Mechanisms of ferroptosis in nonalcoholic fatty liver disease and therapeutic effects of traditional Chinese medicine: a review.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive accumulation of fat in hepatocytes (nonalcoholic fatty liver (NAFL)), and lobular inflammation and hepatocyte damage (which characterize nonalcoholic steatohepatitis (NASH) are found in most patients). A subset of patients will gradually develop liver fibrosis, cirrhosis, and eventually hepatocellular carcinoma, which is a deadly disease that threatens human life worldwide. Ferroptosis, a novel nonapoptotic form of programmed cell death (PCD) characterized by iron-dependent accumulation of reactive oxygen radicals and lipid peroxides, is closely related to NAFLD. Traditional Chinese medicine (TCM) has unique advantages in the prevention and treatment of NAFLD due to its multicomponent, multipathway and multitarget characteristics. In this review, we discuss the effect of TCM on NAFLD by regulating ferroptosis, in order to provide reference for the further development and application of therapeutic drugs to treat NAFLD.

Pluripotent Stem Cell-Derived Hepatocyte-like Cells: Induction Methods and Applications.

The development of regenerative medicine provides new options for the treatment of end-stage liver diseases. Stem cells, such as bone marrow mesenchymal stem cells, embryonic stem cells, and induced pluripotent stem cells (iPSCs), are effective tools for tissue repair in regenerative medicine. iPSCs are an appropriate source of hepatocytes for the treatment of liver disease due to their unlimited multiplication capacity, their coverage of the entire range of genetics required to simulate human disease, and their evasion of ethical implications. iPSCs have the ability to gradually produce hepatocyte-like cells (HLCs) with homologous phenotypes and physiological functions. However, how to induce iPSCs to differentiate into HLCs efficiently and accurately is still a hot topic. This review describes the existing approaches for inducing the differentiation of iPSCs into HLCs, as well as some challenges faced, and summarizes various parameters for determining the quality and functionality of HLCs. Furthermore, the application of iPSCs for in vitro hepatoprotective drug screening and modeling of liver disease is discussed. In conclusion, iPSCs will be a dependable source of cells for stem-cell therapy to treat end-stage liver disease and are anticipated to facilitate individualized treatment for liver disease in the future.

HPF Modulates the Differentiation of BMSCs into HLCs and Promotes the Recovery of Acute Liver Injury in Mice.

Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into hepatocyte-like cells (HLCs) to alleviate acute liver injury (ALI). Herpetfluorenone (HPF), as an active ingredient in the dried, mature seeds Herpetospermum caudigerum Wall, used in Tibetan medicine, has been proven to effectively alleviate ALI. Therefore, the purpose of this study was to determine whether HPF can promote the differentiation of BMSCs into HLCs and promote ALI recovery. Mouse BMSCs were isolated, and the BMSCs' differentiation into HLCs was induced by HPF and hepatocyte growth factor (HGF). Under the induction of HPF and HGF, the expression of hepatocellular specific markers and the accumulation of glycogen and lipids in the BMSCs increased, indicating that BMSCs successfully differentiated into HLCs. Then, the ALI mouse model was established, using carbon tetrachloride, followed by an intravenous injection of BMSCs. Then, only HPF was injected intraperitoneally, in order to verify the effect of HPF in vivo. In vivo imaging was used to detect the homing ability of HPF-BMSCs, and it was detected that HPF-BMSCs significantly increased the levels of serum AST, ALT and ALP in the liver of ALI mice, and alleviated liver cell necrosis, oxidative stress and liver pathology. In conclusion, HPF can promote the differentiation of BMSCs into HLCs and promote the recovery of ALI in mice.

Integrating network pharmacology and pharmacological evaluation to explore the protective mechanism of Ershiwuwei Zhenzhu pill in ischemic stroke.

ETHNOPHARMACOLOGICAL RELEVANCE: Ershiwuwei Zhenzhu Pill (EZP), a representative and classic formula in Tibetan medicine, is commonly used in the treatment of various cerebrovascular diseases, including ischemic stroke (IS). Nevertheless, their efficacy and potential mechanism in treating IS have yet to be investigated. AIM OF THE STUDY: This study aimed to investigate the potential mechanisms of EZP in the treatment of IS based on network pharmacology and experimental verification. MATERIALS AND METHODS: The chemical profile of EZP was characterized using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The targets related to the compounds in EZP were predicted by the Swiss Target Prediction and Target Net platform, and targets of IS were collected from the Gene Cards and OMIM databases. Subsequently, a protein-protein interaction (PPI) network of targets was constructed and analyzed by the STRING database and Cytoscape software, version 3.7.1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed, and an ingredient-target-pathway network was constructed. Ultimately, the middle cerebral artery occlusion (MCAO) model was established to evaluate the anti-IS effects of EZP by detecting the neurological deficit score, HE, Nissl and TCC staining, and inflammatory factors, and the expression of key protein targets was detected by western blotting. RESULTS: A total of 129 components were identified in EZP. Network pharmacology revealed 3136 compound targets and 2826 disease-related targets, and 412 overlapping proteins were obtained as potential therapeutic targets. The PPI network results showed that 6 key targets (AKT1, SRC, VEGFA, TP53, TNF and EGFR) were core targets of EZP in the treatment of IS. Western blotting demonstrated that the expression levels of AKT1, VEGFA, TP53, SRC, TNF and EGFR in the brain tissue of MCAO rats were significantly changed after treatment with EZP compared to the model group. CONCLUSIONS: EZP ameliorated IS in MCAO rats. The underlying mechanism might be associated with inhibiting inflammation and apoptosis, promoting angiogenesis and protecting neurons by regulating multiple targets and pathways.

Mesenchymal Stem Cell-Derived Exosomes: A Promising Therapeutic Agent for the Treatment of Liver Diseases.

Liver disease has become a major global health and economic burden due to its broad spectrum of diseases, multiple causes and difficult treatment. Most liver diseases progress to end-stage liver disease, which has a large amount of matrix deposition that makes it difficult for the liver and hepatocytes to regenerate. Liver transplantation is the only treatment for end-stage liver disease, but the shortage of suitable organs, expensive treatment costs and surgical complications greatly reduce patient survival rates. Therefore, there is an urgent need for an effective treatment modality. Cell-free therapy has become a research hotspot in the field of regenerative medicine. Mesenchymal stem cell (MSC)-derived exosomes have regulatory properties and transport functional "cargo" through physiological barriers to target cells to exert communication and regulatory activities. These exosomes also have little tumorigenic risk. MSC-derived exosomes promote hepatocyte proliferation and repair damaged liver tissue by participating in intercellular communication and regulating signal transduction, which supports their promise as a new strategy for the treatment of liver diseases. This paper reviews the physiological functions of exosomes and highlights the physiological changes and alterations in signaling pathways related to MSC-derived exosomes for the treatment of liver diseases in some relevant clinical studies. We also summarize the advantages of exosomes as drug delivery vehicles and discuss the challenges of exosome treatment of liver diseases in the future.

[Ershiwuwei Shanhu Pills regulate Akt/mTOR/GSK-3ß signaling pathway to alleviate Alzheimer's disease mice].

The present study investigated the mechanism of the Tibetan patent medicine Ershiwuwei Shanhu Pills(ESP) in alleviating Alzheimer's disease in mice via Akt/mTOR/GSK-3ß signaling pathway. BALB/c mice were randomly assigned into a blank control group, a model group, low(200 mg·kg~(-1)), medium(400 mg·kg~(-1)) and high(800 mg·kg~(-1)) dose groups of ESP, and donepezil hydrochloride group. Except the blank control group, the other groups were given 20 mg·kg~(-1) aluminum chloride by gavage and 120 mg·kg~(-1) D-galactose by intraperitoneal injection for 56 days to establish Alzheimer's disease model. Morris water maze was used to detect the learning and memory ability of mice. The level of p-tau protein in mouse hippocampus and the levels of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and total antioxidant capacity(T-AOC) in hippocampus and serum were detected. Hematoxylin-eosin staining and Nissl staining were performed for the pathological observation of whole brain in mice. TdT-mediated dUTP nick-end labeling(TUNEL) staining was employed for the observation of apoptosis in mouse cortex. Western blot was adopted to detect the protein levels of p-mTOR, p-Akt, and GSK-3ß in the hippocampus. Compared with the model group, the ESP groups showcased alleviated pathological damage of the whole brain, decreased TUNEL positive cells, reduced level of p-tau protein in hippocampus, and risen SOD, CAT, and T-AOC levels and declined MDA level in hippocampus and serum. Furthermore, the ESP groups had up-regulated protein levels of p-mTOR and p-Akt while down-regulated protein level of GSK-3ß in hippocampus. Therefore, ESP can alleviate the learning and memory decline and oxidative damage in mice with Alzheimer's disease induced by D-galactose combined with aluminum chloride, which may be related to Akt/mTOR/GSK-3ß signaling pathway.

miR­200b upregulation promotes migration of BEAS­2B cells following long­term exposure to cigarette smoke by targeting ETS1.

Cigarette smoking is the leading cause of all histological types of lung cancer, and the role that microRNAs (miRNAs) serve in its pathogenesis is being increasingly recognized. The aim of the present study was to investigate the role of miR­200b on migration in cigarette smoke­induced malignant transformed cells. In the present study, miR­200b expression was found to be increased in cigarette smoke (CS)­exposed BEAS­2B cells, lung cancer cell lines and tumor tissue samples. Using wound healing and Transwell migration assays, the migratory ability was shown to be increased in miR­200b­overexpressing cells, whereas miR­200b knockdown resulted in reduced migration. Additionally, the expression of E­Cadherin was downregulated, whereas that of N­Cadherin was upregulated in miR­200b mimic­transfected cells, suggesting an increase in epithelial­mesenchymal transition. Downstream, using four target gene prediction tools, six target genes of miR­200b were predicted, amongst which, ETS proto­oncogene 1 transcription factor (ETS1) was shown to be significantly associated with tumor invasion depth and negatively associated with miR­200b expression. The interaction between miR­200b and ETS1 was confirmed using a dual­luciferase reporter assay. Using rescue experiments, the increased migratory ability of the miR­200b­overexpressing cells was reversed by ETS1 overexpression. In summary, this study showed that miR­200b overexpression serves a carcinogenic role and promotes the migration of BEAS­2B cells following long­term exposure to CS by targeting ETS1.

Role of DNA methylation regulation of miR-130b expression in human lung cancer using bioinformatics analysis.

MicroRNAs (miRNAs) are involved in various crucial biological processes including regulation of cell differentiation, proliferation, and migration, and are closely associated with tumor development. This study aimed to investigate miR-130b expression levels in lung cancer patient tissues. Two Gene Expression Omnibus (GEO) databases, including GSE48414 and GSE74190, and two The Cancer Genome Atlas (TCGA) databases including TCGA LUAD and TCGA LUSC, were accessed to obtain information for differential expression analysis and clinical-pathological correlation analysis. The results showed that miR-130b expression levels were significantly increased in lung cancer compared to normal tissues. Data also demonstrated that confounding factors such as tumor clinical stages and tumor invasion depth markedly affected miR-130b expression levels in cancer patients. A total of 169 target genes modified by miR-130b expression were identified by using 4 online websites for target gene prediction. Further enrichment analysis indicated that these 169 target genes were significantly enriched in several cancer-related biological processes and signaling pathways, including wound healing, cell proliferation, Wnt signaling, Ras signaling, and mTOR signaling. It was also of interest to examine the seven sites on the promoter region of miR-130b encoding gene in lung cancer patients and then compare methylation at these loci with miR-130b expression. The correlation analysis between encoding gene methylation and miR-130b expression in TCGA datasets revealed that decreased methylation in the promoter region was significantly associated with elevated miR-130b expression. This phenomenon was markedly dependent upon smoking history and clinical-pathological features. In conclusion, data indicated alterations in the methylation of DNA promoter region of miR-130b encoding gene were associated with disturbances in miR-130b expression in lung cancer patients suggesting that the DNA methylation process and miR-130b expression may serve as biomarkers for detection of lung cancer.

Multi-platform analysis of methylation-regulated genes in human lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most frequent pathological type of lung cancer that has a poor prognosis and high mortality rate. DNA methylation plays a critical role in various biological processes during development, while dysregulation results in pathological consequences. Thus, this study aimed to identify DNA methylation-regulated genes involved in LUAD occurrence. Initially, 300 downregulated and 168 upregulated mRNA expression levels were identified in two databases: Gene Expression Omnibus (GEO) and The Cancer Genome Atlas. In addition, GEO was utilized to detect 243 DNA hyper-methylated sites. Based on our observations, it was possible to correlate downregulation of mRNA expression and DNA hyper-methylation of six genes (ABCA3, COX7A1, HOXA5, SLIT3, SOX17, and SPARCL1). Functional analysis of the six genes indicated that these genes are predominantly enriched in cancer-related pathways and may promote carcinogenesis by regulating epithelialmesenchymal transition processes. In conclusion, our study identified a panel of DNA methylation-regulated genes involved in LUAD and may serve as potential epigenetic markers for this type of carcinoma.

1,25-Dihydroxy Vitamin D3 Attenuates the Oxidative Stress-Mediated Inflammation Induced by PM2.5via the p38/NF-κB/NLRP3 Pathway.

Vitamin D3 is reported to be involved in the regulation of inflammatory processes. In this study, biomarkers related to oxidative stress and inflammation were investigated to clarify the protective effects and possible mechanism of 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3) on PM2.5-induced inflammatory response. In the in vitro study using human bronchial epithelial (HBE) cells, aqueous extracts of PM2.5 could induce oxidative damage which is characterized by significant increases in production of reactive oxygen species, malonaldehyde concentration, and protein expression of HSPA1A and HO-1. Meanwhile, PM2.5 caused secretion of inflammatory factors (IL-6, IL-8) in the culture medium as well as phosphorylation of p38, nuclear factor-kappa B (NF-κB) inhibitor alpha (IκBα), and NF-κB p65 proteins. Increases in NLRP3 expression was also observed in HBE cells after PM2.5 exposure. However, all these biomarkers were remarkably attenuated by a 24-h pretreatment of 1 nM 1,25-(OH)2D3. Furthermore, 1,25-(OH)2D3 also reduced transcriptional activation of NF-κB induced by PM2.5 as indicated by a significant decrease in luciferase activity in HBE cells stably transfected with the NF-κB response element (RE)-driven luciferase reporter. Taken together, our findings provided novel experimental evidences supporting that vitamin D3 could reduce the predominantly oxidative stress-mediated inflammation induced by PM2.5via the p38/NF-κB/NLRP3 signaling pathway.

Cytotoxicity and genotoxicity of nanosilver in stable GADD45α promoter-driven luciferase reporter HepG2 and A549 cells.

OBJECTIVES: The intense commercial application of silver nanoparticles (AgNPs) has been raising concerns about their potential adverse health effects to human. This study aimed to explore the potency of AgNPs to induce GADD45α gene, an important stress sensor, and its relationships with the cytotoxicity and genotoxicity elicited by AgNPs. METHODS: Two established HepG2 and A549 cell lines containing the GADD45α promoter-driven luciferase reporter were treated with increasing concentrations of AgNPs for 48 hours. After the treatment, transcriptional activation of GADD45α indicated by luciferase activity, cell viability, cell cycle arrest, and levels of genotoxicity were determined. The uptake and intracellular localization of AgNPs, cellular Ag doses as well as Ag+ release were also detected. RESULTS: AgNPs could activate GADD45α gene at the transcriptional level as demonstrated by the dose-dependent increases in luciferase activity in both the reporter cells. The relative luciferase activity was greater than 12× the control level in HepG2-luciferase cells at the highest concentration tested where the cell viability decreased to 17.0% of the control. These results was generally in accordance with the positive responses in cytotoxicity, cell cycle arrest of Sub G1 and G2/M phase, Olive tail moment, micronuclei frequency, and the cellular Ag content. CONCLUSIONS: The cytotoxicity and genotoxicity of AgNPs seems to occur mainly via particles uptake and the subsequent liberation of ions inside the cells. And furthermore, the GADD45α promoter-driven luciferase reporter cells, especially the HepG2-luciferase cells, could provide a new and valuable tool for predicting nanomaterials genotoxicity in humans.

Everolimus (RAD001) inhibits the proliferation of rat vascular smooth muscle cells by up-regulating the activity of the p27/kip1 gene promoter.

OBJECTIVE: We investigated whether the inhibitory effect of the immunosuppressant everolimus (RAD001) on vascular smooth muscle cell (VSMC) proliferation is mediated by p27/kip1 gene promoter activity. METHODS: In this experimental study, cultured rat VSMCs were transiently transfected with a recombinant plasmid (pXp27) containing p27/kip1 gene promoter sequence and a chloramphenicol acetyltransferase (CAT) reporter gene. After stimulation with the mitogen platelet-derived growth factor (PDGF-BB, 10 ng/mL) in the presence or absence of RAD001 (10 nM), the promoter activity, mRNA expression, and protein expression of p27/kip1 were examined by CAT assay, RT-PCR, and immunoblotting, respectively. Cell cycle-related changes were detected by flow cytometry. DNA synthesis was determined using 3H-TdR incorporation. RESULTS: Compared with the non-stimulation group, PDGF-BB stimulation induced a significant proliferative response in the VSMCs as indicated by decreased p27/kip1 gene promoter activity, decreased p27/kip1 mRNA and protein expression, increased S-phase and G2/M-phase cells, and increased DNA synthesis. RAD001 intervention increased p27/kip1 gene promoter activity 3.5-fold, promoted p27/kip1 mRNA and protein expression, increased G0-phase cells, reduced DNA synthesis, and, overall, inhibited PDGF-BB-stimulated cell proliferation. CONCLUSION: RAD001 inhibits PDGF-BB-stimulated proliferation of cultured VSMCs by upregulating p27/kip1 gene promoter activity and increasing p27/kip1 mRNA and protein expression.