Mechanisms Of IP3R/Ca2+ Mediated Endoplasmic Reticulum Stress And Mitochondrial Dysfunction In VILI

Part Ⅰ Mechanical stretch-activated IP3R mediates calcium homeostasis imbalance in VILIObjective:To observe the lung injury and inflammatory response,as well as the expression of IP3R and calcium(Ca2+)in mice after mechanical ventilation(MV)with high tidal volume(HTV),and to explore the mechanism of intracellular calcium homeostasis imbalance induced by IP3R activation in VILI.Methods:C57BL/6 mice(body weight 25±2g)were randomly divided into 6 groups.Spontaneous breathing control group(CON group,spontaneous breathing after endotracheal intubation,4h),Low tidal volume group(LTV group,MV with low tidal volume 7ml/kg for 4h),High tidal volume group(HTV group,MV with high tidal volume,20ml/kg for 4h),IP3R agonist carbachol+HTV group(Carbachol group,intraperitoneal injection of 1 mg/kg Carbachol 0.5 h before MV with HTV for 4h,IP3R inhibitor 2-APB+HTV group(2-APB group,intraperitoneal injection of 300μg 2-APB daily for 7 consecutive days,followed by MV with HTV for 4h),calcium chelator BAPTA-AM+HTV group(BAPTA-AM group,Intraperitoneal injection of 2.5 mg/kg BAPTA-AM 0.5 h before MV with HTV for 4h),8 mice in each group.The hematoxylin-eosin(HE)staining and transmission electron microscopy(TEM)were used to evaluate the degree of histopathological lung injury,and the degree of pulmonary inflammation was assessed by measuring the wet/dry(W/D)ratio,bronchoalveolar lavage fluid(BALF)cells and protein counts,and the expression of inflammatory factors(IL-1β,IL-6 and TNF-α)in BALF.The cytoplasm,endoplasmic reticulum(ER)and mitochondrial-associated-membranes(MAMs)were isolated and purified by ultra-high speed differential centrifugation.The expression of IP3R protein was detected by immunofluorescence and Western Blot.Fluo-4AM,Rhod-2 and Mag-Fluo-AM were used to determine Ca2+concentrations in cytoplasm,mitochondria and ER,respectively.MLE12 mouse alveolar epithelial cells and RWA264.7 mouse macrophages were used for vitro experiments.Results:1.MV with HTV for 4h induces lung injury and inflammation in mice,which were manifested as lung histopathological changes,with the up-regulated expression of W/D,total protein volume,cell counts,and inflammatory factors such as IL-1β,TNF-α and IL-6 in BALF.2.Compared with the CON group,the expression of IP3R protein in mice lung tissue in HTV group was significantly increased(P<0.05),and it was mainly expressed in the ER and MAM.3.The formation of MAM increased in alveolar type Ⅱ epithelial cells(AT-Ⅱ)and alveolar macrophages(AM)in HTV group.4.Compared with CON group,cytoplasmic and mitochondrial Ca2+concentrations in HTV group were increased(P<0.05).Carbachol pretreatment further increased cytoplasmic and mitochondrial Ca2+ concentrations,but 2-APB could reverse the increase of cytoplasmic and mitochondrial Ca2+concentrations induced by HTV.On the contrary,compared with CON group,ER Ca2+concentration decreased in HTV group(P<0.05),and the decrease was more significant after carbachol pretreatment.2-APB could reverse the decrease of ER Ca2+concentration induced by HTV.5.Administration of carbachol aggravated HTV-induced lung injury and inflammation while pretreatment with 2-APB or BAPTA-AM largely prevented these effects.6.Carbachol decreased the activity of MLE12 and RAW264.7 cells and promoted the secretion of inflammatory cytokines by these cells.Conclusion:MV with HTV can lead to pathological injury and inflammatory response in mice,and calcium homeostasis imbalance induced by IP3R activation is involved in VILI.Part Ⅱ The role of IP3R/Ca2+mediating endoplasmic reticulum stress in VILI Objective:To observe the expression of endoplasmic reticulum stress(ERS)related proteins and genes in VILI mice lung tissues and in MLE12 and RAW264.7 cells,and to explore the mechanism of ERS induced by IP3R/Ca2+imbalance in VILI mice.Methods:C57BL/6 mice(body weight 25±2g)were randomly divided into 7 groups,CON group,HTV group,2-APB group,ERS agonist Thapsigargin+HTV group(THA,intraperitoneal injection of 0.6 mg/kg Thapsigargin 24h and Oh before MV with HTV),ERS inhibitor TUDCA+HTV group(TUDCA,intraperitoneal injection of 250mg/kg TUDCA 0.5h before MV with HTV for 4h),IRE1α kinase inhibitor KIRA6+HTV group(KIRA6 group,intraperitoneal injection of 10mg/kg KIRA6 0.5h before MV with HTV for 4h),and PERK inhibitor GSK2606414+HTV group(GSK group,intraperitoneal injection of 50mg/kg GSK2606414 0.5h before MV with HTV for 4h),8 mice in each group.The HE staining and TEM were used to evaluate the degree of histopathological lung injury,and the degree of pulmonary inflammation was assessed by measuring the wet/dry(W/D)ratio,bronchoalveolar lavage fluid(BALF)cells and protein counts,and the expression of inflammatory factors(IL-1β,IL-6 and TNF-α)in BALF.Immunofluorescence,Western Blot and RT-PCR were used to detect the protein and gene expression of ERS marker(GRP78,CHOP)in lung tissues and MLE12 cells and RAW264.7 cells.The relative protein expression of IRE1α,PERK and ATF6 pathways in lung tissues were detected.The expression of NF-κB pathway related proteins(p-NF-κB,IκBα)in lung tissues,MLE12 cells and RAW264.7 cells were detected.Results:1.Compared with CON group,protein and gene expressions of ERS markers GPR78 and CHOP in lung tissue of mice in HTV group were increased(P<0.05),and compared with HTV group,GPR78 and CHOP protein and mRNA expressions in THA group were increased(P<0.05).TUDCA could inhibit the high protein and gene expressions of GPR78 and CHOP after MV with HTV.2.TEM results showed aberrant,enlarged ER in alveolar type II epithelial cells(AT-Ⅱs)in the HTV group,and more serious structural changes,including ERs that were largely stripped of ribosomes and disrupted mitochondria,were detected in the THA group.However,these structural injuries were not seen in other groups.3.In addition,Thapsigargin pretreatment exacerbated lung injury and inflammation,but HTV-induced lung injury and inflammation was improved by treatment with TUDCA.4.Compared with CON group,the protein expressions of IRE1α/TRAF2/NF-κB and PERK/eIF2α/NF-κB pathways in HTV mice lung tissues were increased(all P<0.05),and ATF6 was not significantly changed(P>0.05).5.Pretreatment with KIRA6 or GSK2606414 improved lung injury and inflammation after MV with HTV.6.Pretreatment with 2-APB can inhibit the activation of IRE1α and PERK pathways induced by HTV.7.Pretreatment with 2-APB can inhibit the activation of NF-κB pathway induced by HTV.8.Carbachol stimulated ERS and activated NF-κB pathway in MLE12 and RAW264.7 cells.Conclusion:IP3R/Ca2+imbalance activates the endoplasmic reticulum stress IRE1α and PERK pathways as well as the downstream NF-κB pathway,leading to the release of inflammatory cytokines and participate in VILI.Part Ⅲ The role of IP3R/Ca2+mediating mitochondrial dysfunction in VILIObjective:To detect the mitochondrial function and the expression of NLRP3 inflammasome in VILI mice lung tissues and in MLE12 and RAW264.7 cells,and to explore the mechanism of mitochondrial dysfunction induced by IP3R/Ca2+imbalance in VILI mice.Methods:C57BL/6 mice(body weight 25±2g)were randomly divided into 3 groups,CON group,HTV group and 2-APB group.8 mice in each group.ATP levels were determined via firefly luciferase-associated chemiluminescence.Mitochondrial membrane potential(ΔΨm)was assessed using JC-1 staining.Reactive oxygen species(ROS)were detected by 2’,7’-dichlorodihydrofluorescein diacetate(DCFH).Western Blot and RT-PCR were used to detect NLRP3,caspase-1 and ASC protein and gene expression in lung tissues,MLE12 cells and RAW264.7 cells.Results:1.Compared with CON group,ΔΨm and ATP levels in lung tissue of HTV group were decreased,ROS production was significantly increased(all P<0.05),while 2-APB pretreatment inhibited the above changes.2.Compared with CON group,the expressions of NLRP3,Caspase-1 and ASC in lung tissue of HTV group were increased at the protein and mRNA levels(all P<0.05).In addition,2-APB preconditioning significantly inhibited the activation of NLRP3 inflammasomes.3.Exposing MLE12 and RAW264.7 cells to carbachol resulted in mitochondrial dysfunction and increased levels of NLRP3,caspase-1 and ASC mRNA.Conclusion:IP3R/Ca2+mediates mitochondrial dysfunction,increases ROS production,activates NLRP3 inflammasome to trigger inflammatory response,which participate in VILI.