ENO Inhalation Intervention On Hyperoxic Lung Injury Of Neonatal Rats

BackgroundRespiratory diseases, including persistent pulmonary hypertension of newborn (PPHN) and bronchopulmonary dysplasia (BPD), contribute a great deal to the morbidity and mortality associated with neonatal intensive care therapy. New treatment modalities, such as inhaled nitric oxide (iNO), are being developed to prevent both the development and long-term morbidity of these disease processes. iNO acts as a selective pulmonary vasodilator when given to patients with persistent pulmonary hypertension of the newborn (PPHN). However, the pharmacological properties of inhaled nitric oxide are not easy to separate from its toxic effects. INO therapy has some drawbacks such as producing higher oxides of nitrogen (NOx) and a "rebound" increase in pulmonary artery pressure on discontinuing the gas. Unlike inhaled nitric oxide, endogenous nitric oxide synthase (NOSs) do not generate high levels of free NO in the respiratory system. Rather, it has been shown that the NO produced is combined with thiols to form S-nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO), S-nitrosocysteine (CSNO) and S-nitrosohemoglobin (SNO-Hb) in biological system, which are resistant to potentially toxic reaction with O2/O2".Realizing the difference between inhaled nitric oxide and endogenous NO and the advantages of SNOs, Stamler research group selected ENO (ethyl Nitrite , O-Nitrosoethanol) as a candidate gas. ENO can be used as inhalant because of its highvolatility. Inhalation of ENO produced rapid, dose-dependent reductions in PVR and PAP, whereas the drug had no effect on SVR or mean arterial blood pressure. In particular, ENO is stable in 100% 02 and does not generate NOx. Rather ENO reacts preferentially with nucleophiles, of which the most prevalent in the lung are the sulfurs of glutathione and proteins. S-nitrosothiols including GSNO are thus predicted to be the major products of ENO in lung tissues. Compared with iNO, ENO inhalation shows us a light of usage on PPHN based upon current data. However, little is known about the side effect and the adequate concentration of ENO inhalation.NO acts as a neural messenger involved in many kinds of lung injury. When delivered simultaneously with a high inspired concentration of oxygen, NO has been shown to either aggravate or protect against oxidant stress depending on experimental context (time, dose, etc) In hyperoxia induced lung injury, iNO attenuated the oxidative damage with NO increased, but the change of SNOs is unknown. In our present study, we investigated the effect of ENO on hyperoxic lung injury.Surfactant protein has important roles in lung innate immunity, and so does NO. Especially surfactant protein A can increase or decrease nitric oxide production by macrophages. On the other hand, the formation of ONOO" was responsible for SP-A nitration which will decrease the ability to bind lipids and mannose. Therefore we also observed the effect of ENO on lung mannose binding ability.Part 1 Effects of inhaled ENO on anti-oxidation system of hyperoxic lung injury of neonatal ratObjective: To investigate the effects of inhaled ENO on anti-oxidation system of in neonatal rat. To investigate the intervention of inhaled ENO on hyperoxic lung injury of neonatal rat.Methods.1. Synthesis and delivery of ENO: ENO was synthesized in our lab according the citation and was qualitative analyzed by Trace GC-MS 2000 Series. The purified liquid of ENO was delivered to animals used the system built by ourselves. The concentration of ENO was 6ppm, lOppm, 16ppm, 20ppm.2. Neonatal rats with mothers after nature birth 24 hours were assigned randomly to under groups:A. The normal neonatal rats were inhaled ENO/NO and room air (the concentration of oxygen ii 21% in all under groups).(1). control group (C), room air (21%O2). (2). NO group (NO), 10ppmNOx24h. (3). 6ppmENO group (6E), 6ppmENO><24h. (4). lOppmENO group (10E), 10ppmENOx24h. (5). 16ppmENO group (16E), 16ppmENO><24h. (6). 20ppmENO group (20E)> 20ppmENOx24h.B. The neonatal rats with hypeoxic lung injury caused by inhaled >95% oxygen for 21 days were simultaneous inhaled ENO/NO.(1). hyperoxia group (O), >95% O2.(2). hyperoxia+NO group (ONO), hyperoxia with lOppm NOx24h(3). hyperoxia+6ppmENO group (O6E), hyperoxia with 6ppmENOx24h(4). hyperoxia+16ppmENO group (O16E), hyperoxia with 16ppmENOx24h(5). hyperoxia+6ppmENO+SOD group (O6ES), 5mg/kg SOD, hyperoxia with6ppmENOx24h (6). hyperoxia+16ppmENO+SOD group (O16ES), 5mg/kg SOD, hyperoxia with16ppmENOx24h3. The blood, BALF, lung tissue samples of neonatal rats were collected at 2, 3, 7, 10, 14, 21 days after birth. 8 rats were collected in each group at every exposure time points, only 2 of the 8 rats coming from the same mother rat. Lung injury was observed by light microscope with lung tissue section. The MetHb in whole blood, and MDA, SOD and MPO activity in lurfg homogenate were assayed.Results:1. Synthesis and delivery of ENOA. The product was identified as ENO because it had the same molecule structure with ENO analyzed by GC-MS.B. The purified ENO was quantified with 4 kinds of concentration: 6ppm, lOppm, 16ppm, 20ppm.2. The intervention of inhaled ENOA. Histopathological analysis: The lung section of the control group at 2 and 3 days is at sacuular stage of lung development. The protuberances increase at 7 days. At 10 days the section shows mixed saccular and alveolar walls that are much thinner and less cellular than those of 7 days. At 14 days the section shows enlarged air spaces encircled by thin saccular/alveolar walls. At 21 days the section shows the typical alveolar with thin walls, large air spaces, and little interstitial cells. Compared with the control group, there are no significantly differences in NO group, 6ppmENO, lOppmENO and 16ppmENO groups. At the section of 20ppmENO shows slightly hemorrhage and edema at 3 days and 7 days. The lung sections of hyperoxia exposed showed changes the severity of lung injury at 3, 7, 10 days. Hyperoxia exposure leads to substantial alveolar abnormality at 21 days. A mild injury was observed at hyperoxia +6ppmENO group and hyperoxia +6ppmENO+SOD group. It shows more hemorrhage at alveolar wall in 20ppmENO group at 7days.B. MetHb in whole blood: Compared with the control group, MetHb in 20ppmENO group at 2 days increased significantly (P<0.05).C. MDA in homogenate: Compared with the control group, the MDA of 16ppmENO group at 14 days and 20ppmENO group at 10 days were increased significantly (P<0.05). MDA was increased significantly in hyperoxia group compared with the control group at 10, 14 and 21 days (P<0.05). MDA in hyperoxia+NO group and hyperoxia+16ppmENO group were decreased significantly from 14 days compared with hyperoxia group (P<0.05). MDA in hyperoxia+6ppmENO group, hyperoxia+6ppmENO+SOD group and hyperoxia+16ppmENO+SOD were decreased significantly compared with hyperoxia group (P<0.05).D. SOD in homogenate: SOD activity of all room air with ENO/NO inhalation groups had no significant difference compared with the control group at all exposure time points (P>0.05). SOD activity of hyperoxia group at 14 days was increased significantly compared with the control group (P<0.05). SOD activity was increased in hyperoxia+6ppmENO+SOD at 3 days and 7 days compared with hyperoxia group (P<0.05).E. MPO in homogenate: Compared with the control group, the MPO activity of NO groups at 7 days were decreased significantly (PO.05). Compared with NO group, 6ppmENO groups at 3 and 7 days increased significantly (P<0.05). MPO activity in lung homogenate was decreased in hyperoxia group at 7 days (P<0.05). Comparedwith hyperoxia group, MPO activity was decreased in hyperoxia+16ppmENO group at 10 days (P<0.05).Conclusion1. The concentration below 16ppm of ENO inhalation had limited ability to oxidize hemoglobin and had not lung injury effect.2. Low dose ENO inhalation enhanced the anti-oxidation effect and attenuated the hyperoxia induced lung, injury in neonatal rats. SOD enhanced the effect of ENO protection.Part 2 Effects of inhaled ENO on NO metabolism of hyperoxic lung injury of neonatal ratObjective: To investigate the effects of different concentration ENO inhalation on NO metabolism of neonatal rat without and with hyperoxia induced lung injury.Methods: The groups and the method of samples collection were as the same as in part 1. The NO in BALF and lung homogenate, the activity of NOS and the amount of NT in lung homogenate were assayed, SNOs in plasm was assayed by HPLC.Results:1. NO in BALF: Compared with the control group, NO in BALF of lOppmENO group at 10 days, 16ppmENO and 20ppmENO groups at 21 days were increased significantly (P<0.05). NO in BALF was significantly increased at 7 days in hyperoxia group compared with the control group (P<0.05). Compared with hyperoxia group, NO in BALF was increased at 3 days in hyperoxia +16ppmENO group (PO.05).2. NO in lung homogenate: Compared with the control group, the NO in lung homogenous of NO group at 10 days was increased significantly (PO.05). NO in lung homogenate was increased significantly in hyperoxia group compared with the control group at 10, 14 and 21 days (P<0.05). Compared with hyperoxia group, NO was increased in hyperoxia +16ppmENO group at 3, 7 and 10 days (P<0.05).3. NOS: Compared with the control group, the total NOS activity of 20ppmENO group at 2 days and 3 days and NO group at 7 days were significantly decreased (PO.05). The total NOS activity was increased in hyperoxia group at 3 and 10 days compared with the control group but decreased at 7 days (P<0.05). Compared with hyperoxia group, the total NOS activity was decreased in all interventiongroups at 3 and 10 days and hyperoxia +6ppmENO group (PO.05), hyperoxia +6ppmENO+SOD group at 7 days (PO.05).4. iNOS/total NOS: Compared with the control group, the iNOS/total NOS of NO groups at 3, 7, 10, 14 days, and 6ppmENO groups at 3 and 10 days were increased significantly (PO.05). The iNOS/total NOS activity was increased in hyperoxia group at 3, 10, 14 and 21 days (PO.05). Compared with hyperoxia group, iNOS/total NOS was increased significantly in hyperoxia+NO group at 3 and in hyperoxia+16ppmENO+SOD group at 7 days (PO.05). But SOD activity was decreased significantly in hyperoxia+6ppmENO group and hyperoxia+16ppmENO group at 3,7 and 10 days compared with hyperoxia group (PO.05).5. iNOS mRNA: The express of iNOS mRNA was increased significantly in 16ppmENO group, 20ppmENO group at 3 days and hyperoxia group at 3 days at 7 days compared with the control group (PO.05).6. eNOS mRNA: The express of eNOS mRNA was increased significantly in 20ppmENO group at 3 dats and hyperoxia group at 3 days and 7 days compared with the control group (PO.05).7. NT: Compared with the control group, 6ppmENO group at 2 days and 7 days were decreased significantly (PO.05), 16ppmENO group at 3 days and 20ppmENO group at 3, 7 days were increased significantly (PO.05). NT in lung homogenate was increased significantly in hyperoxia group at 2, 3 days compared with the control group (PO.05). Compared with hyperoxia group, NT was decreased significantly at 2 days (PO.05) in hyperoxia+NO group, hyperoxia+6ppmENO group, hyperoxia+16ppmENO group and hyperoxia+6ppmENO+SOD group (PO.05).8. SNOs: Compared with the control group, the SNOs in plasma of all ENO inhalation groups were increased significantly at 2 days (PO.05).Conclusion1. Low dose ENO inhalation enhanced iNOS activity, but higher dose ENO inhalation inhibited iNOS activity simultaneity enhanced the expression of iNOS mRNA.2. Low dose ENO attenuated the level of nitration in lung and protected the lung with hyperoxia.3. Inhaled the concentration bellowed 20ppmENO the amount of SNOs in plasma was increased with the increasing concentration of ENO.Part 3 Effects of inhaled ENO on the express of SP and lung immunity Objective: To investigate the effects of different concentration ENO inhalation on the express of SP mRNA and the immunological function of ^P in neonatal rat with and without hyperoxic lung injury.Methods: The groups and the method of samples collection were as the same as in part 1. SP-A was measured by real time RT-PCR, and SP-D mRNA was measured by RT-PCR. The protein of SP-A was half-quantitative analysis by Western blotting. The mannose binding ability (MBA) of total protein in lung homogenate was assayed by ELISA kit to represent the MBA of SP.Results:'1. Ultrastructure of type II alveolar epithelia cells: Approximately cuboidal cells with short apical microvilli and numerous intracellular lamellar bodies were identified as alveolar type II cells. The cytoplasm was rich in rough endoplasmatic reticulum, free ribosomes and mitochondria at the control group. The amount of these organelles was somewhat higher in neonatal cells, whereas the amount of lamellar bodies appeared to be lower;the neonatal cells regularly had a large Golgi apparatus, frequently multivesicular bodies, and a euchromatin-rich nucleus with a large prominent nucleolus. The majority of cells show edema of mitochondria after hyperoixa exposure at 2 or 3 days. The apical microvilli decreased and nucleus with heterochromosome's emerged in hyperoxia group at 7 days. The fundus membranes were thicker in hyperoxia group at 14 days. The lamellar bodies dissolved and the amount of type II alveolar epithelia cells was decreased in hyperoxia group at 21 days. Compared with the hyperoxia group, the injury of type II alveolar' epithelia cells and lamellar bodies was mild in hyperoxia +6ppmENO+SOD group and hyperoxia +6ppmENO+SOD group.2. SP-A mRNA: The express of SP-A mRNA was decreased significantly in NO group and hyperoxia group but was increased in 6ppmEN0 and 1 OppmENO group at 3 days compared with the control group (P<0.05). Compared with hyperoxia group, SP-A mRNA iwas decreased in hyperoxia +NO group and hyperoxia+10ppmENO group (P>0.05) but increased in hyperoxia +6ppmEN0 group and hyperoxia +6ppmEN0+S0D group (P<0.05).3. SP-D mRNA: The express of SP-D mRNA was decreased significantly in 20ppmENO group and hyperoxia group at 3 days compared with the control group(PO.OS). Compared with hyperoxia group, SP-D mRNA was decreased in andihyperoxia +10ppmENO group and increased in hyperoxia +6ppmEN0 group and hyperoxia +6ppmENO+SOD group (P<0.05).4. Western blot for SP-A: The amount of SP-A was decreased at 3 days and was increased at 7 days in hyperoxia group with significantly difference compared with the control group (P<0.05). Compared with hyperoxia group, SP-A was increased significantly in hyperoxia +6ppmEN0+S0D group and hyperoxia +10ppmENO+SOD group (P<0.05).5. MBA of SP: Compared with hyperoxia group, the MBA was increased in hyperoxia +16ppmEN0 group and hyperoxia +16ppmENO+SOD group with significant difference (.PO.05).Conclusion:1. The express of SP-A, SP-D mRNA was decreased at early of hpyeroxia exposure and was enhanced by ENO inhalation. The protein of SP-A also enhanced by ENO inhalation.2. 16ppmEN0 inhalation enhanced the MBA in lung.