Study On The Mechanism Of Ceramide In Increased Permeability Of Pulmonary Microvascular Endothelial Cells Induced By Lipopolysaccharide

BackgroundAcute respiratory distress syndrome(ARDS)is acute hypoxic respiratory failure caused by many factors,and is a common clinical critical illness.Non-cardiogenic pulmonary edema caused by increased permeability of pulmonary microvascular endothelial cells(PMVECs)is a key link in the pathogenesis of ARDS.When stimulated by malignant factors,PMVECs are the most important target cells in the lung,and the integrity of their structure and function is crucial for the maintenance of normal physiological functions of the lung.Therefore,exploring the mechanism of PMVECs damage and monolayer permeability increase is an important direction to study the occurrence and development of ARDS.Ceramide is reported to be produced at high levels in endothelial cells and is associated with vascular endothelial barrier disruption and high permeability.However,the specificity of the ceramide triggering response depends on the type of ceramide,the enzyme pathway responsible for its upregulation,and the cell type in which the response occurs.In the process of Lipopolysaccharide(LPS)induced high permeability of PMVECs,the enzymatic pathway of ceramide synthesis and the downstream signal transduction pathway in PMVECs have not been reported.Therefore,this study used LPS-induced PMVECs damage as a model to explore the role of ceramide production in LPS-induced PMVECs hyperpermeability.Part 1 Effects of inhibiting ceramide production on LPS-induced apoptosis and hyperpermeability of PMVECsObjective:To study the effect of ceramide production on the apoptosis and hyperpermeability of PMVECs induced by LPS.Methods:Cell experiments:Rat PMVECs were cultured in vitro,assessing PMVECs permeability by measuring transendothelial electrical resistance(TER),fluorescein isothiocyanate-bovine serum albumin(FITC-BSA),and FITC-dextran;Apoptosis of PMVECs detected by Annexin V-FITC/PI double staining;Acid sphingomyelinase(ASMase)kit to detect acid sphingomyelinase activity;The content of ceramide was determined by enzyme Linked immunosorbent assay(ELISA);Western blot was used to detect the protein expressions of Serine palmitoyl transferase(SPT)?Bax and Bcl-2;RT-PCR detection of SPT m RNA expression.Animal experiments:Lung tissue pathological changes were observed by HE staining and pathological scoring;lung tissue wet/dry ratio(W/D)and lung permeability index(LPI)were used to evaluate pulmonary edema and pulmonary vascular permeability;Caspase3 activity was detected by spectrophotometry;Western blot was used to detect the protein expressions of Bax and Bcl-2.Results:1.The morphology of PMVECs was observed under an inverted microscope as paving stones,and after the FITC-BSI binding experiment,it was observed as a halo-like green fluorescence under a fluorescence microscope.2.When LPS concentration was 10?g/m L,The TER value of PMVECs was the lowest and the permeability to FITC-BSA was the highest.When 10?g/m L LPS was applied to PMVECs for different time,the TER value was the lowest at the 16th h,and the permeability of FITC-BSA increased in a time-dependent manner.3.PMVECs were not damaged by 10?g/m L LPS within 8 h,and the apoptosis of PMVECs increased significantly after 12 h,and lasted for 24 h.PMVECs were pretreated with two broad-spectrum caspase inhibitors Q-VD-OPH(Q-VD,50?mol/L)and Z-VAD-FMK(Z-VAD,100?mol/L),which significantly inhibited LPS induced apoptosis of PMVECs.Q-VD and Z-VAD had no effect on the change of TER value of LPS-induced PMVECs,while Q-VD pretreatment significantly reduced the permeability of LPS-induced PMVECs to FITC-BSA and FITC-dextran after 12 h,Z-VAD Pretreatment significantly reduced the permeability of LPS-induced PMVECs to FITC-BSA after 12 h and to FITC-dextran after 16 h.4.LPS(10?g/m L)induced a dramatic increase in ceramide content in PMVECs within4 h and peaked at 16 h;consistent with this,ASMase activity was significantly increased within 4 h,while ASMase-specific inhibition imipramine(50?mol/L)significantly inhibited LPS-activated ASMase activity and ceramide production;GW4869(20?mol/L),a pharmacological inhibitor of NSMase,did not inhibit ceramide production.Myriocin(50 nmol/L)and SPTLC1 si RNA,had no effect on LPS-induced upregulation of early ceramides(within 8 h)in PMVECs,but significantly inhibited ceramide production in the middle and late stages(after 12 h).5.Imipramine(50?mol/L)can significantly improve the LPS(10?g/m L)induced PMVECs TER value decrease and increase the permeability to FITC-BSA;while myriocin(50 nmol/L)and SPTLC1 si RNA have no effect on LPS-induced PMVECs TER value,but can significantly improve LPS-induced PMVECs in the middle and late stages(After 12 h)permeability to FITC-BSA.In addition,further studies showed that myriocin and SPTLC1 si RNA could significantly inhibit the apoptosis of PMVECs induced by LPS within 24 h.6.Myriocin can significantly improve the pathological changes,pulmonary edema and pulmonary vascular permeability of rat lung injury induced by LPS.Meanwhile,Myriocin could significantly inhibit the expression of apoptosis-related proteins and Caspase3 activity in LPS-induced rat lung tissue.conclusion:The rapid upregulation of ceramides by LPS via activation of ASMase leads to early(within 8 h)PMVECs hyperpermeability,and subsequent ceramides synthesized via de-novo pathway lead to apoptosis-dependent mid-to-late(12 h later)PMVECs hyperpermeability.Part 2 Study on the mechanism of LPS induced early PMVECs hyperpermeability through ASMase/Ceramide pathwayObjective:To clarify the specific signaling pathways involved in the activation of ASMase and the subsequent generation of ceramides during LPS-induced early PMVECs hyperpermeability.Methods:Western blot was used to detect the protein expressions of TRPC6,ZO-1 and VE-cadherin;RT-PCR to detect the expression of TRPC6 m RNA;TER and FITC-BSA methods to detect the permeability of PMVECs;Rho A activity detection kit to detect Rho A activation;The intracellular calcium content was detected by flow cytometry;the structural changes of F-actin cytoskeleton and intercellular connexin were detected by immunofluorescence.Results:1.Both imipramine(50?mol/L)and TRPC6 si RNA significantly inhibited the expression of TRPC6 and intercellular connexin in PMVECs induced by LPS(10?g/m L).2.Both imipramine and TRPC6 si RNA significantly inhibited LPS-induced Ca²⁺influx and Rho A activation.3.Both imipramine and TRPC6 si RNA significantly inhibited LPS-induced cytoskeletal rearrangement,disruption of intercellular junctions in PMVECs,and PMVECs hyperpermeability.4.Imipramine could significantly inhibit the production of ceramide in PMVECs induced by LPS,while TRPC6 si RNA had no effect on the production of ceramide in PMVECs.conclusion:During the LPS-induced early PMVECs hyperpermeability stage,ceramides generated by ASMase activation further activated the Rho A pathway by inducing up-regulation of TRPC6 expression and influx of calcium ions.Activation of Rho A further induces cytoskeletal rearrangement and disruption of intercellular junctions in PMVECs,ultimately leading to increased permeability of PMVECs.Part 3 The role and mechanism of ceramide-regulated Txnip expression in LPS-induced PMVECs apoptosisObjective:To explore the relevant molecular mechanisms mediated by ceramide in LPS-induced PMVECs apoptosis.Methods:1.Cell experiments:Apoptosis of PMVECs detected by Annexin V-FITC/PI double staining;The content of ceramide was determined by enzyme Linked immunosorbent assay(ELISA);Western blot was used to detect the protein expressions of Thioredoxin interacting protein(Txnip)?Thioredoxin(Trx)?Bax?Bcl-2?JNK?P-JNK?P38?P-P38?Apoptosis signal-regulating kinase 1(ASK1)and P-ASK1.Co-immunoprecipitation(Co-IP)was used to detect changes in the interaction between Trx-Txnip and Trx-ASK1.RT-PCR detection of Txnip m RNA expression.2.Animal experiments:Lung tissue pathological changes were observed by HE staining and pathological scoring;lung tissue wet/dry ratio(W/D)and lung permeability index(LPI)were used to evaluate pulmonary edema and pulmonary vascular permeability;Caspase3 activity was detected by spectrophotometry;Western blot was used to detect the protein expressions of Txnip?Trx?Bax?Bcl-2?JNK?P-JNK?P38?P-P38?ASK1 and P-ASK1.Results:1.Myriocin(50 nmol/L)and SPT si RNA significantly inhibited LPS(10?g/m L)induced JNK and P38 activation and apoptosis related protein expression in PMVECs.LPS up-regulated the expression of Txnip in PMVECS,promoted the interaction of Txnip and Trx,down-regulated Trx activity and activated ASK1.However,myriocin and SPT si RNA inhibited the above responses.2.SB203580(10?mol/L)and SP600125(20?mol/L)significantly inhibited the activation of P38 and JNK,apoptosis and the expression of apoptosis-related proteins in LPS(10?g/m L)induced PMVECs,but could not inhibit the up-regulation of ceramide content.3.Verapamil(50?mol/L)and Txnip si RNA significantly inhibited the activation of P38and JNK,apoptosis and apoptosis-related protein expression in PMVECs induced by C8-ceramide(10?mol/L).At the same time,C8-ceramide up-regulates the expression of Txnip in PMVECs and promotes the interaction between Txnip and Trx,down-regulating Trx activity,and activated ASK1.However,these can be inhibited by application of Verapamil and Txnip si RNA to inhibit Txnip expression.4.The model of C8-ceramide induced lung injury in rats was simple and stable,and the pathological changes of lung tissue were consistent with the pathological changes of lung injury.Verapamil significantly improved the pathological changes,pulmonary edema and pulmonary vascular permeability of lung injury induced by C8-ceramide in rats.Verapamil also significantly inhibited the expression of apoptosis-related proteins and Txnip proteins,as well as the activation of ASK1 and Caspase3 in lung tissues induced by C8-ceramide.conclusion:In LPS-induced apoptosis-dependent middle and late stages of PMVECs hyperpermeability,ceramide synthesized via the de novo pathway induces apoptotic damage in PMVECs,in which up-regulation of Txnip expression inhibits Trx activity and subsequently activates ASK1,acts upstream of ceramide-induced p38 and JNK activation and apoptosis.