Overexpression Of SIRT3 In Dexmedetomidine Ameliorates Lung Ischemia-Reperfusion Injury:A Study Of Its Mechanism

Part One Lung protective effect of dexmedetomidine on one lung venti-lation in elderly patientsObjective:To evaluate the effects of dexmedetomidine on one lung ventilation related lung injury and transforming growth factorβ1（TGF-β1）/smads signaling pathway in elderly patients undergoing thoracic surgery.Methods:80 elderly patients scheduled for elective thoracoscopic lobectomy were divided into Dex group and Control group（n=40）by a random number table method.In the Dex group,15min before induction of anaesthesia,dexmedetomidine was continuously pumped intravenously with a loading of 0.5μg·kg^-1,following by 0.3μg·kg^-1·h^-1 at a rate continuously pumped to 30min before the operation,and the Control group was given an equal volume of normal saline.Oxygenation index（OI）was calculated by blood gas analysis performed 15 min before anesthesia induction（T₀）,30min of one lung ventilation（T₁）,90min of one lung ventilation（T₂）,and immediately after recovery from two lung ventilation（T₃）,and pulmonary dynamic compliance（Cdyn）was calculated at T_1-3;Peripheral blood levels of clara cell protein 16（CC16）,surfactant protein-D（SP-D）,inflammatory cytokine tumour necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）concentration were measured on T_0-3;Lung structural changes and lung injury scores were observed by HE staining;Meanwhile,the expression of TGF-β1,smad2 and smad3 in peripheral blood monocytes and lung tissue were detected.Results:OI and Cdyn were increased in elderly patients at T_1-3 in the Dex group compared to Control group,and CC16,SP-D,TNF-α,IL-1βof concentrations were significantly lower;On light microscopy,the structure of lung tissue was relatively intact,and the alveolar wall capillary dilation and congestion were mild,accompanied by reduced neutrophil infiltration,less alveolar exudation,and lower lung pathological injury scores.Peripheral blood monocyte TGF-β1 m RNA,smad2 m RNA,and smad3 m RNA were down regulated;The protein expressions of TGF-β1,smad2 and smad3 were down regulated in lung tissue（P<0.05）.Summary:Perioperative continuous pump infusion of dexmede-tomidine in elderly patients undergoing thoracoscopic lobectomy can attenuate the inflammatory response of lung tissue and improve acute lung injury during one lung ventilation,and part of the mechanism may be related to the down-regulation of TGF-β1/smads signaling pathways involved by dexmedetomidine.Part Two Dexmedetomidine attenuates ischemia reperfusion induced lung injury in mice by activating SIRT3 in lung tissueObjective:To determine the effects of dexmedetomidine on ischemia-reerfusion induced lung injury in C57BL/6J mice.Methods:C57BL/6J mice were randomly divided into 4 groups:Control group,dexmedetomidine（DEX）group,pulmonary ischemia-reperfusion（IR）group and DEX+IR group.Lung ischemia-reperfusion model was established.After the successful construction of the model,the arterial blood gas value of mice in each group was analyzed,and the levels of clara cell secretory protein（CC16）,pulmonary surfactant related protein D（SP-D）,inflammatory factor tumor necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）and the levels of reactive oxygen species（ROS）,malondialdehyde（MDA）,glutathione peroxidase（GSH-Px）and superoxide dismutase（SOD）were detected.Hematoxylin-Eosin（HE）staining was used to observe the morphological changes of mice lung tissue.Immunohistochemistry（IHC）and Western blot were used to detect the expression of SIRT3 in lung tissue.Results:1.Compared with the Control group,the indexes of DEX group had no statistical significance;blood gas Pa CO₂,lung injury marker CC16,SP-D and inflammatory factor TNF-α,IL-1βin peripheral blood of IR group.The levels of Pa O₂,GSH-Px and SOD in blood gas decreased significantly（P<0.05）;DEX+IR group could significantly improve the above indexes（P<0.05）.2.Compared with the Control group,there were no significant changes in lung pathological sections in Dex group under light microscope;In IR group,a large number of inflammatory cells were infiltrated,the alveolar structure was seriously damaged,and the lung W/D ratio was significantly increased;DEX+IR group significantly reduced these changes（P<0.05）.3.Compared with control group,IHC staining and Western blot showed that the expression of SIRT3 in lung tissue of IR group was lower;IHC staining and Western blot showed that the expression of SIRT3 in lung tissue was increased in Dex+IR group（P<0.05）.Summary:Dexmedetomidine can reduce the injury caused by pulmonary ischemia-reperfusion in mice,improve arterial blood gas index,reduce the levels of lung injury markers CC16 and SP-D,and reduce the inflammatory factor in ischemia-reperfusion TNF-α,IL-1β.At the same time,it can also reduce the levels of ROS and MDA,increase the levels of antioxidants GSH-Px and SOD,and improve the morphological changes of lung tissue in ischemia-reperfusion mice.Part of the reason may be related to the increased expression of SIRT3 in lung tissue.Part Three Dexmedetomidine achieves lung protective effects by regulating mitochondrial function and apoptosisObjective:Previous studies have found that dexmedetomidine can improve acute lung injury induced by ischemia-reperfusion in mice,in this part of the experiment,by applying dexmedetomidine,we observed the mitochondrial function of lung tissue and the expression of its related proteins in mice with pulmonary ischemia-reperfusion,and clarified whether dexmedetomidine can achieve the effect of protecting lung tissue by regulating mitochondrial function and cell apoptosis.Methods:Healthy clean grade male C57BL/6J mice were divided into five groups of 6 mice each:control（Control）,lung ischemia-reperfusion（IR）,dexmedetomidine（DEX+IR）,inhibitor+dexmedetomidine（3TYP+DEX+IR）,and inhibitor alone（3TYP+IR）.Referring to the previous experiment,the pulmonary ischemia-reperfusion model was prepared.After the success of model construction,the mice were sacrificed,and the lung tissue samples were immediately removed to detect the expression of Bcl-2,Bax,cleaved caspase-3,Nrf2,HO-1 and cytochrome C by Western blot.The changes of mitochondrial membrane potential（MMP）and ATP content in lung tissue were detected,the apoptosis was detected by TUNEL,and the expression of Nrf2 was observed by immunofluorescence（IF）.Results:1.Compared with the Control group,Western blot showed that post ischemia reperfusion in the lungs of mice in the IR group could significantly downregulate Bcl-2 expression,as well as enhance Bax,cleaved caspase-3expression and increase the TUNEL positive rate（P<0.05）;Compared with the IR group,the DEX+IR group significantly promoted Bcl-2 expression,inhibited Bax,cleaved caspase-3 expression,and decreased the percentage of TUNEL positive cells after pulmonary ischemia-reperfusion（P<0.05）;In contrast to the DEX+IR group,the effect of dexmedetomidine to inhibit apoptosis was curtailed due to the inhibition of SIRT3 in the 3TYP+DEX+IR group（P<0.05）.2.Compared with the Control group,mice in the IR group showed decreased levels of MMP and ATP（P<0.05）due to pulmonary ischemia-reperfusion;However,the DEX+IR group prevented the decrease in MMP and ATP levels and improved mitochondrial function（P<0.05）;However,the inhibition of SIRT3 expression in the 3TYP+DEX+IR group partially blocked dexmedetomidine ameliorated mitochondrial function after pulmonary ischemia-reperfusion（P<0.05）.Compared with the Control group,mice in the IR group could undergo significant translocation of cytochrome C from the mitochondria to the cytoplasm as a result of pulmonary ischemia-reperfusion（P<0.05）;Interestingly,the DEX+IR group signifi-cantly reversed the shift of cytochrome C,whereas the 3TYP+DEX+IR group weakened the dexmedetomidine inhibition of cytochrome C shift（P<0.05）.3.IF and Western blot showed increased nuclear accumulation of Nrf2and increased expression levels of Nrf2 and HO-1 in IR group due to pulmonary ischemia-reperfusion compared with Control group（P<0.05）;Compared with the IR group,the nuclear accumulation of Nrf2 was further increased,and the protein expression levels of Nrf2 and HO-1 were further increased in the lungs of DEX+IR mice（P<0.05）;Although SIRT3 was inhibited in the 3TYP+DEX+IR group,the protein expression of Nrf2 and HO-1 was not significantly different from that in the DEX+IR group（P>0.05）.Summary:Lung ischemia-reperfusion can cause mice lung tissue mitochondrial function disorder and appear apoptosis,and dexmedetomidine may alleviate pulmonary ischemia-reperfusion injury partly by up-regulating SIRT3 expression,alleviating oxidative stress stimulation,improving lung tissue mitochondrial function,and inhibiting apoptosis.Part Four Effect of SIRT3/SOD2 signaling pathway on hypoxia/reoxygenation injury in alveolar type II epithelial cellsObjective:In this study,we aimed to simulate the ischemia/reperfusion process induced by one lung ventilated non ventilated side lung from an in vitro cytological perspective,and to investigate the mechanism of SIRT3/SOD2 signaling pathway on alveolar type II epithelial cells injury induced by hypoxia/reoxygenation,in the hope of providing a new preventive therapeutic idea on one lung ventilation induced lung injury in thoracic surgery patients.Methods:Type II alveolar epithelial cell（A549 cell）culture system was established,A549 cells were transfected with empty plasmid（pcdna3.1-NC）and SIRT3 overexpression plasmid（pcdna3.1-SIRT3）,respectively,and hypoxia/reoxygenation induction was performed.The experiments were divided into four groups:control（Control）,hypoxia/reoxygenation（HR）,empty transfection+hypoxia/reoxygenation（NC）,and SIRT3overexpression+hypoxia/reoxygenation（SIRT3）.RT-PCR and Western blot were used to detect SIRT3 expression levels in each group,CCK-8 assay was used to evaluate cell viability,flow cytometry was used to detect mitochondrial membrane potential（MMP）,intracellular ATP content,complex I activity,apoptosis was detected by TUNEL,apoptosis related proteins and the expression of SIRT3/SOD2 signaling pathway.Results:Compared with the Control group,after hypoxia/reoxygenation,the viability of A549 cells decreased,the function of mitochondria decreased:the MMP,the content of ATP and the activity of complex I decreased,the expression of cleaved caspase-9 and cleaved caspase-3 increased,the apoptosis rate increased,the expression level of SIRT3 decreased,and the ratio of ac-SOD2/SOD2 increased in HR group（P<0.05）;Compared with HR group,the activity of A549 cells in SIRT3 group increased,the content of MMP,ATP and the activity of complex I decreased,the expression of cleaved caspase-9 and cleaved caspase-3 decreased,the apoptosis rate decreased,the expression level of SIRT3 increased,and the ratio of ac-SOD2/SOD2decreased（P<0.05）.Summary:SIRT3 overexpression attenuates hypoxia/reoxygenation injury in alveolar type II epithelial cells,and the mechanism may be related to the inhibition of SOD2 deacetylation.Conclusion:1.Perioperative application of dexmedetomidine can inhibit inflammatory response and improve lung injury caused by one lung ventilation in elderly patients.The mechanism may be related to the down-regulation of TGF-β1/smads signaling pathway.2.Part of the mechanism of dexmedetomidine reducing lung injury in lung ischemia-reperfusion mice may be related to the up regulation of SIRT3expression.3.In vitro cell experiments showed that overexpression of SIRT3 could ameliorate the damage of type II alveolar epithelial cells caused by hypoxia/reoxygenation,improve the function of mitochondria and inhibit apoptosis.Its mechanism may be related to the inhibition of SOD2 deacetylation.