Study On The Role Of ERM Protein And N6-methyladenosine In Acute Respiratory Distress Syndrome

BackgroundAcute respiratory distress syndrome（ARDS）are important causes of morbidity and mortality in severe patients.ARDS is a syndrome characterized by pulmonary edema and acute inflammation.At present,there is no specific treatment for ARDS,so new treatment methods are needed.The application of animal and cell models provides an important research basis for exploring the pathogenesis,diagnosis,treatment and prognosis of human diseases,determining new biomarkers and discovering new targets in drug research and development.Lipopolysaccharides（LPS）-induced rupture of the barrier of pulmonary microvascular endothelial cells（PMVECs）leads to increased permeability,which is an important pathogenesis of ARDS.However,the molecular mechanism of LPS induced high permeability of PMVECs is not fully understood.The membrane proteins of Ezrin-Radixin-Moesin（ERM）family are important binding factors connecting filamentous actin and plasma membrane,which are widely distributed.ERM proteins can bind to cytoplasmic signaling molecules to integrate membrane transport and signaling pathways.Our previous studies have confirmed that ERM proteins are involved in the permeability change of PMVECs caused by endothelial cytoskeleton remodeling.As a member of ERM protein family,Moesin is widely expressed in endothelial cells,it considered as a bonding bridge between membrane protein and actin skeleton protein.Another study indicated that Moesin can be phosphorylated as threonine substrate and participate in the permeability change of PMVECs induced by TNF-α.Therefore,Moesin has great meaning in the study of the pathogenesis of ARDS,especially to explore the role of ERM protein in ARDS,Moesin can be selected as the insertion point.N6-methyladenosine（m6A）methylation modification is the methylation at the sixth nitrogen of adenosine.It plays important regulatory roles in RNA splicing,translation,stability and epigenetic effects between m RNA and non-coding RNA.It is also involved in a variety of cellular biopathological processes.m6 A can participate in the occurrence and development of many diseases,for example,the abnormal expression of m6 A was observed in sepsis-induced myocardial injury and renal injury.However,the role of m6 A methylation in the occurrence and development of LPS induced ARDS is still lack of research data.Therefore,this study will explore the mechanism of ERM protein in the high permeability of Rat PMVECs（RPMVECs）induced by LPS in vitro,and further explore the role of m6 A methylation in LPS induced ARDS animal model.ObjectiveTo investigate whether ERM protein is involved in the high permeability of RPMVECs induced by LPS and its underlying mechanism;To investigate whether m6 A affects LPS-induced lung injury in ARDS mice.Methods1.To study the effect of LPS on the permeability of RPMVECs,RPMVECs were cultured in Transwell upper-chamber and stimulated with LPS at concentrations of 0,0.1,1 and 10 mg/L,respectively.The permeability of RPMVECs was measured by transendothelial electrical resistance（TER）within 0-2.5 h.2.To study the effect of LPS on the threonine phosphorylation level of ERM protein in RPMVECs.Western-blot was used to detect the expression of ERM threonine phosphorylated protein in RPMVECs stimulated by different concentrations of LPS（0,0.1,1 and 10 mg/L）for 30 min,or at the same concentration of LPS（10 mg/L）for different times（0,5,15,30,60,90,120 and 180 min）.3.To study the effect of LPS on the expression of Ras related C3 botulinum toxin substrate 1-GTP（Rac1-GTP）and Rac1-total protein in RPMVECs,western-blot was used to detect the expression of Rac1-GTP and Rac1-total protein in RPMVECs stimulated by LPS（10 mg/L）at different times（0,15,30,60,90 and 120 min）.To clarify the role of Rac1 in the high permeability of RPMVECs induced by LPS,si RNA was further used to interfere with the expression of Rac1,the si RNA specifically targeting Rac1 gene（si Rac1）was transfected into RPMVECs,and the phosphorylation level of ERM protein threonine in RPMVECs was detected by western-blot.4.To study the effect of O-Me-c AMP activating Rac1 signaling pathway on the permeability of RPMVECs increased by LPS,RPMVECs were treated with LPS（10mg/L）for 2 h,then co-cultured with O-Me-c AMP（200 mmol/L）for 2 h.The permeability of RPMVECs was detected by TER.After that,si Moesin was transfected into RPMVECs to establish the knocking down cell model.Using TER to detect the permeability of RPMVECs and western-blot for the expression of Rac1-GTP and Rac1-total protein in RPMVECs.5.ARDS mouse model was established by LPS induction.Male C57BL/6 mice were randomly divided into NC group（normal group,no treatment）,VC group（no-load control group,normal saline modeling）,LPS-3 h group（LPS treatment for 3h）,LPS-6 h group（LPS treatment for 6 h）,LPS-12 h group（LPS treatment for 12 h）and LPS-24 h group（LPS treatment for 24 h）.The body weight and whole lung of mice in each group were weighed.The left lower lobe（LD）of mice in each group was harvested and fixed with paraformaldehyde solution,then sections were stained with HE.Pannoramic 250 digital slice scanner was used to observe the pathological state of mouse lung tissue and collect the images.6.In order to clarify the changes of m6 A methylation in LPS-induced lung tissue,after the animals were killed,harvested the left upper lobe（LU）/ right upper lobe（RU）/ right lower lobe（RD）and extracted the tissue RNA or protein,19 m6 A related factors（METTL3,METTL14,ALKBH5,FTO,WTAP,RBM15 B,METTL16,e IF3 a,hn RNP A2/B1,IGFBP1,IGFBP2,IGFBP3,KIAA1429,RBM15,YTHDF1,YTHDF2,YTHDF3,YTHDC1,YTHDC2）were detected by q RT-PCR;western-blot was used to detect the expression of seven m6 A related proteins（METTL16,FTO,YTHDC1,IGFBP3,YTHDF1,YTHDF3,WTAP）in lung tissue samples of mice in NC group,VC group,LPS-3 h group,LPS-6 h group,LPS-12 h group and LPS-24 h group;The immunohistochemical characteristics of five m6 A related proteins（FTO,METTL16,YTHDF1,YTHDF3,YTHDC1）in the above different groups were detected by immunohistochemistry.7.To further analyze the overall m6 A and m5 C modification level,TRNzol was used to extract the total RNA from the lung tissue of mice in each group,and the changes of the overall modification level of m6 A and m5 C in the lung tissue of mice in each group were detected by UPLC-UV-MS.Result1.Compared with the control group,the permeability of RPMVECs was increased gradually in a dose-and time-dependent manner,suggesting LPS could affect the permeability of RPMVECs.2.RPMVECs were induced by LPS（10 mg/L）.The expression level of ERM threonine phosphorylation was increased gradually with the increase of stimulation time,peaked at 30 min.After 30 min,ERM threonine phosphorylation level began to decrease gradually until 180 min.However,with the increase of stimulation time,the expression of total ERM did not increase significantly.These results indicating that with the increase of LPS concentration,the expression level of ERM threonine phosphorylation with continuously increase,while the total ERM expression level did not change.3.After inducing with LPS（10 mg/L）,the expression level of Rac1-GTP gradually decreased in RPMVECs in a time-depended manner.Compared to the control group,the activity of Rac1 was decreased by nearly 76.5% after 120 min of stimulation,but no significant change was observed in the expression level of Rac1-total.Compared to control group,the expression level of ERM phosphorylation in si Rac1 group was increased,but the expression level of ERM did not change in RPMVECs.Compared with LPS group,the expression level of ERM phosphorylation in RPMVECs in LPS+si Rac1 group increased,while there was no significant change in the expression level of ERM in RPMVECs in LPS+si Rac1 group.4.Compared with the control group,the permeability of RPMVECs in LPS group and LPS+O-Me-c AMP group was increased,and O-Me-c AMP could restore the decrease of TER caused by LPS.Compared with si Control group and si Moesin group,the permeability of LPS+si Control group,LPS+si Moesin group and LPS+si Moesin+si Rac1 group was increased.The increase range of permeability in LPS+si Moesin+si Rac1 group was between LPS+si Control group and LPS+si Moesin group.Compared with si Control group,the expression level of Rac1-GTP in si Moesin group was increased,while it was decreased in LPS+si Control and LPS+si Moesin groups.At the same time,there was an increase of the expression level of Rac1-GTP in RPMVECs in LPS+si Moesin group,compared with LPS+si Control group;There was no difference in the expression of Rac1-total among si Control group,si Moesin group,LPS+si Control group and LPS+si Moesin group.5.The results of animal experiments showed that compared to control group,the change of lung weight of mice at each time point after LPS treatment was not obvious,but the body weight of mice was loss after LPS treatment for 12 h and 24 h.6.HE staining was used to observe the pathological state of mice.It was found that in VC group,the bronchial structure at all levels of lung was relatively normal,the bronchial ciliated epithelium was arranged orderly,and no obvious cell abscission was found.Alveolar epithelial cells were relatively normal,1 case（1/2）had alveolar hemorrhage and more red blood cell overflow,and 2 cases（2/2）had a very small amount of inflammatory cell infiltration in the lung stroma,mainly neutrophils.No other obvious pathological changes were found.LPS-6 h group: the pleural structure of visceral layer was normal,the wall was thin,and there was no proliferation and thickening of connective tissue.The bronchial structures are complete and clear with normal epithelial cells.There is no obvious degeneration,necrosis or abscission.Type I and type II alveolar epithelial cells were normal without obvious degeneration and necrosis.A small amount of inflammatory cell infiltration was observed in the stroma of2 cases（2/2）,mainly rod-shaped nuclear neutrophils.No other obvious pathological changes were found.The lung pathological injury in LPS-6 h group was more serious,compared with VC group.For NC group,the bronchial structures and alveolar epithelial cells were relatively normal,the bronchial ciliated epithelium was arranged neatly,without obvious degeneration,necrosis and thickening of alveolar septum.However,a small number of macrophages can be seen in alveolar cavity,no other obvious pathological changes.Group LPS-3 h: alveolar septum thickened was observed in some parts of lung tissue.Neutrophil infiltration in lobulated nuclei was seen in alveolar interstitium.Deep round lymphocytes infiltrated in endoplasm and perivascular interstitium.Foam cells infiltrated in alveolar cavity.In LPS-12 h group,more lymphocytes and neutrophils were observed in alveolar interstitium,while more lymphocytes in perivascular interstitium,and more macrophages in alveolar cavity.To sum up,compared with NC group,the tissues in LPS-3 h and LPS-12 h groups had pathological changes in varying degrees,of which the degree of lesion in LPS-3 h group was relatively severe.7.The expression levels of 19 m6 A related factors were detected by q RT-PCR in lung tissue of NC group,VC group,LPS-3 h group,LPS-6 h group,LPS-12 h group and LPS-24 h group.The results showed that the expression levels of 7 m6 A methyltransferases METTL3,METTL14,WTAP,KIAA1429,METTL16,RBM15 and RBM15B decreased after LPS treatment,KIAA1429 and RBM15 were significantly downregulated after LPS induction for 24 h.Among them,METTL16 was most significantly downregulated after LPS induction for 6 h.The expression of two m6 A demethylases FTO and ALKBH5 showed a downward trend,in which the expression of FTO decreased significantly in a time-dependent manner,while ALKBH5 decreased significantly at 24 h.The expression of m6 A recognition factors e IF3 a,YTHDF2,YTHDF3,YTHDC2 and IGFBP2 showed a downward trend.YTHDF1 and YTHDC1 were significantly up-regulated after 6 h of induction.The expression of other factors had no obvious change.8.Western-blot results showed that among seven m6 A related proteins,the protein levels of METTL16 and FTO exhibited an upward trend in LPS-induced lung tissue,YTHDF1 and IGFBP3 showed a downward trend as well as YTHDF1 and YTHDF3 at24 h,while no change in WTAP expression.9.Immunohistochemical results showed that afte r LPS induction,the immunohistochemical characteristics of five m6 A related proteins in mouse lung tissue were changed significantly.Among them,the expression of FTO protein was increased significantly in LPS induced lung tissue,METTL16 was also increased remarkably at12 h and 24 h.However,the expressions of YTHDF1 and YTHDC1 were decreased.10.The overall modification levels of m6 A and m5 C in lung tissue of mice treated with LPS were detected by UPLC-UV-MS.The results showed that the overall expression of m5 C was stable in different groups,which increased slightly after LPS treatment,but decreased slightly compared with NC group.However,the expression level of overall m6 A modification was changed significantly among the groups.Firstly,the overall m6 A level was increased significantly after LPS induction for 6 h,and then it showed a downward trend,suggesting that m6 A methylation was increased in lung tissue of ARDS mice after LPS induction for 6 h.Conclusion1.The TER of RPMVECs decreased in a dose-and time-dependent manner by LPS induction,with the increase of threonine phosphorylation of ERM and inactivation of Rac1.Rac1 and Moesin are involved in the mediation of high permeability of RPMVECs induced by LPS,while the signal pathway of high permeability mediated by Rac1 and Moesin can regulate each other.Phosphorylated ERM mediates the increase of permeability of RPMVECs induced by LPS by negatively regulating Rac1 activity.2.LPS induced lung tissue of ARDS mice had obvious inflammatory features,including neutrophil and macrophage infiltration.At the same time,the methylation level of m6 A was increased,the m RNA and protein levels of METTL16,FTO,YTHDC1 and YTHDF1 were changed in varying degrees,suggesting m6 A methylation induced by LPS plays an important role in lung injury of ARDS mice,it may become a potential target for the diagnosis or treatment of ARDS.