Effect Of DNA Nanotube Carrying Rab26-siRNA On Endothelial Barrier Function In Pulmonary Microvascular Endothelial Cells And Its Mechanism

Background:Acute lung injury(ALI)is a complex clinical syndrome with common dynamic changes,and it’s the early stage of acute respiratory distress syndrome(ARDS).ALI can be caused by severe infection,shock,trauma,sepsis,etc.ALI is mainly manifested as shortness of breath,respiratory distress,decreased pulmonary compliance,non-cardiac pulmonary fluid and intractable hypoxemia,and then one or more organ and tissue injuries develop into ARDS.The mortality of acute lung injury is high,and its incidence has been increasing in recent years,but its pathogenesis is still uncovered,and there is no effective prevention and treatment to reduce its mortality strategy significantly,so the research of the pathogenesis of ALI is now great significance for its prevention and treatment.The basic pathophysiological changes of ALI are known to be non-cardiogenic pulmonary edema caused by increased permeability of alveolar epithelium and pulmonary microvascular endothelium.A large amount of edema rich in inflammatory cells and pro-inflammatory factors is accumulated in alveoli and interstitial tissue.In addition,fibroblasts and alveolar epithelial cells can also produce lots of cytokines,these factors are all together aggravating the inflammatory response.Therefore,the destruction of endothelial barrier and pulmonary microvascular endothelial cells is the key inducing factor of ALI.Rab protein is a kind of small molecule GTP-binding protein which exists in plasma membrane and organelle membrane,and it is the largest subfamily of Ras-like family.It has GTP enzyme activity that can bind GTP and hydrolyze it,and regulate the fusion of vesicles through GTP-GDP cycle.It also participates in the docking process of transporting vesicles to the cell membrane together with Rab effector.Different Rab proteins have different functions: Rab1 can recruit adhesion factor P115 to participate in vesicle budding and regulate the anchoring of vesicles on Golgi bodies.Rab5 is associated with early phagosome formation,regulates endocytosis of extracellular signaling molecules,and also regulates subcellular localization of β2AR.In our early study,Rab1 and Rab5 protein were participated in controling the function of endothial barrier through regulating receptors tranport.Whether the Rab26,a kind of small molecule GTP-binding protein can regualte the function of endothial barrriers and its mechanism is still clear.Gene therapy refers to the introduction of exogenous gene with normal functions into recipient cells to replace or supplement the defective genes,so as to achieve the purpose of treating diseases.And the introduction of exogenous genes into biological cells must rely on certain technical methods or carriers.The Gene vector can be divided into virus vector and non-virus vector.The transfection efficiency of viral vector is high,but there are a lot of safety problems,and non-viral vectors generally have low transfection efficiency.Therefore,it is of great significance to make a breakthrough in gene therapy to explore a vector which can deliver the target gene safely and efficiently.With the development of Nanomedicine,nanomaterials as gene carriers have become a significant research direction.Compared with traditional carriers just like virus carriers,cationic liposomes,macromolecular polymers and various nanomaterials,DNA as nanomaterials has lots of advantages such as nature,non-immunogenicity,great targeting,high transfection efficiency,low toxicity or non-toxicity.Besides,self-assembled nanomaterials followed the base complementary pairing principle have the advantages of low cost,easy assembly and programming,modifiable,etc.It can also carry small interfering RNA and carry genes to achieve gene silencing through DNA-RNA hybridization technology.In this study,we combined gene silencing technology(Rab26 siRNA)and DNA nanotechnology to study PMVECs’ s barrier function,and provide a new idea for the treatment of ALI.Research purposes1.Synthesize DNA nanotube carrying Rab26 siRNA2.Characterize the DNA nanotube,detect its intracellular transfection efficiency and biological effects.3.Study the effects of Rab26 on the endothelial barrier function of PMECs and the mechanism preliminarily.Research methods1.According to the principle of complementary base pairing,Rab26 siRNA-NT were designed and synthesized by a step-wised annealing process.The DNA nanotubes were characterized by Native-PAGE and DLS.2.We incubated the DNA nanotubes with A549/DDP cells and then used the CLSM to observe the time and dose dependent cellular uptake of the DNA nanotubes.Flow cytometry was used to detect the transfection efficiency.Then incubated the DNA nanotubes with human lung adenocarcinoma cisplatin resistance strain cell(A549/DDP)to validate its gene silencing efficiency and the effect of cell killing.3.LPS was used to stimulate HPMECs,then used WB to detect Rab26 expression and used Transwell to detect HPMECs permeability.Transwell assay was used to detect the effect of DNA nanotubes with Rab26 siRNA on the permeability of HPMVECs under different conditions(DMSO/LPS).4.LPS was used to stimulate HPMECs,then used WB to detect P62,LC3-II/I expression.HPMECs were treated with autophagy agonists and inhibitors respectively,then used WB to determine the expression of P62,LC3-II/I to study the effect of DNA nanotubes on HPMECs autophagy.5.DNA nanotubes with Rab26 siRNA were transfected with HPMECs,and the expression of β2-AR and TLR4 was detected by CLSM.The TLR4 siRNA,β2-AR siRNA and TLR4 siRNA+β2-AR siRNA were transfected with HPMECs respectively,then use WB to detect the expression of TLR4 and β2-AR on the cell membrane,used Transwell to detect HPMECs permeability.6.Wild-type C57 mice were intraperitoneally injected with LPS to establish a mouse model of acute lung injury,then used the same conditions of LPS and PBS to treat the Rab26 knockout mice and Wild-type C57 mice.ALI in wild-type C57 mice and Rab26 knockout mice under PBS/LPS was compared by observing lung tissue HE staining,measuring pulmonary vascular permeability,lung wet-dry ratio and TNF,il-6 inflammatory factor expression.Research results1.Designed and synthesized the DNA nanotube with Rab26 siRNA successfully by the regular annealing,it can be observed a dominant band which represents the DNA nanotube on Native-PAGE.The result of DLS shows that the DNA nanotube particles were uniform in size and particle distribution.2.The results of CLSM indicated that the cellular uptake ofRab26 siRNA-NT in A549/DDP cells was time-and concentration-dependent;The results of flow cytometry suggested that Rab26 siRNA-NT transfected A549/DDP cells had high transfection efficiency and the transfection efficiency reached over 60% after 6 h;Rab26 siRNA-NT transfected A549/DDP cells can inhibit the expression of Rab26 and enhance the killing effect of cisplatin on A549/DDP cells.3.The expression of Rab26 was inhibited and the permeability was increased in HPMECs treated with LPS.Compared with the NC group,the permeability of HPMVECs in the Rab26 siRNA-NT group was significantly increased by LPS treatment.4.LPS treatment of HPMECs can inhibit autophagy;Rab26 siRNA-NT transfection inhibited autophagy in pulmonary vascular endothelial cells.5.Rab26 siRNA-NT was transfected with HPMECs,and CLSM results showed that the expression of β2-AR was inhibited and the expression of TLR4 was enhanced.The β2-AR siRNA and TLR4 siRNA were transfected with HPMECs,compared with control group,the WB results shows that β2-AR’s expression was increased in the TLR4 siRNA group,the expression of TLR4 was increased in the β2-AR siRNA group and the expression of TLR4 and β2-AR were inhibited in the TLR4 siRNA + β2-AR siRNA group.The result of transwell showed that compared with control group the permeability of HPMECs in the experimental group was meaningfully higher;The permeability of HPMECs in the TLR4 siRNA group was significantly lower than that in the β2-AR siRNA group and the permeability of HPMECs in the TLR4 siRNA + β2-AR siRNA group was significantly higher than that in the β2-AR siRNA group.6.After LPS stimulation,the alveolar wall of Rab26 knockout mice was thicker than that of wild-type C57 mice,and the alveolar atrophy and collapse were more obvious with a large number of red blood cells and inflammatory cell infiltration,and pulmonary vascular permeability,the expression of inflammatory factors were significantly increased,showing more serious lung damage.Conclusions1.Designed and synthesized the DNA nanotube with Rab26 siRNA successfully,the efficient cell transfection can be achieved,which can effectively inhibit the gene expression of Rab26.2.The expression of Rab26 was inhibited and the permeability was increased in HPMECs.Inhibiting Rab26 expression,can lead to the inhibition of autophagy,β2-AR and TLR4 receptor balance is also broken,thus influence the microvascular endothelial barrier function.3.Inhibiting Rab26 expression may increase the severity of ALI,Rab26 plays an important role in ALI protection.