Mechanism of Delayed Neutrophil Apoptosis in Acute Lung Injury
Condition: Acute Lung Injury/Acute Respiratory Distress Syndrome (ARDS); Neutrophils; Apoptosis; Immunosuppression; Pathogenesis
MeSH terms: Respiratory Distress Syndrome; Respiratory Distress Syndrome, Newborn; Acute Lung Injury; Lung Injury; Wounds and Injuries
Study type: Observational

Summary: 
      Literature basis Acute respiratory distress syndrome (ARDS) is a clinical syndrome
      characterized by respiratory distress and progressive hypoxemia, which is caused by diffuse
      alveolar and pulmonary interstitial edema caused by various pulmonary and extrapulmonary
      factors other than cardiogenic factors. ARDS incidence rate is as high as 75 /10 000 per
      year, and sepsis and pulmonary infection are the most common causes. In the past, it was
      generally believed that excessive immune activation is the core of the pathophysiology of
      ARDS, and neutrophils are recognized as the core driver of inflammatory hyperactivity and
      lung injury in ARDS. Although some progress has been made in the epidemiology, pathogenesis
      and pathophysiology of ARDS in the past 50 years, and the clinical outcomes of some patients
      with ARDS have been improved by optimizing the mode of mechanical ventilation and fluid
      treatment, as well as prone ventilation and the use of muscle relaxants, ARDS is still one of
      the most common causes of death and disability in intensive care units, The mortality rate of
      the disease is currently as high as 30-40%. There is still a lack of effective drugs for the
      treatment of ARDS in clinic, and even glucocorticoids applied for immune overactivation have
      not achieved good results. This is related to the unclear pathogenesis of ARDS. Therefore, it
      is still a hot and difficult point to further explore the pathogenesis and progression of
      ARDS and find new therapeutic targets.

      In the past, mature PMN in peripheral blood was generally considered as a functional cell in
      the end stage, but it is widely involved in different innate immune responses (including
      inflammation, infection, tumor, autoimmunity, etc.) and can adopt very different effector
      mechanisms. Therefore, with the deepening of research, neutrophil subtypes with different
      functions (such as immune regulation and repair) have been identified in recent years:
      cd16dimcd62lbrightpmn and cd16brightcd62ldimmpmn. In the steady state of healthy people, the
      classic mature neutrophils (cd16brightcd62lbright) in peripheral blood account for more than
      98% of the total PMN, and the proportion of the two neutrophil subtypes is relatively low. In
      the inflammatory state, the proportion of cd16dimcd62lbright and cd16brightcd62ldim
      neutrophils increased significantly. Proteomic analysis showed that there were significant
      differences between the two subtypes of neutrophils. The nucleus of cd16dimcd62lbright
      neutrophil subgroup is banded, which is released from bone marrow after being stimulated by
      lipopolysaccharide (LPS). It accounts for 20% - 25% of PMN in whole blood in LPS infection
      model. The apoptosis rate is significantly reduced, and the bacteriostatic effects such as
      oxidative burst and phagocytosis are significantly enhanced; On the contrary,
      cd16brightcd62ldim neutrophil subgroup has reduced antibacterial ability and shows
      immunosuppressive phenotype. It is a newly discovered neutrophil subtype with
      immunosuppressive function in recent years, which can inhibit T cell proliferation, which is
      related to immunosuppression in the experimental human endotoxemia model.

      In our previous studies, we have successfully obtained a new amino acid derivative of
      ocotillol ginsenoside, which may have the pharmacological activities of ocotillol ginsenoside
      and glycine, and has a potential role in improving the delay of apoptosis and
      immunosuppression of ARDS neutrophil subtypes, and has the potential of new drug development
      for the treatment of ARDS.

      The experimental steps are as follows:

      Firstly, the peripheral blood of ARDS patients in ICU was collected, and neutrophils were
      isolated from the peripheral blood. The proportion of neutrophil subtypes and the degree of
      apoptosis were detected by flow cytometry. Co culture with human T lymphocytes in vitro to
      observe its ability to inhibit T cell proliferation. Then, the neutrophils of ARDS patients
      were cultured with different doses of ginsenoside glycine derivatives, and the detection of
      the above indexes was repeated again. Finally, the mechanism of neutrophils in the
      pathogenesis and progression of ARDS was discussed.
    

Detailed description: 
      1. Literature basis cute respiratory distress syndrome (ARDS) is a clinical syndrome
      characterized by respiratory distress and progressive hypoxemia, which is caused by diffuse
      alveolar and pulmonary interstitial edema caused by various pulmonary and extrapulmonary
      factors other than cardiogenic factors. ARDS incidence rate is as high as 75 /10 000 per
      year, and sepsis and pulmonary infection are the most common causes. In the past, it was
      generally believed that excessive immune activation is the core of the pathophysiology of
      ARDS, and neutrophils are recognized as the core driver of inflammatory hyperactivity and
      lung injury in ARDS. Although some progress has been made in the epidemiology, pathogenesis
      and pathophysiology of ARDS in the past 50 years, and the clinical outcomes of some patients
      with ARDS have been improved by optimizing the mode of mechanical ventilation and fluid
      treatment, as well as prone ventilation and the use of muscle relaxants, ARDS is still one of
      the most common causes of death and disability in intensive care units, The mortality rate of
      the disease is currently as high as 30-40%. There is still a lack of effective drugs for the
      treatment of ARDS in clinic, and even glucocorticoids applied for immune overactivation have
      not achieved good results. This is related to the unclear pathogenesis of ARDS. Therefore, it
      is still a hot and difficult point to further explore the pathogenesis and progression of
      ARDS and find new therapeutic targets.

      In the past, mature PMN in peripheral blood was generally considered as a functional cell in
      the end stage, but it is widely involved in different innate immune responses (including
      inflammation, infection, tumor, autoimmunity, etc.) and can adopt very different effector
      mechanisms. Therefore, with the deepening of research, neutrophil subtypes with different
      functions (such as immune regulation and repair) have been identified in recent years:
      cd16dimcd62lbrightpmn and cd16brightcd62ldimmpmn. In the steady state of healthy people, the
      classic mature neutrophils (cd16brightcd62lbright) in peripheral blood account for more than
      98% of the total PMN, and the proportion of the two neutrophil subtypes is relatively low. In
      the inflammatory state, the proportion of cd16dimcd62lbright and cd16brightcd62ldim
      neutrophils increased significantly. Proteomic analysis showed that there were significant
      differences between the two subtypes of neutrophils. The nucleus of cd16dimcd62lbright
      neutrophil subgroup is banded, which is released from bone marrow after being stimulated by
      lipopolysaccharide (LPS). It accounts for 20% - 25% of PMN in whole blood in LPS infection
      model. The apoptosis rate is significantly reduced, and the bacteriostatic effects such as
      oxidative burst and phagocytosis are significantly enhanced; On the contrary,
      cd16brightcd62ldim neutrophil subgroup has reduced antibacterial ability and shows
      immunosuppressive phenotype. It is a newly discovered neutrophil subtype with
      immunosuppressive function in recent years, which can inhibit T cell proliferation, which is
      related to immunosuppression in the experimental human endotoxemia model.

      In our previous studies, we have successfully obtained a new amino acid derivative of
      ocotillol ginsenoside, which may have the pharmacological activities of ocotillol ginsenoside
      and glycine, and has a potential role in improving the delay of apoptosis and
      immunosuppression of ARDS neutrophil subtypes, and has the potential of new drug development
      for the treatment of ARDS.

      2. The experimental steps are as follows:

        1. Peripheral blood samples from patients with ARDS were collected to detect inflammatory
           markers;

        2. Neutrophils were isolated from peripheral blood of patients with ARDS;

        3. The proportion of neutrophil subtypes was detected by flow cytometry, and the neutrophil
           subtypes were isolated;

        4. Neutrophil subtypes were co cultured with peripheral blood T lymphocytes to detect their
           ability to inhibit T cell proliferation and the apoptosis of neutrophil subtypes;

        5. Peripheral blood neutrophils and different doses of ginsenoside glycine derivatives in
           patients with ARDS (2050100 μ m) After incubation for 20h, the apoptosis rate was 28h.

        6. Bax, Mcl-1 and cleaved caspase-3 were detected by Western blot.

        7. High throughput PCR and mapp-4 were used to detect the expression of apoptosis related
           genes in akg38k pathway.

        8. Metabolomic analysis: collect the peripheral blood of ARDS patients for metabolomic
           analysis.

      3. Research methods

        1. Diagnostic criteria: refer to the 2012 Berlin standard for the diagnostic criteria of
           ARDS.

        2. Inclusion criteria: (1) age ≥ 18 years old; (2) Diagnosis of acute lung injury within 24
           hours (Berlin diagnostic criteria for acute lung injury)

        3. Exclusion criteria: (1) age < 18 years; (2) Discharge or death within 48 hours after
           admission; (3) Pregnancy or puerperium; (4) Patients with immunosuppression.

      4. Test procedure

        1. Methods: ICU patients who met the inclusion criteria were selected

        2. Informed consent process: sign the informed consent form after communicating with the
           patient or legal representative

      5. Start and end of the experiment Informed consent was signed in January 2020

      6. Data security and monitoring plan The patient's medical records (study medical records /
      CRF, test forms, etc.) will be kept completely in the hospital. The doctor will record the
      results of laboratory tests and other examinations in his medical record. The researchers,
      the ethics committee and the drug administration will be allowed access to his medical
      records. No public report on the results of the study will reveal the individual identity of
      the patient. We will make every effort to protect patient information to the extent permitted
      by law. According to medical research ethics, except for personal privacy information,
      experimental data will be available for public inquiry and sharing, and the query and sharing
      will be limited to the electronic database based on the network to ensure that no personal
      privacy information will be disclosed.

      7. Abide by ethical principles and relevant laws and regulations Whether or not to
      participate in the study depends entirely on the wishes of patients and their legal
      representatives. Patients and their families may refuse to participate in the study or
      withdraw from the study at any time during the course of the study. This will not affect the
      relationship between the patient and his doctor, nor will it affect the loss of medical or
      other benefits for the patient.

      8. Statistical analysis plan SPSS15.0 software was used for statistical analysis. The
      measurement data were expressed as mean ± standard deviation, t test or nonparametric test
      was used for comparison between groups, chi square test was used for counting data, and
      linear correlation analysis was used for the relationship between variables, P < 0.05 was
      considered as statistically significant.
    

Intervention name(s): Ocotillol Type Ginsenoside Derivatives
Intervention type(s): Drug
Intervention description(s): Peripheral blood PMN of patients with ARDS were taken. The neutrophils isolated from peripheral blood of ARDS patients were incubated with different doses of OCT glycine derivatives (20,50,100 UM) for 20 h
Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        ICU patients admitted to the first hospital of Jilin University in Changchun City, Jilin
        Province, China
      
Biospecimen description: 
      The peripheral blood of the patient was centrifuged and the supernatant was reserved for
      cryopreservation