The Effect Of The Proteasome Inhibitor MG-132 On Lung Injury Induced By Hyperoxia In Rats And Its Mechanism

Backgroud:Oxygen,one of the most abundant elements in our world,is essential for the oxidation of organic compounds to generate the energy needed to sustain life.Under ambient conditions,reactive oxygen species(ROS) are generated at a low level in lung cells during aerobic metabolism.To minimize the oxidant injury that is a consequence of aerobic life,the human lung is endowed with an integrated antioxidant system,which detoxifies reactive products.However,excessive ROS may overwhelm the antioxidant system and result in damage to major cellular components.The pathophysiologic consequences of this injury may include cell death, and tissue inflammation and damage.The bronchial epithelium is particularly vulnerable to the effects of airborne oxidative stress as the moist mucosal surface of the airway is in direct contact with the environment.Oxidative damage of proteins and loss of biological function is one defined end-point of hyperoxic injury.In the past,significant attention has focused on clarifying the role of antioxidant enzymes such as superoxide dismutases,catalase,or glutathione peroxidases in mitigating airway damage resulting from hyperoxia.In this context,we hypothesized that in the absence of increased protection against oxidative injury,clearance pathways for oxidatively damaged proteins may have a central role in the early response of cells to maintain the homeostatic integrity of the epithelial layer.Substantial evidenceindicates that the ubiquitin-proteasome system is responsible for degrading altered proteins in the cytoplasm,nucleus,and endoplasmic reticulum of cells. Substantial evidenceindicates that the ubiquitin-proteasome system is responsible for degrading altered proteins in the cytoplasm,nucleus,and endoplasmic reticulum of cells.Some researches suggested the NF-κB way which can be actived by the UPS,play an important role in the pathological changes of hyperoxia injury of lungs.It is uncleared wheather UPS contribute on the pathological process of hyperoxia injuries of lungs directly.During onset of hyperoxia lung injury,the relationgship between oxidative stress and the activity of ubiquitin-proteasome pathway,wheather proteasome inhibitors can influence the pathophysiological progress of hyperoxia lung injury mediated by multiple mediators of inflammation through inhibiting the ubiquitin-proteasome pathway,are also uncleared.At present, there is no report about MG-132,as one kind of proteasome inhibitors,being used to the study of hyperoxia lung injury.Therefore,we hypothesized that inhibiting the ubiquitin-proteasome pathway's reactive may reduce the activition of inflammatory factors and hyperoxia injurie,if the ubiquitin-proteasome pathway related with pathophysiological progress of hyperoxia injuries.Objective(1) Observe the effects of hyperoxic exposure on lungs in early stages.(2) The histomorphology and oxidative stress index were observed to evaluated the extent of lung injury induced by hyperoxia.Observe the activity of ubiquitin-proteasome pathway in pathophysiological progress of hyperoxia lung injury.Discuss the effects of proteasome inhibitor MG-132 on hyperoxic lung injury.(3) Observe the infiltration of leucocyte neutrophil and the changes of NF-κB/inflammatory factor access,then discuss the potential mechanism of proteasome inhibitor MG-132.Methods:(1) Establish hyperoxia lung injury rats model.(2) Adopt completely random design.30 SD rats were randomly divided into 3 groups:Exposure to O₂ was performed in a airtight plastic cage.O₂ was provided continuly.(1) control group with air:The O₂ concentration exiting the cages was analyzed with a O₂ monitor and O₂ concentration remained 21%for 72 hours, intraperitoneal injection of DMSO 0.25 ml every day for three days from the first day of experiment;(2)hyperoxia group:the condition changed into high-density oxygen(Fi02＞0.95) for 72hours,;(3)hyperoxia add to MG-132 group:Intraperitoneal injection of MG-132(10mg/kg,dissolved in DMSO 0.25 ml) every day for three days from the first day of experiment.O₂ exposure procedure is same as hyperoxia group.Observe heart rate and the breath condition of the rats respectively at 0,24,48,72 hour point during the experiment,detect other indexes in 72 hour。(3) To estimate the rats model,the heart rate and the breath conditions of the rats were observed by the electrocardiogram,the contents linked morphology as pathological classification in gross finding,Pathological score of lung injury in Light Micros and the index of pneumonedema—the ratio of moist to dry weight of lungs were measured.(4) Determin the concentration of malondialdehyde(MDA) and the activity of the Speroxide dismutase(SOD) in lung homogenate which reflect the oxidative damage and oxidative stress of lung tissue indirectly.(5) Detect the expressions of ubiquitin protein(Ubiquitin/Ubiquitin-Conjugates) and the activity of proteasome 20S and estimate the active statement of Ubiquitin-proteasome pathway by immunohistochemistry and West blot methods.(6) Detect the activity of myeloperoxidase in lung homogenate,the expression of NF-κB p56 by immunohistochemistry methods and cytokine TNF-αand IL-6 by RT-PCR in lung tissue and then the activity conditions on NF-κB/inflammatory access were reflected by it.Result(1) The hyperoxia lung injury rat model was successfully duplicated.(2) In hyperoxia group,the rate of resperation and heart became fast gradually and pulmonary edema with increased ratio of moist to dry weight of lungs can be found(P＜0.01).Compared with hyperoxia group,the rate of resperation and heart of the rats reduced relativly(P＜0.05)and pulmonary edema mitigated with reduced ratio of moist to dry weight of lungs(P＜0.01) at 72 hours point in hyperoxia add to MG-132 group. (3) Macroscopic observation:bright red and full-stacked lung tissue,foliated or lacal hemorrhage on the surface,few Pleural Effusion were observed in hyperoxia group.There were statistically significance on pathological classification in gross finding(P＜0.01) between hyperoxia group and control with air group.Light microscope observation:swelled alveolar epithelium,widen alveoli wall,capillary engorgemen and telangiectasis,obvious edema in interstitial tissue of pulmonary aveolus and alveolar space,increased inflammatory cells were observed in hyperoxia group.The findings of pathological score of lung injury indicated more serious injure than control group(P＜0.01).The proteasome inhibitor MG-I32 can mitigate hyperoxia lung injury and there are significance on pathological score of lung injuryand pathological classification in gross finding(P＜0.01) between hyperoxia group and hyperoxia add to MG-132 group pathological evaluation.(4) compared the control with air group,the lever of MDA and the activity of SOD both increased(P＜0.01) in lung homogenate in hyperoxia group but there was no statistically significant compared with hyperoxia add to MG-132 group.(5) The increased expression of ubiquitin protein in lung tissue was discoved by immunohistochemistry and West blot findings after hyperoxia exposuring 72 hours.(p＜0.01) and that can be induced by Proteasome inhibitor MG-I32.(p=0.000in immunohistochemistry,p=0.022 in westen blot)(6) The activity of proteasomes 20S in hyperoxia group is higher than in the control with air group(P＜0.01) and that can be inhibited by Proteasome inhibitor MG-I32(P＜0.01)(7) The activity of MPO and the expression of NF-κB and inflammatory factor as TNF-α,IL-6 were both enhanced compared with the control with air group P＜0.01. Proteasome inhibitor MG-I32 inhibited the infiltration of inflammatory cells, activitity of MPO(P＜0.05) and the expression of NF-κB(P＜0.05) and TNF-α(P＜0.05),IL-6(P＜0.05).Conclusion:(1)The hyperoxia lung injury rat model was successfully duplicated. (2)The mainly pathological changes of lung are generated by hyperoxic exposure 72 hours,including alveolar epithelial cell and vascular endothelial cell injury disffusly,inflammatory cell infiltration and pulmonary edema.(3)Active the ubiquitin-proteasome pathway related with the pathophysiological process of lung injury in the initial stage of e hyperoxia-exposure.(4)The proteasome inhibitor MG-I32 can mitigate the lung injury induced by hyperoxia.(5) The protection of the proteasome inhibitor MG-I32 on hyperoxia lung injury may owe to inhibitting the access of NF-κB/inflammatory factor.(6)UPS may be one of the new targets for prevention or treatment hyperoxia lung injury.