EGFR promotes the apoptosis of CD4+ T lymphocytes through TBK1/Glut1 induced Warburg effect in sepsis.

INTRODUCTION: Sepsis-induced apoptosis leads to lymphopenia including the decrease of CD4+ T cells thus favoring immunosuppression. OBJECTIVES: Although epidermal growth factor receptor (EGFR) inhibitors significantly improve the survival rate of septic mice, the effect of EGFR on the function and metabolism of CD4+ T cells in sepsis remained unknown. METHODS: CD4+ T cells from septic mice and patients were assessed for apoptosis, activation, Warburg metabolism and glucose transporter 1 (Glut1) expression with or without the interference of EGFR activation. RESULTS: EGFR facilitates CD4+ T cell activation and apoptosis through Glut1, which is a key enzyme that controls glycolysis in T cells. EGFR, TANK binding kinase 1 (TBK1) and Glut1 form a complex to facilitate Glut1 transportation from cytoplasm to cell surface. Both the levels of membrane expression of EGFR and Glut1 and the activation levels of CD4+ T cells were significantly higher in patients with sepsis as compared with healthy subjects. CONCLUSION: Our data demonstrated that through its downstream TBK1/Exo84/RalA protein system, EGFR regulates Glut1 transporting to the cell surface, which is a key step for inducing the Warburg effect and the subsequent cellular activation and apoptosis of CD4+ T lymphocytes and may eventually affect the immune functional status, causing immune cell exhaustion in sepsis.

Glycopyrrolate versus atropine for preventing bradycardia induced by neostigmine injection after general anesthesia surgery: a randomized open, parallel-controlled multicenter clinical trial.

OBJECTIVE: With atropine as a positive control, randomized controlled clinical trials were conducted to verify the efficacy of glycopyrrolate injection in preventing bradycardia caused by neostigmine. METHOD: Patients undergoing elective general anesthesia and non-cardiac surgery were randomly divided into an experimental group (129 cases) and control group (127 cases) (ChiCTR2100046022, http://www.chictr.org.cn/showproj.aspx?proj=126075). At the end of the operation, the test group was given glycopyrrolate 6 ug/kg + neostigmine 0.04 mg/kg, and the control group was given atropine 0.016 mg/kg + neostigmine 0.04 mg/kg, bolus time 1 min, to antagonize muscle residual effects of relaxants. We compared the area under the time curve (AUC) of the difference between heart rate and baseline heart rate within 15 minutes of administration, the measured value of heart rate per minute, and the change in heart rate compared with baseline. We verified the safety of glycopyrrolate injection through laboratory tests, clinical symptoms, signs, and adverse events/serious adverse events. RESULTS: The AUC of the experimental group's heart rate within 15 minutes after the administration was lower than the baseline heart rate change value, (P<005). The measured value of the heart rate at each time changed less than the control group; the experimental group's heart rate remained at the baseline level for longer than the control group (P<005). There was no significant difference in the incidence of adverse reactions between the two groups of patients (P>005). CONCLUSION: Glycopyrrolate and atropine are safe to prevent heart rate slowing induced by the non-depolarizing muscle relaxant antagonist neostigmine, and glycopyrrolate is more conducive to maintaining a stable heart rate in patients.

[Effects of microRNA-294 on inflammatory factor of sepsis by targeting triggering receptor expressed on myeloid cells-1].

OBJECTIVE: To investigate the effects of microRNA-294 (miR-294) on tumor necrosis factor-α (TNF-α), interleukin -6 (IL-6) and high mobility group box 1 (HMGB1) secretion in sepsis by targeting triggering receptor expressed on myeloid cell-1 (TREM-1). METHODS: miRNA-294 was predicted to regulate TREM-1 specially through bioinformatics analysis. Mice macrophage cell lines RAW264.7 were cultured in vitro, the cells were divided into non-inflammatory stage and inflammatory stage, and the cells in the two stages were subdivided into five groups as follows: normal control (NC), NC mimic transfection (NCm), NC inhibitor transfection (NCi), miR-294 mimic transfection (miR-294 m) and miR-294 inhibitor transfection (miR-294i) groups. The ability of miR-294 was confirmed with dual-luciferase activity assay. At non-inflammatory stage, the cells were transfected with mimic or inhibitor of miR-294 or NC using TurboFect™ siRNA Transfection Reagent for 48 hours, mRNA expression of TREM-1 was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR). At inflammatory stage, 6 hours after stimulation by lipopolysaccharide (LPS, 1 mg/L), the concentrations of TNF-α, IL-6 and HMGB1 were determined by enzyme linked immunosorbent assay (ELISA), the protein expression of TREM-1 was determined by Western Blot. RESULTS: (1) Dual-luciferase activity assay demonstrated that TREM-1 was the target of miR-294. (2) In non-inflammatory stage, the expression of TREM-1 mRNA (2(-ΔΔCt) ) in miR-294m group was significantly lower than that of the NC and NCm groups (0.673 ± 0.049 vs. 1.000 ± 0.003, 0.915 ± 0.039, t1=2.184, t2=5.421, both P<0.001), the expression of TREM-1 protein (gray scale) was (50.00 ± 1.19)% of NCm group (t=41.586, P<0.001). (3) In inflammatory stage, the concentrations of TNF-α (ng/L) in miR-294 m group was significantly lower than that of the NC group (1 547.18 ± 47.18 vs. 2 702.11 ± 327.20, t=4.212, P=0.010), the concentrations of IL-6 (ng/L) was significantly lower than that of the NC and NCm groups (505.28 ± 33.33 vs. 837.66 ± 69.43, 918.72 ± 119.39, t1=4.382, P1=0.015; t2=5.451, P2=0.021), the level of TREM-1 protein(gray scale) was (51.33 ± 0.88)% of NCm group (t=63.368, P<0.001). CONCLUSIONS: miR-294 reduce TNF-α and IL-6 secretion in LPS-induced RAW264.7 through inhibiting the expression of TREM-1 specifically.