Autophagy affects hepatic fibrosis progression by regulating macrophage polarization and exosome secretion.

BACKGROUND: In this study, the role of autophagy in hepatic fibrosis and its effects on macrophage polarization and exosomes (EVs) were verified by establishing hepatic fibrosis model and co-culture model, providing evidence for treatment. METHODS: In this study, CCL4 was used to establish hepatic fibrosis model. The morphology and purity of exosomes (EVs) were verified by transmission electron microscopy, western blotting (WB), and nanoparticle tracing analysis (NTA). Real-time quantitative PCR (qRT-PCR), WB and enzyme-linked immunoadsorption (ELISA) were used to detect hepatic fibrosis markers, macrophage polarization markers and liver injury markers. Histopathological assays were used to verify the liver injury morphology in different groups. The cell co-culture model and hepatic fibrosis model were constructed to verify the expression of miR-423-5p. RESULTS: Hepatic fibrosis model showed that CCL4 promoted early autophagy increase but inhibited autophagy flux in liver. mRFP-GFP-LC3 detection showed that both LPS group and Baf group inhibited autophagy flux. This inhibitory effect was reversed by Rap combination therapy. The M1/M2 markers of macrophage polarization were further tested, and it was found that LPS and Baf could promote M1 polarization and inhibit M2 polarization. Rap processing reverses this phenomenon. These data suggest that autophagy can regulate the polarization process of liver macrophages. WB and NTA showed that LPS induced EVs generation. In addition, LPS-induced EVs could promote HSC proliferation, cell cycle, migration, and the expression of fibrosis markers. Macrophage-EVs could affect the fibrosis process of stellate cells through the secretion of miR-423a-5p expression. The hepatic fibrosis model was further established to verify the regulation of autophagy and EVs on the fibrosis process. CONCLUSION: This study was showed that autophagy could regulate fibrosis by promoting HSC activation by regulating macrophage polarization and exosome secretion.

Immunological characteristics of human umbilical cord mesenchymal stem cells after hepatogenic differentiation.

BACKGROUND: Acute liver failure is one of the most intractable clinical problems. The use of bioartificial livers may solve donor shortage problems. Human umbilical cord mesenchymal stem cells (hUCMSCs) are an excellent seed cell choice for artificial livers because they change their characteristics to resemble hepatocyte-like cells (HLCs) following artificial liver transplantation. OBJECTIVE: This study aimed to determine whether the immunological characteristics of hUCMSCs are changed after being transformed into hepatocyte-like cells. METHODS: HUCMSCs were isolated by the adherent method. The following hUCMSC surface markers were detected using flow cytometry: CD45, CD90, CD105, CD34, and octamer-binding transcription factor 4 (OCT-4). Functional detection of adipogenic differentiation was performed. The hUCMSCs were cultured in complete medium (control group) or induction medium (induction group), and flow cytometry was used to detect cell surface markers. Peritoneal lavage fluid was collected after intraperitoneal injection of 1 × 106 cells/mouse over 40 minutes. The leukocyte count, labeled CD45, CD3, CD4 and CD8 antibodies, and flow detection of T lymphocyte subsets were determined using the peritoneal lavage fluid. RESULTS: Using phenotypic and functional identification, hUCMSCs were successfully isolated using a two-step induction method. The surface markers of the hUCMSCs cells changed after HLC induction. In vivo immune results showed that hUCMSCs and HLsC induced leukocyte production. CONCLUSION: Hepatic induction of hUCMSCs changes their cell surface markers. Both HLCs and hUCMSCs cause leukocytosis in vivo, but the immune response induced by HLCs is slightly stronger.

Mechanism of HBV-positive liver cancer cell exosomal miR-142-3p by inducing ferroptosis of M1 macrophages to promote liver cancer progression.

Background: Exosomes are becoming an important mediator of the interaction between tumor cells and the microenvironment. Ferroptosis is a newly discovered type of cell death. However, its role in the progression of liver cancer is largely unknown. The aim of the presents study was to analyze the mechanism by which hepatitis B virus (HBV)-positive liver cancer secretes exosomes to mediate the iron death of M1 macrophages, thereby promoting the development of liver cancer. Methods: Liver cancer tissues and peripheral blood with positive and negative clinical HBV infection were collected, and M-type macrophages, miR-142-3p, and recombinant solute carrier family 3, member 2 (SLC3A2) expressions were detected in the samples. CD80+ M1 macrophages and CD163+ M2 macrophages were isolated from the 2 tissues, and levels of miR-142-3p, SLC3A2, and ferroptosis markers were detected. Exosomes of HBV-positive hepatocellular carcinoma (HCC) cells were isolated and co-cultured with M1 macrophages to observe their effect on the invasion ability of HCC cells. Results: The expression of miR-142-3p significantly increased in the exosomes extracted from the peripheral blood of patients with HBV-positive liver cancer. Genes related to intracellular iron metabolism and homeostasis, such as ferritin heavy chain 1 (FTH1), transferrin receptor 1 (TfR1), recombinant glutathione peroxidase 4 (GPX4), and activating transcription factor 4 (ATF4), had abnormal expression levels in M1 macrophages. HBV-positive HCC exosomes treated with M1-type macrophages had a weakened inhibitory effect on the invasion of HCC cells, but ferroptosis inhibitors could reverse the effect of HBV-positive HCC exosomes treated M1-type macrophages on HCC cells. Knockdown of the expression of miR-142-3p can also weaken the invasive ability of liver cancer cells. Conclusions: The results of the present study confirmed that HBV-positive liver cancer cell exosomal miR-142-3p can promote the progression of liver cancer by inducing iron death of M1-type macrophages.

Exosomal miR-142-3p secreted by hepatitis B virus (HBV)-hepatocellular carcinoma (HCC) cells promotes ferroptosis of M1-type macrophages through SLC3A2 and the mechanism of HCC progression.

Background: Most patients with hepatitis B virus (HBV) infection will develop hepatocellular carcinoma (HCC). This study aimed to explore the potential mechanism of miR-142-3p in HCC caused by HBV infection. Methods: HepG2 cells and M1 macrophages were cocultured and then infected with HBV to establish an in vitro model. MicroRNA (miRNA) and messenger RNA (mRNA) expression was analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The protein expressions of COX2, ACSL4, PTGS2, GPX4, and NOX1 were analyzed by Western blot. Flow cytometry and TUNEL assays were used to assess cell reactive oxygen species (ROS) and ferroptosis, respectively. Cell invasion and migration were measured by Transwell assay. To evaluate the ferroptosis of M1-type macrophages, glutathione (GSH), malondialdehyde (MDA), and Fe2+ content was detected by corresponding kits. Dual luciferase reporter gene detection verified the targeting relationship between miR-142-3p and SLC3A2. Results: MiR-142-3p was highly expressed in HBV-infected HCC patients and HBV-infected M1-type macrophages. Inhibition of miR-142-3p or overexpression of SLC3A2 reversed ferroptosis and inhibited the proliferation, migration, and invasion of HCC cells. Conclusions: Our findings indicated that miR-142-3p promoted HBV-infected M1-type macrophage ferroptosis through SLC3A2, affecting the production of GSH, MDA, and Fe2+ and accelerating the development of HCC. The regulation of miR-142-3p and its target genes will help to clarify the pathogenesis of HCC induced by HBV infection and provide new theoretical foundations and therapeutic targets.