氢气在高氧肺损伤修复中的作用及相关机制研究
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摘要
背景
氧疗是临床常用的一种治疗手段，但持续高浓度氧疗易引起机体氧中毒，在新生儿特别是早产儿易导致慢性肺疾病（CLD）或支气管肺发育不良（BPD）的发生，严重影响患儿健康，目前尚无确切有效的防治方法。肺泡上皮损伤的正常修复主要依赖肺泡Ⅱ型上皮细胞（AECⅡ）的增殖与分化，高氧导致AECⅡ氧化应激性损伤，并抑制AECⅡ增殖是BPD发生的主要机制之一。传统抗氧化剂在减轻高氧肺损伤的同时干扰了正常肺发育。氢气（H_2）的选择性抗氧化作用和相对安全性使H_2成为治疗多种疾病的研究热点，但其具体分子机制不清。叉头框蛋白O（FoxO）是一类与氧化应激、凋亡、增殖、发育等密切相关的转录因子，其活性受丝裂原活化蛋白激酶（MAPKs）和磷酸酰肌醇3激酶/蛋白激酶B（PI3K/Akt）等多种信号途径调控，而业已证实H_2对MAPKs信号通路中重要的关键酶细胞外调节蛋白激酶（ERK）、c-Jun氨基末端激酶（JNK）、P38等的活性和PI3K/Akt信号通路中Akt活性均具有调控作用。我们推测H_2可能通过调控FoxO信号途径对高氧肺损伤发挥保护作用。深入研究H_2在高氧肺损伤中的作用及其FoxO信号机制可能为临床防治BPD带来新突破，为H_2的机制研究带来新进展。
目的
1.分离培养高纯度、高活力的原代早产大鼠AECⅡ细胞；建立高氧致AECⅡ细胞损伤模型；建立高氧致新生鼠肺损伤动物模型。为后续H_2干预实验及机制研究奠定基础。
2.观察H_2对高氧致AECⅡ细胞损伤的作用，探讨其作用是否与FoxO信号途径有关。
3.观察H_2对高氧致新生鼠肺损伤的作用，探讨FoxO信号途径在其中的可能机制。
方法
1. SPF级孕19d Sprague-Dawley(SD)大鼠，水合氯醛麻醉后剖宫产取出胎鼠，分离肺脏，剪碎，胰蛋白酶联合胶原酶消化肺组织细胞，制成细胞悬液，差速离心和反复贴壁纯化AECⅡ，含10%胎牛血清（FCS）的DMEM/F12培养基培养细胞。台盼蓝染色法检测细胞活力，改良巴氏染色法检测细胞纯度，透射电镜鉴定细胞，倒置相差显微镜下观察细胞的生长情况。
2.原代AECⅡ体外培养24h后，随机分为空气组和高氧组，高氧组细胞置于氧体积分数为95%（95%浓度氧）的细胞氧仓中，氧仓与空气组细胞一并放于细胞培养箱中。24h后观察细胞形态，MTT检测细胞增殖，流式细胞仪检测细胞的凋亡及存活情况。
3. SD新生大鼠随机分为空气组和高氧组，高氧组大鼠置于氧体积分数为95%的动物氧仓中，与空气组大鼠置于同一室内。3d，7d，14d，21d后取出肺组织行病理学检查，辐射状肺泡计数（RAC），化学比色法测定肺组织羟脯氨酸（HYP）含量。
4.原代分离培养的AECⅡ随机分为空气组、高氧组、空气+H_2组、高氧+H_2组。H_2组细胞用富氢培养基干预。24h后观察AECⅡ的形态变化；检测MTT、细胞周期和增殖细胞核抗原（PCNA）蛋白表达观察细胞增殖情况；检测细胞线粒体膜电位（△Ψ）和凋亡率观察细胞损伤情况；检测细胞内活性氧（ROS）和超氧化物阴离子（O-2）水平，细胞培养上清丙二醛（MDA）水平和超氧化物歧化酶（SOD）活性，观察细胞氧化损伤和抗氧化能力；Western Blot检测细胞总FoxO3a、β-catenin蛋白和p-FoxO3a、p-β-catenin蛋白的表达。
5. SD新生大鼠随机分为空气组、空气+富氢生理盐水组、空气+H_2组、高氧组、高氧+富氢生理盐水组和高氧+H_2组。各高氧组大鼠均置于氧体积分数为95%的动物氧仓中。H_2干预：富氢生理盐水组大鼠予腹腔注射富氢生理盐水10mL/kg，每天2次；H_2组大鼠予腹腔注射H_2气体10mL/kg，每天2次。非H_2干预组大鼠则腹腔注射等量生理盐水。14d后，取肺组织做病理学检查；检测大鼠血清MDA水平和SOD活力；测定肺组织HYP含量；免疫组化法测定肺组织α-平滑肌激动蛋白（α-SMA）表达；Western blot检测肺组织总FoxO3a、β-catenin蛋白和p-FoxO3a、p-β-catenin蛋白的表达。
结果
1.原代培养的AECⅡ产量较高，每只早产大鼠肺组织可获得(8.5±1.8)×106AECⅡ，细胞活力为(95.0±2.1)％，细胞纯度为（94.3±2.5）％。电镜可见AECⅡ的特征性结构--细胞膜表面的微绒毛和胞浆内的板层小体。AECⅡ体外培养12h左右开始贴壁生长，至18h绝大部分细胞已贴壁伸展，24-48h细胞生长良好，增殖活跃，处于对数生长期，72h后细胞状态逐渐变差，丧失功能。
2. AECⅡ予95%浓度氧刺激24h后，细胞出现皱缩变形，细胞间隙增大，增殖较空气组明显受抑，凋亡率明显增加，存活率明显降低。
3. SD新生大鼠高氧暴露3d和7d后肺组织出现肺泡上皮细胞肿胀，间质充血水肿，炎性细胞浸润，肺结构紊乱，7d更明显。14d和21d可见纤维增生，肺泡间隔明显增宽，肺组织HYP含量较空气组显著增高，21d更为明显。高氧组RAC值于7d，14d，21d显著低于空气组。
4.与空气组比较，空气+H_2组细胞总FoxO3a蛋白表达增加，p-FoxO3a蛋白表达降低，其余各指标均无显著差异。与空气组比较，高氧组细胞OD492值和PCNA蛋白表达明显降低，G1期细胞比例增多而S期细胞比例减少；细胞△Ψ降低，凋亡率增加；细胞内ROS和O-2水平增高；细胞上清MDA含量增高，SOD活性下降；细胞总FoxO3a蛋白表达增加，p-FoxO3a蛋白表达降低；总β-catenin蛋白表达降低，p-β-catenin蛋白表达增高。与高氧组比较，H_2干预可减轻高氧引起的上述改变。
5.与空气组比较，空气+富氢生理盐水组和空气+H_2组大鼠肺组织均有FoxO3a与β-catenin的轻微激活，其余各指标均无显著差异；与空气组比较，高氧组肺发育受阻，RAC值降低；肺组织间隔增宽，纤维化明显，肺组织HYP含量增高，α-SMA表达增高；血清MDA水平升高，SOD活力降低；肺组织总FoxO3a蛋白表达增加，且较H_2干预空气组显著，p-FOXO3蛋白表达降低；总β-catenin蛋白表达增加，p-β-catenin蛋白表达亦增高。与高氧组比较，高氧+富氢生理盐水组和高氧+H_2组肺损伤均有所减轻，均一定程度恢复了高氧引起的上述改变。高氧+H_2组血清MDA水平和肺组织HYP含量较高氧+富氢生理盐水组低，差异有统计学意义，其余指标两组间无差异。
结论
1.采用胰酶联合胶原酶消化、差速离心和反复贴壁的方法获得的原代AECⅡ产量、纯度和活力均较高，可满足细胞学实验研究的需要。AECⅡ体外培养24-48h生长状态最佳，适合做体外研究。
2.95%浓度氧可诱导AECⅡ的损伤、凋亡并抑制其增殖，也可导致新生鼠肺损伤和肺发育受阻，成功建立高氧致AECⅡ细胞损伤模型和高氧致新生鼠肺损伤动物模型。
3.富氢培养基能一定程度减轻高氧导致的AECⅡ凋亡、氧化损伤，并促进其增殖，而对正常AECⅡ的增殖无明显作用。
4.腹腔注射富氢生理盐水和H_2气体均可有效减轻高氧导致的肺损伤，减轻肺纤维化，腹腔注射H_2气体的效果略佳。H_2对正常肺组织无明显作用。
5. H_2对高氧导致的细胞和肺损伤的保护作用可能与抑制高氧导致的FoxO3a蛋白过度活化并激活β-catenin蛋白有关。
Background
Oxygen 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 (H_2) 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 H_2can regulate the activities of ERK, JNK, P38and Akt which are thekey enzymes of MAPKs and PI3K/Akt signaling pathway.We speculatethat the H_2may play a protective role in the hyperoxia-induced lung injuryby regulating the FoxO signaling pathways. Study of H_2inhyperoxia-induced lung injury and the FoxO signaling mechanisms maybring new breakthroughs in the prevention and treatment of BPD and newprogress in mechanism research of H_2.
Objective
1. 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 H_2intervention experiments and the mechanism research.
2. Observe the effect of H_2on the hyperoxia-induce AECⅡ injury andexplore if the effect is related with the FoxO signaling pathway.
3. Observe the effect of H_2on the hyperoxia-induced lung injury innewborn rat and explore the possible mechanisms of FoxO signalingpathway in it.
Methods
1. 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+H_2 group and hyperoxia+H_2group. Intervene the cells of H_2group 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+H_2group, hyperoxia group, hyperoxia+hydrogen-richsaline group and hyperoxia+H_2group. Put the rats of the hyperoxia groupsin the animal oxygen chamber in which the oxygen concentration kept at95%. H_2intervention: Intraperitoneal inject hydrogen-rich saline10mL/kg,2times per day in rats of hydrogen-rich saline groups; Intraperitoneal injectH_2gas10mL/kg,2times per day in rats of H_2groups; Intraperitonealinject saline10mL/kg,2times a day in rats of non-H_2intervention 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.
Results
1. 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, H_2intervention 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, H_2intervention 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+H_2groups, 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+H_2group were extenuated. The above-mentioned changesinduced by hyperoxia were reduced by H_2intervention. The serum MDAlevel and the HYP content of lung tissue were little lower in hyperoxia+H_2group than that in hyperoxia+hydrogen-rich saline group, and otherindicators had no difference.
Conclusions
1. 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 H_2gas caneffectively reduce the hyperoxia induced lung damage, reduce pulmonaryfibrosis. Intraperitoneal injection of H_2gas obtained better effects. Therewas no significant influence of H_2intervention 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.
氧疗是临床常用的一种治疗手段，但持续高浓度氧疗易引起机体氧中毒，在新生儿特别是早产儿易导致慢性肺疾病（CLD）或支气管肺发育不良（BPD）的发生，严重影响患儿健康，目前尚无确切有效的防治方法。肺泡上皮损伤的正常修复主要依赖肺泡Ⅱ型上皮细胞（AECⅡ）的增殖与分化，高氧导致AECⅡ氧化应激性损伤，并抑制AECⅡ增殖是BPD发生的主要机制之一。传统抗氧化剂在减轻高氧肺损伤的同时干扰了正常肺发育。氢气（H_2）的选择性抗氧化作用和相对安全性使H_2成为治疗多种疾病的研究热点，但其具体分子机制不清。叉头框蛋白O（FoxO）是一类与氧化应激、凋亡、增殖、发育等密切相关的转录因子，其活性受丝裂原活化蛋白激酶（MAPKs）和磷酸酰肌醇3激酶/蛋白激酶B（PI3K/Akt）等多种信号途径调控，而业已证实H_2对MAPKs信号通路中重要的关键酶细胞外调节蛋白激酶（ERK）、c-Jun氨基末端激酶（JNK）、P38等的活性和PI3K/Akt信号通路中Akt活性均具有调控作用。我们推测H_2可能通过调控FoxO信号途径对高氧肺损伤发挥保护作用。深入研究H_2在高氧肺损伤中的作用及其FoxO信号机制可能为临床防治BPD带来新突破，为H_2的机制研究带来新进展。
目的
1.分离培养高纯度、高活力的原代早产大鼠AECⅡ细胞；建立高氧致AECⅡ细胞损伤模型；建立高氧致新生鼠肺损伤动物模型。为后续H_2干预实验及机制研究奠定基础。
2.观察H_2对高氧致AECⅡ细胞损伤的作用，探讨其作用是否与FoxO信号途径有关。
3.观察H_2对高氧致新生鼠肺损伤的作用，探讨FoxO信号途径在其中的可能机制。
方法
1. SPF级孕19d Sprague-Dawley(SD)大鼠，水合氯醛麻醉后剖宫产取出胎鼠，分离肺脏，剪碎，胰蛋白酶联合胶原酶消化肺组织细胞，制成细胞悬液，差速离心和反复贴壁纯化AECⅡ，含10%胎牛血清（FCS）的DMEM/F12培养基培养细胞。台盼蓝染色法检测细胞活力，改良巴氏染色法检测细胞纯度，透射电镜鉴定细胞，倒置相差显微镜下观察细胞的生长情况。
2.原代AECⅡ体外培养24h后，随机分为空气组和高氧组，高氧组细胞置于氧体积分数为95%（95%浓度氧）的细胞氧仓中，氧仓与空气组细胞一并放于细胞培养箱中。24h后观察细胞形态，MTT检测细胞增殖，流式细胞仪检测细胞的凋亡及存活情况。
3. SD新生大鼠随机分为空气组和高氧组，高氧组大鼠置于氧体积分数为95%的动物氧仓中，与空气组大鼠置于同一室内。3d，7d，14d，21d后取出肺组织行病理学检查，辐射状肺泡计数（RAC），化学比色法测定肺组织羟脯氨酸（HYP）含量。
4.原代分离培养的AECⅡ随机分为空气组、高氧组、空气+H_2组、高氧+H_2组。H_2组细胞用富氢培养基干预。24h后观察AECⅡ的形态变化；检测MTT、细胞周期和增殖细胞核抗原（PCNA）蛋白表达观察细胞增殖情况；检测细胞线粒体膜电位（△Ψ）和凋亡率观察细胞损伤情况；检测细胞内活性氧（ROS）和超氧化物阴离子（O-2）水平，细胞培养上清丙二醛（MDA）水平和超氧化物歧化酶（SOD）活性，观察细胞氧化损伤和抗氧化能力；Western Blot检测细胞总FoxO3a、β-catenin蛋白和p-FoxO3a、p-β-catenin蛋白的表达。
5. SD新生大鼠随机分为空气组、空气+富氢生理盐水组、空气+H_2组、高氧组、高氧+富氢生理盐水组和高氧+H_2组。各高氧组大鼠均置于氧体积分数为95%的动物氧仓中。H_2干预：富氢生理盐水组大鼠予腹腔注射富氢生理盐水10mL/kg，每天2次；H_2组大鼠予腹腔注射H_2气体10mL/kg，每天2次。非H_2干预组大鼠则腹腔注射等量生理盐水。14d后，取肺组织做病理学检查；检测大鼠血清MDA水平和SOD活力；测定肺组织HYP含量；免疫组化法测定肺组织α-平滑肌激动蛋白（α-SMA）表达；Western blot检测肺组织总FoxO3a、β-catenin蛋白和p-FoxO3a、p-β-catenin蛋白的表达。
结果
1.原代培养的AECⅡ产量较高，每只早产大鼠肺组织可获得(8.5±1.8)×106AECⅡ，细胞活力为(95.0±2.1)％，细胞纯度为（94.3±2.5）％。电镜可见AECⅡ的特征性结构--细胞膜表面的微绒毛和胞浆内的板层小体。AECⅡ体外培养12h左右开始贴壁生长，至18h绝大部分细胞已贴壁伸展，24-48h细胞生长良好，增殖活跃，处于对数生长期，72h后细胞状态逐渐变差，丧失功能。
2. AECⅡ予95%浓度氧刺激24h后，细胞出现皱缩变形，细胞间隙增大，增殖较空气组明显受抑，凋亡率明显增加，存活率明显降低。
3. SD新生大鼠高氧暴露3d和7d后肺组织出现肺泡上皮细胞肿胀，间质充血水肿，炎性细胞浸润，肺结构紊乱，7d更明显。14d和21d可见纤维增生，肺泡间隔明显增宽，肺组织HYP含量较空气组显著增高，21d更为明显。高氧组RAC值于7d，14d，21d显著低于空气组。
4.与空气组比较，空气+H_2组细胞总FoxO3a蛋白表达增加，p-FoxO3a蛋白表达降低，其余各指标均无显著差异。与空气组比较，高氧组细胞OD492值和PCNA蛋白表达明显降低，G1期细胞比例增多而S期细胞比例减少；细胞△Ψ降低，凋亡率增加；细胞内ROS和O-2水平增高；细胞上清MDA含量增高，SOD活性下降；细胞总FoxO3a蛋白表达增加，p-FoxO3a蛋白表达降低；总β-catenin蛋白表达降低，p-β-catenin蛋白表达增高。与高氧组比较，H_2干预可减轻高氧引起的上述改变。
5.与空气组比较，空气+富氢生理盐水组和空气+H_2组大鼠肺组织均有FoxO3a与β-catenin的轻微激活，其余各指标均无显著差异；与空气组比较，高氧组肺发育受阻，RAC值降低；肺组织间隔增宽，纤维化明显，肺组织HYP含量增高，α-SMA表达增高；血清MDA水平升高，SOD活力降低；肺组织总FoxO3a蛋白表达增加，且较H_2干预空气组显著，p-FOXO3蛋白表达降低；总β-catenin蛋白表达增加，p-β-catenin蛋白表达亦增高。与高氧组比较，高氧+富氢生理盐水组和高氧+H_2组肺损伤均有所减轻，均一定程度恢复了高氧引起的上述改变。高氧+H_2组血清MDA水平和肺组织HYP含量较高氧+富氢生理盐水组低，差异有统计学意义，其余指标两组间无差异。
结论
1.采用胰酶联合胶原酶消化、差速离心和反复贴壁的方法获得的原代AECⅡ产量、纯度和活力均较高，可满足细胞学实验研究的需要。AECⅡ体外培养24-48h生长状态最佳，适合做体外研究。
2.95%浓度氧可诱导AECⅡ的损伤、凋亡并抑制其增殖，也可导致新生鼠肺损伤和肺发育受阻，成功建立高氧致AECⅡ细胞损伤模型和高氧致新生鼠肺损伤动物模型。
3.富氢培养基能一定程度减轻高氧导致的AECⅡ凋亡、氧化损伤，并促进其增殖，而对正常AECⅡ的增殖无明显作用。
4.腹腔注射富氢生理盐水和H_2气体均可有效减轻高氧导致的肺损伤，减轻肺纤维化，腹腔注射H_2气体的效果略佳。H_2对正常肺组织无明显作用。
5. H_2对高氧导致的细胞和肺损伤的保护作用可能与抑制高氧导致的FoxO3a蛋白过度活化并激活β-catenin蛋白有关。
Background
Oxygen 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 (H_2) 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 H_2can regulate the activities of ERK, JNK, P38and Akt which are thekey enzymes of MAPKs and PI3K/Akt signaling pathway.We speculatethat the H_2may play a protective role in the hyperoxia-induced lung injuryby regulating the FoxO signaling pathways. Study of H_2inhyperoxia-induced lung injury and the FoxO signaling mechanisms maybring new breakthroughs in the prevention and treatment of BPD and newprogress in mechanism research of H_2.
Objective
1. 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 H_2intervention experiments and the mechanism research.
2. Observe the effect of H_2on the hyperoxia-induce AECⅡ injury andexplore if the effect is related with the FoxO signaling pathway.
3. Observe the effect of H_2on the hyperoxia-induced lung injury innewborn rat and explore the possible mechanisms of FoxO signalingpathway in it.
Methods
1. 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+H_2 group and hyperoxia+H_2group. Intervene the cells of H_2group 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+H_2group, hyperoxia group, hyperoxia+hydrogen-richsaline group and hyperoxia+H_2group. Put the rats of the hyperoxia groupsin the animal oxygen chamber in which the oxygen concentration kept at95%. H_2intervention: Intraperitoneal inject hydrogen-rich saline10mL/kg,2times per day in rats of hydrogen-rich saline groups; Intraperitoneal injectH_2gas10mL/kg,2times per day in rats of H_2groups; Intraperitonealinject saline10mL/kg,2times a day in rats of non-H_2intervention 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.
Results
1. 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, H_2intervention 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, H_2intervention 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+H_2groups, 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+H_2group were extenuated. The above-mentioned changesinduced by hyperoxia were reduced by H_2intervention. The serum MDAlevel and the HYP content of lung tissue were little lower in hyperoxia+H_2group than that in hyperoxia+hydrogen-rich saline group, and otherindicators had no difference.
Conclusions
1. 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 H_2gas caneffectively reduce the hyperoxia induced lung damage, reduce pulmonaryfibrosis. Intraperitoneal injection of H_2gas obtained better effects. Therewas no significant influence of H_2intervention 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.
引文
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[1] Liu Y,Sadikot RT,Adami GR,et al.FoxM1mediates the progenitor function of type IIepithelial cells in repairing alveolar injury induced by Pseudomonas aeruginosa[J]. JExp Med.2011,208(7):1473-84.
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