Effect Of Hydrogen On Hyperoxia-induced Lung Injury And Repair And The Mechanism Research

BackgroundOxygen therapy is commonly used in clinical treatment, butcontinuous oxygen therapy with high concentration can cause oxygentoxicity and easily lead to chronic lung disease （CLD） orbronchopulmonary dysplasia （BPD） in newborns, especially prematurechildren, which seriously threaten to the children’s health. There are noeffective prevention or cure methods to CLD and BPD currently. Thenormal repair of alveolar epithelial injury is mainly dependent on theproliferation and differentiation of alveolar type Ⅱ epithelial cells （AECⅡ）.Hyperoxia lead to the AECⅡ oxidative stress damage and inhibit theproliferation of AECⅡ is one of the main mechanism of BPD occurrence.Traditional antioxidants can reduce hyperoxia-induced lung injury, but alsointerfere the normal development of lung. Hydrogen （H₂） is the focus of avariety of diseases research for its selective antioxidation and the relativesafety, but the molecular mechanisms of its fuction is unclear. FoxO are aclass of transcription factors which are closely associated with oxidative stress, apoptosis, proliferation and development. MAPK and PI3K/Akt andother signaling pathways can regulate their activities. It has been proventhat H₂can regulate the activities of ERK, JNK, P38and Akt which are thekey enzymes of MAPKs and PI3K/Akt signaling pathway.We speculatethat the H₂may play a protective role in the hyperoxia-induced lung injuryby regulating the FoxO signaling pathways. Study of H₂inhyperoxia-induced lung injury and the FoxO signaling mechanisms maybring new breakthroughs in the prevention and treatment of BPD and newprogress in mechanism research of H₂.Objective1. Separate and cultivate primary premature rat AECⅡ withhigh-purity and high vitality; Establish stable hyperoxia-induced celldamage model; Establish an animal model of hyperoxia-induced lunginjury in neonatal rats. Lay the foundation for the subsequent H₂intervention experiments and the mechanism research.2. Observe the effect of H₂on the hyperoxia-induce AECⅡ injury andexplore if the effect is related with the FoxO signaling pathway.3. Observe the effect of H₂on the hyperoxia-induced lung injury innewborn rat and explore the possible mechanisms of FoxO signalingpathway in it.Methods1. SPF level Sprague-Dawley （SD） rats with19days gestational age, chloral hydrate anesthetize it. Take out the fetal rats. Isolate lungs and cutinto pieces. Digest the lung tissue and cells to cell suspension with trypsinand collagenase. Purify the AECⅡ with the methods of differentialcentrifugation and repeated attachment. Culture the cells with DMEM/F12medium containing10%fetal bovine serum. Assess the cell viability bytrypan blue staining and evaluate the cells purity by modified papanicolaoustaining. Identify the cell by transmission electron microscopy. Observe thecellular growth status under inverted phase contrast microscope.2. Randomly divide the primary AECⅡ cultured24h in vitro into airgroup and hyperoxia group. Place the hyperoxia group cells in oxygenchamber which filled with a concentration of95%oxygen. Then place thecell in the cell incubator together with the cells of air group.24h later,observe the cell morphology, detect cell proliferation by MTT and detectthe apoptosis and survival by flow cytometry.3. Randomly divide the SD neonatal rats into air group and hyperoxiagroup. Put the rats of hyperoxia group in the animal oxygen chamber inwhich the oxygen concentration kept greater than95%.Then put the rats oftwo groups in the same room.3d,7d,14d,21d later, remove the lungtissue for pathological examination, count RAC（Radial alveolar count）values and detect the content of hydroxyproline （HYP） in the lung tissue bychemical colorimetric method.4. Randomly divide AECⅡ into air group, hyperoxia group, air+H₂ group and hyperoxia+H₂group. Intervene the cells of H₂group withhydrogen-rich medium.24h later, observe the AECⅡ morphologicalchanges; Evaluate the cell proliferation by detecting MTT, cell cycle andproliferating cell nuclear antigen （PCNA） protein expression; Assess thedegree of cell damage by detecting mitochondrial membrane potential （△Ψ） and apoptosis rate; Evaluate the cellular oxidative damage andantioxidant capacity by detecting intracellular level of oxygen （ROS） andsuperoxide anion （O-2）, and the malondialdehyde （MDA） level andsuperoxide dismutase （SOD） activity of cell culture supernatant; Detect theprotein expression of total FoxO3a and β-catenin protein, also thep-FoxO3a and p-β-catenin by Western blot.5. Randomly divide SD neonatal rats into air group, air+hydrogen-richsaline group, air+H₂group, hyperoxia group, hyperoxia+hydrogen-richsaline group and hyperoxia+H₂group. Put the rats of the hyperoxia groupsin the animal oxygen chamber in which the oxygen concentration kept at95%. H₂intervention: Intraperitoneal inject hydrogen-rich saline10mL/kg,2times per day in rats of hydrogen-rich saline groups; Intraperitoneal injectH₂gas10mL/kg,2times per day in rats of H₂groups; Intraperitonealinject saline10mL/kg,2times a day in rats of non-H₂intervention groupsas control.14d later, remove the lungs for pathological examination;Detect the MDA level and SOD activity of serum; Determine the HYPcontent of the lung tissue; Detect the α-SMA protein expression of lung tissue by immunohistochemical method; Detect the total andphosphorylated FoxO3a and β-catenin protein expression of lung tissue byWestern blot.Results1. The yield of primary cultured AECⅡ is good and each premature ratlung can obtain the number of AECⅡ about （8.5±1.8）×106. The cellviability is about （95.0±2.1）%and the purity is about （94.3±2.5）%. Themicrovillus on the cell membrane and the lamellar bodies in cytoplasmwhich are the characteristic structure of AECⅡ were observed underelectron microscope. AECⅡ start to attach the bottom after cultured12hand most of the cells closed to the bottom and stretched. After cultured24-48h, cells were in exponential growth phase. After cultured72h,AECⅡ became flat and decreased attachment.2. After cultured24h in vitro, then stimulated by95%oxygen, AECⅡshrunk and the cell gap increased. Compared to air group, the OD492valueand survival rate decreased and the apoptosis rate increased significantly.3. After hyperoxia-exposed3d and7d, pathological examinationshowed changes including alveolar epithelial cell swelling, interstitialhyperemia and edema, inflammatory cell infiltration, lung structuraldisorder, and the changes were more obvious in7d. Afterhyperoxia-exposed14d and21d, fibrosis was visible, alveolar septawidened significantly, and the HYP content of the lung tissue was significantly higher than that of the air group, and the changes were moreobvious in21d.The RAC values of hyperoxia groups were significantlylower than air groups at7d,14d,21d.4. Compared with air group, H₂intervention up-regulated the totalFoxO3a, down-regulated the p-FoxO3a of AECⅡ and had no obvious effecton the other indicators. Compared with air group, hyperoxia exposuredecreased the OD492values and PCNA protein expression; increased thecell proportion of G1phase and reduced the cell proportion of S phase;reduced the cell△Ψ and increased the apoptosis rate; improved theintracellular ROS and O-2levels; increased the MDA content and reducedthe SOD activity of cell supernatant; up-regulated the total FoxO3a andp-β-catenin protein expression; down-regulated the p-FoxO3a and totalβ-catenin protein expression. Compared with hyperoxia group, H₂intervention reduced the above-mentioned change induced by hyperoxia.5. Compared with air group, FoxO3a and β-catenin of lung tissueswere activiated in air+hydrogen-rich saline groups and air+H₂groups, andthere was no significant difference in other indicators. Compared with airgroup, hyperoxia exposure induced lung stunting; widened the lung tissueinterval; induced obvious fibrosis; increased the HYP content of the lungtissue; up-regulated the expression of α-SMA; elevated the serum MDAlevels and decreased the SOD activity; improved the total FoxO3a proteinexpression in lung tissue and suppressed the p-FoxO3a which were more obviously than that induced by hydrogen; increased total β-catenin andp-β-catenin protein expression in lung tissue. Compared to hyperoxia group,the lung damages of hyperoxia+hydrogen-rich saline group andhyperoxia+H₂group were extenuated. The above-mentioned changesinduced by hyperoxia were reduced by H₂intervention. The serum MDAlevel and the HYP content of lung tissue were little lower in hyperoxia+H₂group than that in hyperoxia+hydrogen-rich saline group, and otherindicators had no difference.Conclusions1. The yield, purity and vitality of primary AECⅡ which cultured bythe method of trypsin joint collagenase digestion, differential centrifugationand repeated adherent were high enough to meet the need of the cytologyexperimental research. AECⅡ were in best status cultured24-48h in vitroand were suitable for studies.2.95%oxygen concentration can significantly induce AECⅡ damage,apoptosis and inhibit their proliferation, also can cause the lung tissue ofnewborn rats damage and fibrosis. The hyperoxia-induced cells andanimals damage models were successfully established.3. Hydrogen-rich medium can alleviate the hyperoxia-induced AECⅡapoptosis, oxidative damage and inhibition of proliferation to some extent,and hydrogen-rich medium had no effect on the proliferation of normalAECⅡ. 4. Intraperitoneal injection of hydrogen-rich saline or H₂gas caneffectively reduce the hyperoxia induced lung damage, reduce pulmonaryfibrosis. Intraperitoneal injection of H₂gas obtained better effects. Therewas no significant influence of H₂intervention on normal lung tissue.5. The protective effect of hydrogen on the cell and animal injury maythrough inhibiting the excessive activation of FoxO3a protein caused byhyperoxia and activiating the β-catenin protein.