The Mechanism Of Electrical Vagus Nerve Stimulation On Alleviating Lung Injury In A Murine Model Of ARDS

Introduction : To investigate the effect and mechanism of electrical vagus nerve stimulation in lipopolysaccharide(LPS)induced acute respiratory distress syndrome(ARDS)in mice.Methods:(1)SPF grade male C57BL/6 mice were selected and ARDS model was induced by intratracheal instillation with LPS(3 mg/kg).The right cervical vagus nerve of mice was isolated and transected,then stimulated with different voltage(5V,10V)following intratracheal instillation of LPS 。 After 4h,mice were sacrificed to investigate whether electrical vagus nerve stimulation could mitigate LPS-induced inflammatory lung injury.(2)To determine the role of the alpha 7acetylcholine nicotinic receptor(α7nAChR).In vivo,mice were intraperitoneally injected with different doses of PNU-282987,α7nAChR agonist(1mg/kg or 10mg/kg)for 30 min,then intratracheally instilled with LPS.Mice in each group were divided into two parts,and sacrificed respectively after given LPS for 4h and 24 h 。 Lung tissues were taken for HE staining to observe the histopathological changes.Alveolar lavage fluid(BALF)was collected from mice to detect the total protein concentration and the expression of pro-inflammatory cytokines TNF-α,IL-6.In vitro,the murine alveolar macrophage cell line MH-S was selected and stimulated by LPS(100ng/ml)to constructed the inflammatory response model.ELISA was used to detect the expression of IL-6 induced by LPS after preconditioning PNU-282987 with different concentration(0-1000μM).(3)To investigate the effect of PNU-282987 on the phosphorylation level of STAT3,the expression of p-STAT3 at different time points(0h,1h,2h,3h,4h)were evaluated by Western Blotting.The optimal time pointwas selected to detect the effect of PNU-282987 at different concentration(0-1000μM)on LPS-induced p-STAT3 expression.Results:(1)Compared with the model group,the low voltage(5V)of vagus nerve stimulation effectively improved the extent of lung tissue damage,mitigated the infiltration of inflammatory cells and reduced the concentration of TNF-α、IL-6 and total protein in BALF.(2)In vivo,the low dose(1mg/kg)of PNU-282987 improved the alveolar integrity,inhibited the aggregation of inflammatory cells,and down-regulated the expression of pro-inflammatory cytokines TNF-α 、 IL-6 after instilled with LPS for 4h in mice.In vitro,PNU-282987 reduced the expression of LPS-induced inflammatory cytokine IL-6 in a dose-dependent manner.(3)After given PNU-282987 for 1h,the expression of p-STAT3 was inhibited and decreased significantly at 2h.Meanwhile,PNU-282987 dose-dependently inhibited STAT3 phosphorylation induced by LPS.Conclusions: Electrical vagus nerve stimulation attenuates LPS-induced lung injury by inhibiting STAT3 phosphorylation via α7nAChR in a murine model of ARDS.