The Role And Mechanism Of Peroxiredoxin 6 In High-Volume Ventilation Induced Lung Injury

Peroxiredoxin 6（PRDX6）is a multifunctional protein with multiple active sites.Its most important functions include peroxidase activity and phospholipidase A2（PLA2）activity.PRDX6 is the only protein with single-cysteine selenium-independent PRDXs,and its phospholipase A2 activity is not calcium-dependent.PRDX6 plays an important role in maintaining the redox balance of lung tissue,repairing membrane damage caused by oxidative stress,and participating in the regulation of lung inflammation and injury.PRDX6 mainly functions through the following two aspects:On the one hand,PRDX6eliminates excessive ROS in the lungs through its GSH-Px enzyme function,maintains lung homeostasis,and has a positive protective effect.On the other hand,When a variety of lung pathological injuries induce oxidative stress,the i PLA2 enzyme activity of PRDX6 will up-regulated,which is an important reason why PRDX6 mediates cytotoxicity and tissue damage.So far,whether PRDX6 is involved in the pathogenesis of VILI has not been reported.In this study,wild-type and PRDX6 knockout rats were used as the research object.Ventilation-induced lung injury（VILI）rat model was constructed through long-term high-volume ventilation to observe the role of PRDX6 in VILI model of rats and look for potential prevention targets about PRDX6.First,through histopathological staining,the detection of inflammatory factor transcription levels and the detection of oxidative stress-related products,we found that compared with wild-type（WT）rats,PRDX6 KO rats may have mild spontaneous inflammation under physiological conditions reaction.After high-volume ventilation,the lung tissue damage of KO rats was more severe than that of WT rats.This suggests that PRDX6 is predominantly protective in VILI,and this protective effect is guessed to be achieved by exerting its peroxidase activity.In order to further clarify the role of the two enzyme activities of PRDX6 in this model,we injected PLA2 inhibitor-MJ33 into the abdominal cavity before ventilation.The results showed that MJ33 intervention significantly reduced pathological indicators,relieved lung inflammation,and reduced oxidative stress products.This result suggests that after using MJ33,PRDX6 can exert a better protective effect on VILI through its peroxidase activity.This result also suggests that PLA2 may be a potential target for preventing lung injury from mechanical ventilation.Objective:1.To observe the the role of PRDX6 in ventilation-induced lung injury in rats;2.To explore the molecular mechanism of PRDX6 in VILI;3.To explore the preventive measures for VILI with PRDX6 functional activity as the target.Methods:The wild-type SD rats will randomly divide to three groups:sham group,model group and MJ33 intervention group.The PRDX6 knockout rats randomly divide to two groups:sham group and model group.Two sham groups undergo tracheotomy as a control,model groups and MJ33 group will be ventilated with high volume after tracheotomy for3 hours to construct ventilation-induced lung injury model.The intervention group was intraperitoneally injected with phospholipase A2 inhibitor MJ33 before ventilation.Polymerase chain reaction（PCR）was detected to verify the PRDX6 gene knockout successfully.Hematoxylin-eosin staining（HE）staining,tissue wet-to-dry weight ratio,and cell counting in bronchoalveolar lavage fluid were used to evaluate the degree of lung injury in each group.Western blotting（WB）and immunohistochemical staining（IHC）were used to observe the changes in PRDX6 protein levels,IHC to detect the levels of related inflammatory factors,fluorescent quantitative q PCR to detect the transcription levels of inflammatory factors,and enzyme reduction to detect the levels of oxidative stress-related products H₂O₂.Results:1.High-volume ventilation will cause lung tissue damage in rats and increase the level of PRDX6 protein;In the high-volume ventilation model,we used HE staining,lung tissue wet-to-dry weight ratio,bronchoalveolar lavage fluid（BALF）cell count and protein quantification to determine the successful establishment of the VILI model and assess the degree of lung tissue damage.Western blotting and IHC staining were used to observe the expression of PRDX6 protein in lung tissue.The results showed that the lung tissue of the model group was significantly damaged,the wet-to-dry ratio increased,the cell count in BALF increased,and the expression of PRDX6 protein in the lung tissue increased,compared with the sham group.2.The lung injury caused by high-volume ventilation increased after knocking out the PRDX6 gene;In order to explore the significance of up-regulated PRDX6 protein levels,we constructed PRDX6 knockout（KO）rats.We found that after knockout of PRDX6 gene,the lung tissue of the KO sham group rats developed mild spontaneous inflammation.It shows that the lung tissue damage in the KO rats model group is more severe than that of the WT rats.3.The degree of lung injury caused by high-volume ventilation in rats is relieved after using MJ33.To further explore the mechanism of PRDX6 lung protection,we used PRDX6phospholipase A2（PLA2）activity inhibitor MJ33 to intervene.The results showed that after intraperitoneal injection of MJ33,the lung tissue damage in the model group was reduced,and the oxidative stress level was relieved.This result suggests that after inhibiting the phospholipase A2 activity of PRDX6,PRDX6 can better protect lung tissue from mechanical damage by alleviating oxidative stress and inflammation.Conclusions:1.The level of endogenous PRDX6 protein in lung tissue increases during VILI;2.Endogenous PRDX6 up-regulation has a protective effect in VILI;3.The peroxidase activity of PRDX6 protect lung injury in VILI.