Effect Of Remimazolam On Gene Expression And Function Of T Lymphocyte Subsets

BackgroundAs a new type of benzodiazepine used in clinical anesthesia,remimazolam has the advantages of fast binding to corresponding receptors,short half-life,fast metabolism and excretion,and rapid recovery of patients.It has been widely used in various examinations and clinical anesthesia.The receptor that remimazolam acts on is the gamma-aminobutyric acid(GABA)receptor,which has been identified not only in the central nervous system,but also in immune cells,especially T lymphocytes.The first choice for the treatment of early tumors is surgical resection,which in turn requires clinical anesthesia as the basis and premise of surgery.Whether the anesthetics used during surgical resection affect the patient’s immune function will directly affect the prognosis of surgical patients.To date,the effect of remimazolam on T lymphocyte function has not been reported.ObjectiveThe purpose of this study was to study the effect of remimazolam on gene expression and function of T lymphocyte subsets.Methods1.Jurkat T cells were respectively treated with PMA/Ionomycin and PMA/Ionomycin/remimazolam,and normal cultured cells served as control.Then total RNA was extracted for transcriptome sequencing analysis to detect the effect of Jurkat T cell activation on the expression of GABA receptor family genes.2.Jurkat T cells were respectively treated with PMA/Ionomycin and PMA/Ionomycin/remimazolam,and normal cultured cells served as control.Then the effect of remimazolam on T cell activation was analyzed by flow cytometry.3.The TGFBI gene regulated by remimazolam in Jurkat T cells was knocked out by CRISPR/Cas9 technology,and the effect of remimazolam treatment on the resting state and activation state of TGFBI-deficient Jurkat T cells was analyzed.4.C57BL6/N mice were injected intraperitoneally with normal saline and remimazolam,respectively.Then the spleen of the mice were collected at different time points after injection.Flow cytometry was used to analyze the effect of remimazolam on the composition of T cell subsets and CD69 expression.5.After 4h and 8h of intraperitoneal injection of remimazolam into mice,the spleen of the mice were collected for sorting and purification of T cells,and single-cell transcriptome sequencing was performed to analyze the composition of T cell subsets.Results1.Through transcriptome sequencing analysis and q RT-PCR verification,it was found that after remimazolam treatment,the expression level of GABA receptor family gene GABBR1 was significantly increased along with the activation of Jurkat T cells,while the expression level of GABRB3 was significantly decreased,indicating that remimazolam treatment may affect T cell function.2.Flow cytometry analysis showed that remimazolam treatment could significantly inhibit the activation of Jurkat T cells.3.Transcriptome sequencing analysis and q RT-PCR verification found that remimazolam treatment could significantly up-regulate the expression level of TGFBI.Knockout of TGFBI effectively blocked the inhibition of Jurkat T cell activation by remimazolam,including decreased expression of T cell activation marker CD69 and increased the phosphorylation level of ERK.4.Intraperitoneal injection of remimazolam into mice did not affect the composition of T cell subsets and CD69 expression,but single-cell sequencing analysis showed that injection of remimazolam affected the single-cell transcriptome level of T cells,among which the proportion of phe 3 T cell subset increased significantly.ConclusionThe use of remimazolam may inhibit the activation of T cells by regulating the upregulated expression of TGFBI through GABA receptor,and knockout of TGFBI in T cells can reverse the inhibitory effect of remimazolam.