The Effects And Mechanisms Of Polyunsaturated Fatty Acids Omega-3 On Hyperoxia-induced Pulmonary Hypertension In Rats

Bronchopulmonary dysplasia(BPD)is one of the most common and serious chronic lung disease of preterm infants,characterized by alveolar and vascular impairment.It is predominantly observed in very low or extremely low birth weight infants who subjected to high ventilator pressures and oxygen concentrations.Recent evidence has shown that pulmonary hypertension(PH)and right ventricular hypertrophy(RVH)affect 16-25%of premature infants with BPD,contributing significantly to perinatal morbidity and mortality.The pathophysiology of PH in BPD is complex and may result from multiple risk factors,including prenatal factors,such as prenatal infection,preeclampsia and intrauterine growth restriction(IUGR),as well as postnatal factors,such as oxidative stress and infection.At present,the pathophysiology of PH in BPD has not yet been fully defined.The lung development of premature infants is still at the stage of vesicle at birth,exposure to hyperoxia as well as other pathogenic factors would interrupt the alveolar and pulmonary vascular development process,leading to pulmonary dysfunction.The lung development of newborn rats is also at the stage of vesicle at birth,which is equivalent to the characteristics of lung structure of human at the age of 26-28 weeks gestation.At this time,the plasticity and remodling ability are active and hyperoxia exposure is more likely to cause direct damage and inhibit lung development.On the basis that the microvascular maturation stage is postnatal days 14-20 in rats,hyperoxia stimulation at this time can further damage the structure and function of the alveolar and pulmonary microvessels,thus providing a theoretical basis for the establishment of the BPD-PH model.In recent years,much attention has been paid to the regulatory role of microRNAs(miRNAs)in the pathogenesis of PH.It is reported that miR-21 plays a key role in the regulation of pulmonary vasoconstriction and remodeling in the PH through VEGF and PPAR-y signaling pathways of endothelial,smooth muscle cells and fibroblasts of the pulmonary vessels.But in the BPD-PH model,whether miR-21 is involved in the regulation of target genes related to vascular development remains unclear.The intervention measures such as mechanical ventilation and oxygen therapy result in adverse reactions such as lung inflammation in the infants of BPD,leading to a reduction in the cross-sectional area of the pulmonary vasculature,which ultimately results in an increase in pulmonary vascular resistance and pulmonary arterial pressure,right heart failure and even death.To date,despite significant advances in neonatal critical care,preventive or treatment options for BPD infants with PH have not been developed.In addition,follow-up studies showed that the adverse effects of BPD-PH survivors in early life would persist to youth and even adulthood.Therefore,the strategies on early life interventions and to prevent later deterioration of PH should be focus on targeting signaling pathways which mediate the BPD-PH.In recent years,omega-3 polyunsaturated fatty acids(PUFA ω-3)have been studied for its beneficial effects in immune system,inflammatory response and cardiovascular system.PUFA ω-3 is an essential fatty acid and must be obtained from diet.PUFA ω-3 mainly includes eicosapentaenoic acid(EPA;C20:5 n-3)and docosahexaenoic acid(DHA;C22:6 n-3),which are enriched in seafood and fish oil.It was reported that PUFA ω-3 could regulate angiogenesis and vascular development via VEGF/NO signaling pathway,attenuate the increased pulmonary aterial pressure and reverse pulmonary vascular remodeling in a MCT-PH adult rat model,and activate PPAR-y/NF-KB pathway to blunt inflammatory response.However,the effects of early life PUFA ω-3 supplementation in BPD-PH and its long-term outcomes are unknown.The aim of the present study was to evaluate the short-and long-term effects of PUFAω-3 on alveolar and pulmonary vascular development in a hyperoxia-induced rat model of PH.Part Ⅰ The establishment of hyperoxia-induced pulmonary hypertension model in ratsObjective:1.To establish BPD-PH rat model and assess pulmonary hemodynamics and morphometry.2.To observe VEGF/NO and ANGPT/TIE-2 signaling pathways,NF-kB p65 and proinflammatory cytokines,as well as vascular regulation related miRNA expression in lung tissues of BPD-PH rats.Methods:1.BPD-PH model:neonatal rats were exposed to continuous hyperoxia(FiO2=85%)for 20 days.2.Assessment of model:right ventricular systolic pressure(RVSP)was evaluated after 20 days of hyperoxia exposure.Fulton’ s Index(ratio of right ventricular weight to the left ventricular + septum weight)and the ratio of right ventricular weight to body weight were used to assess the right ventricular hypertrophy(RVH).Brain natriuretic peptide(BNP)concentration in the plasma was measured.3.Lung development assessment:lung weight and lung to body weight ratio were measured.Alveolar structure was evaluated by performing radial alveolar counts(RAC),and by measuring the mean linear intercept(MLI),secondary septa count,and septal wall thickness.4.Pulmonary vascular remodeling:the medial wall thickness fraction and medial wall area fraction were determined by positive a-SMA stained arteries.Collagen deposition was visualized on Picrosirius red-stained lung sections and expressed as a percentage of the vessel area5.Pulmonary vascular density:The number of small pulmonary vessels was counted on von-Willebrand Factor(vWF)-stained lung sections6.The changes of related genes in angiogenesis signaling pathway:Real-time RT-PCR and ELISA were performed to assess the mRNA and protein expression of VEGFA,VEGFR1,VEGFR2,eNOS,ANGPT-1,ANGPT-2,TIE-2 in lung tissue.The NO content of lung tissue and plasma was detected by Griess method.7.Real-time RT-PCR and ELISA were used to detect the expression and activity of NF-κB p65 in the nucleus and the levels of cytokine IL-1β,IL-6 and TNF-a.The infiltration of neutrophils and macrophages were evaluated by MPO and ED1 immunohistochemical staining respectively.8.The different miRNA expression profiles in lung tissue were examined by miRNA sequencing screening.Real-time RT-PCR was performed to validate the results.Results:1.Hyperoxia exposed rats showed growth retardation and increased RVSP,RVH,and BNP concentration in the plasma.2.The total lung weight and the lung to body weight ratio increased in BPD-PH rats.Air space area increased and alveolar structure was simplified.RAC and secondary septa count decreased,MLI and septal wall thickness increased significantly.3.Pulmonary arterioles medial wall thickening and collagen deposition in the adventitia were observed in BPD-PH rats.4.Pulmonary vascular density of BPD-PH rats was significantly reduced.5.The mRNA and protein levels of VEGFA,VEGFR-1,VEGFR-2,ANGPT-1,TIE-2 and eNOS in lung tissue decreased significantly,the expression of ANGPT-2 increased in hyperoxia exposed rats.NO content in lung tissue and plasma was also reduced in BPD-PH rats.6.The mRNA and protein expression of NF-κB p65 and proinflammatory cytokines IL-1β,IL-6,TNF-a in lung tissue of BPD-PH rats increased significantly,accompanied by neutrophils and macrophages infiltration in hyperoxia exposed rats.7.Comparision of miRNA expression profiles between air kept rats and hyperoxia exposed rats:12 upregulated miRNAs with more than 2 changes and 18 downregulated miRNAs with less than 0.5 changes were discovered in lung tissue of BPD-PH rats.8.The expression of miRNA-21 in BPD-PH rats was significantly higher than that in control group,which was consistent with the results obtained by sequencing screening.Conclusion:1.BPD-PH rat model was successfully established.2.VEGF/NO and ANGPT/TIE-2 signaling pathways and inflammatory response were involved in the pathogenesis of BPD-PH in rats.3.The expression of miRNA-21 was significantly increased in hyperoxia exposed rats than that in room air controls,which may participate in the regulation of lung development in BPD-PH rats.Part II The effects and mechanisms of Omega-3 polyunsaturated fatty acids on hyperoxia-induced pulmonary hypertension in ratsObjective:To explore whether the supplementation of omega-3 polyunsaturated fatty acids(PUFA co-3)in early life could improve the lung development in BPD-PH rats by regulating angiogenesis,inhibiting the inflammatory response,and affecting the expression of microRNA.Methods:PUFA co-3(0.2ml/day)was supplemented from late pregnancy(embryonic day 15)to the end of lactation(postnatal day 20),the control groups were simultaneously given the same amount of normal saline or PUFA ω-6.The groups were stratified as follows:Control/Air,Control/02,PUFA co-6/Air,PUFA ω-6/02,PUFA ω-3/Air and PUFAω-3/O2.RVSP and RVH were assessed.HE staining was used to observe lung development.Pulmonary arteriole remodeling and pulmonary microvascular density were assessed.GC-MS was used to determine the fatty acid content in the lung tissue,plasma and milk.Real-time RT-PCR and ELISA were used to evaluate the expression of PPAR-γ,VEGF/NO and ANGPT/TIE-2 signaling pathways,the levels of IκB-α and NF-κB p65 in the nucleus as well as proinflammatory cytokines concentration in the lung tissue,NO content in lung tissue and plasma were evaluated by Griess method.Immunohistochemical staining was used to observe inflammatory cells infiltration.The expression of miRNA-21 in lung tissue was performed by real-time RT-PCR.Results:1.The mean body weight of the newborn rats was not significantly different at birth.Hyperoxia exposure impaired growth from their sixth(for Control/O2 and PUFAω-6/O2 groups)or twelfth(for PUFA ω-3/O2 group)day of life until day 20.The survival rate decreased to 66.7%and 62.5%in Control/O2 and PUFA ω-6/O2 groups,respectively.Only four pups(11.1%)in PUFA ω-3/O2 died before the sacrifice day.2.The RVSP was higher in hyeroxia exposed rats.PUFA ω-3 supplementation to the dams improved hyperoxia-induced pressure augmentation in their pups in comparison with control rats(19.87 ± 0.43 mmHg(PUFA ω-3/O2)vs,27.57 ± 0.86 mmHg(Control/O2)and vs.27.87 ± 1.48 mmHg(PUFA ω-6/O2).3.Hyperoxia exposed rats had severe RVH.Maternal supplementation with PUFA co-3 following postnatal hyperoxia exposure led to significant reduction in Fulton’s Index and improvement in the RVH.Similar observations were found when the RVH was assessed as ratio of right ventricular weight to body weight.4.There was no significant difference between the lung weight and lung to body weight ratio at birth.After 20 days exposure,the lung weight and lung to body weight ratio increased significantly compared with the air control group.Compared with the Control/O2 and PUFA ω-6/O2 group,the lung weight and lung to body weight ratio of the PUFA ω-3/O2 group decreased,RAC and secondary septa count increased significantly,MLI and alveolar septal wall thichness and the air space area and perimeter of the alveoli decreased.5.Pulmonary arterial medial wall thickness and collagen deposition in adventitia were decreased in PUFA ω-3/O2 group compared with Control/O2 and PUFA ω-6/O2 group.6.Pulmonary vascular density was increased in PUFA ω-3/O2 group compared with Control/O2 and PUFA ω-6/O2 group.7.PUFA ω-3 supplementation significantly increased the contents of EPA,DPA and DHA in the lung tissue and plasma of offspring rats and reduced the content of PUFA ω-6(AA)and the ratio of ω-6 to ω-3.The concentration of EPA and DHA increased in the PUFA ω-3 supplemented milk,accompanied by a decrease in the ratio of ω-6 to ω-3.8.There were no differences in the expression of PPAR-γ between room air controls regardless of maternal supplementation.The mRNA and protein expression of PPAR-y was significantly higher in PUFA ω-3/O2 group than that of Control/O2 and PUFA ω-6/O2 group.9.There were no differences in the expression of VEGFA,VEGFR-1,VEGFR-2,ANGPT-1,ANGPT-2,TIE-2 and eNOS between room air controls regardless of maternal supplementation.The mRNA and protein expression of VEGFA,VEGFR-2,ANGPT-1,TIE-2 and eNOS was significantly higher and ANGPT-2 was lower in PUFA ω-3/O2 group than that of Control/O2 and PUFA ω-6/O2 group.10.There were no differences in the NO content between room air controls regardless of maternal supplementation.The NO content of lung tissue in PUFA ω-3/O2 group was significantly higher than that of the PUFA ω-6/O2 group,but was no statistical different compared with the Control/O2 group,and the NO content of the PUFA ω-3/O2 group was significantly higher than that in the Control/O2 and PUFAω-6/O2 group.11.There were no differences in the expression of IκB-α and NF-κB p65 between room air controls regardless of maternal supplementation.The mRNA and protein expression of IκB-α in the lung tissue was significantly higher in PUFA ω-3/O2 group than that of Control/O2 and PUFA ω-6/O2 group,the mRNA and protein expression of NF-κB p65 in the nucleaus was significantly lower in PUFA ω-3/O2 group than that of Control/O2 and PUFA ω-6/O2 group.12.There were no differences in the expression of IL-1β,IL-6 and TNF-α between room air controls regardless of maternal supplementation.The mRNA and protein expression of IL-1β,IL-6 and TNF-α in lung tissue were significantly lower in PUFAω-3/O2 group than that of Control/O2 and PUFA ω-6/O2 group.13.There were no differences in the inflammatory cells infiltration between room air controls regardless of maternal supplementation.Less infiltration of neutrophils and macrophages were observed in PUFA ω-3/O2 group.14.The expression of miRNA-21 was significantly lower in PUFA ω-3 supplemented newborn rats at birth and in PUFA ω-3/O2 group after 20 days’ hyperoxia exposure.Conclusion:1.PUFA ω-3 supplementation during late pregnancy and lactation could increase PUFA ω-3 content and decrease the ratio of ω-6 to ω-3 in the lung tissue,thus significantly improve lung development of BPD-PH rats.2.PUFA ω-3 supplementation could attenuate PH through improving pulmonary vascular remodeling and microvascular growth development,which is likely via the regulation of miRNA-21 and associated with VEGF/NO and ANGPT/TIE-2 signaling pathways and PPAR-γ/IκB-α/NF-κB pathway in the lung tissue.Part Ⅲ The effects and mechanisms of Omega-3 polyunsaturated fatty acids on hyperoxia-induced pulmonary hypertension in adult male ratsObjective:To explore whether the beneficial effects of PUFA ω-3 in BPD-PH could be sustained into adulthood,so as to improve the adverse effects in later life of the population.Methods:Using the established BPD-PH with supplementation rat model,all the male rats were kept and recovered in the room air for 6 weeks after weaned.The groups were stratified as follows:Control/Air,Control/O2,PUFA co-3/Air and PUFA ω-3/O2.Mean pulmonary arterial pressure(mPAP)and RVH were assessed.BNP concentration in the plasma was measured.Pulmonary arteriole remodeling was evaluated.Real-time RT-PCR and ELISA were performed to evaluate the expression of PPAR-y,of VEGF/NO and ANGPT/TIE-2 signaling pathways,the levels of IκB-α and NF-κB p65 in the nucleus as well as proinflammatory cytokines concentration in the lung tissue.Results:1.The mean body weight of PUFA ω-3 groups were higher thant those supplementaed with normal saline.Hyperoxia exposed rats had lower weight gain from the first week of life until adult and didn’t show catch-up after weaned.2.The mPAP and RVH were higher in hyperoxia exposed rats at 9 weeks of age.PUFA ω-3.supplementation to the dams improved hyperoxia-induced pressure augmentation and RVH in their pups in adulthood in comparison with control rats.3.Pulmonary arterial medial wall thickness and collagen deposition in adventitia were decreased in PUFA ω-3/O2 group compared with Control/O2 group at 9 weeks of age.4.The mRNA and protein expression of PPAR-y was significantly higher in PUFAω-3/O2 group than that of Control/O2 at 9 weeks of age.5.The mRNA and protein expression of VEGFA,VEGFR-2,ANGPT-1,TIE-2 and eNOS was significantly higher and ANGPT-2 was lower in PUFA ω-3/O2 group than that of Control/02 at 9 weeks of age.6.The mRNA and protein expression of IκB-a in the lung tissue was significantly higher in PUFA ω-3/O2 group than that of Corntrol/O2,the mRNA and protein expression of NF-κB p65 in the nucleaus was significantly lower in PUFA ω-3/O2 group than that of Control/O2.7.The mRNA and protein expression of proinflammatory cytokines IL-1β,IL-6 and TNF-α in the lung tissue were significantly lower in PUFA ω-3/O2 group than that of Control/O2 at 9 weeks of age.Conclusion:1.The hyperoxia-induced PH could persistent into adulthood,PUFA ω-3 supplementation during late pregnancy and lactation had long-term protective effects on BPD-PH survivors and might attenuate the increased PAP.2.PUFA ω-3 supplementation in early life could improve pulmonary vascular remodeling in adulthood,which is likely associated with VEGF/NO and ANGPT/TIE-2 signaling pathways and PPAR-γ/IκB-κ/NF-κB pathway in the lung tissue.