Downregulation Of Dopamine Receptor Contributes To Mechanical Stretch-induced Lung Endothelial Barrier Dysfunction

Objectives:Dopamine（DA）is a common neurotransmitter that has been known to regulate behavior,movement,cardiovascular,endocrine and gastrointestinal functions,but also functions as an important molecule engaging in the immune systems to possess anti-inflammatory effects.However,its role in ventilator-induced lung injury（VILI）is still unclear.Herein,this study aimed to investigate the therapeutic efficacy of dopamine on ventilation-induced lung endothelial barrier dysfunction and explore the possible underlying molecular mechanisms.Methods:We have analyzed gene expression data from the clinical lung tissue and have correlated DA receptor expression with Ac-α-tubulin.The mice were anesthetized,established a tracheotomy with a 20-G intravenous catheter and then subjected to mechanical ventilation（30 ml/kg）.The lung tissues were harvested after0.5 h、1 h、2 h、3 h、4 h.For histological assessment of DA synthetases and receptors,the lungs were harvested and stained with DAB.Total RNA and protein were isolated to determine the change of DA synthetases and receptors.Dopamine（50 mg/kg）were intraperitoneally administered before the onset of 4 hours’ventilation.For si RNA research,mice were intratracheally instilled with control or DRD1/DRD2 si RNA（2mg/kg）using in vivo-jet PEI polymer as a transfecting agent.DRD1 knockout(DRD1^-/-)mice were subjected to mechanical ventilation（30 ml/kg）for 4 h.Bronchoalveolar lavage（BAL）was performed after ventilation and measured the cell count and protein concentration.Lung W/D ratio was measured as an index of pulmonary edema.Pulmonary vascular permeability was analyzed by using Evans blue-labeled albumin extravasation into the lung tissue.The left lung was removed for histopathologic examination using hematoxylin and eosin staining.Total RNA and protein were isolated to determine the change of NLRP3 and Ac-α-tubulin.Content of dopamine in the lung tissues and plasma were determined by ELISA（Enzyme-linked immuno sorbent assay）.Mouse lung vascular endothelial cells（MLVECs）were subjected to high-magnitude（20%linear elongation,sinusoidal wave,30 cycles/min）cyclic stretch for 4 h.MLVECs were treated with DA,adenylate cyclase（AC）agonist forskolin,AC inhibitor KH7,glycogen synthase kinase3（GSK-3β）inhibitor SB216763,histone deacetylase6（HDAC6）inhibitor tubacin,NAD⁺-dependent deacetylase sirtuin-2（SIRT2）inhibitor AGK-2,protein kinase A（PKA）inhibitor H89,Exchange protein directly activated by c AMP（Epac）agonist 8-p CPT-2′-O-Me-c AMP and then stimulated with cyclic stretch.Total protein was isolated to determine the changes of these pathways.Main results:1.Mechanical ventilation downregulated pulmonary DRD1 expression,while the DA synthetases and DRD2 had no changes.Mechanical ventilation downregulated pulmonary dopamine D1 receptor expression in a time-dependent manner both in the clinical samples and mice lung tissues after ventilation.2.DA treatment attenuated VILI as shown in decreased cell counts and protein content levels in BAL fluid.DA treatment also attenuated the development of VILI as assessed by histopathological examination,lung W/D and measurement of Evans blue leakage into the lung tissue.DRD1 si RNA,but not DRD2 si RNA,blocked the protective effect of DA against mechanical ventilator-induced acute lung injury.DRD1 deficiency also abrogated the protective effect of DA against mechanical ventilator-induced acute lung injury.DRD1 agonist,but not DRD2 agonist,attenuated mechanical ventilator-induced acute lung injury.3.In human tissue sample,we found ventilation-induced D1 downregulation was correlated with the downregulation of Ac-α-tubulin.In addition,we found that DA treatment significantly attenuated mechanical ventilation-inducedα-tubulin deacetylation both in vivo and in vitro.DRD1 si RNA,but not DRD2 si RNA,blocked the protective effect of dopamine against mechanical ventilator-inducedα-tubulin deacetylation both in vivo and in vitro.DRD1 deficiency abrogated the protective effect of dopamine against mechanical ventilator-inducedα-tubulin deacetylation.Microtubule stabilizer Taxol protected VILI,while microtubule inhibitor nocodazole aggravated VILI as measured by cell counts and protein content levels in BAL fluid,histopathological examination,lung W/D and measurement of Evans blue leakage into the lung tissue.4.Primary cultured MLVECs were treated with HDAC6 inhibitor Tubacin or SIRT2 inhibitor AGK-2 and then stimulated with cyclic stretch,we found HDAC6,but not SIRT2,contributed to cyclic stretch-inducedα-tubulin deacetylation.GSK-3βinhibitor blocked cyclic stretch-induced HDAC6 phosphorylation.CS inhibited AKT and activated GSK-3β,these effects were ameliorated by DA treatment.The increase of the c AMP levels with AC activator forskolin attenuates mechanical stretch-inducedα-tubulin deacetylation,similar with DA treatment.Importantly,DA-induced inhibition ofα-tubulin deacetylation could be blocked by KH7,an AC inhibitor.The inhibition of PKA with H89 had no effect on DA-induced inhibition ofα-tubulin deacetylation,while Epac agonist attenuated mechanical stretch-induced microtubule disassembly,similar with DA treatment.5.DA treatment inhibited the activation of NLRP3 inflammasome and subsequent caspase-1 cleavage as well as interleukin（IL）-1βsecretion via DRD1 both in vivo and in vitro.In addition,microtubule stabilizer Taxol inhibited the activation of NLRP3 inflammasome,while microtubule inhibitor nocodazole blocked the inhibitory effect of DA.Conclusions:1.Mechanical ventilation suppressed DRD1 expression in a time-dependent manner.2.DA attenuated ventilation-associated lung vascular hyperpermeability via DRD1.3.DA inhibited ventilation-associatedα-tubulin deacetylation via DRD1.4.DRD1 signaling attenuated mechanical stretch-inducedα-tubulin deacetylation via c AMP/Epac-mediated inactivation of HDAC6.5.DA alleviated mechanical stretch-induced activation of NLRP3 inflammasome via DRD1.Selective activation of DRD1 may be a novel therapeutic option for VILI.