阅读说明：本技术 血浆外泌体中组蛋白h3作为中暑患者预后生物标志物的应用 (Application of histone H3 in plasma exosome as prognosis biomarker of heatstroke patient ) 是由 李悦 苏磊 文强 钱晶 万露露 于 2021-08-12 设计创作，主要内容包括：本发明公开了血浆外泌体中组蛋白H3作为中暑患者预后评判生物标志物的应用。本发明研究了中暑患者血浆外泌体中组蛋白H3水平与器官功能和疾病严重程度之间的相关性,结果表明,组蛋白H3在不同病情严重程度的HS患者的血浆外泌体中存在差异表达,健康对照组、存活组和死亡组中外泌体组蛋白H3含量分别为161.1±52.49pg/100μg,249.3±104.6pg/100μg,500.4±216.8pg/100μg。入院第4天与第1天相比,死亡组外泌体组蛋白H3含量增高,而存活组降低。此外,外泌体组蛋白H3含量与脏器损伤指标有显著相关性。因此,血浆外泌体组蛋白H3可作为HS预后的可靠生物标志物。(The invention discloses application of histone H3 in plasma exosomes as a prognosis evaluation biomarker of heatstroke patients. The invention researches the correlation between the level of histone H3 in plasma exosomes of heatstroke patients and organ functions and disease severity, and the result shows that histone H3 has differential expression in the plasma exosomes of HS patients with different disease severity, and the histone H3 content of exosomes in a healthy control group, a survival group and a death group is 161.1 +/-52.49 pg/100 mu g, 249.3 +/-104.6 pg/100 mu g and 500.4 +/-216.8 pg/100 mu g respectively. The exosome histone H3 content was increased in the dead group and decreased in the surviving group on day 4 of admission compared to day 1. In addition, the content of the exosome histone H3 has a significant correlation with the organ injury index. Therefore, plasma exosome histone H3 can be a reliable biomarker for HS prognosis.)

1. Application of histone H3 in plasma exosomes as a prognostic biomarker for heatstroke patients.

2. The use according to claim 1, wherein the level of histone H3 in plasma exosomes is considered in the use.

3. The use according to claim 1 or 2, wherein the difference in plasma exosomes of patients with different prognosis of heatstroke is histone H3, with survivors 249.3 ± 104.6pg/100 μ g and deaths 500.4 ± 216.8pg/100 μ g.

4. The use of claim 1 or 2, wherein the amount of histone H3 in plasma exosomes predicts the risk of mortality in heatstroke patients with an area under the operating characteristic curve of 0.9668[ 95% confidence interval, 0.9231-1.014] with the best predicted sensitivity and specificity at a break point of 307pg/100 μ g.

5. The use according to claim 1 or 2, wherein the level of plasma exosome histone H3 in the deceased persons is increased and the level of plasma exosome histone H3 in the survivors is decreased as the course of the disease progresses.

6. The use according to claim 1 or 2, wherein the level of exosome histone H3 content is indicative of the extent of organ damage.

7. The plasma exosomes and histones in the plasma exosomes can be used as potential clinical treatment targets for relieving HS-induced organ injury.

Technical Field

The invention relates to application of histone H3 in plasma exosomes as a prognosis biomarker for heatstroke patients.

Background

Heatstroke (HS) is a disease characterized by elevated core body temperature >40 ℃ and central nervous system disorders. HS is considered to be a serious and widespread disease, possibly leading to secondary systemic inflammatory responses and Multiple Organ Dysfunction Syndrome (MODS). Epidemiological studies have shown that the average mortality rate for Chinese HS patients is 10-15% and the mortality rate for severe HS patients is even as high as 40%. Therefore, early identification of critically ill patients and timely and effective intervention are critical to improving the survival rate of HS patients. However, there is currently no reliable method of predicting HS severity and prognosis.

Recent studies have shown that exosomes play an important role in the pathogenesis of various diseases, including pathogenic immune responses, inflammation, tumors, and infection. Exosomes are vesicle-like structures with diameters of 30-150 nanometers, containing a variety of bioactive molecules, including proteins, nucleic acids, and lipids. According to previous studies, the number of plasma exosomes differed between healthy humans and patients with various diseases (such as sepsis, cardiogenic shock and alcoholic hepatitis), and the expression profile of the exosome contents was highly correlated with and disease-specific. In addition, compared with the free substances in the blood plasma, the exosome protected by the exosome double-layer membrane has more stable content, is easier to transport in a long distance in a circulatory system without being degraded, and has a wider detection time window. In conclusion, plasma exosomes and their contents can be used as novel and more reliable disease biomarkers.

Disclosure of Invention

The present invention is intended to discuss monitoring whether histone H3 levels in plasma exosomes can be used to predict HS prognosis. Proteomic analysis of HS-induced exosomes revealed that histone H3 was identified as one of the top 10 proteins with the most pronounced upregulation in HS exosomes. The invention aims to explore new biomarkers and potential clinical therapeutic intervention targets of HS prognosis by investigating changes of histone H3 levels in plasma exosomes of HS patients and evaluating the correlation between the changes and organ dysfunction, disease severity and death risk.

Blood samples were collected from HS patients admitted to an intensive care unit (36 survivors and 8 deaths) at admission and 4 days after admission, and 15 additional healthy volunteers from a physical examination center were used as controls. Plasma exosomes were separated using ultra-high speed differential centrifugation. Correlations between histone H3 levels and organ function and disease severity were examined. The results showed that there was differential expression of the exosome histone H3 in HS patients plasma exosomes (survivors, 249.3. + -. 104.6 pg/100. mu.g; deaths, 500.4. + -. 216.8 pg/100. mu.g; healthy controls, 161.1. + -. 52.49 pg/100. mu.g; P < 0.05). The increase in exosome histone H3 expression was associated with disease severity and disease progression and was significantly correlated with the organ dysfunction index (P < 0.0001). The area under the ROC curve distinguishing survivors from deaths was 0.9668, with the best sensitivity (95%) and specificity (91.67%) to predict risk of death at the break point where exosome histone level was 307pg/100g, indicating that histone H3 level in plasma exosomes is likely to be a reliable biomarker for HS prognosis.

Drawings

Figure 1 is a characterization of HS patient plasma exosomes in healthy control versus survival and death groups. (FIG. 1A) morphology of plasma exosomes under transmission electron microscopy. White arrows indicate vesicles with a bilayer circular or elliptical structure of about 100nm in diameter (scale bar 100 nm). (FIG. 1B) nanoparticle trace analysis of the detected exosome size distribution. (FIG. 1C) Western blot analysis detected the expression of the characteristic exosome surface marker proteins CD9, CD63 and CD81, all experiments were repeated three times.

Figure 2 is the plasma exosomes and free histone H3 levels in plasma of healthy control groups and HS patients (survivor and dead groups) at day 1 and day 4.

Figure 3 is a ROC curve for histone H3 in plasma exosomes to distinguish survivors and deaths in HS patients.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

The present study was directed to a study to investigate whether the detection of histone H3 levels in plasma exosomes can be used to predict HS prognosis.

Materials and methods of investigation

1. Patient recruitmentAnd clinical data collection: during the period of 2016 to 2019, 6 months, 44 patients admitted to the Intensive Care Unit (ICU) of the general hospital in the southern military of the liberated military of Chinese people within 24 hours after HS occurred were included in the study. The standard for determining HS is according to the general consensus of Chinese experts on diagnosis and treatment of heat-shooting disease (2019), promulgated by the heatstroke prevention and treatment experts group of the people's liberation army and the committee on critical illness. The diagnostic criteria were as follows: 1) exposure to high temperature and high humidity environments or 2) a history of high intensity exercise; the clinical manifestations are as follows: 1) central nervous system dysfunction (e.g., coma, convulsions, delirium, or abnormal behavior), 2) core body temperature>40 ℃, 3) multiple (2 or more) organ dysfunction, which cannot be explained by other causes. Patients with malignant tumors, chronic liver or kidney diseases, chronic cardiac insufficiency (grade 3-4 in new york order of cardiac function), chronic pulmonary insufficiency, underlying central nervous system diseases or metabolic disorders were excluded. Patients were divided into survivors (36 cases) and deaths (8 cases). Healthy subjects (15) from the physical examination center were included as a control group. Baseline characteristics were recorded on day 1 of admission to the ICU and core body temperature was measured with an ear thermometer. Blood samples were collected on ICU days 1 and 4 and examined by clinical laboratory for biochemical parameters reflecting organ function [ lactic acid (Lac), Albumin (ALB), alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), urea nitrogen (BUN), Creatine Kinase (CK), creatine kinase-isozyme (CK-MB), creatinine (Cr), troponin I (cTnI), Fibrin Degradation Products (FDP), fibrinogen (Fib), International Normalized Ratio (INR), Myoglobin (MYO), arterial blood oxygen partial pressure (PaO)₂) Procalcitonin (PCT), Platelets (PLT), Prothrombin Time (PT), total bilirubin (TBil), white blood cell count (WBC) and D-dimer (D-dimer) levels]While recording Heart Rate (HR), demand for Mechanical Ventilation (MV), vasoactive drugs and continuous hemofiltration (CRRT), inspired oxygen concentration (FiO)₂) Mean Arterial Pressure (MAP), Glasgow Coma Score (GCS), acute physiological and chronic health score (APACHE) II and Sequential Organ Failure Assessment (SOFA) score. All patients or their families signed written informed consent, and the study was approved by the ethical review committee of general hospital medical in the southern war of the people's liberation army of China.

2. Blood sample collection and exosome isolation: for each patient and control group of healthy subjects, 10 ml of peripheral venous blood was collected with a blood collection tube containing ethylenediaminetetraacetic acid anticoagulant. The blood collection tube was left standing at 22-27 ℃ for 30 minutes. The whole blood was then centrifuged at 2500 Xg for 10 minutes at 4 ℃ to separate the plasma. Each plasma sample was diluted with an equal amount of Phosphate Buffered Saline (PBS) and centrifuged three times (2500 Xg for 30 min, 12000 Xg for 45 min, 110000 Xg for 2 h) at 4 ℃. Finally, the pellet (exosomes) was resuspended in 50-200 μ L of PBS and stored at-80 ℃.

3. The exosome morphology was observed by transmission electron microscopy: exosome samples were obtained from healthy controls, mild HS (not combined with multiple organ failure) patients and severe HS (combined with multiple organ failure) patients (randomly selected), and exosome morphology was observed under Transmission Electron Microscopy (TEM). The exosomes were immobilized on loaded copper mesh: resuspend 100,000g of centrifuged exosomes into 50-100 μ L of 2% formalin. mu.L of exosome suspension was added to a Formvar-carbon loaded copper mesh. 2-3 copper meshes were prepared for each exosome sample. The lid was closed and the Formvar membrane was allowed to absorb for 20min in a dry environment. Or 5-10 mu L of exosome suspension can be dripped on the sealing film, and the copper mesh Formvar film is placed on the suspension face down; 100 μ L of PBS was added to the sealing film. Washing the copper mesh (Formvar membrane face down) on the PBS drop with tweezers, keeping the Formvar membrane face wet in all steps, and drying the other face; placing copper net on 50 μ L of 1% glutaraldehyde drop for 5 min; the copper mesh was washed in 100. mu.L ddH2O for 2min (8 times total); placing the copper net on 50 μ L uranium oxalate uranyl drop for 5 min; placing the copper net on 50 μ L of methylcellulose-UA liquid drop for 10min, and operating on ice; the copper mesh was removed with a stainless steel ring and excess liquid was gently blotted onto filter paper, leaving a thin layer of cellulose film, the thickness of the methyl cellulose film being controlled to a suitable range, which affects the contrast of the image. The stainless steel ring is fixed on a 1ml blue gun head; the copper mesh is still on the stainless steel ring and is dried in the air for 5-10 min; after drying, the blue-gold interference fringes indicate that the thickness of the methyl cellulose film is proper and uniform; the copper mesh was placed in a box and an electron micrograph was taken at 80kV using a Philips CM10 transmission electron microscope (model: JEM-2100F; Philips healthcare).

4. Nanoparticle Tracking Analysis (NTA): the number and size distribution of isolated exosomes was determined (Malverpa family; Spectris plc). Briefly, exosome samples were diluted to a final ratio of 1:5000 in sterile PBS and each sample was analyzed three times for 60 seconds each using the autosampler setup of NanoSight.

5. Western blot analysis: proteins were extracted from plasma exosomes using lysis buffer (RIPA: PMSF: CK ═ 100:1: 1). Protein concentration was determined using BCA assay kit (Biosharp Life Sciences; cat. No. bl507 a). Proteins were loaded and separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10 μ g/lane) and electrotransferred to 0.2 μm nitrocellulose membrane. Non-specific binding sites were blocked with bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) and incubated at 25 ℃ for 1 hour, the membrane was incubated with primary antibody (dilution 1:1000) overnight at 4 ℃ and then with secondary antibody (Goat Anti-Mouse IgG H & L; cat No. aba6789; 1:5,000) at 25 ℃ for 1 hour. The primary antibody used was as follows: anti-CD 63(Abcam, cat. No. ab216130), anti-CD 9(Abcam, cat. No. ab223052), anti-CD 81(Abcam, cat. No. ab155760), anti-Tsg-101 (Abcam, cat. No. ab30871), and anti-GADPH (Abcam, cat. No. ab8245).

6. Detection of exosome histone H3 level: a commercial Enzyme-linked immunoassay kit (Human Histone H3 ELISA kit; cat. No. MM-51796H; Jiangsu Enzyme Free Industrial Co., Ltd.) was used.

7. Statistical analysis: the kurtosis test is used to check the normal distribution of data. Normal distribution data are expressed as mean. + -. standard deviation, while non-normal distribution data are expressed as median. + -. interquartile range. Statistical comparisons between the two groups were performed using the two-tailed Student's t test (for normal distribution data), the Kruskal-Wallis H test (for non-normal data), and the Fisher's exact test (for categorical data). For the comparison of more than two groups, two-way ANOVA analysis and post hoc test (simple main effects with a Bonferroni correction) were used, while for the classification data McNemar's test was used. The correlation analysis used the Spearman's rank test. The efficacy of histone H3 in plasma exosomes was examined for discrimination between survivors and deaths by Receiver Operating Characteristic (ROC) curve analysis and compared to plasma ALT and AST. P values <0.05 were considered statistically significant. All statistical analyses were performed using GraphPad software version 7.0 (GraphPad software inc).

Results

1. Comparison of baseline characteristics of healthy control and HS patients

All HS patients and healthy controls were male, with no prior basic medical history. The mean ages of healthy control and HS patients were 25 + -6 years (17-40) and 21 + -4 years (16-38), respectively. Clinical characteristics and test data are summarized in tables 1 to 3. The 28-day mortality rate for HS patients was 18.2% (8/44). The ICU survival time was significantly longer for the deceased than for the survivors (median: 10 days vs.7 days; P < 0.001). The body temperature at admission was not statistically different between survivors and deaths (38.58 + -1.62 deg.C and 39.11 + -1.81 deg.C; P >0.05, respectively), while both survivors and deaths were significantly higher than healthy controls (36.42 + -0.46 deg.C; P < 0.001). Most patients were cooled before admission and the cooling strategy is shown in table 2. Significant regression of hyperthermia was observed on day 4 in both survivors and deaths (P <0.001 compared to day 1; Table 1).

TABLE 1 comparison of patient characteristics on day 1 and day 4 of ICU

APACHE, acute physiological and chronic health assessment; SOFA, sequential organ failure assessment. Day 1 compared to healthy controls:^aP<0.05，^bP<0.001; compared to day 4 with healthy controls:^cP<0.05，^dP<0.001: compared to day 1 of the death group:^eP<0.001; compared to day 4 of the surviving group:^fP<0.001。

TABLE 2 comparison of patient hospitalization time and pre-hospitalization treatment

ICU, intensive care unit; IQR, quartile range; CRRT, continuous renal replacement therapy; HS, heatstroke.

2. Comparison of function index of each organ in healthy control group, HS survival group and death group

TABLE 3 comparison of clinical characteristics and visceral function indices of Sunstroke patients and healthy controls

ALB, albumin; ALT, glutamic-pyruvic transaminase; AST, aspartate aminotransferase; BUN, urea nitrogen; CK, creatine kinase; CK-MB, creatine kinase-isozyme; CNS, central nervous system; cr, creatinine; cTnI, troponin I; FDP, fibrin degradation products; fib, fibrin; FiO₂The inspired oxygen concentration; GCS, glasgow coma scale; HR, heart rate; INR, international normalized ratio; MAP, mean arterial pressure; MV, mechanical ventilation; MYO, myoglobin; PaO₂Arterial oxygen partial pressure; PCT, procalcitonin; PLT, platelets; PT, prothrombin time; TBil, total bilirubin; WBC, white blood cell count; lac, lactic acid. Compared with the healthy control group on day 1:^aP<0.05，^bP<0.01，^cP<0.001; compared with the healthy control group on day 4:^dP<0.05，^eP<0.01，^fP<0.001; compared to survivors day 1:^gP<0.05，^hP<0.01，ⁱP<0.001; compared to the deceased on day 1:^jP<0.05，^kP<0.01，^lP<0.001; compared to survivors on day 4:^mP<0.05，ⁿP<0.01，^oP<0.001。

as can be seen from Table 3, on day 1, conventional biochemistry reflecting organ dysfunctionThe parameters were hardly significantly different between the deceased and the survivors. Including hemodynamic instability (increased demand for vasoactive drugs and Lac levels), respiratory dysfunction (demand for mechanical ventilation), need for continuous renal replacement therapy, and blood coagulation disorders [ prolonged INR, FDP, and Fib levels are reduced]Rhabdomyolysis (increased CK and myoglobin MYO levels) and central nervous dysfunction (decreased GCS score). At the same time, survivors and deaths were in HR, MAP, PaO₂/FiO₂There were no significant differences in the ratios, liver dysfunction (TBil, ALT and AST levels), kidney dysfunction (Cr, BUN levels and urine volume), PT and D-dimer levels, myocardial injury index (cTnI) and PCT levels as the inflammation marker. The MYO levels in HS survivors were significantly higher than in healthy controls, but those in deaths were lower than survivors. Notably, there were no significant differences in TBil levels, renal function parameters, and D-dimer and cTnI levels between the deceased and survivors before day 4. On day 1, APACHE II and SOFA scores used to assess overall disease severity were not significantly different between deaths and survivors, whereas SOFA scores were significantly higher in deaths than survivors only on day 4. The hemodynamic indices, MV rates, TBil levels and renal function parameters of the deaths were significantly worsened on day 4 compared to day 1, while the remaining organ function indices remained unchanged. In survivors, most variables were not significantly different between day 1 and day 4 except for WBC and PCT levels (tables 1-3).

3. Identification of plasma exosomes

Figure 1 shows the plasma exosomes characteristic of healthy control versus HS surviving and dead patients. (FIG. 1A) plasma exosome morphology visible under transmission electron microscopy. White arrows indicate bilayer circular vesicles of about 100nm in diameter (scale bar 100 nm). (FIG. 1B) nanoparticle trace analysis of the detected exosome diameter size distribution. (FIG. 1C) Western blot analysis of expression of the characteristic exosome surface marker proteins CD9, CD63 and CD81, all experiments were repeated three times.

Transmission electron microscopy was performed on isolated plasma exosome samples obtained from HS patients of healthy controls, survivors and deaths. Observed in each set of samplesBilayer membrane vesicle-like structures of about 100nm in diameter (FIG. 1A). According to the NTA result, the plasma exosome quantity of survivors and deaths is higher than that of the control group (6.10 multiplied by 10)⁹And 3.37X 10⁹vs.0.66×10⁹Particles/ml) and the diameter distribution among the three groups is similar, the peak value of the healthy control group is 76nm, the survivor group is 78nm, and the dead group is 67 nm; the proportion of particles with diameters ranging between 30-200 nm in the healthy control group was 94.7%, while the surviving group was 78.2% and the dead group was 97.1% (fig. 1B). Diameter of>The 200nm particle fraction was higher in the HS group than in the control group, probably due to increased cell damage/death leading to increased contaminant particles/microbubbles in the HS group. Western blot analysis showed that the characteristic exosome surface markers CD9, CD63 and CD81 were significantly highly expressed in both healthy controls as well as in the surviving and dead groups (fig. 1C). In summary, the extracted vesicles are identified in many ways to meet the characteristics of plasma exosomes.

4. Comparison of Histone H3 levels in plasma exosomes of healthy control, HS-surviving and dead groups

Figure 2 shows plasma exosomes and free histone H3 levels in plasma of healthy control group and HS patients (surviving and dead) at day 1 and day 4. On days 1 and 4, the plasma exosomes of the dead group had significantly higher levels of histone H3 than the healthy control and the live group. The level of histone H3 in the dead group plasma exosomes was significantly higher on day 4 than on day 1. The plasma exosome histone H3 levels were significantly reduced in the surviving group compared to day 1 on day 4, while there was no significant change in the healthy control group. The plasma free histone H3 levels were elevated in the dead group and kept unchanged in the surviving group on day 4 compared to day 1. P <0.05, P <0.01, P < 0.0001.

As shown in fig. 2, the levels of histone H3 in plasma exosomes of HS-dead group were significantly higher than those of HS-alive group and healthy control group (P was <0.001) both at day 1 and 4 after admission. At admission, plasma exosome histone H3 levels were increased 2.0-fold in the deceased compared to the surviving group. However, there was no significant difference in plasma exosome histone H3 levels between the surviving and healthy controls (P > 0.05). Plasma exosome histone H3 levels were significantly increased in the dead group (P <0.05) and significantly decreased in the live group (P <0.05) at day 4 compared to day 1. The level of free histone H3 in plasma was significantly elevated in the dead group (P <0.001) compared to day 4 compared to day 1, but not significantly changed in the live group (P >0.05) (fig. 2). It can be seen that plasma exosome histone H3 levels are more responsive to the evolution of the disease course and the relevance to prognosis than free histone H3 levels.

Correlation of plasma exosome histone H3 levels with organ function and disease severity in HS patients

Histone H3 levels in plasma exosomes at intensive care unit admission were significantly positively correlated with a number of organ function parameters, including heart rate, lactate levels, glutamate-pyruvate transaminase levels, creatinine levels, urea nitrogen levels, prothrombin levels, INR and D-dimer levels, and APACHE II and SOFA scores on days 1 and 4; glutamate oxaloacetate transaminase, total bilirubin, myoglobin, and troponin-1 levels on day 1; and sodium fructose diphosphate level on day 4. In contrast, plasma exosome levels of histone H3 were compared to PaO at day 1 and day 4₂/FiO₂The ratio, urine volume, fibrinogen level, platelet count and GCS score and albumin level at day 4 were negatively correlated (table 4). However, in addition to D-dimer levels (r ═ 0.78), the association between plasma histone H3 exosome levels and organ function indices at day 1 was poor (mean r)<0.6), whereas the correlation between plasma histone H3 exosome levels and organ function indicators improved on day 4.

TABLE 4 correlation of plasma exosome Histone H3 levels at day 1 with day 1 and day 4 visceral function indices

ALB, albumin; ALT, glutamic-pyruvic transaminase; AST, aspartate aminotransferase(ii) a BUN, urea nitrogen; CK, creatine kinase; CK-MB, creatine kinase-isozyme; CNS, central nervous system; cr, creatinine; cTnI, troponin I; FDP, fibrin degradation products; fib, fibrin; FiO₂The inspired oxygen concentration; GCS, glasgow coma scale; HR, heart rate; INR, international normalized ratio; MAP, mean arterial pressure; MV, mechanical ventilation; MYO, myoglobin; PaO₂Arterial oxygen partial pressure; PCT, procalcitonin; PLT, platelets; PT, prothrombin time; TBil, total bilirubin; WBC, white blood cell count; lac, lactic acid; APACHE, acute physiological and chronic health score; SOFA, sequential organ failure assessment.

6. Efficacy of plasma exosome histone H3 levels to distinguish HS survivors from deaths

FIG. 3 is a ROC curve showing plasma exosome histone H3 levels distinguishing survivors and deaths of HS patients, and compared to ROC curves for plasma AST and ALT.

As shown in fig. 3, the area under the ROC curve for plasma exosome histone H3 levels to distinguish deaths from survivors was 0.9668[ 95% Confidence Interval (CI), 0.9231-1.014; p <0.001], and high resolution efficiency. The sensitivity and specificity of the plasma histone H3 exosome content at the break point of 307pg/100 μ g predicted mortality risk were 95% and 91.67%, respectively (fig. 3). The plasma exosome histone H3 had higher area under the ROC curve than plasma AST (0.7882; 95% CI, 0.5602-1.016; P ═ 0.01158) and ALT (0.9028; 95% CI, 0.8121-0.9935; P <0.001, fig. 3).

Therefore, the present invention aims to evaluate the changes in the plasma exosome histone H3 level of HS patients, its correlation with organ function and disease severity and its prognostic value. The mortality rate of severe HS patients is up to 18.2%, and the deaths are characterized by prolonged ICU time, more severe hyperthermia at the time of admission, and a higher incidence of subsequent multi-organ dysfunction. Histone H3 was enriched in plasma exosomes of HS patients, and was expressed at higher levels in the deceased than in the survivors. As the course of the disease evolves, the abundance of plasma exosome histone H3 decreases in survivors but increases in deaths. There was a significant correlation between plasma exosome histone H3 levels and organ dysfunction (assessed by the SOFA score) and disease severity (assessed by the APACHE II score). The plasma exosome histone H3 level was highly effective in distinguishing between deceased and survivors (area under ROC curve: 0.9250). At the break point of 307 pg/100. mu.g, the sensitivity and specificity of predicting the risk of death is optimal.

In the invention, the value of histone H3 level in plasma exosome as a potential prognostic evaluation index of HS is clinically verified. The extracellular form of histones is usually passively released due to chromatin destruction after disintegration by cell death in the late phase of severe injury, whereas the release of exosomes is a relatively early event, occurring in the initial stages of injury through an active mode independent of cell death. Therefore, histone levels in exosomes are more sensitive than their plasma free state as diagnostic marker.

Histones are highly conserved intranuclear proteins that normally do not function to maintain the structural conformation and stability of chromatin. But under different types of pathological stress (e.g., injury or infection), it can be released extracellularly, acting as an endogenous injury-associated molecular pattern, inducing cell death, inflammatory response, and tissue damage. Administration of sub-lethal doses of histone to mice resulted in intra-alveolar hemorrhage of the lungs and neutrophil infiltration. Histones are the major mediators of endothelial dysfunction and organ failure in sepsis. In the concanavalin a and acetaminophen liver injury models, histones are key mediators in the induction of hepatocyte death. In contrast, antagonizing histone reduces mortality in animal models of acute organ injury.

In the research of the invention, the rising of the histone level of exosomes of HS patients and the correlation between the histone level and the severity of diseases also indicate that exosomes and the histone thereof are potential targets for reducing HS-induced organ injury. There are three current therapeutic strategies that can antagonize the deleterious effects of histone proteins: block the release of histones, neutralize circulating histones, and inhibit intracellular signal transduction. In animal models, these strategies have been shown to be beneficial in reducing acute organ damage associated with sepsis, trauma, and toxicity among others. However, the drugs used in these studies are directed against circulating histones or neutrophil extracellular trap-associated histones, and interference with intracellular signaling pathways may disrupt DNA structure or function, possibly leading to catastrophic side effects. The results of this study indicate that blocking histone in exosomes may provide a new therapeutic target, and inhibition of exosome production with GW4869 has been shown to alleviate sepsis-associated cardiac injury.

In summary, current studies indicate that histone H3 in plasma exosomes may be a novel and effective marker for severe HS patient disease severity stratification and prognosis prediction, and may have potential clinical applications.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.