Licorice flavonoid ameliorates ethanol-induced gastric ulcer in rats by suppressing apoptosis via PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is the dry roots and rhizomes of Glycyrrhiza uralensis Fisch., Glycyrrhiza glabra L. and Glycyrrhiza inflata Bat., which was first recorded in Shengnong's herbal classic. Licorice flavonoid (LF) is the main compound isolated from licorice with an indispensable action in treating gastric ulcer (GU). However, the underlying mechanisms need to be further explored. AIM OF THE STUDY: This study aimed to investigate and further elucidate the mechanisms of LF against ethanol-induced GU using an integrated approach. MATERIALS AND METHODS: The anti-GU effects of LF were evaluated in an ethanol-induced gastric injury rat model. Then, the metabolomics approach was applied to explore the specific metabolites and metabolic pathways. Next, the network pharmacology combined with metabolomics strategy was employed to predict the targets and pathways of LF for GU. Finally, these predictions were validated by molecular docking, RT-qPCR, and western blotting. RESULTS: LF had a positive impact on gastric injury and regulated the expression of GU-related factors. Upon serum metabolomics analysis, 25 metabolic biomarkers of LF in GU treatment were identified, which were primarily involved in amino acid metabolism, carbohydrate metabolism, and other related processes. Subsequently, a "components-targets-metabolites" network was constructed, revealing six key targets (HSP90AA1, AKT1, MAPK1, EGFR, ESR1, PIK3CA) that may be associated with GU treatment. More importantly, KEGG analysis highlighted the importance of the PI3K/AKT pathway including key targets, as a critical route through which LF exerted its anti-GU effects. Molecular docking analyses confirmed that the core components of LF exhibited a strong affinity for key targets. Furthermore, RT-qPCR and western blotting results indicated that LF could reverse the expression of these targets, activate the PI3K/AKT pathway, and ultimately reduce apoptosis. CONCLUSION: LF exerted a gastroprotective effect against gastric ulcer induced by ethanol, and the therapeutic mechanism may involve improving metabolism and suppressing apoptosis through the PI3K-AKT pathway.

Comparison of seven CD19 CAR designs in engineering NK cells for enhancing anti-tumour activity.

Chimeric antigen receptor-natural killer (CAR-NK) cell therapy is emerging as a promising cancer treatment, with notable safety and source diversity benefits over CAR-T cells. This study focused on optimizing CAR constructs for NK cells to maximize their therapeutic potential. We designed seven CD19 CAR constructs and expressed them in NK cells using a retroviral system, assessing their tumour-killing efficacy and persistence. Results showed all constructs enhanced tumour-killing and prolonged survival in tumour-bearing mice. In particular, CAR1 (CD8 TMD-CD3ζ SD)-NK cells showed superior efficacy in treating tumour-bearing animals and exhibited enhanced persistence when combined with OX40 co-stimulatory domain. Of note, CAR1-NK cells were most effective at lower effector-to-target ratios, while CAR4 (CD8 TMD-OX40 CD- FcεRIγ SD) compromised NK cell expansion ability. Superior survival rates were noted in mice treated with CAR1-, CAR2 (CD8 TMD- FcεRIγ SD)-, CAR3 (CD8 TMD-OX40 CD- CD3ζ SD)- and CAR4-NK cells over those treated with CAR5 (CD28 TMD- FcεRIγ SD)-, CAR6 (CD8 TMD-4-1BB CD-CD3ζ 1-ITAM SD)- and CAR7 (CD8 TMD-OX40 CD-CD3ζ 1-ITAM SD)-NK cells, with CAR5-NK cells showing the weakest anti-tumour activity. Increased expression of exhaustion markers, especially in CAR7-NK cells, suggests that combining CAR-NK cells with immune checkpoint inhibitors might improve anti-tumour outcomes. These findings provide crucial insights for developing CAR-NK cell products for clinical applications.

Mesothelin CAR-engineered NK cells derived from human embryonic stem cells suppress the progression of human ovarian cancer in animals.

CAR-NK cell therapy does not require HLA matching and has minimal side effects. However, traditional methods of engineering CARs into human tissue-derived NK cells exhibit heterogeneity, low transduction efficiency, and high manufacturing costs. Here, we provide a reliable approach for generating large-scale and cryopreserved mesothelin (MSLN) CAR-NK cells from human embryonic stem cells (hESCs) as an alternative cell source. We first constructed MSLN CAR-expressing hESCs to reduce CAR engineering costs and subsequently differentiated these stem cells into MSLN CAR-NK cells via an efficient organoid induction system. The MSLN CAR-NK cells exhibit the typical expression patterns of activating receptors, inhibitory receptors, and effector molecules of NK cells. In the presence of tumour cells, the MSLN CAR-NK cells show increased secretion of IFN-γ and TNF-α, as well as elevated CD107a expression level compared with induced NK cells. We cryopreserved the MSLN CAR-NK cells in liquid nitrogen using a clinical-grade freezing medium (CS10) for more than 6 months to mimic an off-the-shelf CAR-NK cell product. The thawed MSLN CAR-NK cells immediately recovered after 48-72-h culture and effectively eliminated ovarian tumour cells, including human primary ovarian tumour cells from patients. The thawed MSLN CAR-NK cells efficiently suppressed ovarian tumour development in vivo and prolonged the survival of tumour-bearing mice. Our study provides insights into the clinical translation of hESC-derived MSLN CAR-NK cells as a promising off-the-shelf cell product.

Requirements for human natural killer cells.

'Requirements for Human Natural Killer Cells' is the latest set of guidelines on human NK cells in China, jointly drafted and agreed upon by experts from the Standards Committee of Chinese Society for Cell Biology. This standard specifies requirements for the human natural killer (NK) cells, including the technical requirements, test methods, test regulations, instructions for use, labeling requirements, packaging requirements, storage and transportation requirements, and waste disposal requirements of NK cells. This standard is applicable for the quality control of NK cells, derived from human tissues, or differentiated/transdifferentiated from stem cells. It was originally released by the Chinese Society for Cell Biology on 30 August, 2022. We hope that the publication of these guidelines will promote institutional establishment, acceptance, and execution of proper protocols and accelerate the international standardization of human NK cells for applications.