CN117517679A - Use of apolipoprotein H as biomarker in diagnosing, prognosticating or monitoring the progression of sepsis in children - Google Patents

Abstract

The invention discloses an application of apolipoprotein H as a biomarker in diagnosing, predicting or monitoring the progress of sepsis in children, and belongs to the technical field of biomarkers. The invention discovers that the APOH can be used as a diagnosis marker of the sepsis of children, has better prospect, can specifically detect the serum APOH level in the infected patients, is used for prompting whether the sepsis of the pediatric patients occurs or not and provides a basis for the prognosis of the patients; the invention has strong specificity, predicts the occurrence of sepsis by detecting the expression condition of the peripheral blood APOH of a patient through ELISA means, can be directly applied to clinic, and has practical clinical use value. The invention discovers that supplementing APOH in vivo can reduce the sepsis inflammatory level and tissue injury, and blocking APOH can aggravate the sepsis inflammatory level and tissue injury, which suggests that APOH can be used as a medicament for treating sepsis injury.

Description

Use of apolipoprotein H as biomarker in diagnosing, prognosticating or monitoring the progression of sepsis in children

Technical Field

The invention relates to the technical field of biomarkers, in particular to application of apolipoprotein H as a biomarker in diagnosing, predicting or monitoring the progress of sepsis in children.

Background

Sepsis is an organ dysfunction syndrome caused by deregulation of the host's response to infection, causing systemic multi-organ, multi-system damage with high mortality. It is estimated that there are 4890 ten thousand new sepsis cases and 1100 ten thousand sepsis-related deaths worldwide in 2017, and their management and treatment remain a major challenge for the medical community. The World Health Organization (WHO) announces that improving the prevention, identification and treatment of sepsis is a global health priority. Sepsis is thought to involve excessive inflammatory responses and immunosuppression, ultimately involving systemic inflammatory responses and worsening of organ function. This result explains the cause of high mortality in sepsis patients. Thus, much work is currently focused on identifying immune mediators involved in interfering with the host immune response, which may indicate a new direction for the treatment of sepsis.

Sepsis is not only a process of systemic inflammatory response or immune dysfunction, but it also involves changes in the function of multiple organs in the body. For example, at the cellular and molecular level, the pathogenesis of sepsis is extremely complex, including pathophysiological processes such as inflammatory reaction imbalance, immune dysfunction, mitochondrial damage, coagulation dysfunction, abnormal neuroendocrine immune network, endoplasmic reticulum stress, autophagy, etc., ultimately leading to organ dysfunction. Therefore, finding a target that effectively inhibits inflammatory responses in sepsis is a currently a problem that is in need of clinical resolution.

Apolipoproteins (APO) are important components of lipoproteins, involved in the binding of lipoproteins to their receptors, and in lipoprotein metabolism. At present, there is no report on the relationship between APOH and sepsis.

Disclosure of Invention

The object of the present invention is to provide the use of apolipoprotein H as biomarker for diagnosing, predicting or monitoring the progression of sepsis in children, to solve the problems of the prior art described above.

In order to achieve the above object, the present invention provides the following solutions:

the present invention provides the use of apolipoprotein H as a biomarker in the preparation of a product for diagnosing, prognosticating or detecting or staging the progression of sepsis in children.

Preferably, the product takes peripheral blood of children to be detected as a sample to be detected.

Preferably, the product comprises a reagent or kit.

The invention also provides a detection reagent or a kit for children sepsis, which takes the apolipoprotein H as a detection object.

The invention also provides application of the apolipoprotein H in preparing medicines for treating sepsis.

Preferably, the sepsis comprises high cecal ligation perforation-induced sepsis.

The invention also provides a medicine for treating children sepsis, which comprises apolipoprotein H.

Preferably, the composition further comprises other pharmaceutically acceptable auxiliary materials.

The invention also provides application of the apolipoprotein H in preparing medicaments for relieving tissue injury.

The invention also provides application of the apolipoprotein H in preparing medicines for reducing inflammatory reaction.

Based on the technical scheme, the invention has the following technical effects:

the invention has the advantages that: 1. the invention discovers that the APOH can be used as a diagnosis marker of the sepsis of children, has better prospect, can specifically detect the serum APOH level in the infected patients, is used for prompting whether the sepsis of the pediatric patients occurs or not and provides a basis for the prognosis of the patients; 2. the invention has strong specificity, predicts the occurrence of sepsis by detecting the expression condition of the peripheral blood APOH of a patient through ELISA means, can be directly applied to clinic, and has practical clinical use value. 3. The invention discovers that supplementing APOH in vivo can reduce the sepsis inflammatory level and tissue injury, and blocking APOH can aggravate the sepsis inflammatory level and tissue injury, which suggests that APOH can be used as a medicament for treating sepsis injury.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows differences in the expression levels of APOH in different groups of children, where a is the difference in the relative protein abundance of APOH in healthy children and in sepsis children, b is the difference in the relative protein abundance of APOH in sepsis surviving children and sepsis dying children, c is the predictive value of APOH levels between sepsis patients and healthy controls, d is the prognostic value between sepsis patients survival and death;

FIG. 2 shows the differences in the expression levels of APOH in children in different groups, wherein a is the expression level of APOH in serum of healthy children and children with sepsis, b is the differential expression of APOH levels during the progression of sepsis, c is the predicted value of APOH expression levels between the children with sepsis and children in healthy control group, d is the predictive ability of serum APOH levels and CRP, PCT, pSOFA scores to mortality of patients with sepsis for 28 days;

FIG. 3 is the predictive capacity of serum APOH levels in combination with CRP, PCT, pSOFA scores for mortality in sepsis patients for 28 days;

FIG. 4 shows the difference of the expression level of APOH in the sepsis mouse model and the sham operation group, wherein a is the difference of the expression level of APOH in the sepsis mouse model and the sham operation group, b and c are the correlation analysis of the expression of APOH and inflammatory factor IL-6, and d is the detection of the expression level of APOH in lung, liver and kidney tissues of the sham operation group and the sepsis mouse by Western blot;

FIG. 5 is a graph showing the differences in the expression levels of relative quantification of APOH levels in lung, liver and kidney tissues of sham-operated groups and sepsis mice;

FIG. 6 shows the effect of in vivo addition of APOH on survival of CLP model mice, wherein a and b are schematic diagrams of construction of severe CLP model mice and administration, and c is the effect of supplementation of rAPOH on survival of CLP model mice;

FIG. 7 shows the effect of supplementation of recombinant protein APOH on tissue and organ inflammatory injury, wherein a is the effect of hematoxylin-eosin staining method on lung, liver and kidney tissue injury of sepsis mice detected by supplementation of bovine serum albumin (bovine serum albumin, BSA) as a control agent and recombinant protein APOH for therapeutic use; b is the quantitative detection of lung, liver and kidney tissue injury of a sepsis mouse by supplementing BSA and recombinant protein APOH;

FIG. 8 shows the difference in the expression level of inflammatory cytokines in serum and peritoneal lavage fluid (peritoneal lavage fluid, PLF) of mice supplemented with BSA and recombinant protein APOH, wherein a is the expression level of inflammatory cytokines in serum and b is the expression level of inflammatory cytokines in peritoneal lavage fluid sample;

FIG. 9 shows the effect of in vivo blocking of APOH on survival rate of CLP model mice, wherein a and b are non-severe CLP model construction and administration modes, and c is the survival rate difference between anti-APOH+CLP group sepsis mice and control group;

FIG. 10 shows the difference in lung, liver and kidney injury in anti-APOH+CLP mice, wherein a is the effect of hematoxylin-eosin staining on lung, liver and kidney tissue injury in sepsis mice supplemented with IgG and APOH antibodies as controls; b is the quantitative detection of lung, liver and kidney tissue injury of a sepsis mouse by supplementing BSA and recombinant protein APOH;

FIG. 11 shows the difference in the levels of inflammatory factors in serum and peritoneal lavage fluid of anti-APOH+CLP mice and IgG mice, wherein a is the difference in the levels of inflammatory factors in serum of anti-APOH+CLP mice and IgG mice, and b is the difference in the levels of inflammatory factors in peritoneal lavage fluid of anti-APOH+CLP mice and IgG mice.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

The technical scheme of the invention is conventional in the field, and the reagents or raw materials are purchased from commercial sources or are disclosed.

The data quality control method comprises the following steps:

data are expressed as mean ± Standard Deviation (SD). The group comparisons used either a t-test (Mann-Whitney U test) or a one-way analysis of variance (Tukey's multiple comparison test). Survival analysis used Log-rank (Mantel-Cox) test and correlation analysis used non-parametric Spearman rank correlation coefficients. The ability of APOH to identify healthy control and sepsis patients was analyzed using a subject work character (ROC) Curve and the Area Under the Curve (AUC) and its 95% Confidence Interval (CI) was calculated. All statistical tests were performed using GraphPad Prism 6.02 (GraphPad Software, san Diego, CA). The difference p <0.05 is statistically significant.

Example 1 differences in the expression level and Properties of APOH in different groups

The infants taken in the study are all from serious medical departments of auxiliary children hospitals in Chongqing medical university, peripheral blood of the infants meeting the group-entering standard is collected, 2mL of peripheral blood of patients is respectively collected at 24h,48h and 72h of sepsis diagnosis, and the separated frozen serum is frozen at-80 ℃ for subsequent detection. And collecting general conditions of the infant, including patient name, gender, age, critical disease score, viscera function index, breathing machine parameter, etc., and collecting clinical test indexes of the infant, including blood routine, liver and kidney function, blood coagulation routine, blood lactic acid, etc.

The invention establishes the following group standard (see Fang Na standard in detail) according to the international guidelines for sepsis in children. The healthy children control population is a physical examination center of a subsidiary child hospital of the university of Chongqing medical science for carrying out a healthy physical examination, after informed consent of the subject and the guardian is obtained, the residual peripheral blood after physical examination detection is collected, and frozen serum is separated for carrying out the next experiment at the temperature of minus 80 ℃. The specific experimental details are as follows:

(1) nano-row standard

Experimental group:

inclusion criteria: a. meets the international consensus on sepsis for children in 2005: (1) Reaching two or more systemic inflammatory response syndrome standards; (2) a confirmed or suspected invasive infection; (3) There is cardiovascular dysfunction, acute respiratory distress syndrome, or two or more organ dysfunctions; b. age range 29 days to 18 years; c. informed consent and complete study data were available.

Exclusion criteria: a. is not in accordance with the international consensus on sepsis for children in 2005; b. age ranges from 28 days to 18 years of age are not satisfied; c. informed consent and complete study data were not available.

Control group:

inclusion criteria; a. the physical examination center of the auxiliary children hospital of Chongqing medical university detects the children suffering from physical examination; b. age range 28 days to 18 years; c. informed consent and complete study data were available.

Exclusion criteria: a. age ranges from 28 days to 18 years of age are not satisfied; b. informed consent and complete study data were not available.

(2) Sample collection and processing: blood samples of the infants who are incorporated into the standard test group at 24h,28h and 72h after hospitalization are 2mL each, the children in the control group collect 2mL of blood samples on the day of physical examination, centrifuge treatment (3500 rpm, 8 min) is carried out within 1 hour, serum is separated, and 200 mu L/tube is sub-packaged and then stored at ultralow temperature of-80 ℃.

(3) Sample detection: the collected samples of the experimental group and the control group are subjected to detection of APOH expression level by adopting a liquid chromatography-mass spectrometer (Liquid Chromatograph Mass Spectrometer, LC-MS) and an enzyme-linked immunosorbent assay (ELISA), and clinical data such as gender, age, white Blood Cell (WBC) count at 24 hours of hospital admission, procalcitonin (PCT), C-reactive protein (CRP), sequential organ failure score of children (pediatric Sequential Organ Failure Assessment score, pSOFA), critical case score of children (Pediatric Critical Illness Score, PCIS), systemic inflammatory response syndrome score (Systemic Inflammatory Response Syndrome, SIRS) and the like are collected.

The results are shown in fig. 1-3, and the expression level of APOH in healthy children and infants suffering from sepsis is verified by establishing a test point queue through a liquid chromatograph-mass spectrometer. A total of 68 sepsis patients (42 surviving and 26 dying infants) and 26 healthy controls were included. The APOH level in the sepsis infant was significantly lower than in the healthy control group (fig. 1 a), and the APOH expression level in the sepsis-dead infant was significantly lower than in the sepsis-surviving infant (fig. 1 b). The predictive value of APOH levels between sepsis patients and healthy control group (c in fig. 1), and the prognostic value between sepsis patient survival and death (d in fig. 1) was analyzed using an area under subject work profile (Receiver Operating Characteristic curve, ROC) assessment. The study results showed that APOH predicted AUC of sepsis occurrence to be 0.98 ([ 95% CI ] 0.9379-1.032, p < 0.001) and predicted death to be 0.88 ([ 95% CI ]0.7021-1.076, p < 0.005).

Further, the present example verifies the expression level of APOH in healthy children and in sepsis-affected infants by establishing a verification queue. A total of 36 sepsis patients and 69 healthy controls were included. The array uses enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) to detect the level of APOH expression in each group. The results are shown in fig. 2, and the APOH expression level in the serum of the infant with sepsis is significantly reduced compared with the healthy control group (a in fig. 2). This example observes a gradual decrease in APOH levels during subsequent sepsis progression (b in fig. 2). The predictive and prognostic value of APOH for infants with sepsis was assessed using the area under the subject's working curve (ROC) (AUC). AUC detection results show that the predicted area of APOH expression level between sepsis infant and healthy control group children is 0.928 ([ 95% CI ]0.8771-0.9796, p < 0.0001) (c in FIG. 2). According to the expression level of APOH in healthy control group and infant with sepsis, the sensitivity of APOH for predicting infant with sepsis is 100% and specificity is 85.5% according to AUC curve and about Deng index. In addition, the predictive ability of 28-day mortality in sepsis patients was further scored by comparing serum APOH levels to CRP, PCT, pSOFA. The results showed that APOH AUC was 0.644 ([ 95% CI ] 0.423-0.866, p=0.270) higher than PCT (auc=0.538, [95% CI ]0.285-0.791, p=0.772) and PSOFA (auc=0.571, [95% CI ]0.285-0.85, p=0.772) with comparable capacities to CRP (auc=0.698, [95% CI ]0.496-0.899, p=0.135) (d in fig. 2).

The AUC (auc=0.759, [95% CI ]0.575-0.944, p=0.05) of APOH combined CRP was higher than APOH combined PCT (auc=0.673, [95% CI ]0.436-0.910, p=0.192) and PSOFA (auc=0.623, [95% CI ]0.363-0.884, p=0.35) (fig. 3).

Example 2 differences and characteristics in expression levels of APOH in sepsis mice models and sham operated groups

10C 57/6 mice were selected, 5 in each group, randomly divided into the following groups: CLP and Sham groups were sacrificed after 24 hours, and blood and tissues and organs were collected for detection of APOH expression levels and inflammatory factors.

C57BL/6 mice were purchased from university of Daqing medical science, kept in a controlled environment (20-24 ℃ C., 12h light/dark cycle), fed standard feed and water. A model of sepsis was established using Cecal Ligation and Perforation (CLP), and pre-operative intraperitoneal injection of 75mg/kg sodium pentobarbital was anesthetized. After skin disinfection, the middle abdomen was cut about 1cm, the cecum was exposed, and 60% was ligated (severe CLP, 20-40% survived), and a 21G puncture needle was used. The cecum is then placed back into the abdominal cavity, and the incision is closed. Physiological saline (0.05 mL/g body weight) was injected intraperitoneally for resuscitation, and water and feed were recovered after surgery. Wherein a Sham group (Sham) group mouse model was constructed as follows: preoperative intraperitoneal injection of 75mg/kg sodium pentobarbital for anesthesia. After skin sterilization, the incision was made about 1cm in the midabdominal region, the cecum was exposed, and then the cecum was returned to the abdominal cavity, and the incision was closed. Physiological saline (0.05 mL/g body weight) was injected intraperitoneally for resuscitation, and water and feed were recovered after surgery.

FIGS. 4-5 are the protective effects of APOH on the systemic inflammatory response of CLP model mice. To detect APOH levels in serum of sepsis model mice, changes in APOH expression during sepsis progression were observed. During the subsequent sepsis progression, the APOH concentration in CLP mice was significantly reduced compared to sham mice (a in fig. 4). Furthermore, APOH expression in CLP mice was significantly inversely correlated with inflammatory factor IL-6 (B, C in fig. 4). Meanwhile, APOH expression in lung, liver and kidney, which are important tissues and organs of CLP mice, was reduced compared to sham-operated groups (D in fig. 4 and fig. 5).

EXAMPLE 3 in vivo protection of systemic inflammatory response in CLP model mice by APOH addition

14C 57/6 mice were selected, 7 in each group, randomly divided into the following groups: clp+0.1% bsa and clp+rapoh were intervened for 24 hours, and blood and tissues were taken for inflammatory factor and histomorphometric detection, respectively.

The mice were divided into clp+0.1% Bovine Serum Albumin (BSA) and clp+recombinant murine apolipoprotein H (rAPOH) groups as in example 2. Mice in the clp+rapoh group were intraperitoneally injected with 20 μg of mouse rAPOH in 100 μl of Phosphate Buffer (PBS), and mice in the clp+0.1% bsa group were intraperitoneally injected with 0.1% bsa in 100 μl of PBS. The survival condition is monitored every day in the 2 nd to 14 th days after operation, and serum and tissues are collected for detecting inflammatory factors and tissue injury.

FIGS. 6-8 are the protective effects of in vivo APOH addition on the systemic inflammatory response of CLP model mice. A, B in FIG. 6 is a schematic of the construction of a severe CLP mouse model (60% of the cecum of the ligated mice, as shown in FIG. 6A, with yellow at the end of the cecum and green lines at the site of ligation). The effect of 5-20 μg recombinant protein APOH on the survival rate of severe sepsis mice was evaluated, and the results showed that supplementation with rAPOH (20 μg) significantly increased CLP mice survival rate (C in fig. 6) compared to sepsis mice treated with 0.1% BSA. Supplementation with rAPOH significantly reduced the levels of inflammatory cytokines including Interleukin (IL) -6, tumor necrosis factor alpha (TNF alpha), interleukin (IL) -1 beta and IL-10 in tissue and organ inflammatory lesions (FIG. 7) and serum and peritoneal lavage fluid samples (FIG. 8) compared to the 0.1% BSA control group.

Example 4 in vivo blocking of APOH aggravates the systemic inflammatory response in CLP model mice

14C 57/6 mice were selected, 7 in each group, randomly divided into the following groups: clp+0.1% bsa and clp+rapoh were intervened for 24 hours, and blood and tissues were taken for inflammatory factor and histomorphometric detection, respectively.

The method for raising mice and constructing models is the same as in example 2, and the mice are divided into a CLP+isogypic IgG group and a CLP+anti-APOH group. To neutralize APOH activity in CLP mice, 10 μg of clp+ anti-Mouse APOH neutralizing monoclonal antibody (protein Mouse IgG1,66074-1-Ig, china) was dissolved in 100 μl PBS, and another group of mice was injected with Mouse IgG antibody as a control. The survival condition is monitored every day in the 2 nd to 14 th days after operation, and serum and tissues are collected for detecting inflammatory factors and tissue injury.

FIGS. 9-11 are in vivo blockade of APOH exacerbates the systemic inflammatory response in non-severe CLP model mice. The non-severe CLP model was constructed (40% of the cecum of the ligating mice, yellow at the end of the cecum, green line at the site of ligation) and the mode of administration was as shown (A, B in fig. 9) to investigate the potential detrimental effects of blocking APOH in vivo with murine anti-APOH antibodies. The study results showed that the survival rate of the anti-apoh+clp group sepsis mice was significantly lower than that of the IgG control group (C in fig. 9). Lung, liver, kidney injury was significantly aggravated in anti-apoh+clp mice compared to isotype IgG group (fig. 10). Also, the inflammatory factor levels in serum and peritoneal lavage fluid were significantly higher in anti-apoh+clp mice than in isotype IgG group (fig. 11).

In conclusion, the present invention finds that APOH plays a central role in the pathogenesis of sepsis. First, the present invention found that APOH expression levels in serum of infants with sepsis were significantly reduced compared to healthy controls and maintained at lower levels during the progression of sepsis. In addition, the diagnostic and prognostic AUCs for APOH were 0.98 and 0.88, respectively, suggesting that continuous detection of APOH may be helpful in predicting the occurrence and prognosis of sepsis. APOH has a higher predictive efficacy for 28-day mortality in ICU infants compared to PCT and PSOFA. Secondly, APOH can reduce tissue damage by reducing the expression of inflammatory factors IL-1 beta and TNF-alpha, thereby reducing the mortality of sepsis mice. Furthermore, neutralization of APOH activity with anti-APOH antibodies exacerbates CLP mouse mortality and tissue damage in non-severe sepsis models.

The invention first determines a stable and sustained decrease in serum APOH levels in pediatric sepsis patients, correlated with disease severity and mortality. In animal experimental studies, administration of recombinant APOH may alleviate severe sepsis, whereas anti-APOH treatment may exacerbate non-severe sepsis. The study reveals the diagnostic ability of APOH in infants suffering from sepsis and the therapeutic effect of APOH in sepsis infection, and provides a new target for the treatment of sepsis and other infectious diseases.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (10)

1. Use of apolipoprotein H as biomarker for the preparation of a product for diagnosing, prognosticating or detecting or staging the progression of sepsis in children.

2. The use according to claim 1, wherein the product is intended for testing the peripheral blood of a child.

3. The use according to claim 1, wherein the product comprises a reagent or a kit.

4. A detection reagent or a kit for children sepsis, which is characterized in that apolipoprotein H is used as a detection object.

5. Use of apolipoprotein H in the preparation of a medicament for the treatment of sepsis.

6. The use of claim 5, wherein the sepsis comprises high cecal ligation perforation-induced sepsis.

7. A medicament for treating sepsis in children, comprising apolipoprotein H.

8. The medicament of claim 7, further comprising other pharmaceutically acceptable excipients.

9. Use of apolipoprotein H in the manufacture of a medicament for reducing tissue damage.

10. Use of apolipoprotein H for the preparation of a medicament for reducing inflammatory reactions.