Study Of Rapamycin Inhibiting Type Ⅱ Alveolar Epithelial Cells Ferroptosis Through P62-Keap1-Nrf2 Pathway On The Pathogenesis In Bronchopulmonary Dysplasia

Objective: With the development of NICU treatment technology,the survival rate of premature infants with small gestational age has been continuously improved,however,the incidence of bronchopulmonary dysplasia has increased year by year.BPD is one of the common respiratory diseases of premature infants.It is mainly characterized by alveolar dysplasia.Its main pathological features are small number of alveoli,large size,simple structure,and associated with pulmonary microvascular dysplasia.The long-term outcomes mainly include repeated respiratory infections,airway hyperresponsiveness,and abnormal lung function.Therefore,it is urgent to explore the key mechanism of alveolar dysplasia.Type II alveolar epithelial cells(AECII),as "stem cells" in the alveolar epithelium,can differentiate into AECI during lung injury,jointly maintain the stability of the alveolar structure,ensure the alveolar surface area,and play an important role in the process of lung tissue repair.During the observation of the ultrastructure of BPD model in the early stage of our research group,we found that the mitochondria of AECII have undergone changes such as smaller volume,outer membrane rupture,and disappearance of crest structure.This characteristic change was consistent with a new type of cell death mode-Ferroptosis.Ferroptosis is a new type of cell death caused by Fe-dependent oxidative damage.This process of cell death is different from apoptosis,necrosis and autophagy in morphological characteristics,hysiological and biochemical.The current research on ferroptosis mainly focuses on tumors,neurodegenerative diseases,ischemia-reperfusion injury,etc.The mechanism of ferroptosis,such as the imbalance of iron ion metabolism,oxidative stress and abnormal glutamate metabolism,has more in common with the pathogenesis of BPD.However,it is not clear whether this form of death is involved in the occurrence of BPD.At present,it is considered that oxidative stress injury caused by imbalance of oxidative / antioxidant system is the main form of BPD injury.Nrf2 is the core gene of the anti-oxidation system in the organism,and it is widely expressed in the body.A variety of downstream target genes can participate in the regulation of ferroptosis.At present,the application of Keap1-Nrf2 antioxidant system protection mechanism in many treatments has been carried out to the pre-clinical research stage,but the conclusion of the research on the application of Nrf2 agonists in the prevention and treatment of BPD is still controversial,so it is crucial to explore the clear mechanism of Nrf2 participation in the development of BPD important.Autophagy is an important mechanism for maintaining the homeostasis of organisms,and is a necessary biological behavior in lung development.It also plays an important role in the antioxidant response.Previous studies have shown that autophagy and Keap1-Nrf2 pathway cross through p62 regulation,and excessive autophagy has been shown to participate in the occurrence of BPD,however,the autophagy agonist rapamycin can repair alveolar dysplasia,the mechanism is not yet clear.Therefore,this study intends to verify the effect of AECII ferroptosis on alveolar dysplasia;to study the role of Keap1-Nrf2 pathway in regulating AECII ferroptosis;and to explore whether rapamycin can regulate AECII ferroptosis by affecting Keap1-Nrf2 pathway.It is expected to elucidate the molecular regulatory mechanism of AECII ferroptosis in BPD and provide a new target for the effective prevention and treatment of BPD..Methods: Part 1: 1.Preparation of BPD animal model.The new Sprague Dawley(SD)rats were randomly divided into two groups: model group(Fi O2=0.8)and control group(Fi O2=0.21).On the 3rd,7th,10 th and 14 th day,eight pups in each group were randomly selected to collect lung tissue samples,eight of them were randomly selected to extract the primary AECII,the pathological changes were observed by HE staining at the tissue level,and the morphological changes of mitochondria were observed by transmission electron microscopy.Western blot was used to detect the protein expression of PTGS2,GPx4 and FTH1,and RT-PCR was used to detect the m RNA expression of PTGS2,GPx4 and FTH1.2.Inhibition of ferroptosis in BPD rats.The newborn SD rats were randomly divided into three groups: the ferroptosis inhibitor group(Fi O2=0.8,Liproxstein-1,10mg/kg,QD,IP),the model group(Fi O2=0.8)and the control group(Fi O2=0.21).On the 14 th day,eight pups in each group were randomly selected to collect lung tissue samples,the pathological results were observed by HE staining and the morphological changes of mitochondria were observed by transmission electron microscopy.Western blot was used to detect the protein expression of PTGS2,GPx4 and FTH1,the proliferation of lung tissue was observed by Ki67 staining,and the injury of lung tissue was observed by γ-H2 AX staining.3.Establishment of hyperoxia exposed cell model.Extract the primary AECII of SD rats within 12 hours after birth,and divide it into three groups: ferroptosis inhibitor group(Fi O2=0.8,lip-1,200nm),model group(Fi O2=0.8)and control group(Fi O2=0.21).Harvest the cells after 48 hours of culture,Western blot was used to detect the protein expression of PTGS2,GPx4 and FTH1,the proliferation of cells was detected by Ki67 staining,and DNA damage was detected byγ-H2 AX staining.Part 2: Preparation of BPD animal model.The new Sprague Dawley(SD)rats were randomly divided into two groups: model group(Fi O2=0.8)and control group(Fi O2=0.21).On the 3rd,7th,10 th and 14 th day,eight pups in each group were randomly selected to collect lung tissue samples,eight of them were randomly selected to extract the primary AECII.Western blot was used to detect the protein expression of p62,p62(p-S351),Keap1,Nrf2,HO-1,NQO1 and GCLC,and RT-PCR was used to detect the m RNA expression of Keap1,Nrf2,HO-1,NQO1 and GCLC.The expression of Nrf2 protein in nucleus and cytoplasm were detected by nuclear plasma separation technique.The expression and localization of Nrf2,HO-1,NQO1 and GCLC were detected by immunohistochemistry.The co-localization of Nrf2-Keap1,p62-Keap1 and p62(p-S351)-Keap1 was detected by immunofluorescence double staining.Part3: Promotes autophagy in BPD rats.The newborn SD rats were randomly divided into three groups: the autophagy agonist group(Fi O2=0.8,Rapamycin,10mg/kg,QOD,IP),the model group(Fi O2=0.8)and the control group(Fi O2=0.21).On the 14 th day,eight pups in each group were randomly selected to collect lung tissue samples,and the pathological results were observed by HE staining.The expression and localization of Nrf2 was detected by nuclear plasma separation technique and immunohistochemistry.Western blot was used to detect the protein expression of PTGS2,GPx4,FTH1,p62,p62(p-S351),Keap1,Nrf2,HO-1,NQO1 and GCLC.The co-localization of Nrf2-Keap1,p62-Keap1 and p62(p-S351)-Keap1 was detected by immunofluorescence double staining.Results: Part 1: 1.Morphological and ultrastructural changes of lung tissue: The results of HE staining showed that the RAC value of model group was significantly lower than that of control group(P<0.05)on the 7d and 10 d,and the difference was more significant on the 14d(P<0.01);the thickness of alveolar septum was significantly higher on the 7d and 10d(P<0.05),and the difference was more significant on the 14d(P<0.01).In the model group,the volume of mitochondria decreased,the crista structure disappeared,the outer membrane shrunk and broke.2.Expression of ferroptosis related protein and m RNA in lung tissue and AECII:At the protein level,compared with the control group,PTGS2 in the lung tissue of the model group began to increase at 7d,GPx4 began to decrease at 7d,and FTH1 continued to be significantly lower than that of the control group(P<0.05).Compared with the control group,the expression of PTGS2 in the primary AECII of the model group began to increased significantly at 10 d,GPx4 began to decreased significantly at 7d,and FTH1 continued to be significantly lower than that of the control group(P<0.05).At the m RNA level,compared with the control group,the PTGS2 expression in the lung tissue of the model group continued to increase,GPx4 began to decrease on the 7d,and the FTH1 level was significantly lower than that of the control group(P<0.05).Compared with the control group,the expression of PTGS2 began to increase on the 7d,GPx4 began to decrease significantly on the 7d,and FTH1 in the model group was significantly lower than that in the control group(P<0.05).3.The effect of Liproxstatin-1 on the lung tissue and AECII of BPD: After applying ferroptosis inhibitor,compared with the model group,the RAC value of the lung tissue in the 14 d intervention group increased(P<0.05),and the alveolar space became thinner(P<0.05).Under transmission electron microscope,the mitochondrial structure of AECII in the intervention group was more complete than that in the model group.The protein expression of PTGS2 decreased(P<0.05),the protein expression of GPx4 and FTH1 increased(P<0.05),the ratio of Ki67 and γ-H2 AX positive cells decreased(P<0.05),and there was no significant difference between the intervention group and control group.After applying the ferroptosis inhibitor,compared with the model group,the expression of PTGS2 protein in the intervention group was reduced(P<0.05),the expression of GPx4 and FTH1 protein was increased(P<0.05),and the ratio of Ki-67 positive marker cells was significantly increased(P<0.05),the ratio ofγ-H2 AX positive marker cells decreased significantly(P<0.05),and there was no significant difference between intervention group and the control group.Part2: The expression of p62-Keap1-Nrf2 pathway in BPD lung tissue and AECII:Compared with the control group,the expression of total Nrf2 protein in the lung tissue and AECII of the model group was increased(P<0.05),and the Nrf2 nucleoprotein increased at 3d,7d and 10d(P<0.05),and the expression of Nrf2 plasma protein increased at 7d,10 d and 14d(P<0.05).Compared with the control group,the protein expression of HO-1 and NQO1 in the lung tissue of the model group increased(P<0.05),the m RNA expression of HO-1 in the lung tissue continued to increase(P<0.05),and the m RNA expression of NQO1 was increased at 3d,7d and 10d(P<0.05),GCLC m RNA expression increased at 3d and 7d(P<0.05);HO-1,NQO1,GCLC protein expression increased in AECII of the model group(P<0.05).Compared with the control group,the Keap1 protein expression in the lung tissue and AECII of the model group increased from 7 days(P<0.05),while the Keap1 m RNA expression in the lung tissue of the model group was lower than that of the control group from 7 days(P<0.05).Compared with the control group,the expression of p62 in the lung tissue of the model group increased at 7d and 10 d,the expression of p62(p-S351)increased from 7d,and the ratio of p62(p-S351)/ p62 increased from 10d(P<0.05);In AECII,p62 expression increased at 7d and 14 d,p62(p-S351)expression increased from 3d,and p62(p-S351)/ p62 ratio increased from7d(P<0.05).Immunofluorescence double staining and co-localization scattergram analysis showed that the co-localization of Nrf2-Keap1,p62-Keap1,p62(p-S351)-Keap1 in the 7d model group was significantly increased compared with the control group(P<0.05).Part3: Effect of rapamycin on BPD lung tissue: After applying the autophagy agonist rapamycin,compared with the model group,the RAC value of the lung tissue in the 14 d intervention group increased(P<0.05),and the alveolar interval became thinner(P<0.05).In the intervention group,the expression of Nrf2 protein in the nucleus increased significantly(P<0.01),the expression of Nrf2 in the plasma decreased(P<0.05),and there was no significant change in the total protein.In the intervention group,HO-1 protein expression decreased(P<0.01),and NQO1 and GCLC protein expression increased significantly(P<0.01).In the intervention group,Keap1,p62,p62(p-S351)protein expression decreased(P <0.05),and p62(p-S351)/ p62 ratio decreased(P<0.05).The co-localization of Nrf2-Keap1,p62-Keap1,p62(p-S351)-Keap1 in the intervention group was weaker than that in the model group(P<0.05).In the intervention group,the expression of PTGS2 protein in lung tissue decreased(P <0.05),and the expression of GPx4 and FTH1 protein increased(P<0.05).Conclusions: 1.In the BPD neonatal rat model,there is an AECII ferroptosis,and inhibiting ferroptosis in newborn BPD rats can improve the alveolar development outcome of BPD,suggesting that ferroptosis is involved in the development of BPD.2.In the BPD neonatal rat model,the Nrf2-ARE pathway is activated,but the degradation of the p62-Keap1 complex causes Nrf2 cytoplasmic retention,making the activated Nrf2-ARE pathway insufficient to reverse the alveolar dysplasia in BPD rats.3.3.In the BPD neonatal rat model,rapamycin can promote the degradation of p62-Keap1 complex,upregulate Nrf2 nuclear transport,inhibit ferroptosis,improve the alveolar development of BPD rats,and provide a new theoretical basis for the prevention and treatment of BPD in the future.