The Mechanism of lncRNA NEAT1 in Alleviating Acute Respiratory Distress Syndrome Through miR-27b Regulated Nrf2 Pathway

Study Overview
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Brief Summary
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The acute respiratory distress syndrome, formerly known as the acute lung injury (ARDS/ALI), is a critical illness with high mortality due to the lack of effective treatment. The pathogenesis of ARDS/ALI has not been fully elucidated. Nuclear factor E2-related factor 2 (Nrf2) plays a key role in regulating lung inflammation and oxidative stress which are closely related to lung injury in ARDS/ALI, but its regulatory mechanism remains unclear. The investigator's provious study shown that microRNA-27b (miR-27b) downregulated Nrf2 to aggravate lung inflammation and histological injury. Furthermore, in lipopolysaccharide (LPS)-induced cell (J774A.1) inflammation model, miR-27b was upregulated while the long non-coding RNA (lncRNA) NEAT1 was downregulated, the putative binding sites of lncRNA NEAT1 and miR-27b were successfully predicted by bioinformatics approach. Thus, the investigators propose that NEAT1 plays as a competing endogenous RNA (ceRNA) to adsorb miR-27b and liberate Nrf2, therefore, to attenuate lung inflammation and related lung injury in ARDS/ALI. This project aims to explore the role of the lncRNA NEAT1/ mir-27b /Nrf2 signal axis in the development and treatment of ARDS/ALI in patients, as well as in LPS-induced ALI animal and cell models by using bioinformatics, molecular biology, histomorphology and clinical phenotype approaches, and to clarify the new mechanism in ARDS/ALI development and to provide new therapeutic targets.

Detailed Description
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Collect blood and BALF from 400 ARDS patients at different time (at check-in, 24, 48 and 72 h after check-in the hospital) and 25 gender and age matching healthy controls. Use RT-PCR to detect the expression of lncRNA NEAT1、miR-27b and Nrf2 in blood and BALF of ARDS patients and health controls. The expressions of inflammatory and oxidative stress associated factors (NLRP3、NF-κB-P65、 p-P65、IκB、p-IκB、HO-1、NQO1、caspase-1、IL-1β、IL-6、IL-18、TNF-α) will be detected by western blot、ELISA and RT-PCR. Moreover, flow cytometry will be adopted to measure the numbers and kinds of cells in BALF. Then, analyze the differences of the expressions of lncRNA NEAT1、miR-27b and Nrf2 in the groups. To explore the correlation of expressions of lncRNA NEAT1、miR-27b and Nrf2 with inflammation and oxidative stress in the groups. Finally, to declare the relative of lncRNA NEAT1、miR-27b and Nrf2 with the time of mechanical ventilation, severity and mortality in 28 days of ARDS patients.

Official Title
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The Mechanism of lncRNA NEAT1 in Alleviating Acute Respiratory Distress Syndrome Through miR-27b Regulated Nrf2 Pathway

Conditions
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Acute Respiratory Distress Syndrome, Inflammation

Intervention / Treatment
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* Other: no intervention


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: We included patients with acute respiratory distress according to 2012 ARDS Berlin new definition (Acute Respiratory Distress Syndrome: The Berlin Definition. JAMA, 2012, 307(23):2526). Acute or progressive dyspnea within 1 week with identify cause; Chest radiograph/chest CT showed double lung infiltration, which could not be fully explained by pleural effusion, atelectasis, or nodules; Respiratory failure cannot be fully explained by heart failure and fluid overload; Hypoxemia, partial pressure of oxygen in arterial blood (PaO2)/oxygen fraction in air (FIO2) <150 mm Hg under PEEP ≥5 cm H2O, (mild ARDS: 200mmHg<PaO2/FiO2≤300mmHg, moderate ARDS: 100mmHg<PaO2/FiO2≤200mmHg, severe ARDS: PaO2/FiO2≤100mmHg); 18 70 years old; Agree to participate in the trial, and sign the informed consent. Exclusion Criteria: Age less than 18 years old; Time of hospital stay <24 h; Pregnancy; Using V-V ECOM; Cardiac index <1.5L·ml.min-1.m-2; Pulmonary resection; Pulmonary embolism ; Refused to participate in the study.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 70 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Control group<br>25 gender and age matching healthy controls | Other: no intervention<br>* no intervention<br>|
| ARDS group 1<br>100 ARDS patients at the time of check in hospital | Other: no intervention<br>* no intervention<br>|
| ARDS group 2<br>100 ARDS patients at the time of 24h after check in hospital | Other: no intervention<br>* no intervention<br>|
| ARDS group 3<br>100 ARDS patients at the time of 48h after check in hospital | Other: no intervention<br>* no intervention<br>|
| ARDS group 4<br>100 ARDS patients at the time of 72h after check in hospital | Other: no intervention<br>* no intervention<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| The expression of lncRNA NEAT1 in blood and BALF in all groups | Use RT-PCR to measure the expression of lncRNA NEAT1 in blood and BALF in all groups | up to 24 day | 
| The expression of miR-27b in blood and BALF in all groups | Use RT-PCR to measure the expression of miR-27b in blood and BALF in all groups | up to 3 day | 
| The expression of Nrf2 in blood and BALF in all groups | Use RT-PCR and Wsetern blot to measure the expression of Nrf2 in blood and BALF in all groups | up to 3 day | 
| The expression of inflammatory factors(IL-1β、IL-6、IL-18、TNF-α) in blood and BALF in all groups | Use RT-PCR and ELISA to measure the expression of inflammatory factors(IL-1β、IL-6、IL-18、TNF-α) in blood and BALF in all groups | up to 3 day | 
| The expression of oxidative stress associated factors in blood and BALF in all groups | Use Western blot to measure the expression of oxidative stress associated factors(NLRP3、NF-κB-P65、 p-P65、IκB、p-IκB、HO-1、NQO1、caspase-1) in blood and BALF in all groups | up to 3 day | 
| The numbers and kinds of inflammatory cells in BALF and blood in all groups | Use flow cytometry to detect the number of inflammatory cells in BALF and blood in all groups | up to 3 day | 
| The kinds of inflammatory cells in BALF and blood in all groups | Use flow cytometry to detect the kinds of inflammatory cells(neutrophile、macrophage、 lymphocyte) in BALF and blood in all groups | up to 3 day | 
| The time of mechanical ventilation of patients in ARDS groups | Record the time of mechanical ventilation of patients in ARDS groups | up to28 day | 
| The severity of ARDS patients in ARDS groups | Record the severity(PaO2/FiO2、OI、S/F、OSI) of ARDS patients in ARDS groups | up to 28 day | 
| the mortality in 28 days of ARDS patients | Record the mortality in 28 days of ARDS patients | up to 28 day | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| The differences and correlation of the expressions of lncRNA NEAT1、miR-27b and Nrf2 in the groups | Analyse the differences of the expressions of lncRNA NEAT1、miR-27b and Nrf2 in the groups, and to explore the relations between the three(lncRNA NEAT1、miR-27b and Nrf2) in different groups. | up to 28 day | 
| The correlation of expressions of lncRNA NEAT1、miR-27b and Nrf2 with inflammation and oxidative stress in the groups. | To explore the correlation of expressions of lncRNA NEAT1、miR-27b and Nrf2 with inflammation and oxidative stress in the groups. | up to 28 day | 
| The relative of lncRNA NEAT1、miR-27b and Nrf2 with the time of mechanical ventilation, severity and mortality in 28 days of ARDS patients | To declare the relative of lncRNA NEAT1、miR-27b and Nrf2 with the time of mechanical ventilation, severity and mortality in 28 days of ARDS patients | up to 28 day | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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long non-coding RNA NEAT1, microRNA-27b, nuclear factor E2-related factor 2