Honokiol Protects Pulmonary Microvascular Endothelial Barrier Against Lipopolysaccharide-Induced Ards Via The Sirt3/AMPK Signaling Pathway

Background and objective:Acute respiratory distress syndrome(ARDS)is a common form of critical respiratory diseases characterized by acute hypoxemia with diffuse alveolar damage and pulmonary osmotic edema.Sirtuin 3(Sirt3),mainly expressed in the matrix of mitochondria,maintains mitochondrial homeostasis and participates in the physiological and pathological processes of energy metabolism,stress response,aging and tumorigenesis.Honokiol(HKL),a traditional Chinese medicine monomer extracted from magnolia,possesses multiple properties,including anti-oxidation,anti-inflammation and anti-vascular damage.Besides,HKL has also been verified as an agonist of Sirt3.Angiopoietin-2(Ang-2)has been implicated in pulmonary osmotic edema and severity of ARDS.However,whether HKL regulates pulmonary microvascular endothelial barrier or inhibits the expression of Ang-2 in ARDS,and the underlying mechanisms of its action have not been reported.This study was aimed at exploring the effects of HKL on the pulmonary microvascular endothelial barrier in ARDS and its potential mechanisms,and providing a new basis for the diagnosis and treatment of ARDS.Methods: Circulating Ang-2 levels in the plasma of healthy controls and patients with ARDS were measured within 24 h of admission to assess the correlationship between Ang-2 and the severity of ARDS.Mice were intraperitoneally injected with 2.5 or 5 mg/kg HKL 30 min after intratracheal administration of LPS.Lung tissue inflammatory cytokine,indicators of pulmonary permeability and the survival curve were determined to explore the protective effects of HKL on ARDS.Human pulmonary microvascular endothelial cells(HPMECs)were pretreated with HKL 2 h before LPS stimulation to investigate the effects of HKL on the cell viability,apoptosis,the expression of intercellular adherent junction proteins,Sirt3 and Ang-2.Nicotinamide(NAM)and Compound C(CMC)were applied to inhibit the expression of Sirt3 and AMPK respectively,to further explore whether the Sirt3/AMPK signaling pathway plays a critical role in the protection of HKL against HPMECs stressed with LPS.Results:1.The clinical study showed that compared with the healthy controls,circulating Ang-2 levels were significantly higher in the patients with ARDS.Moreover,the subgroup analysis showed that the Ang-2 levels notably increased in parallel with the severity of ARDS,and were obviously higher in nonsurvivors than in survivors.Furthermore,the correlation analysis showed that plasma Ang-2 levels in patients with ARDS were positively correlated with white blood cells(WBC)count and procalcitonin(PCT)levels.2.In vivo experiments showed that HKL intervention notably alleviated lung inflammatory injury,pulmonary microvascular endothelial barrier damage and pulmonary osmotic edema,which promoted survival of ARDS mice.Furthermore,HKL increased Sirt3 expression and inhibited Ang2 expression at the genes and protein levels.3.In vitro experiments showed that pretreatment with HKL improved ECs survival,inhibited the apoptosis of ECs under LPS stimulation and restored the normal physiologic function of mitochondria.In addition,Nicotinamide and Compound C partially antagonized the effects of HKL via the inhibition of the Sirt3/AMPK signaling pathway.4.Both in vivo and in vitro experiments showed that HKL protected pulmonary microvascular endothelial barrier stressed by LPS and decreased the microvascular leakage,which may partially through the activation of Sirt3/AMPK pathway and inhibition of Ang-2 expression.Conclusion:Our findings suggest that the levels of Ang-2 significantly increased in the plasma of ARDS patients and mouse lung tissue.HKL protects against LPS-induced ARDS via inhibiting inflammatory lung injury and antagonizing pulmonary microvascular hyperpermeability,and promoting the survival of ARDS mice.These protective effects could be probably through activation of the Sirt3/AMPK signaling pathway and inhibition of Ang-2 expression.Therefore,to restore pulmonary microvascular barrier may be a new therapeutic strategy for the treatment of ARDS.