| BackgroundInhalation injury is a kind of respiratory and lung damage caused by inhalation of hot or toxic gases.It was usually born with extensive burns,especially those with head facial burns.Inhalation damage is a mixture of heat and chemicals.One of the main causes of inhalation damage is the thermal effect,with the respiratory and alveoli as the mainly damage target.Inhalation injury can cause serious complications,such as systemic inflammatory response and chronic obstructive pulmonary disease.Extensive burns accompanied by inhaled injury can lead to acute respiratory failure,and the mortality rate is far higher than that of simple burns.At present,in inhalation injury,there are so many research about the effect of smoke and chemicals on the tissue and less about the effect of single heat.In the inhalation injury after burn,the injury can induce the lungs inflammatory cascade reaction,thus causing lung inflammation,systemic inflammatory response the initial mechanism of sepsis and organ failure.At the same time,heat stress and inflammatory response also cause the apoptosis and death of the bronchocells through different molecular mechanisms,further aggravating the damage to the lungs.In the case of aspiration injury,the phenomenon and mechanism of inflammation and apoptosis in bronchial epithelial cells are still unclear.Mitochondria are recoginaze as the cell power factory,and the mitochondrial calcium channel protein(MCU)can affect the metabolism of cells.In an inflammatory response,it is still not clear how the MCU can feel the inflammatory response and affect the cellular biological mechanism through Ca2 +.In our research,we performed animal model of inhalation injury in vivo and heat induced human bronchial epithelial cells injury in vitro to observe the effects of heat stress on inflammatory response and apoptosis,and then use a variety of methods to study the different molecular mechanisms.Finally,after observing the mitochondrial changes and generate ROS caused by the heat,then we focuses on how can MCU feel ROS in inflammatory reaction and regulate cell metabolism,illustrating the new mechanism.Part I The CFTR regulated MAPK/ NF-κB pathway in pulmonary inflammation in thermal inhalation injuryMethod1.We established heat induced cell injury model in vitro,treating the cell with 52 ℃ for 5 minutes.RealtimePCR and westerning boltting was used to detect the CFTR and COX2 expression changes at 0 hour 8 hours,1 day,2 day,5 days after heat treatment.2.We established thermal inhaltion injury animal model in vivo.After heat treatment,immunohistochemical and westerning boltting was used to detect the CFTR and COX2 expression changes at 0 hour 8 hours,1 day,2 day,5 days after heat treatment.3.Westerning boltting was used to detect the MAPK and p-Iκ Bα expression changes at 0 hour 8 hours,1 day,2 day,5 days after heat treatment.Immunocytochemistry was used to detect the location change of the NF-κB.4.NF-κB inhibiter(BAY11),ERK inhibiter(PD98059)and JNK inhibiter(SP600125)was used to treat the 16 HBE cells 4 hours and then performed the heat treatment.Westerning boltting was used to detect the COX2 expression changes.Elisa was used to detect the PGE2 in the supernatant.5.16 HBE cells was tranfeted with Rib plasmid to downregulate the CFTR expression,with PEF group as the control.Westerning boltting was used to detect the COX2,NF-κB,PERK,PJNK expression changes.NF-κB inhibiter(BAY11),ERK inhibiter(PD98059)and JNK inhibiter(SP600125)was used to treat the 16 HBE cells transfected with Rib or PEF plasmid and then performed the heat treatment.Westerning boltting was used to detect the COX2 expression changes.Elisa was used to detect the PGE2 in the supernatant.6.16 HBE cells was tranfeted with p-hCFTR plasmid or VX-809 to upregulate the CFTR expression.Westerning boltting was used to detect the COX2,NF-κB,PERK,PJNK expression changes.Elisa was used to detect the PGE2 in the supernatant.7.In the heat induced cell injury model,ERK,JNK inhibiter(PD98059,SP600125)was used to treat the cells and then check the expression of p-IκBα.At the same time,NF-κB inhibiter(BAY11)was used to treat the cells and then check the expression of p-ERK and p-JNK.8.16 HBE cells was tranfeted with Rib plasmid to downregulate the CFTR expression,with PEF group as the control.NF-κB inhibiter(BAY11),ERK inhibiter(PD98059)and JNK inhibiter(SP600125)was used to treat the 16 HBE cells transfected with Rib or PEF plasmid and then performed the heat treatment.Elisa was used to detect the IL-8 in the supernatant.9.16 HBE cells was treated with curcumin 0.5h and 8h before heat treatment,Elisa was used to detect the PGE2 and IL-8 in the supernatant.In the thermal inhalation animal model,the rat was intranasally injected with curcumin(10mg/kg/d).HE and MPO immunohistochemical was used to lung inflammation.10.In the thermal inhalation animal model,the rat was intranasally injected with curcumin(10mg/kg/d).westerning boltting and immunohistochemical was used to detect the CFTR and COX2 expression changes.Elisa was used to detect the IL-8 and PGE2 in the supernatant.Results:1.Aftre treatment with 52 ℃ for 5 minutes,the expression of CFTR was decreased at 0 hour,8 hours,day1 and day2,with lowest expression on day1 and return to normal on day5.The expression of COX2 was increased at 0 hour,8 hours,day1 and day2,with highest expression on day1 and return to normal on day5.2.In the thermal inhalation animal model,after heat treatment,the expression of CFTR or COX2 in the lung was decreased or increased at 8 hour and day1,returning back to normal on day5.3.Aftre treatment in vitro,the expression of P-ERK and P-JNK reach to the peak at 0 hour,with no effection on the RK,JNK and P38,PP38.Immunocytochemistry indicated that the location of the NF-κB was transferred from cytoplasm to nucleus.4.For the protein level,either alone or mixed with BAY11,PD98059,and SP600125 inhibitors can decrease the COX2 expression induced by the heat.The ELISA results showed that the BAY11,PD98059,and SP600125 inhibitors alone were able to suppress the inflammation factor PGE2 secreted by cells.5.In normal 16 HBE cells,the Rib transfection can increase the COX2,NF-κB,PERK,PJNK expression compared with the PEF group.And ERK,JNK,nf-kb’s specific inhibitors(BAY11,PD98059,SP600125)can reduce inflammatory factor COX2 and PGE2 expression.6.Westerning blotting showed COX2,p-Iκ Bα,P-ERK,P-JNK was decreased after CFTR overexpression in the p-hcftr transfection group,compared with the PC3(control plasmid)group.The vx-809 processing group(1um or 3um)can also make the CFTR expression bounce back after the heat stimulus,and also reduce the expression of the COX2 after the heat treatment.The ELISA results also showed a significant decrease in PGE2 after the warming was processed in VX809(1um or 3um).7.In the heat treated cell model,ERK,JNK specific inhibitors(PD98059,SP600125)can decrease the p-IκBα expression indueced by the heat.However,NF-κB specific inhibitors(BAY11)has no effect on the PERK and PJNK induced by the heat.8.Realtime PCR and Elisa indicated that,after heat treatment and RIB plasmid transfection,IL-8 was increased.And ERK,JNK,NF-κB,COX2 inhibitor(BAY11,PD98059,SP600125,NS-398)can reduce the expression of IL – 8.VX809 also reduces the production of il-8 inflammatory factors caused by heat or CFTR downregualtion.9.In vitro heat treatment of 16 HBE cells,which are treated with curcumin 0.5 h before and 8h after heat treatment.curcumin can reduce PGE2 of IL-8.In the meantime,we used curcumin to treat the rats with inhaled injury,and HE showed that curcumin was able to significantly reduce the inflammatory response associated with heat.The myeloperoxidase(MPO)positive cells of the curcumin treated group were significantly lower than the heat group.10.In termal inhalation injury animal model,immunohistochemical and western blotting showed that curcumin(10 mg/kg/day)can increase CFTR expression,and at the same time also can reduce the expression of COX2,eventually reducing the secretion of IL-8 and PGE2.Part II The heat induces respiratory epithelial cell inflammationthrough the endoplasmic reticulum stress and autophagymethod:1.Cell heat model was established.The reactive oxygen species in the cellswere stained with DCFH-DA and detected by immunofluorescence at 0 hours,12 hours,1 day and 2 days.2.In cellwarming model,GRP78 and CHOP,PERK / eIF2α,IRE1 / XBP1 s and ATF6 in endoplasmic reticulum stress were detected by protein electrophoresisat 0 hours,12 hours,1 day and 2 days.3.After heat stress,LC3,P62,and Beclin1 were examined by protein electrophoresisat 0 hours,12 hours,1 day,2 days,and 5 days.The autophagy flux in 16 HBE cells after heat stress were detected by LC3-GFP-mRFP adenovirus.4.After heat stress,TNF-ɑ,IL-6,IL-10 and TGF-β were detected by realtime-PCR and ELISA at 0 hours,12 hours,1 day,2 days and 5 days.5.In thecell warming model,16 HBE cells were treated with active oxygen scavenger(NAC).GRP78 and CHOP and endoplasmic reticulum stress pathway PERK / eIF2α were detected by protein electrophoresis.(NAC),the inhibitor of endoplasmic reticulum stress(GSK2656157),eLF2ɑ siRNA and the endoplasmic reticulum stress activator tunicamycin were used to treat 16 HBE cells.LC3,P62 and Beclin1 were detected by protein electrophoresis.6.Active oxygen scavenger(NAC),endoplasmic reticulum stress inhibitor GSK2656157,eLF2ɑ siRNA,CHOP siRNA and ATF4 siRNA were used to inhibit endoplasmic reticulum stress.Beclin1 siRNA,and ATG5 siRNA were used to inhibit autophagy,respectively.Realtime-PCR and ELISA were used to detect changes in heat-induced inflammatory factors.Result:1.In the cell warmingmodel,the content of reactive oxygen species was significantly increased at 0h,12 h and 1d after heat stress in 16 HBE cells.But the active oxygen content level has returned to normal on the second day.2.Thermal stimulation increased the expression of GRP78 and CHOP,and reached the peak at 12 hours,and the level gradually decreased to normal on day 2.Phosphorylated PERK and elF2α also peaked at week 12 and were gradually restored on the second day.However,the protein expression of IRE1,XBP1 s and ATF6 did not have significantchange after thermal stimulation.3.After heat stress,autophagy indicators LC3Ⅱ / LC3 I and Beclin1 were significantly elevated on day 1 and day 2 and reached their normal level on day 5.In contrast,the expression of P62 gradually decreased with the time of temperature treatment,reached the lowest value on day 1 and day 2,and reached the highest value on day 5.Laser confocal results also showed that autophagy was significantly higher on day 1 and day 2 after heat treatment and decreased to the normal range on day 5.4.After the thermal stress,Realtime-PCR results showed that the expression of TNF-ɑ,IL-6,IL-10 and TGF-β reached the highest level on day 1 and day 2,gradually returned to the normal level on day5.In the supernatant of cell culture,TNF-ɑ,IL-6,IL-10 and TGF-β were significantly increased on the first day and the second day,and decreased to normal on day 5.5.After thermal stress,the active oxygen scavenger(NAC)reduced the expression of GRP78 and CHOP in endoplasmic reticulum stress as well as the endoplasmic reticulum stress pathway PERK / eIF2α activation.Reactive oxygen scavenger(NAC),endoplasmic reticulum stress inhibitor(GSK2656157),eLF2ɑ siRNA reduced the increased LC3 Ⅱ/LC3Ⅰ,Beclin1 and the decreased P62 expression induced by heat stress.6.Realtime-PCR and ELISA showed thatthe expression of TNF-ɑ,IL-6,IL-10 and the expression of TGF-β and cytokine secretionwere reduced by NAC,GSK2656157,eLF2ɑ siRNA,CHOP siRNA,ATF4 siRNA,Beclin1 siRNA and ATG5 siRNA after heat stress.Part III Heat induced apoptosis through the activation of mitochondrial calcium channel protein(MCU)in bronchial epithelial cellmethod:1.Cell warming model was established.cell viability was detected by CCK8 kit at 0 hours,1 day,2 days,and 5 days after heat stimulation.Apoptosis was determined by flow cytometry.Apoptotic protein Bcl2 and Bax were detected by protein electrophoresis.2.Mitochondria was stained with mitochondrial marker mitotracker.Mitochondrial morphology after thermal stimulation was observed underlaser confocal.3.MCU channel activitywas detected with Patch clamp after thermal stimulation.Mitochondrial MCU calciumuptaking was determined byLaser confocal real-time dynamic detection.4.The mitochondrial basal Ca2 + in16 HBE cells was labeled with Rhod-2 AM +,and the basal Ca2 + storage was detected by laser confocal microscopy.Ca2 + uptake and basal calcium content were measured by PTI(Photon Technology International)instrument.5.ATP change in 16 HBE after thermal stimulation was detected by ATP kit.Oxygen consumption and maximum oxygen consumption after thermal stimulation were detected by Seahorse.6.Mitochondrial ROS was stained with Mitosox and the change of mitochondrial ROSafter thermal stimulation and RU360 treatment was detected under laser confocal microscopy.The levels of nitrotyrosine,4-Hydroxynonenal,catalase and superoxide dismutase(SOD)in 16 HBE were detected by ELISA.7.The mitochondrial membrane permeability was labeled withCalesin-AM,and the changes of mitochondrial membrane permeability were detected under laser confocal microscopy.Mitochondrial membrane potential were stained with TMRE and detected under laser confocal microscopy.8.The cells were treated with RU360 inhibitor or mitochondrial ROS inhibitor Mito Tempo treatment,after heat stimulation,cell viability was detected by CCK8 kit.Apoptosis was detected by Flow cytometry.Apoptosis protein Bcl2 and Bax were detected by protein electrophoresis.Result:1.Cell viability after heat treatment was significantly reduced on day1 and 2,returned to normal on day 5.Flow cytometry showed that the proportion of apoptotic cells reached the peak on days 1 and 2,and returned to normal on day 5.The ratio of Bcl-2 / Bax was reduced to the lowest level on days 1 and 2,and returned to normal on day 5.2.Laser confocal results showed that thermal stimulation can induced mitochondria stress,the matrix of linear strip became round and arranged in a loose.3.Compared with untreated group,the intensity of IMCU significantly increased after heat treatment,indicating that the heat can improve the strengthof mitochondrial MCU channel.The uptake slope of Ca2 + in 16 HBE cells after heat treatment was significantly higher than that in untreated group and RU360 treated group,as determined by laser confocal real-time dynamic detection.4.The Ca2 + fluorescence intensity in 16 HBE cells after heat treatment was significantly stronger than that in untreated group.PTI showed that the Ca2 + uptakein 16 HBE cells was significantly higher than that in untreated group and RU360 group.The storage capacity of Ca2 + in 16 HBE cells was significantly higher than that in untreated group and RU360 group after heat treatment.5.After heat treatment,ATP metabolism was significantly reduced,and the decreased ATP will rise again after RU360 inhibitortreatement.Similarly,after heat treatment,the basic oxygen consumption and maximum oxygen consumption were significantly reduced,and the basic oxygen consumption and maximum oxygen consumption increased in RU360 inhibitor treatment.6.Compared with untreated group,mitoSOX was significantly higher in heat treated group,but it was significantly reduced when RU360 was added.After heat stress,the contents of nitrotyrosine,4-Hydroxynonenal,catalase and superoxide dismutase(SOD)in 16 HBE cells was significantly higher than that in untreated group and RU360 treated group.7.After heat treatment,the mitochondrial membrane potential in 16 HBE cells was significantly decreased and thedegree opening of mitochondrial permeability transition pore was significantly increased.After treated with RU360 inhibitor or mitochondrial ROS inhibitor Mito Tempo,the mitochondrial membrane potential was significantly increased and the degree opening of mitochondrial permeable transition hole was reduced.8.After inhibting the mitochondrial ROS production by using RU360 or Mito Tempo,CCK8 showed a significant increase in cell viability,a significant decreased level of apoptosis in flow cytometry,and decreased ratio of the Bcl-2 / Bax in protein electrophoresis.Conculsion1.In 16 HBE cells treated by heat and thermal inhalation injury animal model,heat stress can induce inflammatory factors such as COX2,PGE2,TNF-ɑ,IL-6,IL-10 and TGF – β。2.Heat stress can induce inflammation by the CFTR/MAPK/ NF-κB/COX2 signal pathway.3.Heat stress regulates the production of inflammatory factors through the ROS/endoplasmic reticulum stress/autophagy pathway.4.Heat stress can activate the MCU,causing the cell’s mitochondrial calcium overload,mitochondrial membrane potential and energy metabolism changes to cause apoptosis. |
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