The Protective Effects And Mechanisms Of Hepatocyte Growth Factor On Neonatal Rats With Hyperoxia-induced Chronic Lung Disease

The Protective effects and mechanisms of hepatocyte growth factor on neonatal rats with hyperoxia-induced chronic lung diseaseWith the development of perinatology, perfection of mechanical ventilation and application of surface active substance, the survival rate of premature infant especially very-low-birth-weight children increases constantly, meanwhile the disease incidence of chronic lung disease (CLD) elevates concomitantly year by year. CLD not only influences the life quality of premature infant severely, but also is one of major diseases that disable neonates. The pathological characteristics of CLD transform from the view of severe airway injuries, over aeration of pulmonary alveoli and pulmonary fibrosis in the past to the present recognition of alveolar capillary dysplasia, enlargement and structure simplification of pulmonary alveoli, impairments of them and structural abnormalities of the capillaries. But the pathogenesy of CLD is not completely clear yet, but the lack of effective therapeutic measures. Study in this field has become a hot topic currently.The occurrences of CLD may be reactive injuries of immature lung that are probably caused by multiple factors. Inhalation of high concentration oxygen is among the major factors that cause the CLD injuries of the premature infants. The existing research has proved that ROS is the initiation factor of the apoptosis of alveoli epithelial cellâ…¡(AECâ…¡) caused by hyperoxia, and that the proliferation, migration of AECâ…¡and transformation to AECâ… take the key roles in repairing procedures of hyperoxia pulmonary injury. What's more, the apoptosis and abnormal proliferation of AECâ…¡is possibly one significant factor that causes the growth and remodeling abnormalities of the lung hyperoxia in the critical developing phase. Therefore, we presume that preventing the apoptosis of AECâ…¡, improving the proliferation, migration and transformation to AECâ… of it, and inducing pulmonary angiogenesis could obviate or improve the CLD of premature infants caused by hyperoxia.Hepatocyte growth factor (HGF) is a multifunction cytokine. It not only improves the pulmonary development, but also contributes to the lung recovery from damage, facilitates the proliferation and reparation of the pulmonary epithelial cells after injuries, is effective in obviating or facilitating pulmonary fibrosis, induces pulmonary angiogenesis and inhibits the endotheliocyte apoptosis induced by ischemic-reperfusion.To be closer to clinical conditions, we explored the pathogenesy of hyperoxia-induced lung injury in vivo and vitro on full term neonatal rats and premature rats to provide experimental foundation and theoretical evidence to more effective prevention and cure of CLD in clinic. Chap oneEffect of prolonged hyperoxia on the expression of HGF, histology and oxidative stress reaction in neonatal rat lung, and the relationship between themPart 1 The effect of prolonged hyperoxia on histology and oxidative stress reaction in neonatal rat lungObjective Observing the change of lung histology and oxidative stress reaction in neonatal rat exposed to hyperoxia to determine the effect of prolonged hyperoxia on the development of neonatal rat lung. At the same time, duplicate animal model of hyperoxia-induced CLD in neonatal rats. Methods The Full-term newborn rats were randomly assigned into a hyperoxia group(group HO) and an Air group(group Air). The group HO was continuously given a high concentration of oxygen (FiO2＞0.90) and the group Air received oxygen with the FiO2 of 0.21 after birth. The lung histological change, collagen area and the concentration of the 8-isoprostane2α, a specific marker of lipid peroxidation in vivo, were monitored in lung on day 3,7,14 and 21 in two groups. Results Compared with the group Air,the arrest of lung development was accompanied by interstitial fibrosis and increased collagen deposition, which was evident after 7d of oxygen exposure, the impairment kept increasing through 21d, resembled to the histology of chronic lung disease, CLD which appeared as fewer and bigger alveolar; The level of 8-isoprostane2αincreased at 3d and reached the peak at 7d, then decreased, but still remained at a higher level on the 21st day compared with that of the group Air. Conclusions: Prolonged hyperoxia exposure appears to lead to arrest of lung development. The lung oxidative stress reaction is closely related to the hyperoxia -induced lung injury.Part 2 HGF dynamic expression in neonatal rat lung tissue exposured to prolonged hyperoxia and the relationship between cell apoptosis and HGFObjective Observing cell apoptosis and protein, mRNA dynamic expression of hepatocyte growth factor (HGF) and its receptor cMet in neonatal rats lung tissue exposed to prolonged hyperoxia, and the relationship of cell apoptosis and dynamic expression of HGF and discussing the function of HGF pathogenesy of hyperoxia-induced CLD in neonatal rats. Methods The neonatal rats were randomly assigned into group Air and group HO (90ï¼…oxygen), getting the lung tissue specimen after 3 d, 7 d, 14 d, 21d respectively. Using TUNEL to observe the change of cell apoptosis, transmission electron microscope to observe ultra structural changes of AECâ…¡, immunity tissue method to test the protein levels expression of HGF and cMet, RT-PCR to test mRNA levels expression of HGF and cMet. Resultsâ‘ HGF protein mainly expressed in mesenchyme and bronchioles epithelium, while cMet protein mainly expressed in bronchioles and pulmonary alveoli epithelium.â‘¡Ultra structural changes of AECâ…¡: emptied LB on 3d, increased matachromatic granules of nuclei on 7d, disappeared organelle, dissolved nuclei on 21d.â‘¢Compared with group Air, prolonged hyperoxia exposure led the number of apoptotic cells, the expression levels (protein and mRNA) of HGF and cMet enhanced significantly, and kept rising till 21d.â‘£In group HO, changes of the levels of protein of HGF and cMet correlated positively with the levels of mRNA of HGF and cMet respectively, and correlated positively with the number of apoptotic cells.Conclusion:1. Apoptosis of pneumonocyte is involved in the formation of CLD, which is the main cause of arrest of lung development induced by hyperoxia.2. HGF and cMet are involved in lung development of neonatal rats.3. Abnormal expression of HGF may contribute to the pathogenesis of CLD induced by hyperoxia via its effects on apoptosis. Chap twoThe protection effects and mechanisms of HGF on hyperoxia exposed typeâ…¡alveolar epithelial cells isolated from premature rat lungPart 1 Isolation and purification and primary culture of lung cells from premature rat lungs and establishing hyperoxiaexposed cell modelobjective To develop a reliable method of isolation, purification and primary culture of typeâ…¡alveolar epithelial cells (AECâ…¡S) from premature rat lung, further more, to establish hyperoxia-exposed cell model for providing a basis for the future investigation of hyperoxia induced premature lung injury and lung diseases of premature infant in cellular and molecular level. Methods Cell proliferating vitality was examined by MTT assay after treatment with HGF at various concentrations. The lung of 20-day fetal rat was digested by trypsin, DNAase and collagenase under sterile condition. AECâ…¡s were isolated and purified in different centrifugal force and different adherence, then cultured. The nature of the cultures was identified by cytokeratin, vimentin and SPA staining and electron micrograph. For establishing hyperoxia-exposed cell model, purified AECâ…¡s were cultured for 48 hours after culture flasks were filled with 95ï¼…oxygen-5ï¼…CO2 at 5 L/min for 10 min, and then sealed. Oxygen concentrations were tested in CYS-1 digital oxygen monitor and supernatants were harvested after 48 hours of exposure. Lactate dehydrogenase (LDH) activity and 8-iso-PF2αof supernatants were examined by chronometry and ELISA respectively. The example was discarded if its oxygen concentration was＜90ï¼…. Results Excellent yields of highly purified, cultural AECâ…¡could be obtained from 20-day fetal lungs. The expression of cytokeratin and SPA in AECâ…¡s was positive and that of vimentin negative by immunocytochemistry. Lamellar bodies in purified AECâ…¡s were revealed by ultrastructural examination with electron micrograph. The results showed that the method assured the oxygen concentrations of culture bottles to be more than 90ï¼…. LDH activity of supernatants of hyperoxia groups was elevated in comparing with that of air groups, however, there was no significant difference. It suggested that the cells exposed to more than 90ï¼…oxygen during 48 hours still reserve cell activity and integrity. But the level of 8-iso-PF2αin supernatants of hyperoxia groups were elevated significantly compared with that of air groups. It suggested that the products of oxidation metabolism were changed for hyperoxia exposed. Conclusion AECâ…¡s of premature rats were isolated and purified and cultured successfully, and hyperoxia-exposed cell model was established in this experiment. Part 2 Effects of HGF on proliferation, apoptosis and function of hyperoxia exposed typeâ…¡alveolar epithelial cells isolated from premature rat lungObjective Observing the effects of hepatocyte growth factor on proliferation, apoptosis and function of hyperoxia exposed typeâ…¡alveolar epithelial cells isolated from premature rat lung to explore the mechanism of protective effects of hepatocyte growth factor HGF on hyperoxia-induced lung injury. Methods Typeâ…¡alveolar epithelial cells (AECâ…¡) were gained by primary culture from 20-days-old fetal rat lung. Cell proliferating vitality was examined by MTT assay after treatment with HGF at various concentrations. After being purified, AECâ…¡was randomly assigned to four groups: Air group (Air), hyperoxia group (HO), Air plus hepatocyte growth factor group (Air+HGF), hyperoxia plus hepatocyte growth factor group (HO+HGF). Groups HO,HO+HGF were flushed with 95ï¼…oxygen, 5ï¼…CO₂ at 5 L / min for 10 min into the culture flask, then were sealed and cultured for 24 hours. Groups Air+HGF, HO+HGF were added with 25 ng / ml of hepatocyte growth factor at the same time. All groups were in CO₂ culture chamber (37℃, 5ï¼…CO₂) for 24 hours, cells were harvested and extracted for total RNA by Trizol reagent, mRNA levels of SP were measured by reverse transcription polymerase chain reaction (RT-PCR). The proliferation and apoptosis of AECâ…¡were analyzed with flow cytometry. Resultsâ‘ HGF at the concentration rang from 5 ng/ml to 25 ng/ml stimulated the proliferation of AECâ…¡in a dose-dependent manner.â‘¡Compared with group Air, the apoptosis rate in group HO increased significantly, while G2/M phase percentage decreased significantly; the S phase percentage in group Air+HGF, increased significantly.â‘¢Compared with group HO, the apoptosis rate and G0 /G1 phase percentage in group HO +HGF decreased significantly, S phase, G2/M phase percentage increased significantly.â‘£SPs mRNA levels significantly decreased in group HO compared with group Air; After hepatocyte growth factor was added, SPs mRNA levels increased in group HO +HGF and group Air+HGF compared to group HO.Conclusion Hyperoxia inhibited the proliferation, increased apoptosis rate and decreased SPs mRNAs levels in premature rat AECâ…¡in vitro, while HGF could partly inhibit the changes of SPs mRNAs levels and cell proliferation of AECâ…¡resulted from hyperoxia and HGF may play protective role in hyperoxia-induced lung injury.Part 3 The molecule mechanism and signal transmission of cytoprotective effect of HGF against hyperxia-induced AECâ…¡isolated from premature rat apoptosis and proliferation depression objective To explore the molecular mechanism and signal transmission of cytoprotective effect of HGF against hyperxia-induced typeâ…¡alveolar of premature rat apoptosis and proliferation depression through examinig p-AKT, P21^CIP1, PCNA protein and Bcl-2, P21^CIP1, PCNA gene expression, as well as the cell cycle change after utilizing phosphorylation blocking agent LY294002 of PI-3KAKt/ signal transmission on hyperxia impaired AECâ…¡model of premature rat with RT-PCR, flow cytometry and western blot and so on. Methods AECâ…¡s isolated from 20d premature rat were divided into 4 groups (each group of 10 bottles): group of Air(Air), group of hyperxia(HO), group of Air+HGF(Air+HGF) and group of hyperxia+HGF(HO+HGF). Each group draw-off at random 5 bottles to be treated with ordinary culture fluid, another 5 bottles are treated with culture fluid with blocking agent(LY294002). Groups HO,HO+HGF were flushed with 95ï¼…oxygen, 5ï¼…CO2 at 5 L/ min for 10 min into the culture flask, then sealed and cultured for 24 hours. Groups Air+HGF, HO+HGF were added with 25 ng / ml of hepatocyte growth factor at the same time. All groups were in CO₂ culture chamber (37℃, 5ï¼…CO₂) for 24 hours, cells were harvested. The mRNA levels of Bcl-2, P21^CIP1, and PCNA were measured by RT-PCR. The protein expression levels of Bcl-2, P21^CIP1, PCNA were measured by Western blot. And the proliferation and apoptosis of AECâ…¡were analyzed with flow cytometry assay. Results â‘ Compared to group Air, the apoptosis rate and GO /G1 phase percentage in group HO increased up obviously, G2/M phase percentage decreased obviously, S phase percentage had no obvious difference, the apoptosis rate and G0/G1, S, G2/M phase percentage in group Air+HGF had no significant difference; Compared to group HO, the apoptosis rate and G0/G1, S, G2/M phase percentage in group HO+HGF had no significant difference.â‘¡Compared to group Air, the mRNA expression and protein expression levels of PCNA in group HO and group HO+HGF all decreased obviously before and after blocking, while the mRNA expression and protein expression levels of P21^CIP1 all increased obviously before and after blocking; the mRNA and protein expression levels of PCNA in group Air+HGF increased obviously, the mRNA and protein expression levels of P21^CIP1 decreased obviously before blocking, but neither had remarkable difference after blocking, while the mRNA and protein expression levels of PCNA and P21^CIP1 in group Air+HGF had no remarkable difference. Before blocking, the mRNA expression and protein expression levels of PCNA in group HO+HGF increased obviously and the levels of P21^CIP1 decreased obviously compared to group HO, but had no remarkable difference after blocking. Before and after blocking, all the mRNA expression levels of Bcl-2 in group HO and group HO+HGF were lower than group Air; Before blocking, the mRNA expression levels of Bcl-2 in group HO+HGF were obviously increased than those in group HO, but had no remarkable difference after blocking.â‘¢Before blocking, all p-Akt protein expression in group HO, HO+HGF and Air+HGF obviously elevated compared with group Air; While compared with group HO, p-Akt protein expression in group HO+HGF elevated obviously. After blocking, the p-Akt protein expression was not detected in any group.Conclusion:1. Hyperxia up-regulates P21^CIP1, down-regulates PCNA duplication and the translation level of expression, thus is possibly related to its causing suppression of AECâ…¡multiplication.2. Hyperxia declines the Bcl-2 duplication level of expression; it is likely related to causing AECâ…¡apoptosis.3. HGF can partially reverse the influence of hyperxia on the expression of P21^CIP1, PCNA, and Bcl-2.4. HGF partially reverses the influence of hyperxia on multiplication and apoptosis of AECâ…¡via PI3-K/The Akt signal conduction possibly.