| Acute Respiratory Distress Syndrome(ARDS)is a common and severe clinical syndrome.The pathological feature is the severe damage to the alveolar epithelium and capillary endothelium,resulting in diffuse and heterogeneous pulmonary interstitial and alveolar edema.The patient was clinically characterized by refractory hypoxemia and progressive respiratory distress.ARDS is a serious life-threatening disease with a prevalence of 1 to 5 per 10,000 in the community and a mortality rate of up to 40%.The treatment of ARDS still mainly relies on ventilator-assisted ventilation,strict volume management,and symptomatic support,and there is still no specific drug that can effectively control the progression of ARDS.Therefore,in-depth study of the molecular mechanism of the occurrence and development of ARDS and the exploration of specific therapeutic targets are very important to reduce mortality and improve the prognosis of ARDS patients.The pathogenesis of ARDS is extremely complex,and the uncontrolled inflammatory response is considered to be a key factor in the occurrence of ARDS.The damage of the vascular endothelial cell barrier is casual in the development of ARDS.Impaired vascular endothelial cell barrier function can cause the outflow of protein-rich body fluids,while the entry of edema fluid into the alveolar space can activate inflammatory cells such as neutrophils and alveolar macrophages,resulting in the release of inflammatory cytokines and inflammatory mediators,causing inflammation reaction,oxidative stress,and pyroptosis,etc.,ultimately affect respiratory function and endanger the life of patients.Therefore,how to effectively reduce vascular endothelial barrier function impairment,especially to improve the barrier function of pulmonary microvascular endothelial cells,has been a research focus of ARDS.With the development of genomics and molecular sequencing technology,a large number of studies have shown that both Long non-coding RNA(LncRNA)and MicroRNA(miRNA)are involved in the occurrence and development of ARDS.LncRNA is a class of RNA usually composed of more than 200 nt,which does not encode a protein or may have limited peptide encoding ability.LncRNA is widely involved in a variety of important life activities,including gene replication,gene transcription,protein translation,and transport,and can regulate cell proliferation and apoptosis,inflammatory response,oxidative stress,and many other biological processes.MircroRNA is a class of non-coding RNAs composed of about 21-25 bases,which participate in the regulation of post-transcriptional levels of genes by binding to specific target mRNA.Regulation of miRNA expression is one of the main mechanisms of LncRNA,which can reduce the binding between miRNA and target mRNA by"sponging" certain sequences of miRNA,thus regulating the expression level of downstream genes.LncRNA OIP5-AS1(Opa-interacting protein 5 antisense RNA 1)is transcribed from the genes encoding OIP5 in the antisense direction.Its homologous genes are widely present in mammals.LncRNA OIP5-AS1 has been reported to promote inflammatory response and apoptosis in a variety of cells,such as epithelial cells,macrophages,and vascular endothelial cells,and is involved in the occurrence and development of various diseases,such as atherosclerosis,diabetic nephropathy,hip arthritis,cerebrovascular diseases and so on.However,it has not been reported whether LncRNA OIP5-AS1 is involved in the regulation of ARDS.A variety of miRNAs are abnormally expressed in acute lung injury.The study found that in ALI,the inhibition of miR-223 expression can aggravate the degree of pulmonary inflammatory damage.miR-223 can significantly reduce the inflammatory response of ALI by inhibiting NLRP3 inflammasome-mediated pyroptosis and inhibiting the expression of pro-inflammatory factors IL-1? and IL-18 in alveolar macrophages and epithelial cells.However,whether miR-223 can inhibit the NLRP3 inflammasome pathway in pulmonary microvascular endothelial cells has not been studied yet.Bioinformatics studies have found that LncRNA OIP5 AS1 can target miR-223.Therefore,we speculated that LncRNA OIP5 AS1 can regulate the expression of inflammatory proteins and participate in biological processes such as oxidative stress,inflammatory response,and pyroptosis by targeting miR-223.It plays an important role in the occurrence and development of ARDS.Furthermore,inhibiting LncRNA OIP5 AS1 by targeting miR-223 can treat or alleviate the progression of ARDS.To verify this scientific hypothesis,this study firstly clarified the expression of LncRNA OIP5 AS1 and miR-223 in ARDS patients in clinical samples.Then,human lung microvascular endothelial cells(HPMECs)treated with lipopolysaccharide were used to study the mechanism of LncRNA OIP5 AS1-miR-223 in cells.Finally,The therapeutic effects of inhibition of LncRNA OIP5 AS1 and overexpression of Mir-223 in ARDS were preliminarily explored through the rat ARDS model.Part one Expression of LncRNA OIP5-AS1 and miR-223 in ARDS patientsObjective:To compare the expression of LncRNA OIP5-AS1 and miR-223 in ARDS patients and healthy controls,and to explore the correlation between the expression of LncRNA OIP5-AS1 and miR-223.Methods:A total of 20 ARDS patients admitted to the Intensive Care Unit of the First Affiliated Hospital of Anhui Medical University from January 2019 to December 2019 were enrolled,and 20 age-and sex-matched healthy controls were recruited from the physical examination population during the same period.The expression levels of LncRNA OIP5-AS1 and miR-223 in the two groups were detected by quantitative PCR,and their correlation was analyzed.Results:1.Compared with the healthy control group,the expression level of LncRNA OIP5-AS1 in peripheral blood of ARDS patients was significantly increased,and the expression level of miR-223 was significantly decreased.2.Correlation analysis showed that LncRNA OIP5-AS1 expression was significantly negatively correlated with miR-223 expression.Conclusion:The expression level of LncRNA OIP5-AS1 was significantly increased and the expression level of miR-223 was significantly decreased in ARDS patients,and the two were significantly negatively correlated,suggesting that LncRNA OIP5-AS1 and miR-223 were involved in the occurrence and development of ARDS.Part Two The mechanism of LncRNA OIP5-AS1 in LPS-induced HPMECs injury by targeting miR-223/NLRP3Objective:To investigate the effects of LncRNA OIP5-AS1 and miR-223 on cell proliferation,apoptosis,pyroptosis,inflammatory response and oxidative stress response in LPS-induced HPMECs injury by transfecting HPMECs with si-OIP5-AS1 and miR223 mimics/inhibitor.Methods:1.Quantitative PCR was used to detect the transcription levels of LncRNA OIP5-AS1 and miR-223 in LPS-treated HPMECs.2.LncRNA OIP5-AS1 was knocked down and miR-223 was overexpressed in HPMECs.Proliferation and apoptosis of HPMECs after LPS treatment were analyzed by MTT method and flow cytometry.The expression levels of NLRP3,ASC,GSDMD-N,Caspase-1,Caspase-3,Bax and Bcl-2 were detected by Western blot,and the expression levels of IL-1?,IL-6,IL-18 and IL-10 were detected by ELISA kit.ROS,SOD and MDA products were detected with corresponding kits.3.Dual luciferase activity assay confirmed the targeting relationship between LncRNA OIP5-AS1 and miR-223,miR-223 and NLRP3.Results:1.The expression level of LncRNA OIP5-AS1 increased and the level of miR-223 decreased in HPMECs treated with LPS,which was consistent with the changes in ARDS patients;2.LncRNA OIP5-AS1 knockdown in HPMECs induced cell proliferation,while pyroptosis,inflammatory response and oxidative stress response were inhibited by LPS treatment,which showed decreased expression of NLRP3 inflammasome and proinflammatory factors IL-1?,IL-6 and IL-18,the expression of anti-inflammatory factor IL-10 increased,ROS and MDA levels decreased,and SOD level increased.3.miR-223 is a target of LncRNA OIP5-AS1.Inhibition of miR-223 can cause apoptosis,partially abolish the effect of knockdown of LncRNA OIP5-AS1 on HPMECs,while overexpression of miR-223 promotes cell proliferation,inhibited cell pyroptosis,inflammatory response,and oxidative stress response,consistent with the knockdown of LncRNA OIP5-AS1 phenotype;4.NLRP3 is a target of miR-223,and overexpression of NLRP3 can eliminate the effect of miR-223 overexpression on HPMECs;Conclusion:LPS treatment of HPMECs can increase the expression of LncRNA OIP5AS1 and decrease the expression of miR-223.Inhibition of LncRNA OIP5-AS1 or overexpression of miR-223 can promote cell proliferation,inhibit cell apoptosis and pyroptosis,and reduce inflammation and oxidative stress response.There is a targeting relationship between LncRNA OIP5-AS1 and miR-223,miR-223 and NLRP3.LncRNA OIP5-AS1 participates in LPS-induced HPMECs injury by activating NLRP3 signaling pathway through adsorption of miR-223.Part Three Therapeutic effect of LncRNA OIP5-AS1 and miR-223 in ARDS ratsObjective:To investigate the therapeutic effects of LncRNA OIP5-AS1 and miR-223 in LPS-induced ARDS rats by knocking down LncRNA OIP5-AS1 and overexpressing miR-223 by adenovirus vector.Methods:1.Establish the LPS-induced ARDS model in rats,and use adenovirus vectors to carry the target gene of siRNA and miR-223,respectively,to reduce the expression level of LncRNA OIP5-AS1 and overexpress miR-223;2.The expression levels of NLRP3,ASC,GSDMD-N,Caspase-1,Caspase-3,Bax and Bcl-2 were detected by Western blot.The expression levels of IL-1?,IL-6,IL-18 and IL-10 were detected by ELISA kits,and the products of ROS,SOD and MDA were detected by corresponding kits.Results:1.In the LPS-induced rat ARDS model,successfully knocked down LncRNA OIP5-AS1 and overexpressed miR-223.2.Knockdown of LncRNA OIP5-AS1 and overexpression of miR-223 can alleviate LPSinduced lung tissue damage in rats.3.Knockdown of LncRNA OIP5-AS1 and overexpression of miR-223 reduced the expression of NLRP3 inflammasome,the expression of pro-inflammatory factors IL-1?,IL-6 and IL-18,and the expression of anti-inflammatory factor IL-10 in lung tissue increased,the levels of ROS and MDA decreased,and the level of SOD increased.Conclusion:Knockdown of LncRNA OIP5-AS1 and overexpression of miR-223 inhibited pulmonary inflammatory response,oxidative stress response and cell pyroptosis,alleviated lung injury in LPS-induced ARDS rats through LncRNA OIP5AS1/miR-223/NLRP3 signaling pathway.Summary:1.LncRNA OIP5-AS1 and miR-223 are involved in the development of ARDS.2.Inhibition of LncRNA OIP5-AS1 or overexpression of miR-223 can promote cell proliferation,inhibit cell apoptosis and pyroptosis,and reduce inflammation and oxidative stress response.There is a targeting relationship between LncRNA OIP5-AS1 and miR-223,miR-223 and NLRP3.LncRNA OIP5-AS1 participates in LPS-induced HPMECs injury by activating NLRP3 signaling pathway through adsorption of miR-223.3.Knockdown of LncRNA OIP5-AS1 and overexpression of miR-223 inhibited pulmonary inflammatory response,oxidative stress response and cell pyroptosis,alleviated lung injury in LPS-induced ARDS rats through LncRNA OIP5-AS1/miR-223/NLRP3 signaling pathway. |
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