| ALI / ARDS,always a hot spot in critically ill diseases,the basic pathophysiology of which is clearly the injury of pulmonary microvascular endothelial cells and alveolar epithelial cells,leads to reduced alveolar surfactant,increased cell permeability,polymorphonuclear leukocyte aggregation,inflammatory activation,and ultimately the formation of extensive pulmonary edema and other pathological manifestations.As one of the main causes of the development of critical diseases,the primary cause of acute lung injury and acute respiratory distress syndrome(ALI / ARDS)is the damage of pulmonary microvascular endothelium.Β2-AR and TLR4 receptors exist in the PMECs in balance,maintaining various cellular activities.Normally,β2-AR receptor is activated to play biological role.Under pathological states when PMECs respond to inflammatory stimuli such as lipopolysaccharide,the expression and function of the two receptors will be changed,with TLR4 receptor activated and β2-AR receptor damaged,thus breaking the balance of the two types of receptors’ expression and mediation in inflammation,leading to runaway inflammation and eventually ALI.Rab26 is a small G protein belonging to the Ras superfamily.In HEK293 cells,ERK1 / 2 activated by α2-AT,Rab26 regulates the expression of α(2A)-AR and α(2B)-AR on the cell surface and the binding protein of Rab3-interacting molecule(Rim1),which is a key component of neurotransmitter release,and the transcription factor MIST1,which is important for maturation of secretory granules in cells.However,little is known about Rab26 in lung tissue so far.Therefore in this study,first using LPS to stimulate pulmonary microvascular endothelium,we observed the expression of β2-AR and TLR4.Then using si RNAs to silence or agonists to thrill the expression,we observed the change of β2-AR and TLR4 expression.We further explored the imbalance of these two receptors in inflammation and the regulatory effect of Rab26 plasmide.Methods:1.PMECs were transfected with β2-AR and TLR4 si RNA,and corresponding m RNA and protein levels were detected.2.The PMECs were treated with β2-AR and TLR4 agonists,and the expression of relative receptor gene and protein production were observed.3.The transfection efficiency of Rab26 plasmid in PMECs was detected by flow cytometry,and RT-PCR and Western blot were used to asses the expression.4.Expression of inflammatory cytokines in PMECs was detected by ELISA.5.The permeability of PMECs was determined by pervasion biont-BSA.Result:1.After LPS treatment of HPMECs,β2-AR on the cell surface was decreased,while the TLR4 expression was increased,which is the preliminary proof of receptors imbalance.2.The expression of IL-6,IL-8 and TNF-α was up-regulated by β2-AR si RNA,down regulated by TLR4 si RNA.The expression of TLR4 was increased by β2-AR si RNA,and the expression of β2-AR was increased by TLR4 si RNA.3.According to the results of concentration-time curve,the optimal time of epinephrine(EPI)and LPS,which are β2-AR and TLR4 agonists,is 6h,with optimal concentration 50pmol/L and 2ng/ul respectively.The results showed that the expression of β2-AR increased and TLR4 decreased after EPI treatment.Two agonists used simultaneously showed certain reversal effect.4.The expression of Rab26 m RNA and protein production were decreased after LPS.The expression of Rab26 mRNA and protein production and β2-AR were decreased,while the expression of TLR4,IL-6,IL-8 and TNF-α and cell permeability were upregulated after GDP active plasmid transfection.The expression of Rab26 m RNA and protein production and β2-AR were increased,while the expression of TLR4,IL-6,IL-8 and TNF-α and cell permeability were down-regulated after GTP active plasmid transfection.Conclusion:1.LPS stimulation of pulmonary microvascular endothelial cells results in the downregulation of β2-AR and upregulation of TLR4 receptors.2.LPS-induced imbalance of β2-AR and TLR4 receptor is modulated through Rab26 mediated β2-AR trafficking in PMEC cells. |
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