this procedure. MATERIAL AND METHODS Twenty-four male rat DCD donor livers were randomly and averagely divided into normal, static cold storage (SCS), NMP, and NMP combined with BMMSCs groups. Liver function, bile secretion, and pathological features of DCD donor livers were detected to evaluate the protective effects of NMP and BMMSCs on DCD donor livers. Hydrogen peroxide was used to induce an oxidative stress model of hepatocyte IAR-20 cells to evaluate the protective effects of BMMSCs in vitro. RESULTS Livers treated with NMP combined with BMMSCs showed better liver function, relieved histopathological damage, reduced oxidative stress injury and ferroptosis, and the mechanism of reduction was associated with downregulation of intracellular reactive oxygen species (ROS) and free Fe²âº levels. BMMSCs showed significant protective effects on the ultrastructure of DCD donor livers and ROS-induced injury to IAR-20 cells under electron microscopy. BMMSCs also significantly improved the expression level of microtubule-associated protein 1 light chain 3 (LC3)-II in both DCD donor livers and ROS-induced injured IAR-20 cells, including upregulating the expression of ferritin. CONCLUSIONS BMMSCs combined with NMP could reduce the level of ROS and free Fe²âº in oxidative stress damaged rat DCD donor livers, potentially reduce the ferroptosis in hepatocytes, and repair both morphology and function of DCD donor livers.

Recurrent broad ligament leiomyosarcoma with pancreatic and thigh metastasis: a case report.

BACKGROUND: Leiomyosarcoma (LMS) is an uncommon mesenchymal neoplasm, which infrequently metastasizes to pancreas and thigh. Clinical presentation and imaging findings of metastatic broad ligament LMS are often nonspecific. Complete excision plays an important role in treatment of patients with localized LMS. CASE PRESENTATION: Here, we report a case of a 33-year-old woman with recurrent broad ligament LMS metastasizing to pancreas and thigh. Previously, she was diagnosed with broad ligament LMS and underwent hysterectomy, bilateral salpingo-oophorectomy. The disease-free interval was 2.5 years until metastases were found. Computerized tomography (CT) of abdomen and thighs, magnetic resonance imaging (MRI) of thighs and whole-body 18-fluorodeoxyglucose positron emission tomography - computed tomography (PET-CT) performed, revealed pancreatic and thigh metastasis. Ultrasonography-guided biopsy and histological examinations confirmed LMS at both the sites. Pancreatic metastasis was completely resected first. Then the patient underwent surgical resection of thigh metastasis when both chemotherapy and radiotherapy failed. She recovered well and remained free of disease recurrence in the 2 years follow-up. CONCLUSIONS: Though imaging lacks specificity, it is a valuable asset in assessing the burden of disease and characterizing lesions while histological examination with immunohistochemistry is helpful for the diagnosis of LMS. Complete surgical resection of all metastatic sites where-ever feasible should be strongly considered in a treated case of broad ligament LMS with a durable disease-free interval.

MiR-200b in heme oxygenase-1-modified bone marrow mesenchymal stem cell-derived exosomes alleviates inflammatory injury of intestinal epithelial cells by targeting high mobility group box 3.

Heme Oxygen-1 (HO-1)-modified bone marrow mesenchymal stem cells (BMMSCs) are effective to protect and repair transplanted small bowel and intestinal epithelial cells (IECs); however, the mechanism and the role of HO-1/BMMSCs-derived exosomes is unclear. In the present study, we aimed to verify that exosomes from a HO-1/BMMSCs and IEC-6 cells (IEC-6s) co-culture system could reduce the apoptosis of IEC-6s and decrease the expression of the tight junction protein, zona occludens 1, in the inflammatory environment. Using mass spectrometry, we revealed that high mobility group box 3 (HMGB3) and phosphorylated c-Jun NH2-terminal kinase (JNK), under the influence of differentially abundant proteins identified through proteomic analysis, play critical roles in the mechanism. Further studies indicated that microRNA miR-200b, which was upregulated in exosomes derived from the co-culture of HO-1/BMMSCs and IEC-6s, exerted its role by targeting the 3' untranslated region of Hmgb3 in this biological process. Functional experiments confirmed that miR-200b overexpression could reduce the inflammatory injury of IEC-6s, while intracellular miR-200b knockdown could significantly block the protective effect of HO-1/BMMSCs exosomes on the inflammatory injury of IEC-6s. In addition, the level of miR-200b in cells and exosomes derived from HO-1/BMMSCs stimulated by tumor necrosis factor alpha was significantly upregulated. In a rat small bowel transplantation model of allograft rejection treated with HO-1/BMMSCs, we confirmed that the level of miR-200b in the transplanted small bowel tissue was increased significantly, while the level of HMGB3/JNK was downregulated significantly. In conclusion, we identified that exosomes derived from HO-1/BMMSCs play an important role in alleviating the inflammatory injury of IECs. The mechanism is related to miR-200b targeting the abnormally increased expression of the Hmgb3 gene in IECs induced by inflammatory injury. The reduced level of HMGB3 then decreases the inflammatory injury.

Heme oxygenase-1-modified bone marrow mesenchymal stem cells combined with normothermic machine perfusion to protect donation after circulatory death liver grafts.

BACKGROUND: Donation after circulatory death (DCD) liver grafts have a poor prognosis after transplantation. We investigated whether the outcome of DCD donor organs can be improved by heme oxygenase 1 (HO-1)-modified bone marrow-derived mesenchymal stem cells (BMMSCs) combined with normothermic machine perfusion (NMP), and explored its underlying mechanisms. METHODS: BMMSCs were isolated, cultured, and transduced with the HO-1 gene. An NMP system was established. DCD rat livers were obtained, preserved by different methods, and the recipients were divided into 5 groups: sham operation, static cold storage (SCS), NMP, BMMSCs combined with NMP, and HO-1/BMMSCs combined with NMP (HBP) groups. Rats were sacrificed at 1, 7, and 14 days after surgery; their blood and liver tissue samples were collected; and liver enzyme and cytokine levels, liver histology, high-mobility group box 1 (HMGB1) levels in monocytes and liver tissues, and expression of Toll-like receptor 4 (TLR4) pathway-related molecules were evaluated. RESULTS: After liver transplantation, the SCS group showed significantly increased transaminase levels, liver tissue damage, and shorter survival time. The HBP group showed lower transaminase levels, intact liver morphology, prolonged survival time, and decreased serum and liver proinflammatory cytokine levels. In the NMP and SCS groups, HMGB1 expression in the serum, monocytes, and liver tissues and TLR4 pathway-related molecule expression were significantly decreased. CONCLUSIONS: HO-1/BMMSCs combined with NMP exerted protective effects on DCD donor liver and significantly improved recipient prognosis. The effect of HO-1/BMMSCs was greater than that of BMMSCs and was mediated via HMGB1 expression and TLR4 pathway inhibition.