The Study On Effects Of Losartan On Lung Fibrosis Of Hyperoxic Induced Neonatal Rats CLD

IntroductionChronic lung disease (CLD) of prematurity is a major long-term pulmonary consequence of preterm birth, in which pulmonary immaturity, oxygen toxicity, baro/volutrauma as a consequence of mechanical ventilation and/or infections are involved. Bronchopulmonary dysplasia (BPD) is one of a chronic lung disease first described in 1967 as a complication of therapy for premature infants with hyaline membrane disease, and treatment with high concentrations of oxygen was thought to be a major contributor to its development. Administration of supplemental oxygen as one of supportive therapies employ to elevate oxygen concentrations, which put them at risk for encountering pulmonary oxygen toxicity, cause significant lung damage, even develop to CLD.With neonatal medicine development, the incidence of premature CLD is at a rising tendency continously (reaching 30%～40% abroad). There is no ideal prevention and perfect therapeutic strategies due to unclear mechanism. Hyperoxic induced premature CLD is one of the most difficult problem in neonatal intensive care unit(NICU).So far the mechanism research on CLD has been one of the hot topics all around the world, the accurate mechanism hasn't been identified although a great deal of clinical researches and animal experiments have been done on oxidative stress, inflammation, apoptosis, mechanism of collagen degradation and arrested lung development induced by the lackness of some growth factors to clarify the incidence and the development of CLD. However no matter what the mechanism is, histopathologic characteristics of the lung injury in CLD are arrested lung development and pulmonary interstitial fibrosis. At present, there are no effective treatment for CLD. Corticosteroids facilitate extubation and decrease neonatal respiratory support and oxygen exposure. However, more and more clinical research indicate glucocorticoids in premature infants has raised a host of concerns about effects on lung, brain and somatic growth, and substantially worse neuromotor and developmental outcomes in early childhood. Surfactant replacement therapy in early time after birth may improve pulmonary function, but cannot cut down incidence of CLD. So study the pathogenesy of CLD to explore how to reverse lung development arrest, to relieve lung fibrosis, decrease incidence of premature CLD become a urgent problem.The vasoactive octapeptide, angiotensin II (AngII), has a well- described role in the control of systemic blood pressure and volume homeostasis. Local rennin angiotensin systems (RAS) have been described for a number of tissues in which AngII production is independent of circulating precursors. There are some Experimental evidence to suggest an important role for AngII in the fibrotic response to tissue injury. Ang II can stimulate lung fibroblast proliferation via activation of the AT1 receptor and involves the autocrine action of TGF-β1, and induces AEC apoptosis in response to Ang II is mediated by receptor subtype AT(1); some study consider that collagen remodeling may be facilitated by Ang II to effects on MMPs/TIMPs and collagen expression via the AT1 receptor to modulates profibrotic effects. Moreover local RAS take part in lung injury by increase vascular permeability, vascular tone and so on. Angiotensin-converting enzyme (ACE) inhibitors and AT1 receptor antagonist attenuate bleomycin-induced lung fibrosis in a number of animal models. So it is supposed that local RAS may have significant effect in course of lung fibrosis.Li research showed that lung tissue of hyperoxia-exposed induced newborn rats lung injury AngII and ACE content increase signifencently, and Captopril may extenuate lung fibrosis induced by hyperoxia. But the mechanism is not clear completely. In this study we prepared an animal model of CLD with newborn rats induced by continuously inhaling high concentration of oxygen(FiO₂85%)and at the 6th day after hyperoxia losartan (5mg/kg), a selective antagonist of AT1 receptors for Ang II, by intragastric administration until the end of experiment completed. Using special staining technic, morphology, immunoassay, immuno-histochemistry and Reverse transcription polymerase chain reaction (RT-PCR) to observing the dynamic changes of lung development and fibrosis, the MMPs/TIMPs, collagen expression are detected after losartan intervention. We try to provide new way for research pathogenesy of hyperoxia induced CLD.Materials and Methods1. Animal modleThe present study was performed in accordance with the guidelines provided by the Experimental Animal Laboratory of Shengjing Hospital China Medical University. Within 24 hours after birth, pups were randomly redistributed to the newly delivered mothers. Term neonatal Wistar rats were divided randomly into four groups. AIR group served as controls and were kept in air and not subjected to hyperoxia. Other groups were exposed to oxygen. The pups of hyperoxia exposures groups were maintained in glass chambers in which the oxygen was infused continuously to achieve 85% oxygen concentration, with oxygen monitor(OM-25ME, USA) twice daily. CO₂ was removed by soda lime absorption to keep CO₂ levels below 0.5%(Dapex Gas Monitor, USA). Temperature and Humidity was maintained at 20℃～25℃and 50%⁷0%, respectively. Every day, at clocked time of 0.5h opening chambers to exchange nursing mothers betweenin air and O₂-exposed groups every 24 hours to prevent maternal O₂ toxicity and eliminate maternal effects between groups, meanwhile change water, add food, clean dirty cages and record body weight. All animals were raised in the same room and all other conditions were the same. In the oxygen exposed groups, O₂ groups were simple oxygen exposed, AO₂groups and LO₂ were received daily aqua(3ml/kg) and losartan (5mg/kg) by intragastric administration from the sixth day after birth until the end of experiment completed, respectively.2. tissue preparationOn postnatal 1d, 3d 7d, 14d, 21d. Each group of hyperoxia as well as air rats was anesthetized by intraperitoneal injection of 5% chloral hydrate (6ml/kg), and put to death. The thorax was opened and the lungs were resected, the right lung tissue were put in the Rnase-free Ependorf tubes, and immediately frozen in liquid N₂ for mRNA, Enzyme linked immunoadsorbent assay(ELISA), and so on. the left lung tissue were fixed in 4%formaldehydum polymerisatum which contains 0.1% DEPC(0.1MPBS, pH7.0～7.6), then were dehydrated, embedded in wax within 24 hours.3. Experiment Methods1.1 The appearance, survial rate and weight were monitored everyday in every experiment groups. Life table for survival analysis, point survival and survival curve were compared.1.2 The changes of the lung pathology1.2.1 For lung pathology assessment, lung sections from the left lobes were stained with HE (hematoxylin and eosin).1.2.2 The thickness of inter alveolar septa: the hispathological changes of pulmonary fibrosis were evaluated by image analysis.1.2.3 Masson staining: to evaluate degree of collagen fibers deposition in the lung.3.3 Using Immonohistochemistry and RT-PCR to detect protein and mRNA of AT1R expression in the lung.3.4 The measurement of collagen expression in the lung tissue3.4.1 Immonohistochemistry: the detection intensity and position of the protein expression in lung tissue about alpha-SMA3.4.2 Quantitation of total lung collagen: to detect hydroxyproline (HYP) of lung tissue by acidolysis assay, to express of total lung collage content.3.4.3 ELISA: quantitation to detect the protein level of collagen I (ColI) in lung tissue. 3.4.4 RT-PCR: the detection at mRNA level of Coll.3.5 The measurement of MMP-13 and TIMP-1 expression in the lung tissue.3.5.1 Immonohistochemistry: the detection intensity and position of the protein expression in lung tissue about MMP-13 and TIMP-1.3.5.2 RT-PCR: to detect mRNA level of MMP-13 and TIMP-1 in lung tissue of neonate rats .4. Statistical analysisSSPS version 11.5 was used to perform statistical analysis, with all data expressed as (mean±SD). Statistically significant differences in the mean values were analysis using the One-Way ANOVA, Independent-Samples T Test, bivariate. Life table was used to value survival rate, u-test for time comparison. Statistically significant differences set P<0.05.Results1. Survival rate and general status of rats in four groups1.1 The hyperoxia rats almost began to present dyspnea, pale, cyanosis of different degrees after 5-7 days of oxygen exposure, some even depended high oxygen after 10 days of age. LO₂ groups was better than oxygen exposed and AO₂ groups withdraw from oxygen. The AIR groups didn't have the appearance above.1.2 The death of hyperoxia rats increased with the time of oxygen exposure, and survival rate decreased significently (P<0.05); while there is no death after Id in AIR groups, and the survival curves were different in groups from 2 days of age(P<0.01). The survival rate of LO₂ groups increased comparing to oxygen exposure.1.3 There was no difference in average birth weight between the four groups, but from 7 day of oxygen exposure, Body weights of the O₂ groups, AO₂ groups and LO₂ group animals showed a significant decrease compared with air controls at the sams time period(P<0.05). And the difference lasted to 21 days of age and more evident compared with air controls(P<0.01). There was no difference between the LO₂ groups and O₂ groups.2. The pathological changes of the lung tissue2.1 On day 1 of the experiment, it was observed in both room air and O₂ groups that the alveolar structure was irregular, terminal air space size was rather small, and the alveolar septum was thick. On 3 day, the alveolar structure of the each groups was more regular, the size of alveolus was equal, and alveolar septum was thinner, but in O₂ groups, there was a few inflammatory cells exuded out, blooding, interstitial cells increased. On 7 day, in room air rats, the alveolar size was equal, while the terminal air space size of the oxygen-exposed rat became large, there was inflammatory response and more interstitial cells. On 14 day and 21 day, air groups continue to progress alveolization; but in O₂ groups, the terminal air space size grew significantly large, secondary septum decreased, the quantity of alveolar reduced, with alveolus fusion, interstitial cells increased, and alveolar septa was thicker. There was no significant difference between the LO₂ groups and O₂ groups on 7 day. On 14 day d and 21 day, alveolar septum is thinner in LO2 groups, but alveolar space are not deflate obviously, and the sum of secondary septum is still less.2.2 Masson staining: In air group, blue fibres mainly deposited in bronchil and vascular wall, little in alveolar septum. In O₂ groups, on 14 day and 21 day there were more collagen fibres along bronchil and vascular wall and alveolar septum, especially in alveolar septum. In LO₂ groups, the ratio of blue fibres is decreased and rarefied relatively compare with O₂ groups, but it is more than air group.3. The dynamic changes of AT1R expression in the lung tissueImmuonhistochemical studies shows AT1R expression in alveolar epithelial cells, fibroblast, interstitial macrophage and external of vessels and bronchus in control group; AT1R positive expression in endothelial cells and bronchial epithelial cells were also observed in fibrotic lesions in lungs. AT1R expression in air groups lung decreased but increased significantly in hyperoxia-exposed groups on 14 and 21 day.4. The dynamic change of collagen expression in lung tissue 4.1 Immunohistochemical assessment of alpha-SMA expressionIn air groups, alpha-SMA immunostaining was restricted to the smooth muscle of the pulmonary vasculature, airway walls, and openings to the respiratory bronchioles of terminal airway; meanwhile in 7-day-old rats, alpha-SMA positive cells are located in the slender elongated cells in the interstitium of primitive alveolar septa; in 21-day-old rats, alpha-SMA positive cells with a round shape are found at the tip of the secondary septa. Exposure to hyperoxia resulted in its expression a marked increasing in smooth muscle and alveolar septa myofibroblast foci and on the surface of alveolar significantly on 14 day and 21 day. Treatment of the hyperoxia-exposed animals with Losartan result in a significant decrease alpha-SMA expression compared with simple O₂ groups(P<0.01).4.2 detect of HYP content in lung tissue by acidolysis.There were no difference of HYP contents to four groups from 1 day to 7 day of the experiment. On 14 day and 21 day, HYP contents increased significantly in O₂ groups comparing to air groups(P<0.01); and changes of AO₂ groups similar to that of simple hyperoxia; in LO₂ groups it decreased slightly on 14 day; and on 21 day decreased significantly(P<0.01), however higher than that in air groups(P<0.01).4.3 The dynamic change at the protein and mRNA level of ColI expressionThe protein of Col I in the newborn lung tissue decreased with postnatal age rising in air groups; But in the O₂ and AO₂ groups it increased significantly than that in air control group(P<0.01), and its of larger difference on 21 day; in LO₂ groups it decreased slightly on 14 day; and on 21 day decreased significantly(P<0.01), but its higher than that in air group(P<0.01).There was no different about mRNA expression of ColI at each spot in air groups; O₂ and AO₂ groups it increased with oxygen-exposed extending, and reach peak on 21 day. mRNA expression of ColI decreased significantly after Losartan-treated.4.4 There was correlation between alpha-SMA and ColI mRNA in the lung (r=0.64, P<0.05), but none with the protein of ColI (r=0.28, P >0.05). 5.The dynamic changes of MMP-13 and TIMP-1 expression in the lung tissue5.1 The dynamic change MMP-13 expressionImmunohistochemical staining shows, MMP-13 was expressed in the endochylema of epithelial cells, microvascular endothelial cells, interstitial cells and macrophages, in air lung tissues, there was no change of the MMP-13 expression at each time; In O₂ and AO₂ groups, it express at the same level from 1 day to 14 day, which was no difference with the air group. But on 21d, the expression of MMP-13 decreased significantly(P<0.01). In LO₂ group, it increased signifycantly comparing to O₂ group and reach the level of air groups. The curve change of MMP-13 mRNA is the same with that of protein.5.2 The dynamic change TIMP-1 expressionTIMP-1 was expressed in epithelial cells, interstitial cells and pulmonary macrophages, the expression intensity of TIMP-1 protein and mRNA of all the rats were rising, from 7d after oxygen-exposed, its expression obvious increased, and significant on the 14 and 21d. losartan result in a significant decrease of the protein and mRNA expression of TIMP-1 compared with simple hyperoxia-exposed animals(P<0.01), but were higher than that of air group.Concludsion1. pathological findings charactered by the arrested alveolar development and deposited fibres in newborn rats after prolonged hyperoxia-exposed are consistent with those of CLD of prematurity in human.2. Immuonhistochemical studies shows AT1R expression in alveolar epithelial cells, fibroblast, interstitial macrophage and external of vessels and bronchus in control group; AT1R positive expression in endothelial cells and bronchial epithelial cells were also observed in fibrotic lesions in lungs. AT1R expression increased significantly at the late stage of hyperoxia which is consist with the time of lung fibrosis genesis. It is hint that angiotensin II as a ligand of AT1R may take part in CLD genesis.3. Exposure to hyperoxia resulted in a marked increase in smooth muscle, alveolar septa myofibroblast foci and on the surface of alveolar at the late phase of hyperoxia-exposed; however in air control groups, alpha-SMA positive cells with a round shape are found at the tip of the secondary septa, absent expression in alveolar space and mesenchymal. It is suppose that persistent hyperoxia exposed induced the position and intension change of myofibroblast may be one of the most reason of CLD genesis.4. After Losartan administration, alpha-SMA, HYP content, and the protein and mRNA expression of ColI decreased significantly, meanwhile the degree of lung disorganization relieved. It is indicated that Losartan attenuated hyperoxia induced lung injury of neonate. It is also hint that hyperoxia induced lung fibrosis genesis may performed by the pathway Ang II binding to AT1 receptor regulating synthesis of ColI.5. MMP-13 and TIMP-1 expression in the neonatal lung tissue, which hint MMP-13 and TIMP-1 invoved construction of lung morphous and collagen metabolism.6. At the late phase of hyperoxia-exposed, the protein and mRNA expression disbalance of MMP-13/TIMP-1 in lung tissue may play an important role during the remodeling of CLD induced by hyperoxia.7. losartan induced MMP-13 and down regulated TIMP-1 expression in hyperoxia-exposed lung tissue, attenuated extracellular matrix including ColI deposition at the late phase of hyperoxia-exposed, which is indicated that AngII may taked part in lung fibrosis induced by hyperoxia via AT1 receptor regulating synthesis and activity of MMP-13/TIMP-1.