Application of CD24 / Siglec-10 axis related serum marker in prediction of total cause death rate and adverse nervous system prognosis of patient with successful cardio-pulmonary resuscitation

Abstract

The invention discloses an application of a CD24 / Siglec-10 axis related serum marker in prediction of total cause death rate of a patient with successful cardio-pulmonary resuscitation and prognosis of an adverse nervous system, and belongs to the technical field of biology. The research finds that the serum sCD24 and sialic acid levels of non-survivors and the relative activity of neuraminidase are continuously and remarkably increased within one week after the spontaneous circulation is recovered; the rising of the serum sCD24 level is in positive correlation with the relative activity of serum sialic acid and neuraminidase, the score of pro-inflammatory mediators (IL-6, HMGB1 and TNF-alpha), the score of nerve injury markers NSE and the score of nerve injury markers APACHE II; in addition, the increased serum sCD24 level has predictive ability to total cause death rate and poor nervous system prognosis 28 days after ROSC of cardiac arrest patients, has high sensitivity and specificity, and is helpful for clinicians to adjust treatment strategies of patients after successful cardiopulmonary resuscitation according to prognosis judgment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the application of serum markers related to the CD24 / Siglec-10 axis in predicting all-cause mortality and poor neurological prognosis in patients with successful cardiopulmonary resuscitation. Background Art

[0002] In recent years, although a lot of progress has been made in the study of cardiac arrest (CA)-related injury mechanisms and the popularization of cardiopulmonary resuscitation (CPR) technology, there are still a large number of CPR patients with poor prognosis. Globally, the average survival rate of these patients after discharge is about 8.8%; in China, this rate is only about 1.15%. According to a baseline survey of outpatient CA patients in China conducted in 2023, nearly 0.83% of post-CA patients survived discharge and had a good neurological prognosis. The reason for the low survival rate of these patients is related to a complex pathophysiological process called post-CA syndrome. The syndrome includes post-CA brain injury, post-CA myocardial dysfunction, post-CA systemic ischemia / reperfusion response, and continuous induced pathology. It is reported that after spontaneous circulation recovery (ROSC), systemic ischemia / reperfusion injury can trigger systemic inflammatory activation similar to the sepsis process, characterized by elevated levels of inflammatory cytokines in plasma that are unrelated to endotoxin induction.

[0003] Cluster of differentiation 24 (CD24) is a sialylated glycoprotein located on the cell membrane of various tumor and hematopoietic cells. It can bind to sialic acid-binding immunoglobulin-like lectin (Siglec)-10 as an emerging immune checkpoint. Siglecs are expressed in most leukocytes of the immune system and play a key role in immune cell signaling. Siglecs can distinguish non-self molecules by recognizing sialic acid-containing glycans in glycoproteins and glycolipids. The CD24-Siglec interaction is considered to be an innate immune checkpoint that can regulate host responses after tissue damage and cancer immunotherapy. In addition, the CD24-Siglec interaction has been shown to selectively inhibit certain host tissue damage-induced immune responses, including inflammatory responses induced by damage-associated molecular pattern (DAMP) molecules, but not by microbial pathogen-associated molecular pattern (PAMP) molecules, and macrophage-mediated phagocytosis (here CD24 / Siglec-10 acts as a "don't eat me" signal). During DAMP-induced inflammation, CD24 and Siglec-10 form complexes with DAMP molecules, such as high-mobility group protein 1 (HMGB1), thereby inhibiting the activation of the pro-inflammatory HMGB1 / toll-like receptor (TLR) 4 / nuclear factor kappa B (NF-κB) axis.

[0004] The association of CD24 with Siglec-10 depends on an arginine residue in the Siglec-10 V-set domain that is involved in sialic acid recognition. Sialidases, also known as neuraminidase, can hydrolyze sialic acid fragments within the extracellular domain of CD24, resulting in desialylation of CD24, thereby preventing the interaction between CD24 and Siglec G (Siglec-10 in humans) in mice. In mammals, four sialidase isoforms have been identified, each with different substrate specificity and cellular distribution, and are named neuraminidase-1, 2, 3, and 4. Upregulation of neuraminidase-1 after myocardial ischemia-reperfusion can lead to aggravated inflammation by activating monocyte / macrophage invasion, ultimately leading to myocardial damage and cardiac dysfunction. Conversely, sialidase inhibitors can inhibit neuraminidase-mediated desialylation of CD24 and prevent the formation of sepsis in mice.

[0005] The "sepsis-like" inflammatory response triggered by host DAMP molecules (such as HMGB1, etc.) has been shown to cause ischemia-reperfusion injury in vital organs (brain and heart), which is closely related to the severity of disease, neurological dysfunction and high mortality in patients after ROSC. Therefore, the present invention speculates that the CD24 / Siglec-10 axis may be activated, which may be related to the inflammatory response caused by tissue damage and prognosis in these patients. However, the relationship between the CD24 / Siglec-10 axis and the severity of disease, neurological prognosis and all-cause mortality in patients after ROSC is still unclear. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of serum markers related to the CD24 / Siglec-10 axis in predicting all-cause mortality and poor neurological prognosis in patients with successful cardiopulmonary resuscitation.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] Application of serum markers related to the CD24 / Siglec-10 axis in the prediction of all-cause mortality and adverse neurological prognosis in patients with successful cardiopulmonary resuscitation. The serum sCD24 level of patients with successful cardiopulmonary resuscitation was positively correlated with adverse neurological prognosis and all-cause mortality 28 days after restoration of spontaneous circulation.

[0009] Preferably, the serum sCD24 level of patients with successful cardiopulmonary resuscitation is positively correlated with serum sialic acid, neuraminidase, HMGB1, IL-6, TNF-α, NSE, and APACHE II score on days 1, 3, and 7 after the patients' recovery of spontaneous circulation.

[0010] Preferably, the serum sCD24 level in patients with successful cardiopulmonary resuscitation is negatively correlated with the Siglec-10 level.

[0011] Preferably, the serum sCD24 level is positively correlated with CD24 expression, and CD24 is induced by activated NF-κB, resulting in increased CD24 expression.

[0012] Preferably, the damage-associated molecular pattern molecules activate NF-κB, and CD24 is induced by the activated NF-κB, resulting in increased expression of CD24.

[0013] Preferably, a promoter region in CD24 binds to activated NF-κB, promoting CD24 activation and increasing CD24 expression.

[0014] The present invention also provides the use of a CD24 inhibitor in the preparation of a medicine for improving the poor neurological prognosis and mortality of patients who have successfully undergone cardiopulmonary resuscitation.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present study found that within one week after ROSC, the serum sCD24 and sialic acid levels and the relative activity of neuraminidase in non-survivors continued to increase significantly; in addition, compared with survivors, the relative activity of serum sialic acid and neuraminidase in non-survivors increased significantly on the 3rd and 7th days after ROSC, and the serum Siglec-10 level decreased at a delayed rate. Within 1 week after ROSC, the increase in serum sCD24 levels was positively correlated with serum sialic acid, relative activity of neuraminidase, proinflammatory mediators (IL-6, HMGB1, TNF-α), nerve injury marker NSE, and APACHE II score; therefore, the increase in sCD24 levels can also predict all-cause mortality and poor neurological prognosis 28 days after ROSC. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 a flow chart for participant registration;

[0018] Figure 2 Comparison of sCD24 (A), Siglec-10 (B), sialic acid (C), HMGB1 (D), IL-6 (E), TNF-α (F), NSE (G) levels and relative activity of neuraminidase (H) in the serum of healthy volunteers, survivors and non-survivors; * indicates comparison with healthy volunteers, P < 0.05; # indicates comparison with survivors, P < 0.05;

[0019] Figure 3 This is the predictive performance diagram of sCD24 level on 28-day all-cause mortality and adverse neurological prognosis; Figure 3 A is the performance graph for predicting all-cause mortality; Figure 3 B is the performance graph for predicting poor neurological outcome. DETAILED DESCRIPTION

[0020] The following is a description of the embodiments of the present invention. Figures 1 to 3 , clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The embodiment of the present invention provides the application of serum markers related to the CD24 / Siglec-10 axis in the prediction of all-cause mortality and poor neurological prognosis in patients with successful cardiopulmonary resuscitation. The serum sCD24 level of patients with successful cardiopulmonary resuscitation is positively correlated with poor neurological prognosis and all-cause mortality 28 days after recovery of spontaneous circulation.

[0022] In the embodiment of the present invention, the serum sCD24 level of the patient with successful cardiopulmonary resuscitation was positively correlated with the serum sialic acid, neuraminidase, HMGB1, IL-6, TNF-α, NSE, and APACHE II score on the 1st, 3rd, and 7th days after the patient's spontaneous circulation was restored.

[0023] In the embodiment of the present invention, the serum sCD24 level of patients with successful cardiopulmonary resuscitation is negatively correlated with the Siglec-10 level.

[0024] In the examples of the present invention, serum sCD24 levels are positively correlated with CD24 expression, and CD24 is induced by activated NF-κB, resulting in increased CD24 expression.

[0025] In the embodiment of the present invention, the damage-associated molecular pattern molecules activate NF-κB, and CD24 is induced by the activated NF-κB, so that the expression of CD24 increases.

[0026] In the embodiment of the present invention, a promoter region in CD24 binds to activated NF-κB, promoting CD24 activation and increasing CD24 expression.

[0027] The following study examined the correlation between serum markers related to the CD24 / Siglec-10 axis and all-cause mortality and adverse neurological prognosis in patients with successful cardiopulmonary resuscitation.

[0028] 1. Research Methods

[0029] 1. Participant selection criteria

[0030] Inclusion criteria were patients aged ≥18 years who were successfully resuscitated from CA. Exclusion criteria were patients with severe infection, autoimmune disease, long-term use of immunosuppressants or hormones, malignancy, pregnancy or lactation, and other severe systemic diseases such as hematological diseases and cirrhosis. Age- and sex-matched healthy adults were also recruited as healthy volunteers.

[0031] 2. Data Collection

[0032] All enrolled patients were treated according to the 2020 International Consensus on CPR. Throughout the study period, all patients after ROSC used ordinary ice blankets to control or prevent fever.

[0033] Clinical data of the patients were collected, including demographic information, medical history, ICU length of stay, cause of CA, initial cardiac rhythm, witnessed CA events, bystander CPR, CPR duration, treatment, laboratory test results, and patient outcomes. Acute Physiology and Chronic Health Evaluation (APACHE II) scores were calculated on days 1, 3, and 7 after ROSC. The Glasgow-Pittsburgh Cerebral Performance (CPC) score was used to evaluate neurological prognosis on day 28 after ROSC. CPC scores of 1-2 or 3-5 indicate good or poor neurological prognosis, respectively.

[0034] 3. Measurement of serum biomarkers

[0035] Peripheral venous blood samples were collected at 2 hours (day 1), 72 hours (day 3), and 168 hours (day 7) after ROSC or at the time of enrollment of healthy volunteers. The collected peripheral venous blood samples were centrifuged at 1000g for 15 min at 44°C, and the separated serum was subjected to enzyme-linked immunosorbent assay to detect sCD24 (Fine Test, Wuhan, China), sialic acid (FineTest, Wuhan, China), Siglec-10 (Fine Test, Wuhan, China), interleukin (IL)-6 (Elabscience, Wuhan, China), neuron-specific enolase (NSE, CUSABIO, Wuhan, China), HMGB1 (Elabscience, Wuhan, China), and tumor necrosis factor (TNF)-α (Elabscience, Wuhan, China) in the serum. The relative activity of neuraminidase (Beyotime, Shanghai, China) was determined by fluorescence method.

[0036] 4. Statistical analysis

[0037] The enrolled patients were divided into two groups (survivors and non-survivors) according to their 28-day survival results, and healthy volunteers were included in the control group. Data analysis and graphics were performed in SPSS v26 and GraphPadPrism 10. For normally distributed continuous variables, the results were expressed as mean and standard deviation; for non-normally distributed continuous variables, the results were expressed as median (quartile). Categorical variables were compared using Pearson chi-square test or Fisher's exact test. Repeated measures analysis of variance (ANOVA) or Kruskal-Wallis one-way analysis of variance were used to compare the changes in the survival group, death group, and control group at different time points. Subsequently, the Bonferroni test for multiple comparisons or the Mann-WhitneyU test for comparison between two groups was performed. Spearman correlation analysis was used to compare the correlation between sCD24 levels and other indicators. Binary logistic regression was used to analyze whether sCD24 could be used as an independent predictor of poor neurological prognosis or all-cause mortality at 28 days after ROSC. The results were expressed as odds ratios (OR) and their 95% confidence intervals (CI). Receiver operating characteristic (ROC) curves were drawn to explore the predictive efficacy of serum sCD24 levels for poor neurological prognosis or all-cause mortality at 28 days. The area under the ROC curve (AUC) was calculated and compared using the DeLong test. The optimal threshold was determined by the ROC curve, and the prognostic parameters such as sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), Youden index, positive likelihood ratio (LR+), and negative likelihood ratio (LR-) were calculated. P<0.05 was considered statistically significant.

[0038] 2. Results

[0039] 1. Participants’ enrollment status and baseline characteristics

[0040] like Figure 1As shown in the results, a total of 104 CA patients were included, including 30 survivors, 74 non-survivors, and 32 healthy volunteers. The results of the baseline characteristics of the participants are shown in Table 1 below. It can be seen from the results in Table 1 that compared with the survivor group, witnessed CA, bystander CPR, and defibrillable heart rate occurred less frequently in non-survivors, and the ICU stay was shorter; compared with non-survivors, the duration of cardiopulmonary resuscitation in survivors was significantly shorter, and the APACH II score was also lower. 60% (18 / 30) of the surviving patients had a good neurological prognosis, with a CPC score of 1-2 on day 28. Compared with healthy volunteers, patients after ROSC had increased white blood cell counts, neutrophil percentages, procalcitonin, high-sensitivity troponin I, brain natriuretic peptide (BNP), and lactate levels. In addition, the baseline BNP, lactate, and creatinine of non-survivors were significantly higher than those of survivors. Non-survivors received hemodynamic support therapy more often than survivors. Other characteristics were comparable between patients and healthy volunteers, and between survivors and non-survivors.

[0041] Table 1 Baseline characteristics of participants

[0042]

[0043]

[0044] Unless otherwise stated, all data are presented as medians (interquartile range).

[0045] 1 P values ​​are for non-survivors compared with survivors.

[0046] # P<0.05 means that compared with the survivors, * P < 0.05 refers to the comparison with healthy volunteers. APACHE II, acute physiology and chronic health evaluation; BNP, brain natriuretic peptide; CPC, Glasgow-Pittsburgh Cerebral Performance Classification; CPR, cardiopulmonary resuscitation; ICU, intensive care unit.

[0047] 2. Comparison of serum sCD24, Siglec-10, sialic acid, IL-6, HMGB1, and TNF-α levels and relative activity of neuraminidase in healthy volunteers, survivors, and non-survivors

[0048] pass Figure 2It can be seen that on days 1, 3, and 7 after ROSC, the levels of serum sCD24, sialic acid, IL-6, HMGB1, TNF-α, and NSE in patients were significantly higher than those in healthy volunteers; and the relative activity of neuraminidase in patients was also higher than that in healthy volunteers. In addition, on days 1, 3, and 7 after ROSC, the levels of serum sCD24, HMGB1, IL-6, TNF-α, and NSE in non-survivors were significantly higher than those in survivors. However, only on days 3 and 7 after ROSC, the serum sialic acid levels and relative activity of neuraminidase in non-survivors were significantly higher than those in survivors, and the serum Siglec-10 levels in non-survivors were also significantly reduced on days 3 and 7 after ROSC.

[0049] 3. Correlation between serum sCD24 and Siglec-10, sialic acid, relative activity of neuraminidase, HMGB1, IL-6, TNF-α, NSE and APACHEII score

[0050] Table 2 Correlation between serum sCD24 and different biomarkers

[0051]

[0052]

[0053] APACHEII, acute physiology and chronic health score; HMGB1, high-mobility group box 1; IL-6, interleukin-6; NSE, neuron-specific enolase; ROSC, return of spontaneous circulation; sCD24, soluble cluster of differentiation 24; TNF-α, tumor necrosis factor-α.

[0054] The correlation results between the patients' sCD24 levels and other variables are shown in Table 2. It can be seen from Table 2 that the patients' serum sCD24 levels were positively correlated with serum sialic acid, neuraminidase, HMGB1, IL-6, TNF-α, NSE, and APACHE II scores on days 1, 3, and 7 after ROSC, positively correlated with Siglec-10 on day 1 after ROSC, and negatively correlated with Siglec-10 on days 3 and 7 after ROSC.

[0055] 4. Performance of sCD24 levels in predicting 28-day all-cause mortality and adverse neurological outcomes

[0056] pass Figure 3 It can be seen that serum sCD24 levels on days 1, 3, and 7 after ROSC can predict 28-day all-cause mortality ( Figure 3 A) and poor neurological outcome at 28 days ( Figure 3B). Serum sCD24 level on day 7 after ROSC had the largest area under the curve for predicting 28-day all-cause mortality (0.998, 95% CI 0.991–1.000) ( Figure 3 A) and the largest area under the curve for predicting poor neurological outcome at 28 days (0.819, 95% CI 0.695–0.943) ( Figure 3 B) and has the highest sensitivity and specificity. The cut-off value, specificity, sensitivity, NPV, PPV, LR-, LR+, and Youden index of sCD24 at days 1, 3, and 7 after ROSC are shown in Tables 3 and 4.

[0057] Table 3 Predictive effect of serum sCD24 levels on days 1, 3, and 7 after ROSC on 28-day all-cause mortality

[0058]

[0059]

[0060] LR-, negative likelihood ratio; LR+, positive likelihood ratio; NPV, negative predictive value; PPV, positive predictive value; ROSC, return of spontaneous circulation; sCD24, soluble cluster of differentiation 24.

[0061] Table 4 The predictive effect of serum sCD24 levels on days 1, 3, and 7 after ROSC on 28-day neurological outcome

[0062]

[0063] LR-, negative likelihood ratio; LR+, positive likelihood ratio; NPV, negative predictive value; PPV, positive predictive value; ROSC, return of spontaneous circulation; sCD24, soluble cluster of differentiation 24.

[0064] The present invention further uses sCD24, Siglec-10, APACHE II score, initial heart rhythm, CPR time, and eyewitness CA as independent variables, and 28-day all-cause mortality or neurological prognosis as dependent variables for multivariate logistic regression analysis, and the results are shown in Tables 5 and 6. The results of Tables 5 and 6 show that after adjusting other variables, serum sCD24 on day 1 was significantly associated with 28-day all-cause mortality (OR 1.241, 95% CI 1.084-1.421, P=0.002, Table 5) and 28-day poor neurological prognosis (OR 1.482, 95% CI 1.082-2.030, P=0.014, Table 6).

[0065] Table 5 Analysis of variables affecting the prognosis of all-cause mortality within 28 days after ROSC using multivariate logistic model

[0066]

[0067] APACHE II, acute physiology and chronic health evaluation; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation; sCD24 D1 , soluble cluster of differentiation 24 at day 1 after ROSC.

[0068] Table 6 Analysis of variables affecting 28-day neurological prognosis of patients after ROSC using multivariate logistic model

[0069]

[0070] APACHEII, acute physiology and chronic health evaluation; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation; sCD24 D1 , soluble differentiation cluster 24 on day 1 after ROSC.

[0071] In summary, the present invention found that within one week after ROSC, the serum sCD24 and sialic acid levels and the relative activity of neuraminidase in non-survivors continued to increase significantly; in addition, compared with survivors, the relative activity of serum sialic acid and neuraminidase in non-survivors increased significantly on the 3rd and 7th days after ROSC, while the serum Siglec-10 level decreased. Within 1 week after ROSC, the increase in serum sCD24 level was positively correlated with serum sialic acid, relative activity of neuraminidase, proinflammatory mediators (IL-6, HMGB1, TNF-α), nerve injury marker NSE, and APACHE II score.

[0072] The above results indicate that CD24 and Siglec-10 interact and form a tri-molecular complex of CD24, Siglec-10 and HMGB1 with HMGB1, thereby inhibiting the activation of the HMGB1 / NF-κB pro-inflammatory signaling pathway and selectively inhibiting the inflammatory response induced by DAMP molecules. However, the expression of CD24 may be activated through NF-κB, because the promoter region of CD24 may contain NF-κB binding sites. This suggests that there may be a negative feedback between the increased expression of CD24 and the activation of NF-κB. That is, after ROSC, DAMP molecules such as HMGB1 activate NF-κB, which activates the increased expression of CD24, thereby inhibiting HMGB1-induced NF-κB activation and inflammatory response. The increase in serum sCD24 levels is consistent with the increase in CD24 expression. Therefore, the increased serum sCD24 levels are also positively correlated with serum pro-inflammatory mediators, APACHE II scores and brain injury marker NSE, and have predictive power for all-cause mortality and poor neurological prognosis at 28 days after ROSC.

[0073] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. Application of serum markers related to the CD24 / Siglec-10 axis in predicting all-cause mortality and poor neurological prognosis in patients with successful cardiopulmonary resuscitation, characterized in that: Serum sCD24 levels in patients who underwent successful cardiopulmonary resuscitation were positively correlated with adverse neurological outcomes and all-cause mortality 28 days after ROSC.

2. The use according to claim 1, characterized in that The serum sCD24 level of patients with successful cardiopulmonary resuscitation was positively correlated with serum sialic acid, neuraminidase, HMGB1, IL-6, TNF-α, NSE, and APACHE II score on days 1, 3, and 7 after ROSC.

3. The use according to claim 1, characterized in that The serum sCD24 level was negatively correlated with Siglec-10 level in patients with successful cardiopulmonary resuscitation.

4. The use according to claim 1, characterized in that Serum sCD24 levels were positively correlated with CD24 expression, and CD24 expression was increased through the induction of activated NF-κB.

5. The use according to claim 4, characterized in that Damage-associated molecular pattern molecules activate NF-κB, and CD24 is induced by the activated NF-κB, resulting in increased CD24 expression.

6. The use according to claim 5, characterized in that A promoter region in CD24 binds to activated NF-κB, promoting CD24 activation and increasing CD24 expression.

7. Use of CD24 inhibitors in the preparation of drugs for improving poor neurological prognosis and mortality in patients with successful cardiopulmonary resuscitation.

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