The Regulation Of Interstitial Fibrosis And The Effect Of Inhaled Nitric Oxide Or Ethyl Nitrite Intervention In Hyperoxic Lung Injury

BackgroundChronic lung disease (CLD), induced by many causes, is characterized by pulmonary maldevelopment and interstitial fibrosis. The incidence was increased with the use of mechanical ventilation in NICU and increasing survival rate of very low birth weight infants. Increasing evidences suggested that high concentration oxygen inhalation in neonatal period, which caused inflammation and fibrosis (hyperoxic lung injury), was a high risk factor of CLD. Some studies were designed to investigate the mechanism of hyperoxic lung injury, but it was still unclear and no effective therapy was found. Study of the mechanism of high concentration oxygen inhalation, which induces CLD, will help us to understand this disease process and to find an effective therapy of it. Herein, we studied the mechanism of hyperoxic lung injury and investigate the effect of nitric oxide (NO) or ethyl nitrite (ENO) on therapy of hyperoxic lung injury.Part One. Regulation of Interstitial Fibrosis in Hyperoxic Lung InjuryObjectives1. To investigate the effect of Sonic hedgehog (Shh) signaling pathway on hyperoxic lung injury.2. To investigate the matrix metalloproteinase-9 (MMP-9), tissue matrix metalloproteinases inhibitor-1 (TIMP-1), transforming growth factor-β1 (TGF-β1),type I and III collagen homostasis on hyperoxic lung injury.3. To analyze the interactions among these factors and find the possible markers for hyperoxic lung injury.Subjects and MethodsAnimal treatmentHyperoxic group: Fourty neonatal SD rats were exposed to over 95% oxygen in a forced-air environmental chamber for 14 days.Control group: Fourty neonatal SD rats were exposed to 21 % oxygen (room air) for 14 days.All animals were fed by breast milk. The mother-rat was exchanged between the groups every day. Laboratory studiesExperiments were done at day 1(24 h), 3, 5, 7 and 14 respectively. Laboratory studies include the measurement of mRNA and/or protein of Shh, Glil, Gli2, TGF-pi, MMP-9, TIMP-1, type I and HI collagen. Immunohistochemistry was used for the localization of expressions of these factors.In histopathology and transmission electron microscope studies, a part of right low lobes of lung was taken and fixed with 4 % formaldehydum polymerisatum. Tissues were cut into 4um thick sections and stained using HE method for routine histopathologic examination. Other fresh tissue was fixed with 2.5% glutaral for transmission electron microscope examination to observe the ultramicrostructure.In reverse transcriptase-polymerase chain reaction (RT-PCR), total RNA was isolated from 100 mg fresh lung tissue by using Trizol. The total RNA was subjected to reverse transcription and the resulting DNA was amplified with sequence-specific primers and Taq DNA polymerase. Optical density of the bands was analyzed. 0-actin was used as reference.In Western Blot analysis, proteins were extracted and the concentration was measured by Bradford method. A 50-100^g total protein was used for SDS-PAGE electrophoresis. We transfered the protein from polyacrylamide gel to nitrocellulosefilter by electrotransfer, then blocked, combined with frist antibody and second antibody in sequence, and finally used ECL radioautography method. Semiquantitative analysis of protein levels were done by NIH Scion Image software.In immunohistochenustry, tissue sections were fixed in 4 % formaldehydum polymerisatum and embedded in paraffin. The embedded tissues were cut into sections of 4um thickness. Negative, positive and blank controls were used during the study.ResultsThe lung PathologyEffusion and ecchymosis in pleura were found in hyperoxic group. Alveolar space effusion, inflammatory cell infiltration and alveolus mesenchymal thickening were noted in histopathology. Under transmission electron microscope, the destruction of laminar body in type II alveolar cell and the formation of vacuole were found. The expression of Shh and Gli signalsThe Shh mRNA and protein levels of control group in different time pionts had no significant difference (f>0.05 respectively). The Shh mRNA and protein levels of hyperoxic group were increased gradually with the duration of hyperoxic exposure (P<0.05 respectively) and peaked in day 14. The protein levels were increasd from day 3, which was behind the increase of mRNA (day 1). The Shh mRNA expressions in hyperoxic group were significantly increased compared with control group in each time point (P<0.05 respectively). The protein level of hyperoxic group in all time points were significantly increased comparaed with control group (P<0.05 respectively) except for day 1 and day 3 (P>0.05 respectively). Immunohistochemistry showed that Shh was mainly observed in bronchi and alveolar epithelia and strong expression in bronchi epithelia was noted in hyperoxic group. Shh expressions were also found in macrophages, interstitial cells and mesenchyme.The GUI or GH2 mRNA levels in control groups at different time points had no significantly different (P>0.05 respectively). But these levels in hyperoxic group atdifferent time points were significantly different (P<0.05 respectively). The GUI mRNA levels in hyperoxic group increased from day 3, peaked at day 7 and decreased at day 14. The levels of GUI in hyperoxic group were significantly increased comparaed with control group (P<0.05 respectively) except for day 1 (P>0.O5). The expression of GU2 mRNA was similar to GUI. Compared to the control, Gli2 mRNA levels in hyperoxic group were not significantly increased until day 5. The expression of TGF-filThe TGF-p 1 mRNA and protein levels in control group at different time points had no significant difference (P>0.05 respectively). The TGF-P 1 mRNA and protein levels in hyperoxic group were increased gradually with the duration of hyperoxic exposure (P<0.05 respectively), but no significant difference was found between day 1 and 3 (P>0.05). The TGF-p 1 mRNA levels between hyperoxic and control groups at each time points were all significantly different (P<0.05 respectively). The protein levels at all time points in hyperoxic group were higher than that of control (P<0.05 respectively), except for day 1 and 3 (P>0.05 respectively). Immunohistochemistry showed an increased number of TGF-p 1 positive macrophages, interstitial cell and mesenchyme in hyperoxic group but only sporadic positive cells were found in control group. The expression ofMMP-9 and TIMP-1The MMP-9 or TIMP-1 mRNA levels in control group at different time points had no significant difference (P>0.05 respectively) while significant differences were found in hyperoxic group (P<0.05 respectively). Increased MMP-9 or TIMP-1 expressions were found at the early hyperoxic exposure, peaked at day 7 and decreased at day 14. The level of MMP-9 in hyperoxic group at each time points were significantly higher compared with the control group (P<0.05 respectively).The levels of TIMP-1 mRNA in each time points in hyperoxic group were significantly higher than that of control group (P<0.05 respectively) except for day 1 (P>0.05). Immunohistochemistry staining showed an increased number of MMP-9 or TIMP-1 positive macrophages, interstitial cell and mesenchyme in hyperoxic group while only a few positive cells were found in control group.The ratio of MMP-9 to TMP-1 (MMP-9/TIMP-1) at different time points in control group had no significant difference (P>0.05) while significant differences were found in hyperoxic group (P<0.05). In hyperoxic group, a significant increase in MMP-9/TIMP-1 was found at day 1 compared to other time points (P<0.05 respectively). The expression of Type I and III collagenThe* type I or in collagen mRNA levels in control group at different time pionts had no significant difference (P>0.05 respectively) while there were significant differences in hyperoxic group (P<0.05 respectively). Levels of Type I or HI collagen mRNA in hyperoxic group were significant higher than controls from day 3 (P<0.05 respectively). Immunohistochemistry staining showed an increased number of collagen I or DI positive macrophages, interstitial cell and mesenchyme in hyperoxic group while only a few positive cells were found in control group.Conclusion1. Shh signaling pathway play an important role in hyperoxic lung injury and it might be associated with reepithelialization and extracellular matrix remodeling.2. MMP-9, TMP-1, TGF-pi, type I and III collagen also play an important role in hyperoxic lung injury which might associate with ECM remodeling.3. Shh, TGF-pi, I and III collagen may be used as markers for hyperoxic lung injury.Part Two. The Effect of Inhaled Nitric Oxide or Ethyl Nitrite Intervention onHyperoxic Lung InjuryObjectives1. To investigate whether inhaled NO or ethyl nitrite (ENO) has protective effect on hyperoxic lung injury.2. To compare the protective effect of iNO and ENO on hyperoxic lung injury.3. To investigate the adequate dose of inhaled ENO and the dose-effect relationship.Subjects and Methods ENO synthesisENO was synthesized by dissolving of sodium nitrite and ethanol in milli-Q water and carefully titrating 6M sulfuric acid into the mixture. When the ENO gas was produced, it was cooled into liquid phase and collected into flask. ENO was purified by gasifing and cooling repeatedly, and analyzed by GC/MS.. Animal Model and GroupNeonatal SD rats were randomly divided into 8 groups (32 rats in each group). NO or ENO was continuously inhalated during the first 12 hours. Hyperoxia exposure was used continuously until the end of study. Rats were studied at day 1 (24h), 3, 7 and 14.(1) Control group (C group): Same as Part one.(2) Hyperoxic group (O group): Same as Part one.(3) NO group: 21 % oxygen (room air)+5ppmNO(4) ENO group-. 21% oxyen (room air)+0.3% ENO (5ppm) (5)ONOgroup: 95%O2+5ppmNO(6) 0.17ppm ENO group: 95%O2+0.025% ENO (0.17ppm)(7) 2ppm ENO group: 95%O2+0.125% ENO (2ppm)(8) 5ppm ENO group: 95%O2+0.3% ENO (5ppm) NO InhalationThe newborns and the mothers were placed in plexiglas chambers. Oxygen/compressd air and NO were mixed immediately before entry into the chambers and were delivered through a humidified circuit at a flow rate of lOL/min.The rapid flow was used to minimize the transit time of NO in the chambers and thereby lower the concentration of nitrogen dioxide (NO2). FiO2, NO and NO2 were monitored continuously. Adjust the flow of NO to keep the NO concentration of 5ppm and the NO2 concentration lower than 2ppm. ENO InhalationENO is a gaseous nitrosant. Nitrogen (0.6L/min) was passed through a gas washer containing selected concentrations of ENO (dissolved in ethanol) and then blended with a room air gas flow in a peadiatric ventilator or oxygen (lOL/min). Samples were collected and analyzed by GC/MS for ENO content.Laboratory studiesShh, TGF-p 1 and type I collagen mRNA expressions were measured by RT-PCR, which was the same as Part one.ResultsEffect of inhaled NO on hyperoxic lung injuryThe levels of Shh, TGF-pi or type I collagen mRNA at different time points in NO group were not significantly different (P>0.05 respectively), which was similar to control group. The levels of Shh, TGF-p 1 or type I collagen mRNA in ONO group were increasd gradually with the duration of treatment (P<0.05 respectively). The levels of Shh, TGF-p 1 or type I collagen mRNA at different time points in ONO group were increased significantly (P<0.05 respectively). Compared with hyperoxic group, the levels of Shh or TGF-p 1 mRNA in ONO group were significantly decreased at day 1 (P<0.05 respectively). No significant differentce at other time points was found. The levels of type I collagen in ONO group were similar to hyperoxic group (P<0.05 respectively).Effect of inhaled ENO on hyperoxic lung injuryThe levels of Shh, TGF-P 1 or type I collagen mRNA in different time points of ENO group were not significantly different (P>0.05 respectively), which was similar to control group. The levels of Shh, TGF-p 1 or type I collagen mRNA at different time in 0.17ppm ENO group, 2ppm ENO group and 5ppm ENO group were increasd gradually with the duration of the hyporxic exposure (P<0.05 respectively). The levels of these factors in 0.17ppm ENO group were similar to hyperoxic group (P>0.05 respectively).Compared with hyperoxic group, the levels of Shh mRNA in 2ppm ENO or 5ppm ENO group were decreased significantly at day 1, day 3 and day 7 (P<0.05 respectively). In 5ppm ENO group, the levels of Shh mRNA were significantly decreased when compared with 2ppm ENO group at day 5 (P<0.05). No significant differentce at day 14 was found (P>0.05). The levels of TGF-p 1 mRNA in 5ppmENO group were significantly decreased at day 1 and day 3 (P<0.05 respectively). No significant difference was found at day 7 or day 14 (P>0.05 respectively). However, the levels of type I collagen in all three ENO treating doses were similar to hyperoxic group (P>0.05 respectively).Compare the effect of inhaled NO and inhaled ENO on hyperoxic lung injuryThe levels of Shh or TGF-pi mRNA at different time points were not significantly different between ONO group and 2 ppm ENO group (P>0.05 respectively). The levels of Shh mRNA in 5ppm ENO group were lower than these in ONO group, 0.17ppm ENO group or 2ppm ENO group at day 1, 3 or 14 (P<0.05 respectively). The levels of TGF-pl mRNA in 5ppm ENO group were lower than these in ONO group, 0.17ppm ENO group or 2ppm ENO group at dayl, 3 or 14 (P<0.05 respectively). No significant difference in type I collagen mRNA were noted among ONO, 0.17 ppm ENO, 2 ppm ENO and 5 ppm ENO groups (P>0.05 respectively).Conclusions1. Low dose inhaled NO or ENO does not show any toxical effect on lung tissue.2. Low dose inhaled NO has protective effect on hyperoxic lung injury.3. Inhaled ENO at a dose of 2 or 5ppm has protective effect on hyperoxic lung injury.4. The protective effect of ENO on hyperoxic lung injury is dose-dependent.