Biomarker combination for predicting and evaluating illness state or prognosis of ARDS and application

Abstract

The invention discloses a biomarker combination for evaluating the illness state or prognosis of ARDS in the technical field of intensive care medicine, the biomarker combination comprises miR-21-5P and lncRNAgad7, and the biomarker combination can be used for predicting and evaluating the illness state or prognosis of ARDS so as to improve the accuracy of evaluating the illness state or prognosis of ARDS.

Description

Technical Field

[0001] The present invention belongs to the technical field of critical care medicine, and particularly relates to a biomarker combination for predicting and evaluating the condition or prognosis of ARDS and its application. Background Art

[0002] Acute Respiratory Distress Syndrome (ARDS), as a common critical illness in clinical practice, often has an acute onset, manifested as refractory hypoxemia and bilateral pulmonary infiltrates, and is extremely likely to lead to severe respiratory failure. Due to the diverse etiologies and complex pathogenesis of ARDS, such as inflammatory response, oxidative stress, altered cell permeability, coagulation imbalance, etc., as well as the significant heterogeneity of the etiology and pathophysiological process, which result in the destruction of the alveolar-capillary barrier and lung tissue damage. Globally, the incidence and mortality of ARDS remain high. Although certain progress has been made in diagnosis and treatment in recent years, especially in the intensive care unit (ICU), ARDS is one of the main causes of patient death, and survivors often suffer from long-term pulmonary dysfunction and a decline in quality of life. Currently, the existing diagnostic indicators for ARDS still have limitations. The core indicators of the Berlin Definition include the oxygenation index (OI) and chest imaging examinations, which still have high clinical practicality and scientificity in the diagnosis of ARDS. However, the oxygenation index is greatly affected by ventilation strategies, and the interpretation of chest CT may be subjective, resulting in delayed or missed diagnosis of ARDS. Although the new definition of ARDS in 2023 has expanded the diagnostic scope and incorporated new diagnostic criteria, such as SpO2 / FiO2 as a determination index for hypoxemia and appropriate revisions of the diagnostic criteria in resource-limited settings, improving the practicality, it still needs to be applied with caution in clinical practice to avoid missed or misdiagnosis.

[0003] Due to the persistently high incidence and mortality rates of ARDS, it has become one of the important issues urgently to be addressed in the global public health field. Despite the remarkable progress made by modern medicine in mechanical ventilation and intensive care, the treatment of ARDS still faces numerous difficulties. Current treatment strategies mainly focus on supportive treatment and symptomatic management, such as restrictive fluid management, lung-protective ventilation strategies, etc. However, these measures have limited effect on improving the mortality rate of ARDS. Mechanical ventilation is a crucial supportive treatment for ARDS patients, but its improper use and long-term use may trigger ventilator-associated lung injury (VALI) and ventilator-associated pneumonia (VAP), thus exacerbating lung injury and ultimately affecting the survival outcome of patients. In addition, there is a lack of specific drugs for the treatment of ARDS, and the existing treatment effects are limited and there are individual differences. For example, although glucocorticoids have shown effects in improving oxygenation and reducing the mortality rate in some studies, their efficacy is inconsistent in the overall population and may bring side effects such as immunosuppression. In addition, the application effect of surfactant in adult ARDS is not significant, and emerging treatment strategies (such as nebulized inhalation of unfractionated heparin) are still in the research stage and have not been widely applied in clinical practice. Overall, the drug treatment of ARDS still faces problems such as unclear efficacy, risk of side effects, and challenges in individualized treatment.

[0004] Biomarkers are of great significance in ARDS research, and their value is reflected in multiple aspects such as early diagnosis, classification, prognosis assessment, and guiding individualized treatment. First of all, biomarkers can help identify the biological subtypes of ARDS and provide a basis for precise treatment. For example, by detecting inflammation-related markers (such as IL-6, TNF) and vascular endothelial injury markers (such as Ang-2), high-inflammatory and low-inflammatory ARDS patients can be distinguished. Secondly, biomarkers also play an important role in the prognosis assessment of ARDS. The levels of markers such as surfactant protein D (SP-D) and von Willebrand factor (vWF) are closely related to the poor prognosis of patients. In addition, biomarkers can also be used to guide individualized treatment strategies. For example, low-inflammatory patients may benefit from conservative fluid therapy. However, there is still a lack of specific biomarkers in clinical practice, which limits the early diagnosis and precise treatment of ARDS.

[0005] Therefore, seeking a biomarker or combination for the condition assessment and prognosis prediction of ARDS has positive practical significance. Summary of the Invention

[0006] The present invention aims to provide a biomarker combination for assessing the condition or prognosis of ARDS to improve the accuracy of the condition or prognosis assessment of ARDS.

[0007] A biomarker combination for predicting and evaluating the condition or prognosis of ARDS in this solution, characterized in that: the biomarker combination includes miR-21-5P and lncRNA gadd7.

[0008] Further, the samples of miR-21-5P and lncRNA gadd7 are derived from blood, serum, plasma or bronchoalveolar lavage fluid.

[0009] Further, miR-21-5P and lncRNA gadd7 can be used to predict and evaluate the condition or prognosis of ARDS.

[0010] Further, when predicting and evaluating the condition or prognosis of ARDS, the miR-21-5P and lncRNA gadd7 are detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction technology, and the condition or prognosis of individual patients with ARDS is predicted and evaluated based on the expression levels of miR-21-5P and lncRNA gadd7 for predicting and evaluating individual patients with ARDS.

[0011] Further, when predicting and evaluating the condition or prognosis of individual patients with ARDS, based on the expression levels of miR-21-5P and lncRNA gadd7 in ARDS patients, and combined with the APACHE-II score to predict and evaluate the condition or prognosis of individual patients with ARDS.

[0012] Further, miR-21-5P and lncRNA gadd7 can be applied to products related to predicting the condition or prognosis of ARDS patients.

[0013] Further, the product is a reagent, kit, chip or test strip capable of detecting the expression levels of miR-21-5P and lncRNA gadd7 in ARDS patients, or an evaluation system that can automatically output the condition assessment result and / or prognosis risk assessment result according to the detection results of miR-21-5P and lncRNA gadd7 input.

[0014] Further, the evaluation system includes:

[0015] An input unit that obtains data to be processed, and the data to be processed includes the detection results of the expression levels of miR-21-5P and lncRNA gadd7 in the sample;

[0016] An evaluation unit inputs the data to be processed into a disease condition evaluation model or a prognosis risk evaluation model to obtain a disease condition evaluation result or a prognosis risk evaluation result of the data to be processed. The disease condition evaluation model and the prognosis risk evaluation model are pre-trained models. The disease condition evaluation result and the prognosis risk evaluation result include the correlations of miR-21-5P, lncRNA gadd7 with the ARDS disease condition and prognosis risk.

[0017] An output unit outputs the disease condition evaluation result and / or the prognosis risk evaluation result of the data to be processed.

[0018] Furthermore, the data to be processed further includes the APACHE-II system score for ARDS. The disease condition evaluation result or the prognosis risk evaluation result includes the correlations of miR-21-5P, lncRNA gadd7 and the APACHE-II score with the severity or prognosis risk of ARDS.

[0019] Since the combination of miR-21-5P and lncRNA gadd7 expression levels in this application can well evaluate the disease condition and / or prognosis of ARDS, this biomarker combination can be used in the screening of ARDS treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a research flow chart for the present invention to study miR-21-5P and lncRNA gadd7 for predicting and evaluating the disease condition or prognosis of ARDS.

[0021] Figure 2 It is a real-time fluorescence quantitative PCR amplification curve graph of lncRNA gadd7.

[0022] Figure 3 It is a melting curve graph of real-time fluorescence quantitative PCR of lncRNA gadd7.

[0023] Figure 4 It is a real-time fluorescence quantitative PCR amplification curve graph of miR-21-5p.

[0024] Figure 5 It is a melting curve graph of real-time fluorescence quantitative PCR of miR-21-5p.

[0025] Figure 6 It is a comparison graph of the expression levels of serum miR-21-5p and lncRNA gadd7 in patients of the survival group and the death group.

[0026] Figure 7 It is a comparison graph of the expression levels of serum miR-21-5p and lncRNA gadd7 in ARDS patients with different severities.

[0027] Figure 8 ROC curve of serum miR-21-5p, lncRNA gadd7 combined with APACHE II score for predicting 28-day all-cause mortality in ARDS patients. Specific implementation manners

[0028] The following is a further detailed description through specific implementation manners:

[0029] 1. Materials and methods

[0030] 1.1 Main experimental instruments

[0031] Instrument Name Instrument Model Supplier Real-time Fluorescent Quantitative PCR Detection System CFX connect Bio-Rad Ice Maker BLO-20 BLEOV Gel Imaging System Universal hood II Bio-Rad Electrophoresis Apparatus Universal-Power Supply Monad Electrophoresis Tank MM-DNA Monad Low-temperature High-speed Centrifuge 5804R Eppendorf High-pressure Steam Sterilizer MJ3780B Jinan Deqiang Ultra-micro Spectrophotometer NANO ONE Youning Electronic Balance AL104 METTLER TOLEDO Centrifuge Tube 1.5mL, 2.0mL, 0.6mL, 0.2mL Kirgen Pipette F1 Thermo Microwave Oven G70D20CN1P-D2 Galanz

[0032] 1.2 Main experimental consumables / reagents

[0033]

[0034]

[0035] 1.3 Research subjects and methods

[0036] 1.3.1 Research subjects

[0037] This study included 110 patients who were admitted to the Intensive Care Unit of the Affiliated Hospital of Zunyi Medical University from November 2023 to November 2024 and were diagnosed with acute respiratory distress syndrome (ARDS). All patients provided complete clinical medical records. The patients were divided into a death group and a survival group according to the 28-day survival outcome. In addition, they were divided into three groups according to the severity of the disease: mild (200 mmHg ≤ oxygenation index < 300 mmHg), moderate (100 mmHg ≤ oxygenation index < 200 mmHg), and severe (oxygenation index < 100 mmHg).

[0038] This study complied with the standards of medical ethics, was approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University (approval number: KLLY-2023-198), and was registered through ClinicalTrials.gov, registration ID: NCT0682575.

[0039] 1.3.2 Inclusion criteria

[0040] 1) 18 years old ≤ age ≤ 89 years.

[0041] 2) ARDS meets the new global definition in 2023; specific criteria: a. Risk factors and origin of edema: Induced by acute susceptibility risk factors, such as pneumonia, non-pulmonary infection, trauma, blood transfusion, aspiration, or shock; b. Timing: Acute onset or deterioration of hypoxemic respiratory failure within 1 week after the estimated onset of susceptibility risk factors or new or worsening respiratory symptoms; c. Chest imaging examination: Bilateral consolidation on chest X-ray and computed tomography or bilateral B-lines and / or ultrasound consolidation that cannot be fully explained by effusion, atelectasis, or nodules / masses; d. Intubated patients: Mild: 200 < PaO2 / FIO2 ≤ 300 mmHg or 235 < SPO2 / FIO2 ≤ 315 (SPO2 ≤ 97%); Moderate: 100 < PaO2 / FIO2 ≤ 200 mmHg or 148 < SPO2 / FIO2 ≤ 235 (SPO2 ≤ 97%); Severe: PaO2 / FIO2 ≤ 100 mmHg or SPO2 / FIO2 ≤ 148 (SPO2 ≤ 97%); e. Non-intubated patients: When the flow rate of nasal high-flow oxygen therapy is ≥ 30 L / min or PEEP ≥ 5 cmH2O in the CPAP mode of non-invasive ventilator, PaO2 / FIO2 ≤ 300 mmHg or SPO2 / FIO2 ≤ 315 (SPO2 ≤ 97%).

[0042] 3) The patient was diagnosed with ARDS within 48 hours after admission.

[0043] 4) Sign the informed consent form.

[0044] 1.3.3 Exclusion criteria

[0045] 1) Patients who are using immunosuppressants or have received stem cell transplantation..

[0046] 2) Patients with malignant tumors.

[0047] 3) Pregnant women.

[0048] 4) Patients who are discharged within 48 hours after admission to the ICU.

[0049] 5) Patients with a history of previously diagnosed chronic lung diseases (including chronic obstructive pulmonary disease (COPD), bronchial asthma, bronchiectasis, and pulmonary tuberculosis, etc.).

[0050] 6) Patients who are participating in other clinical trials.

[0051] 1.4 Trial process

[0052] 1.4.1 Clinical specimen collection and processing

[0053] For patients meeting the inclusion and exclusion criteria, 5 ml of peripheral blood was collected into an EDTA anticoagulant tube within 48 hours after the diagnosis of ARDS, and gently shaken to ensure sufficient mixing of the blood and the anticoagulant. Subsequently, it was centrifuged at 1300 rpm for 15 minutes at 4°C. After separating the supernatant, it was aliquoted into centrifuge tubes at 500 μl per tube. After numbering the samples, they were placed in a -80°C refrigerator, and the basic information of the patients was recorded.

[0054] 1.4.2 Collection of clinical data

[0055] (1) General information: name, gender, age, APCHE II score and SOFA score in the first 24 hours after admission to the ICU;

[0056] (2) Vital signs (first measurement upon admission to the ICU): heart rate, respiration, body temperature, mean arterial pressure (MAP)

[0057] (3) Laboratory indicators (first measurement upon admission to the ICU): blood routine, electrolytes, liver function, renal function, coagulation function, human TH1 / TH2 lymphocyte subsets, PCT, CRP, lactate, etc.

[0058] (4) Diagnosis and treatment conditions: etiology, severity, oxygen therapy methods (invasive ventilator, non-invasive ventilator, high flow), prognosis (28-day all-cause mortality).

[0059] 1.5 Experimental methods

[0060] 1.5.1 Primer sequences

[0061] Search for the CDS sequence of the target gene on the NCBI website (https: / / www.ncbi.nlm.nih.gov / gene / ), input it into the Primer 5.0 primer design software to design appropriate primers. According to the primer design principles, several pairs of primers were screened for specificity testing on NCBI BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Finally, they were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific sequences of the primers used are as follows:

[0062]

[0063]

[0064] 1.5.2 Plasma RNA extraction

[0065] Use the column-type total blood RNA extraction and purification kit of Sangon Biotech (Shanghai) Co., Ltd. for RNA extraction, with the product number B518653.

[0066] 1) Take 200 μl of anticoagulated blood, add 500 μl of DEPC-treated ddH2O, mix well by shaking, centrifuge at 8,000 rpm at 4 °C for 1 min, and carefully pour off the supernatant.

[0067] 2) Add 200 μl of Buffer Rlysis-RG, immediately mix well by shaking, and let it stand at room temperature for 3 min. Then add 400 μl of Buffer NS and mix well by shaking.

[0068] 3) Centrifuge at 12,000 rpm at 4 °C for 5 min, and transfer the supernatant to a new 1.5 ml RNase-free centrifuge tube.

[0069] 4) Add 1 / 2 volume of absolute ethanol to the supernatant and mix well.

[0070] 5) Place the adsorption column into the collection tube, pipette all the solution into the adsorption column, let it stand for 1 min, centrifuge at 12,000 rpm at room temperature for 1 min, and pour off the waste liquid in the collection tube.

[0071] 6) Place the adsorption column back into the collection tube, add 500 μl of GT Solution, let it stand for 1 min, centrifuge at 10,000 rpm at room temperature for 1 min, and pour off the waste liquid in the collection tube.

[0072] 7) Place the adsorption column back into the collection tube, add 500 μl of NT Solution, let it stand for 2 min, centrifuge at 10,000 rpm at room temperature for 1 min, and pour off the waste liquid in the collection tube.

[0073] 8) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm at room temperature for 2 min.

[0074] 9) Place the adsorption column into a 1.5 ml RNase-free centrifuge tube, add 50 μl of DEPC-treated ddH2O to the center of the adsorption membrane, let it stand for 2 min, centrifuge at 12,000 rpm at room temperature for 2 min, and store the obtained RNA solution at -80 °C for subsequent experiments.

[0075] 1.5.3 Reverse transcription of lncRNA gadd7

[0076] ① cDNA synthesis: Use the extracted total RNA to synthesize the first-strand cDNA. Adjust the addition amount of RNA template in each sample according to the concentration of RNA.

[0077] ② Melt the components in the kit on ice, mix the solution well by gently tapping, centrifuge briefly to sediment the solution to the bottom of the tube, and place it on ice for use.

[0078] ③ Use the Evo M-MLV Reverse Transcription Premix Kit (containing gDNA removal reagent for qPCR) Ver.2 from Acclonix Corporation, catalog number AG11728. According to the method provided in the instruction manual, perform the first-strand cDNA synthesis. The reverse transcription reaction system (20 μl) is shown in Table 1, the reverse transcription reaction component table.

[0079] ④ Pipette and mix well, centrifuge, and perform reverse transcription using a PCR instrument. The reaction conditions are 37°C for 15 min, 85°C for 5 s, and 4°C for 2 h. It can be used directly or stored at -20°C for later use.

[0080] Table 1: Reverse transcription reaction component table

[0081]

[0082] 1.5.4 lncRNA gadd7 real-time quantitative PCR

[0083] (1) Use the 2X SG Fast qPCR Premix from Sangon Biotech (Shanghai) Co., Ltd., catalog number B639271, to perform the qPCR reaction. The specific reaction system and reaction conditions are as follows:

[0084] Table 2: qPCR reaction component table

[0085]

[0086] (2) Place each component of the kit on ice to melt, strictly configure the reaction system according to Table 2, and perform a brief centrifugation.

[0087] (3) Set the qPCR reaction conditions according to the program in Table 3.

[0088] Table 3: qPCR reaction conditions

[0089]

[0090] (4) Perform real-time fluorescence quantitative PCR reaction according to the above conditions. The real-time fluorescence quantitative PCR amplification curve is shown in the appendix Figure 2 as shown, and the melting curve is shown in the appendix Figure 3 as shown.

[0091] 1.5.5 miR-21-5p reverse transcription

[0092] (1) cDNA synthesis: Use the extracted total RNA for the synthesis of the first-strand cDNA. Adjust the addition amount of the RNA template in each sample according to the concentration of the RNA.

[0093] (2) Place the components in the kit on ice to melt, gently tap to mix the solution, perform a brief centrifugation to sediment the solution to the bottom of the tube, and place it on ice for later use.

[0094] (3) Use the miRNA First Strand cDNA Synthesis (Stem-Loop Method) Kit from Sangon Biotech (Shanghai) Co., Ltd., with the product number B532453. According to the method provided in the instruction manual, perform the reverse transcription reaction of miR-21-5p. The reverse transcription reaction system is 20 μl as shown in the following table:

[0095] Table 4: Composition Table of Reverse Transcription Reaction

[0096]

[0097] (4) Pipette and mix well, centrifuge, and perform reverse transcription using a PCR instrument. Incubate the reaction mixture at 16 °C for 30 min, then at 37 °C for 30 min, and heat at 85 °C for 5 min to inactivate the enzyme. Store the obtained cDNA at 4 °C.

[0098] 1.5.6 miR-21-5p Real-Time Quantitative PCR

[0099] (1) Perform qPCR reaction using the miRNA Fluorescent Quantitative PCR Kit (Dye Method) from Sangon Biotech (Shanghai) Co., Ltd., with the product number B532461. The specific reaction system and reaction conditions are as follows:

[0100] Table 5: Composition Table of qPCR Reaction

[0101]

[0102] (2) Melt each component of the kit on ice, strictly configure the reaction system according to Table 2, and centrifuge briefly.

[0103] (3) Set the qPCR reaction conditions according to the program in Table 6.

[0104] Table 6: qPCR Reaction Conditions

[0105]

[0106] (4) Perform real-time fluorescence quantitative PCR reaction under the above conditions. The real-time fluorescence quantitative PCR amplification curve is shown in the appendix Figure 4 as shown, and the melting curve is shown in the appendix Figure 5 as shown.

[0107] 1.5.7 Data Analysis

[0108] Use Stepone software to read the Ct values of each PCR reaction. Subtract the Ct value of the internal reference gene from the Ct value of the target gene to obtain ΔCt; subtract the mean ΔCt of the mild group from the ΔCt of the severe group and the moderate group respectively to obtain their respective ΔΔC t . Use 2 -ΔΔCtCalculate the expression changes of the target genes in each group compared with the mild group.

[0109] 2 Results

[0110] 2.1 General situation of the patients

[0111] A total of 110 patients were included in this study, among whom 32 died within 28 days (29.1%), and 78 survived (70.9%).

[0112] In terms of age, the average age was 50 (38, 62) years old, and the median age of the patients in the death group was significantly higher than that in the survival group (64 [50 - 76] vs. 46 [36 - 55], P < 0.001). In terms of gender distribution, there was no significant difference between the two groups (P = 0.886). The SOFA score and APACHE II score were both significantly higher in the death group than in the survival group (SOFA score: 12.5 [8.8 - 14.0] vs. 8.0 [6.0 - 10.8], P < 0.001; APACHE II score: 29 [22 - 31] vs. 13 [9 - 17], P < 0.001). There were no significant differences in body temperature, heart rate, and respiratory rate between the two groups (P values were 0.830, 0.240, and 0.894 respectively). However, the mean arterial pressure (MAP) in the death group was significantly lower than that in the survival group (87 ± 16 vs. 96 ± 17, P = 0.012).

[0113] There was no significant difference in the severity of ARDS between the two groups (mild: P = 0.103; moderate: P = 0.441; severe: P = 0.305). In terms of the inducing factors, the proportion of ARDS patients induced by pancreatitis in the survival group was significantly higher than that in the death group (78.2% vs. 34.4%, P < 0.001), while the proportion of severe pneumonia in the death group was significantly higher than that in the survival group (21.9% vs. 5.1%, P = 0.013). There were no significant differences in sepsis and trauma between the two groups (P values were 0.071 and 0.076 respectively).

[0114] In terms of comorbidities, the proportions of shock and MODS in the death group were significantly higher than those in the survival group (shock: 40.6% vs. 16.7%, P = 0.007; MODS: 31.3% vs. 11.5%, P = 0.013). There were no significant differences in the distributions of hypertension, diabetes, and AKI between the two groups (P values were 0.387, 0.416, and 0.387 respectively) (see Table 7).

[0115] Table 7: General data of ARDS patients

[0116]

[0117] 2.3 Serum miR-21-5p and lncRNA gadd7 expression levels in the survival and death groups of patients

[0118] The serum miR-21-5p and lncRNA gadd7 expression levels in the survival and death groups of patients are shown in Table 8 and the appendix Figure 6 as follows

[0119] Table 8: Serum miR-21-5p and lncRNA gadd7 expression levels in the survival and death groups of patients

[0120]

[0121] 2.4 Comparison of serum inflammatory factors in the survival and death groups of patients

[0122] In human TH1 / TH2 lymphocyte subsets, the levels of IL-6 and IL-10 were significantly higher in the death group than in the survival group. Specifically, the median level of IL-6 in the death group was 139 (37, 309) pg / mL, significantly higher than 53 (18, 200) pg / mL in the survival group, and the difference was statistically significant (P = 0.019). Similarly, the median level of IL-10 in the death group was 17 (10, 41) pg / mL, significantly higher than 7 (5, 14) pg / mL in the survival group, and the difference was statistically significant (P < 0.001). The differences in other inflammatory factors such as IL-2, IL-4, TNF-α, and IFN-γ between the two groups did not reach statistical significance (P values were 0.808, 0.059, 0.399, and 0.082, respectively).

[0123] Table 9: Inflammatory factor levels

[0124]

[0125] 2.5 General conditions of patients with ARDS of different severities

[0126] According to the severity of ARDS, the patients were divided into mild, moderate, and severe groups. There were no significant differences in age, gender ratio, APACHE II score, 28-day mortality rate, body temperature, respiratory rate, and mean arterial pressure among the groups (P values were all > 0.05). There were differences in SOFA score and heart rate among the three groups (P values were 0.001 and 0.034, respectively).

[0127] Among the inducing factors, the proportions of pancreatitis, severe pneumonia, sepsis, and trauma in each group also showed no statistical significance (P values were all > 0.05). In terms of complications, there were also no significant differences in hypertension, diabetes, MODS, and AKI among the groups (P values were all > 0.05).

[0128] Table 10: General conditions of ARDS patients with different severities

[0129]

[0130]

[0131] 2.6 Expression levels of serum miR-21-5p and lncRNA gadd7 in ARDS patients with different severities

[0132] The expression levels of serum miR-21-5p and lncRNA gadd7 in ARDS patients with different severities are shown in Table 11 and the appendix Figure 7 as follows.

[0133] Table 11: Expression levels of serum miR-21-5p and lncRNA gadd7 in ARDS patients with different severities

[0134]

[0135] The expression levels of lncRNA gadd7 in the mild, moderate, and severe groups were 1.52 (1.01, 2.49), 1.66 (1.07, 2.81), and 2.49 (1.84, 3.54), respectively, showing a gradual increase from the mild to the severe group. Through the Kruskal-Wallis test, significant differences in the expression levels among the three groups were found (H = 8.31, p = 0.016). The expression levels of miR-21-5p in the mild, moderate, and severe groups were 1.14 (1.01, 1.30), 0.82 (0.48, 1.31), and 0.48 (0.36, 0.75), respectively, showing a gradual decrease from the mild to the severe group. The results of the Kruskal-Wallis test showed that there were significant differences in the expression levels among the three groups (H = 23.31, p < 0.001).

[0136] Further pairwise comparisons using the Bonferroni method showed that for lncRNA gadd7, only the difference between the mild and severe groups was statistically significant (p < 0.05), while the differences between the mild and moderate groups and between the moderate and severe groups were not statistically significant. For miR-21-5p, the difference between the mild and severe groups was the most significant (p < 0.001), and there was also a significant difference between the moderate and severe groups (p < 0.01), but there was no significant difference between the mild and moderate groups.

[0137] 2.8 Univariate logistic regression analysis of 28-day all-cause mortality in ARDS patients

[0138] The 28-day disease outcome of ARDS patients was used as the dependent variable (survival = 0, death = 1). Univariate Logistic regression analysis showed that, as shown in Table 12: age, SOFA score, APACHE II score, IL-6, IL-10, IFN-γ, PCT, NLR, lactate, hemoglobin, sodium, albumin, creatinine, INR, high-flow oxygen therapy, invasive ventilation, pancreatitis, severe pneumonia, shock, MODS, lncRNA gadd7, and miR-21-5p were all significantly associated with the risk of 28-day all-cause death (all P < 0.05).

[0139] Table 12: Univariate logistic regression analysis table of 28-day all-cause death in ARDS patients

[0140]

[0141]

[0142] 2.9 Multivariate logistic regression analysis of 28-day all-cause death in ARDS patients

[0143] Multivariate Logistic stepwise regression analysis was performed on the indicators with significance in the univariate analysis to screen out the variables that had a significant impact on the 28-day all-cause death of ARDS patients. Finally, the following factors were included: miR-21-5p, lncRNA gadd7, APACHE II score, albumin, and IL-10. The results showed that a decrease in the expression level of miR-21-5p (OR = 0.15, 95% CI: 0.04 - 0.67, P = 0.013), an increase in the expression level of lncRNA gadd7 (OR = 1.73, 95% CI: 1.03 - 2.93, P = 0.039), and a high APACHE II score (OR = 1.25, 95% CI: 1.12 - 1.40, P < 0.001) were independent risk factors for 28-day all-cause death in ARDS patients. In addition, a decrease in albumin level (OR = 0.87, 95% CI: 0.75 - 1.01, P = 0.059) may be associated with an increased risk of death, but did not reach a significant level; while the level of IL-10 was not significantly correlated with the risk of 28-day all-cause death in ARDS patients (OR = 1.05, 95% CI: 0.98 - 1.12, P = 0.150).

[0144] Table 13: Multivariate logistic regression analysis table of 28-day all-cause death in ARDS patients

[0145]

[0146] 2.10 Value of Serum miR-21-5p and lncRNA gadd7 Combined with APACHE II Score in Predicting 28-day All-cause Mortality in ARDS Patients

[0147] To evaluate the predictive value of lncRNA gadd7, miR-21-5p, and APACHE II for the 28-day prognosis of ARDS patients, this study analyzed by plotting ROC curves and calculating relevant indicators. The results are shown in Table 14 and Appendix Figure 8 As shown, the AUC of lncRNA gadd7 was 0.748 (95% CI: 0.656 - 0.826), the optimal cut-off value was 2.237, the Youden index was 0.462, the sensitivity was 71.87%, and the specificity was 74.36%. The AUC of miR-21-5p was 0.667 (95% CI: 0.571 - 0.754), the optimal cut-off value was 0.793, the Youden index was 0.380, the sensitivity was 68.75%, and the specificity was 69.23%. The AUC of APACHE II was 0.893 (95% CI: 0.820 - 0.944), the optimal cut-off value was 17, the Youden index was 0.663, the sensitivity was 90.62%, and the specificity was 75.64%.

[0148] When lncRNA gadd7, miR-21-5p, and APACHE II were combined for combined diagnosis, the AUC increased to 0.921 (95% CI: 0.854 - 0.964), the Youden index was 0.703, the sensitivity was 84.37%, and the specificity was 85.90%. The AUC of the combined diagnosis was significantly higher than that of single indicators, indicating stronger predictive ability and more accurate identification of the prognosis risk of patients.

[0149] Table 14 Value of Serum miR-21-5p and lncRNA gadd7 Combined with APACHE II Score in Predicting 28-day All-cause Mortality in ARDS Patients

[0150] Item lncRNA gadd7 miR-21-5p APACHE II Combined Diagnosis AUC 0.748 0.667 0.893 0.921 Standard Error 0.0469 0.0547 0.0366 0.0287 95%CI 0.656-0.826 0.571-0.754 0.820-0.944 0.854-0.964 P <0.0001 0.0022 <0.0001 <0.0001 Optimal Cut-off Value 2.237 0.793 17 Youden Index 0.462 0.380 0.663 0.703 Sensitivity 71.87％ 68.75％ 90.62％ 84.37％ Specificity 74.36％ 69.23％ 75.64％ 85.90％ Positive Predictive Value 53.50％ 47.80％ 60.40％ 71.10％ Negative Predictive Value 86.60％ 84.40％ 60.40％ 93.10％ Positive Likelihood Ratio 2.80 2.23 3.72 5.98 Negative Likelihood Ratio 0.38 0.45 0.12 0.18

[0151] 3 Conclusion

[0152] ARDS is a severe lung disease caused by various factors inside and outside the lungs, and its occurrence is closely related to the interaction between environmental factors and individual constitutions. As a complex heterogeneous syndrome, its pathophysiological process involves multiple mechanisms such as inflammation, oxidative stress, apoptosis, autophagy, and tissue fibrosis, and its specific pathogenesis has not been fully clarified yet. Approximately 10% of ICU patients are complicated with ARDS, and the mortality rate of ARDS worldwide is still as high as 40%. Survivors often have long-term pulmonary dysfunction. Therefore, for ARDS patients, early identification of the condition and timely intervention are the keys to effectively improving the prognosis.

[0153] This study shows that the expression levels of serum miR-21-5p and lncRNA gadd7 are significantly correlated with the disease severity and 28-day prognosis of ARDS patients. The two may regulate the disease process and can be used as markers for the diagnosis and treatment of ARDS. Further analysis shows that miR-21-5p, lncRNA gadd7, and the APACHE-II score are all independent predictors of 28-day prognosis. The combination of the three can effectively improve the accuracy of prognosis prediction, which helps to provide a basis for risk stratification and individualized treatment of ARDS patients in the ICU.

[0154] That is:

[0155] (1) The expression levels of serum miR-21-5P and lncRNA gadd7 are significantly different from the 28-day prognosis of ARDS patients. The two are involved in the disease process of ARDS and can be used as potential biomarkers for predicting the prognosis of ARDS patients.

[0156] (2) The expression levels of serum miR-21-5P and lncRNA gadd7 are significantly different in patients with different severities of ARDS and can be used as an indicator for distinguishing the severity of ARDS.

[0157] (3) Serum miR-21-5p, serum lncRNA gadd7, and the APACHE-II score are independent factors affecting the 28-day prognosis of ARDS patients. The combination of the three can improve the accuracy of prognosis prediction.

[0158] The above are only examples of the present invention. Common knowledge such as specific structures and characteristics known in the solution is not described in detail here. It should be noted that for those skilled in the art, without departing from the structure of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be subject to the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. A biomarker combination for predicting and evaluating the condition or prognosis of ARDS, characterized in that: The biomarker combination includes miR-21-5P and lncRNA gadd7.

2. The biomarker combination for predicting and evaluating the condition or prognosis of ARDS according to claim 1, wherein: The sample is derived from blood, serum, plasma or bronchoalveolar lavage fluid.

3. Use of the biomarker combination according to claim 1 or 2 in predicting and evaluating the condition or prognosis of ARDS.

4. Use of the biomarker combination according to claim 3 in predicting and evaluating the condition or prognosis of ARDS, characterized in that: Detect the miR-21-5P and lncRNA gadd7 by real-time fluorescence quantitative reverse transcription polymerase chain reaction technology, and predict and evaluate the condition or prognosis of an individual patient with ARDS based on the expression levels of miR-21-5P and lncRNA gadd7 for predicting and evaluating the condition or prognosis of an individual patient.

5. Use of the biomarker combination according to claim 4 in predicting and evaluating the condition or prognosis of ARDS, characterized in that: Predict and evaluate the condition or prognosis of an individual patient with ARDS based on the expression levels of miR-21-5P and lncRNA gadd7, and in combination with the APACHE-II score.

6. Use of the biomarker combination according to claim 3 in predicting and evaluating the condition or prognosis of ARDS, characterized in that: The use is in a product for predicting and evaluating the condition or prognosis of ARDS.

7. Use of the biomarker combination according to claim 6 in a product for predicting and evaluating the condition or prognosis of ARDS, characterized in that: The product is a reagent, kit, chip, test strip or evaluation system.

8. Use of the biomarker combination according to claim 7 in a product for predicting and evaluating the condition or prognosis of ARDS, characterized in that: The evaluation system includes: An input unit that acquires data to be processed, where the data to be processed includes the detection results of the expression levels of miR-21-5P and lncRNA gadd7 in the sample; An evaluation unit that inputs the data to be processed into a disease condition evaluation model or a prognosis risk evaluation model to obtain a disease condition evaluation result or a prognosis risk evaluation result of the data to be processed. The disease condition evaluation model and the prognosis risk evaluation model are pre-trained models, and the disease condition evaluation result and the prognosis risk evaluation result include the correlations between miR-21-5P, lncRNA gadd7 and the ARDS condition, prognosis risk; An output unit that outputs the disease condition evaluation result and / or the prognosis risk evaluation result of the data to be processed.

9. Use of the biomarker combination according to claim 8 in a product for predicting and evaluating the condition or prognosis of ARDS, characterized in that: The data to be processed further includes the APACHE-II system score for ARDS; the disease condition evaluation result or the prognosis risk evaluation result includes the correlations between miR-21-5P, lncRNA gadd7 and the APACHE-II score and the severity or prognosis risk of ARDS.

10. Use of the biomarker combination according to claim 1 or 2 in screening ARDS treatment drugs.

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