Oxidative damage-induced hyperactive ribosome biogenesis participates in tumorigenesis of offspring by cross-interacting with the Wnt and TGF-β1 pathways in IVF embryos

In vitro fertilization (IVF) increases the risk of tumorigenesis in offspring. The increased oxidative damage during IVF may be involved in tumor formation. However, the molecular mechanisms underlying this phenomenon remain largely unclear. Using a well-established model of oxidatively damaged IVF mouse embryos, we applied the iTRAQ method to identify proteins differentially expressed between control and oxidatively damaged zygotes and explored the possible tumorigenic mechanisms, especially with regard to the effects of oxidative damage on ribosome biogenesis closely related to tumorigenesis. The iTRAQ results revealed that ribosomal proteins were upregulated by oxidative stress through the Nucleolin/β-Catenin/n-Myc pathway, which stimulated ribosomes to synthesize an abundance of repair proteins to correct the damaged DNA/chromosomes in IVF-derived embryos. However, the increased percentages of γH2AX-positive cells and apoptotic cells in the blastocyst suggested that DNA repair was insufficient, resulting in aberrant ribosome biogenesis. Overexpression of ribosomal proteins, particularly Rpl15, which gradually increased from the 1-cell to 8-cell stages, indicated persistent hyperactivation of ribosome biogenesis, which promoted tumorigenesis in offspring derived from oxidatively damaged IVF embryos by selectively enhancing the translation of β-Catenin and TGF-β1. The antioxidant epigallocatechin-3-gallate (EGCG) was added to the in vitro culture medium to protect embryos from oxidative damage, and the expression of ribosome-/tumor-related proteins returned to normal after EGCG treatment. This study suggests that regulation of ribosome biogenesis by EGCG may be a means of preventing tumor formation in human IVF-derived offspring, providing a scientific basis for optimizing in vitro culture conditions and improving human-assisted reproductive technology.

Preparation of EGCG decorated, injectable extracellular vesicles for cartilage repair in rat arthritis

Arthritis is a kind of chronic inflammatory autoimmune disease, which can destroy joint cartilage and bone, leading to joint pain, joint swelling, and limited mobility. Traditional therapies have many side effects or focus too much on anti-inflammation while neglecting joint repair. In this experiment, we combined EGCG (Epigallocatechin gallate) with extracellular vesicles derived from macrophages to treat rheumatoid arthritis. Sustained-release resulted in a significant decrease in chondrocyte expression of HIF-1α, a decrease in apoptosis-related proteins Cytochrome C, Caspase-3, Caspase-9, and Bax. Molecular biological analysis showed that extracellular vesicles-encapsulated EGCG (EVs-EGCG) more significantly upregulated type II collagen expression by about 1.8-fold than EGCG alone, which was more beneficial for arthritis repair. Animal experiments revealed that these EGCG-coated extracellular vesicles significantly reduced swelling, decreased synovial hyperplasia, repaired cartilage, and attenuated arthritis-related pathology scores in arthritic rats. Measurement data showed that EVs-EGCG treatment reduced joint swelling by approximately 39.5% in rheumatoid rats. In vitro studies have shown that this EVs-EGCG can increase the expression of cartilage type II collagen and reduce apoptosis of chondrocytes. Moreover, it was demonstrated in vivo experiments to reduce cartilage destruction in rheumatoid arthritis rats, providing a solution for the treatment of rheumatoid arthritis.

Epigallocatechin-3-Gallate Suppresses BMP-6-Mediated SMAD1/5/8 Transactivation of Hepcidin Gene by Inducing SMILE in Hepatocytes

Hepcidin, a major regulator of systemic iron homeostasis, is mainly induced in hepatocytes by activating bone morphogenetic protein 6 (BMP-6) signaling in response to changes in the iron status. Small heterodimer partner-interacting leucine zipper protein (SMILE), a polyphenol-inducible transcriptional co-repressor, regulates hepatic gluconeogenesis and lipogenesis. Here, we examine the epigallocatechin-3-gallate (EGCG) effect on BMP-6-mediated SMAD1/5/8 transactivation of the hepcidin gene. EGCG treatment significantly decreased BMP-6-induced hepcidin gene expression and secretion in hepatocytes, which, in turn, abated ferroportin degradation. SMILE overexpression significantly decreased BMP receptor-induced hepcidin promoter activity. SMILE overexpression also significantly suppressed BMP-6-mediated induction of hepcidin mRNA and its secretion in HepG2 and AML12 cells. EGCG treatment inhibited BMP-6-mediated hepcidin gene expression and secretion, which were significantly reversed by SMILE knockdown in hepatocytes. Interestingly, SMILE physically interacted with SMAD1 in the nucleus and significantly blocked DNA binding of the SMAD complex to the BMP-response element on the hepcidin gene promoter. Taken together, these findings suggest that SMILE is a novel transcriptional repressor of BMP-6-mediated hepcidin gene expression, thus contributing to the control of iron homeostasis.

Epigallocatechin Gallate (EGCG) Promotes the Immune Function of Ileum in High Fat Diet Fed Mice by Regulating Gut Microbiome Profiling and Immunoglobulin Production

This study aimed to investigate the regulatory effect of epigallocatechin gallate (EGCG) on the composition of the gut microbiome, the transcriptomic profiling of ileum, and their interplay in high fat diet (HFD) induced obese mice. Intragastric administration of EGCG to C57BL/6J mice for 14 consecutive weeks remarkably decreased HFD induced excessive fat deposition (p < 0.001), and the increment of serum TG, TC, HDL-C (p < 0.05), as well as improved glucose tolerance (p < 0.001). EGCG shifted the gut microbiota mainly by elevating the relative abundance of Parasutterrlla, Bacteroides, and Akkermansia (p < 0.01), decreasing that of norank_f_Erysipelotrichaceae, unclassified_f_Ruminococcaceae, Anaerotruncus, Roseburia, norank_Lachnospiraceae, and Lachnospiraceae_UCG_006 (p < 0.01) at the genus level. In addition, EGCG affected the transcriptomic profiling of ileum, and the differentially expressed (DE) genes after HFD or/and EGCG treatment were mostly enriched in the immune reaction of ileum, such as the GO term of “immune effector process” and “phagocytosis, recognition.” Furthermore, the KEGG category of “immune diseases,” “immune system,” and “infection diseases: bacterial” were commonly enriched by the DE genes of the two treatments. Among those DE genes, 16 immunoglobulins heavy chain variable region encoded genes (Ighvs) and other immunity-related genes, such as complement component 2 (C2), interferon-induced transmembrane protein 1 (Iftm1), polymeric immunoglobulin receptor (pigR), and alanyl aminopeptidase (Anpep), were highly correlated with the shifted microbes in the gut (p < 0.05, absolute r > 0.5). Overall, the results suggested that EGCG ameliorated the HFD induced metabolic disorder mainly by regulating gut microbiome profiling and the immunoglobulin production of ileum, while the genes expressed in the ileum, especially Ighvs, C2, Iftm1, pigR, and Anpep, might play important roles in coordinating the immunity of mice regarding the gut microbes and the host interactions.

Cerebral Cortex Apoptosis in Early Aged Hypertension: Effects of Epigallocatechin-3-Gallate

This study aimed to investigate cerebral cortex apoptosis on the early aged hypertension and the effects of green tea flavonoid epigallocatechin-3-gallate (EGCG). Twenty-four rats were divided into three groups: a control Wistar-Kyoto group (WKY, n = 8), a spontaneously early aged hypertensive group (SHR, n = 8), and an early aged hypertension with EGCG treatment group (SHR-EGCG, n = 8; daily oral EGCG 200 mg/kg—94%, 12 weeks). At 48 weeks old, blood pressures (BPs) were evaluated and cerebral cortexes were isolated for TUNEL assay and Western blotting. Systolic, diastolic, and mean blood pressure levels in the SHR-EGCG were reduced compared to the SHR. The percentage of neural cell deaths, the levels of cytosolic Endonuclease G, cytosolic AIF (Caspase-independent apoptotic pathway), Fas, Fas Ligand, FADD, Caspase-8 (Fas-mediated apoptotic pathway), t-Bid, Bax/Bcl-2, Bak/Bcl-xL, cytosolic Cytochrome C, Apaf-1, Caspase-9 (Mitochondrial-mediated apoptotic pathway), and Caspase-3 (Fas-mediated and Mitochondria-mediated apoptotic pathways) were increased in the SHR relative to WKY and reduced in SHR-EGCG relative to SHR. In contrast, the levels of Bcl-2, Bcl-xL, p-Bad, 14-3-3, Bcl-2/Bax, Bcl-xL/Bak, and p-Bad/Bad (Bcl-2 family-related pro-survival pathway), as well as Sirt1, p-PI3K/PI3K and p-AKT/AKT (Sirt1/PI3K/AKT-related pro-survival pathway), were reduced in SHR relative WKY and enhanced in SHR-EGCG relative to SHR. In conclusion, green tea flavonoid epigallocatechin-3-gallate (EGCG) might prevent neural apoptotic pathways and activate neural survival pathways, providing therapeutic effects on early aged hypertension-induced neural apoptosis.

The green tea catechin EGCG provides proof-of-concept for a pan-coronavirus entry inhibitor

The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has emphasized the serious threat to human health posed by emerging coronaviruses. Effective antiviral countermeasures are urgently needed as vaccine development against an emerging coronavirus takes time, even in the best-case scenario. The green tea catechin, epigallocatechin gallate (EGCG), has broad spectrum antiviral activity. We demonstrate here that EGCG prevents murine and human coronavirus infection and blocks the entry of lentiviral particles pseudotyped with spike proteins from bat or highly pathogenic coronaviruses. We show that EGCG treatment reduces coronavirus attachment to target cell surfaces. Our results demonstrate the potential for the development of pan-coronavirus attachment inhibitors.

EGCG promotes PRKCA expression to alleviate LPS-induced acute lung injury and inflammatory response

Acute lung injury (ALI), which could be induced by multiple factors such as lipopolysaccharide (LPS), refer to clinical symptoms of acute respiratory failure, commonly with high morbidity and mortality. Reportedly, active ingredients from green tea have anti-inflammatory and anticancer properties, including epigallocatechin-3-gallate (EGCG). In the present study, protein kinase C alpha (PRKCA) is involved in EGCG protection against LPS-induced inflammation and ALI. EGCG treatment attenuated LPS-stimulated ALI in mice as manifested as improved lung injury scores, decreased total cell amounts, neutrophil amounts and macrophage amounts, inhibited the activity of MPO, decreased wet-to-dry weight ratio of lung tissues, and inhibited release of inflammatory cytokines TNF-α, IL-1β, and IL-6. PRKCA mRNA and protein expression showed to be dramatically decreased by LPS treatment while reversed by EGCG treatment. Within LPS-stimulated ALI mice, PRKCA silencing further aggravated, while PRKCA overexpression attenuated LPS-stimulated inflammation and ALI through MAPK signaling pathway. PRKCA silencing attenuated EGCG protection. Within LPS-induced RAW 264.7 macrophages, EGCG could induce PRKCA expression. Single EGCG treatment or Lv-PRKCA infection attenuated LPS-induced increases in inflammatory factors; PRKCA silencing could reverse the suppressive effects of EGCG upon LPS-stimulated inflammatory factor release. In conclusion, EGCG pretreatment inhibits LPS-induced ALI in mice. The protective mechanism might be associated with the inhibitory effects of PRKCA on proinflammatory cytokine release via macrophages and MAPK signaling pathway.

Epigallocatechin-3-gallate inhibits the growth of three-dimensional in vitro models of neuroblastoma cell SH-SY5Y

The aim of the study is to investigate the potential of using three-dimensional (3D) in vitro neuroblastoma models to mimic the neuroblastoma microenvironment by testing a potential therapeutic compound—the natural extract epigallocatechin-3-gallate (EGCG), and to further elucidate the roles of DYRK1A in the growth and differentiation of neuroblastoma tissue. In vitro models based on a classic neuroblastoma cell line SH-SY5Y were employed, including 3D models with extracellular matrix and co-cultured with vascular endothelial cells. Cell viability was tested using AlamarBlue and Resazurin assay. The growth and differentiation of in vitro models of SH-SY5Y were analysed based on microscopy images obtained from immunofluorescence or real-time imaging. Protein expression level was investigated using immunoblotting analysis. The two-dimensional (2D) in vitro model implies the cytotoxicity and DYRK1A inhibition effect of EGCG and shows the induction of neuronal differentiation marker TuJ1. 3D in vitro models suggest that EGCG treatment compromised the growth of SH-SY5Y multicellular 3D spheroids and the viability of SH-SY5Y cultured in 3D Matrigel matrix. In addition, co-culture of SH-SY5Y with human vascular umbilical vein endothelial cells implied the inhibitory effects by EGCG in a vascularised microenvironment. In this study, novel 3D in vitro models of neuroblastoma were established in the application of testing a potential anti-cancer candidate compound EGCG. In pursuit of the goals of the 3Rs (replacement, reduction and refinement), the usage of these 3D in vitro models has the potential to reduce and eventually replace current animal models used in neuroblastoma research. The DYRK1A inhibiting nature of EGCG, together with the facts that EGCG inhibits the growth and induces the differentiation of neuroblastoma in vitro models, suggests an oncogene role of DRYK1A.

The Major Constituent of Green Tea, Epigallocatechin-3-Gallate (EGCG), Inhibits the Growth of HPV18-Infected Keratinocytes by Stimulating Proteasomal Turnover of the E6 and E7 Oncoproteins

Epigallocatechin-3-gallate (EGCG), the primary bioactive polyphenol in green tea, has been shown to inhibit the growth of human papilloma virus (HPV)-transformed keratinocytes. Here, we set out to examine the consequences of EGCG treatment on the growth of HPV18-immortalised foreskin keratinocytes (HFK-HPV18) and an authentic HPV18-positive vulvar intraepithelial neoplasia (VIN) clone, focusing on its ability to influence cell proliferation and differentiation and to impact on viral oncogene expression and virus replication. EGCG treatment was associated with degradation of the E6 and E7 oncoproteins and an upregulation of their associated tumour suppressor genes; consequently, keratinocyte proliferation was inhibited in both monolayer and organotypic raft culture. While EGCG exerted a profound effect on cell proliferation, it had little impact on keratinocyte differentiation. Expression of the late viral protein E4 was suppressed in the presence of EGCG, suggesting that EGCG was able to block productive viral replication in differentiating keratinocytes. Although EGCG did not alter the levels of E6 and E7 mRNA, it enhanced the turnover of the E6 and E7 proteins. The addition of MG132, a proteasome inhibitor, to EGCG-treated keratinocytes led to the accumulation of the E6/E7 proteins, showing that EGCG acts as an anti-viral, targeting the E6 and E7 proteins for proteasome-mediated degradation.

Epigallocatechin Gallate in Relapsing-Remitting Multiple Sclerosis

ObjectiveTo assess the safety and efficacy of epigallocatechin-3-gallate (EGCG) add-on to glatiramer acetate (GA) in patients with relapsing-remitting multiple sclerosis (RRMS).MethodsWe enrolled patients with RRMS (aged 18–60 years, Expanded Disability Status Scale [EDSS] score 0–6.5), receiving stable GA treatment in a multicenter, prospective, double-blind, phase II, randomized controlled trial. Participants received up to 800 mg oral EGCG daily over a period of 18 months. The primary outcome was the proportion of patients without new hyperintense lesions on T2-weighted (T2w) brain MRI within 18 months. Secondary end points included additional MRI and clinical parameters. Immunologic effects of EGCG were investigated in exploratory experiments.ResultsA total of 122 patients on GA were randomly assigned to EGCG treatment (n = 62) or placebo (n = 60). We could not demonstrate a difference between groups after 18 months for the primary outcome or other radiologic (T2w lesion volume, T1w hypointense lesion number or volume, number of cumulative contrast-enhancing lesions, percent brain volume change), or clinical (EDSS, MS functional composite, and annualized relapse rate) parameter. EGCG treatment did not affect immune response to GA. Pharmacologic analysis revealed wide ranging EGCG plasma levels. The treatment was well tolerated with a similar incidence of mostly mild adverse events similar in both groups.ConclusionIn RRMS, oral EGCG add-on to GA was not superior to placebo in influencing MRI and clinical disease activity over 18 months. The treatment was safe at a daily dosage up to 800 mg EGCG. It did not influence immune parameters, despite indication of EGCG being bioavailable in patients.Classification of EvidenceThis study provides Class II evidence that for patients with RRMS, EGCG added to GA did not significantly affect the development of new hyperintense lesions on T2-weighted brain MRI.Trial Registration InformationClinical trial registration number: NCT00525668.