RESUMO
We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by â¼twofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP- or ATP+ ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain.
Assuntos
Ginsenosídeos/farmacologia , Receptores Purinérgicos P2X4/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Benzoxazóis/metabolismo , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Células HEK293 , Humanos , Ivermectina/farmacologia , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Compostos de Quinolínio/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Sapogeninas/farmacologiaRESUMO
The COVID-19 pandemic continues to expand globally, with case numbers rising in many areas of the world, including the Eastern Mediterranean Region. Lebanon experienced its largest wave of COVID-19 infections from January to April 2021. Limited genomic surveillance was undertaken, with just 26 SARS-CoV-2 genomes available for this period, nine of which were from travellers from Lebanon detected by other countries. Additional genome sequencing is thus needed to allow surveillance of variants in circulation. In total, 905 SARS-CoV-2 genomes were sequenced using the ARTIC protocol. The genomes were derived from SARS-CoV-2-positive samples, selected retrospectively from the sentinel COVID-19 surveillance network, to capture diversity of location, sampling time, sex, nationality and age. Although 16 PANGO lineages were circulating in Lebanon in January 2021, by February there were just four, with the Alpha variant accounting for 97â% of samples. In the following 2 months, all samples contained the Alpha variant. However, this had changed dramatically by June and July 2021, when all samples belonged to the Delta variant. This study documents a ten-fold increase in the number of SARS-CoV-2 genomes available from Lebanon. The Alpha variant, first detected in the UK, rapidly swept through Lebanon, causing the country's largest wave to date, which peaked in January 2021. The Alpha variant was introduced to Lebanon multiple times despite travel restrictions, but the source of these introductions remains uncertain. The Delta variant was detected in Gambia in travellers from Lebanon in mid-May, suggesting community transmission in Lebanon several weeks before this variant was detected in the country. Prospective sequencing in June/July 2021 showed that the Delta variant had completely replaced the Alpha variant in under 6 weeks.