| Backgrounds:Bronchopulmonary dysplasia(BPD)is the most common chronic lung disease of premature infants,especially in extremely low or very low birth weight infants.With the rapid development of the neonatal intensive care unit(NICU),extremely low or very low birth weight infants' survival rate is increasing,while the incidence of BPD rises year by year,becoming the leading cause of premature death and poor prognosis.The effects of BPD on respiratory system can extend from neonatal period to adulthood and even affect alveolar development on their offspring.The mortality rate of BPD is high,and the survivors often combine with complications of respiratory infection,feeding difficulties,and growth retardation,which seriously affect the quality of children's life.The pathogenesis of BPD is complex and not completely clear.Inhalation of high concentration of oxygen,forming a large number of highly active oxygen radicals in body,however,with the imperfect development of anti-oxidation and low level of antioxidants,premature babies cannot clear highly active oxygen radicals in time,leading to severe oxidative stress,inflammatory reaction,pulmonary fibrosis,and eventually BPD,so the antioxidants application is one of hot issues of BPD treatment.Inflammatory injury is the key point to the pathogenesis of BPD,so glucocorticoid usage after birth is most early,widely used in the prevention and treatment of BPD,which is supposed to reducing inflammation,improving pulmonary gas exchange,reducing pulmonary edema,improving lung compliance,and decrease the incidence of BPD.But the side effects of glucocorticoids have also been confirmed.So far,various treatments of BPD can improve the symptoms and prognosis of patients to varying degrees,but the efficacy is uncertain and the incidence of BPD has not been reduced.Therefore,we need to find a more safe and effective prevention.Resveratrol(Res)is considered to be the most important bioactive substances in stilbene monomers,with a variety of biological activity and pharmacological action,and studies have shown that the ability of anti-inflammation of Res equals to hormone,but without side effects.Therefore,Res may be a new drug to treat BPD.This experiment uses res to interfere animals and observe the curative effect,providing theoretical basis for BPD treatment.Objective:1.Establish the BPD animal model of rats and lay the foundation for the follow-up Res research of protection mechanism.2.To detect oxidative stress and inflammatory damage in BPD rat model,and to confirm the role of oxidative stress and inflammatory injury in BPD lung injury.3.To provide Res intervention in the rat model of BPD,furtherly explore whether Res could inhibit the oxidative stress and inflammation through the keapl-nrf2 and NF-kappa B signaling pathway.Methods:1.Establishment of rat model:(1)establishment of BPD rat model through high oxygen induction;(2)the detection index:general situation and weight change,gross observation,lung tissue paraffin section of HE staining,Masson staining,the thickness of the alveolar interval,radial alveolar count(radial alveolar counts,RAC).2.In vivo experiment of rat model:(1)the rats were divided into three groups:high-oxygen RES group(Fi02=60%,24),high-oxygen control group(Fi02=60%,24)and normal control group(24);(2)the oxidative damage index determination,detection of cyclooxygenase 2(cox-2),expression of LOX,ROS level(DCFH-DA),catalase,glutathione(GSH),total superoxide dismutase(T SOD);(3)determination of mRNA expression of Keap-1 and Nrf2;(4)Western Blot was used to determine the protein expression of NF-?B and AMPK in rat lung tissue.Results:1.Establishment,grouping and related indicators of BPD rat model:(1)successfully established the BPD rat model;(2)HE staining:no obvious pathological changes were observed in the control group and the high oxygen group in the 3 days,the alveolar structure was clear,the size was uniform,no liquid and inflammatory exudate.Later,with prolonged exposure to high oxygen,compared with control group,alveolar walls gradually becomes thin,alveolar uneven size,alveolar fusion,volume increased,inflammatory cells,some fibroblasts and collagen increased in high oxygen group,which prompts the development of lung.(3)Masson staining:compared with the control group,with the prolonged exposure to high oxygen,the pulmonary interstite of the rats in the high-oxygen group showed more collagen fibers,showing different levels of collagen fibrosis.(4)comparison of alveolar interval thickness:the thickness of alveolar interval in the control group gradually decreased with the gradual increase of daily age;However,the alveolar septum of the high oxygen group gradually widened(P<0.01).there was no significant difference between the 3d high-oxygen group and the control group.Compared with the control group,the alveolar interval of the 7d group was significantly widened(P<0.05).At 14d,the difference was more significant(P<0.01).(5)radial alveolar count(RAC):Compared with the control group,the RAC was significantly reduced in the 7d,14d high oxygen group,P<0.05,and the difference was statistically significant.2.Inhibition of oxidative stress of Res in mice:(1)the cox-2 and LOX in SD rat model of expression in lung tissue:in 3 d high oxygen control group cox-2,LOX expression began to increase,high oxygen RES group of cox-2,LOX expression,although more than normal control group,but its expression gradually reduce in compare of high oxygen control group,with statistical significance.(2)Detection of each group model of the generation of reactive oxygen,the result shows:the comparison between the three groups,the high oxygen control group is significantly higher than Res group and normal control group(P<0.01),the high oxygen RES group is higher than normal control group(P<0.05),the above results suggest Res has obvious inhibitory effect on reactive oxygen for BPD model rat.(3)Detection of intracellular GSH,H2O2 enzyme,SOD enzyme activity and comparison,the results showed that high oxygen control was significantly lower than normal control group and high oxygen RES group(P<0.05);High oxygen RES group is not higher than control group and has significant difference(P<0.05),suggesting that Res has a promoting effect on GSH,H2O2,SOD activity for BPD rat model.(4)The Keap-1 mRNA level:the high oxygen control group is significantly higher than normal control group(P<0.05),high oxygen RES group is higher than high oxygen control group(P<0.05);Nrf2 mRNA level,the high oxygen control group is significantly lower than normal control group(P<0.05),high oxygen RES group is higher than high oxygen control group(P<0.05).(5)NF-? B protein express level:the high oxygen control group is significantly higher than normal control group(P<0.05),high oxygen RES group is lower than high oxygen control group(P<0.05);AMPK protein express level:the high oxygen control group is significantly lowerer than normal control group,high oxygen RES group is higher than high oxygen control group(P<0.05)Conclusions:1.This study successfully established a rat model of BPD.2.Resveratrol can significantly improve the pathological changes of lung tissues in BPD rats,and it has therapeutic effect on BPD rats.3.The therapeutic mechanism of resveratrol on BPD may promote the degradation of Keapl and the activation of Nrf2 as well as the anti-oxidative stress and the inhibition of nf-kB,and the reduction of inflammatory response.The above results provide a new idea for the diagnosis and treatment of BPD. |
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