Application of cytoplasm small-molecule double-stranded DNA (deoxyribonucleic acid) in preparation of sepsis diagnostic reagent and sepsis therapeutic drug

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of cytoplasm small-molecule double-stranded DNA (scDNA) in preparation of sepsis diagnostic reagents and treatment drugs. According to the application, the scDNA can be used as an immunologic diagnosis and treatment target of sepsis, and a new strategy for clinical diagnosis and treatment of sepsis is developed. On the basis, the invention provides an application of the scDNA as a biomarker in preparation of a sepsis diagnostic reagent, a prognosis reagent or a curative effect evaluation reagent, and also provides an application of the scDNA in preparation of a medicine for improving and / or treating sepsis, so that a brand new theoretical basis and practical guidance can be provided for precise medical treatment of sepsis.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the application of cytoplasmic small molecule double-stranded DNA in the preparation of sepsis diagnostic reagents and therapeutic drugs. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome triggered by infection, commonly seen in patients with severe trauma or infectious diseases. Causes include infections caused by bacteria, fungi, viruses, and parasites, leading to an imbalance in the body's inflammatory response and immune regulation. Its high morbidity and mortality present a significant challenge to clinical treatment. Given the complexity and heterogeneity of this disease, there are currently no gold standard biomarkers that can rapidly distinguish sepsis from common infections in the early stages. Clinical diagnosis relies on a combination of inflammatory markers (such as CRP and PCT) combined with clinical manifestations, but these indicators lack sensitivity and specificity. Therefore, further research is urgently needed into the clinical diagnosis and treatment of sepsis. Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the purpose of this application is to provide the use of small cytoplasmic double-stranded DNA (scDNA) in the preparation of sepsis diagnostic reagents and therapeutic drugs. By using scDNA as an immunotherapy target for sepsis, a new strategy with potential and good application value is provided for the diagnosis, prognosis, efficacy evaluation and treatment of sepsis in clinical practice.

[0004] To achieve the above-mentioned objectives and other related objectives, the first aspect of the present application provides the use of scDNA as a biomarker in the preparation of sepsis diagnostic reagents, prognostic reagents or efficacy evaluation reagents.

[0005] In some embodiments, the reagent is used to determine the concentration of scDNA in a blood sample and / or a body fluid sample.

[0006] In some embodiments, the method for detecting the concentration of scDNA by the reagent includes but is not limited to nucleic acid gel electrophoresis method and real-time fluorescence quantitative PCR (qPCR). In some embodiments, the concentration of scDNA in the blood sample and / or body fluid sample is positively correlated with the severity of sepsis.

[0007] In some embodiments, the blood sample includes but is not limited to serum, plasma, whole blood, etc.

[0008] In some embodiments, the body fluid sample includes but is not limited to urine, cerebrospinal fluid, pleural effusion, ascites, etc.

[0009] In some embodiments, the agent is used to diagnose or monitor the presence and / or course and / or severity and / or prognosis of sepsis.

[0010] The second aspect of the present application provides the use of scDNA in the preparation of a drug for improving and / or treating sepsis.

[0011] In some embodiments, the drug has at least one of the following effects (I) to (IV): (Ⅰ) Inhibit bacterial growth in vivo; (II) Slowing down liver and / or kidney damage; (III) Promote the recruitment of macrophages to the inflammatory site and increase the proportion of macrophages in the inflammatory site; (IV) Enhance the ability of macrophages to phagocytize and kill bacteria.

[0012] In some embodiments, the bacteria include Gram-negative bacteria, including but not limited to Escherichia coli and Pseudomonas aeruginosa.

[0013] In some embodiments, the drug promotes the recruitment of macrophages to the inflammatory site by enhancing the expression of the chemokine CCL2, thereby increasing the proportion of macrophages in the inflammatory site.

[0014] In some embodiments, the active ingredient of the drug comprises scDNA.

[0015] In some embodiments, when the drug is used, the effective dosage of scDNA is 0.1-1.25 mg / kg body weight, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.625, 0.65, 0.7, 0.8, 0.9, 1.0, 1.25 mg / kg body weight, etc.

[0016] In some embodiments, the drug can be a single-ingredient substance or a compound preparation.

[0017] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0018] In some embodiments, the form of the drug is not particularly limited and can be in various forms such as solid, liquid, gel, semi-fluid, aerosol, etc.

[0019] In some embodiments, the drug is mainly targeted at mammals, preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc., and the primates are preferably monkeys, apes or humans.

[0020] In some embodiments, the drug can be administered in a variety of ways, including enteral and parenteral administration. Generally, known routes of administration for each chemical drug are recommended, such as intravenous, intraperitoneal, intramuscular, and subcutaneous injections. In animal experiments, intraperitoneal or tail vein injections are commonly used, as they are simple to administer. For the treatment of sepsis in humans, intravenous or other routes are often used.

[0021] As described above, the application of the cytoplasmic small molecule double-stranded DNA in the preparation of sepsis diagnostic reagents and therapeutic drugs has the following beneficial effects: This application analyzes the expression levels of scDNA in the peripheral blood of sepsis patients and mouse models and finds that scDNA has the potential to serve as a biomarker for the diagnosis of sepsis, especially the early diagnosis of sepsis. At the same time, this application further explores the application potential of scDNA in the treatment of sepsis by verifying the specific mechanism of action of scDNA in sepsis, and thus proposes the development of a new sepsis treatment strategy based on scDNA, providing a new theoretical basis and practical guidance for precision medicine of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 a and 1b show the comparison of the expression levels of scDNA in the peripheral blood of sepsis patients and healthy controls, and scDNA in the serum of sepsis model mice and sham-operated mice in nucleic acid gels in Example 1 of the present application.

[0023] Figure 2 Shown is a statistical graph of the survival rates of sepsis model mice in the scDNA protein treatment group and the PBS control group in Example 2 of the present application.

[0024] Figure 3 a, 3b, 3c and 3d respectively show the comparison of bacterial loads in the blood, peritoneal lavage fluid, liver and kidney of sepsis model mice in the scDNA treatment group and the blank control group in Example 3 of the present application.

[0025] Figure 4 a and Figure 4 b shows a comparison of liver function in sepsis model mice in the scDNA treatment group and the blank control group in Example 4 of the present application, Figure 4 c and Figure 4 d shows a comparison of renal function in sepsis model mice between the scDNA-treated group and the blank control group.

[0026] Figure 5 a, 5b and 5c respectively show the cell recruitment comparison chart of the peritoneal lavage fluid of sepsis model mice in the scDNA treatment group and the blank control group in Example 5 of the present application, the macrophage recruitment percentage statistical chart, and the neutrophil recruitment percentage statistical chart.

[0027] Figure 6 a, 6b, 6c and 6d respectively show the expression of CXCL1 in cardiac blood serum, CCL2 in cardiac blood serum, CXCL1 in peritoneal lavage fluid and CCL2 in peritoneal lavage fluid of sepsis model mice in the scDNA treatment group and the blank control group in Example 6 of the present application.

[0028] Figure 7 a and 7b respectively show the number of E. coli phagocytosed and the percentage of E. coli killed by primary peritoneal macrophages of mice in the scDNA treatment group and the blank control group in Example 7 of the present application, Figure 7 c and 7d show the number of Pseudomonas aeruginosa phagocytosed and the percentage of Pseudomonas aeruginosa killed by primary peritoneal macrophages of mice in the scDNA-treated group and the blank control group, respectively. DETAILED DESCRIPTION

[0029] The following describes the embodiments of the present application through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present application from the disclosure herein. The present application may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present application.

[0030] In this application, unless otherwise specified, the term "plurality" means two or more.

[0031] The character " / " indicates that the preceding and following objects are in an "or" relationship. For example, A / B means: A or B.

[0032] The term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0033] Sepsis involves multiple aspects such as the immune system, inflammatory response, and organ dysfunction. Clarifying the immune regulatory mechanism of sepsis is of great significance for the development of new immune diagnosis and treatment targets. Pattern recognition receptors are key components of the innate immune system, among which cGAS protein is an important inflammatory receptor for macrophage cytoplasmic DNA, and the cGAS-STING signaling pathway is the main pathway for mediating DNA immune responses in the body. "Cell Rep" (PMID: 37481718) recently reported a new type of macrophage cytoplasmic small molecule double-stranded DNA, called scDNA, with a length of 15-50bp, which slows down DNA damage through the cGAS-STING pathway. This application explores the role and mechanism of scDNA in treating sepsis through a series of studies, and can be used as a new biomarker for the diagnosis and treatment of sepsis.

[0034] This application mainly explores the medicinal value of such molecules in animals, and studies the in vitro and in vivo anti-inflammatory mechanism of the new scDNA molecules on the immune hyperinflammatory response of sepsis. In order to further explore the application of such new molecules in clinical diagnosis and treatment, this application first evaluated the value of scDNA in clinical diagnosis and found that the expression level of scDNA in the peripheral blood of patients with sepsis and mouse models was increased, indicating its potential as a molecular diagnostic marker. Secondly, since there are no reports of small molecule therapeutic drugs targeting the cGAS-STING pathway for sepsis, this application injected scDNA into septic mice and found that it can improve the survival rate of septic mice, inhibit bacterial growth in the body and slow down liver and kidney damage. In vivo experiments showed that the target cells of scDNA are macrophages, while enhancing the expression of chemokine CCL2, promoting the recruitment of macrophages to the site of inflammation, and enhancing the ability of macrophages to phagocytose and kill bacteria.

[0035] The following specific examples are given to illustrate the present application in detail. It should also be understood that the following examples are only used to specifically illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present application belong to the scope of protection of the present application. The specific process parameters and the like in the following examples are only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range based on the description herein, and are not limited to the specific values ​​exemplified below.

[0036] The experiments involved in the following examples are described as follows: Study Population: Peripheral blood mononuclear cells (PBMCs) were collected from patients with sepsis and hyperinflammatory response who were treated at the First Affiliated Hospital of Chongqing Medical University between March and June 2025. The experimental group included PBMCs from healthy subjects undergoing physical examinations during the same period. Samples were centrifuged at 1400 × g for 7 minutes at 4°C. Mononuclear cells were separated using Ficoll separation buffer and stored at −80°C. Cytoplasmic DNA was isolated, and the expression level of scDNA in the cytoplasm was detected by nucleic acid gel analysis. All patients met the Sepsis 3.0 diagnostic criteria for sepsis as defined by the International Conference on Sepsis. Patients with malignant tumors, HIV infection, hemoglobin levels below 7 g / mL, active bleeding, or the need for more than two units of red blood cells were excluded. All studies were approved by the Science and Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.

[0037] Experimental Animals: Wild-type C57BL / 6J male mice (WT), weighing 20-22 g and approximately 6-8 weeks old, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. and housed in the SPF (Specific Pathogen Free) laboratory of the Experimental Animal Center of Chongqing Medical University. All mice used in this experiment were SPF-grade experimental animals. All animal experiments strictly adhered to the Guide for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China and were in compliance with the regulations of the Laboratory Animal Ethics Committee of Chongqing Medical University. Laboratory Animal License Number: SCKK (Beijing) 2024-0001.

[0038] Example 1 1. Comparison of scDNA expression in peripheral blood mononuclear cells of patients with sepsis and healthy controls Serum specimens from healthy subjects and peripheral blood specimens from patients in the hyperinflammatory phase of sepsis were collected. Cytoplasmic DNA was isolated according to the cytoplasmic DNA isolation procedure of STARProtoc (PMIB: 38573862), and the expression of scDNA was detected by nucleic acid gel. The optical density values of the test results were determined using Image J software, and graphs and statistical analyses were performed using GraphPad Prism 9.5.0 software.

[0039] Figure 1 a and 1b show the comparison of the expression levels of peripheral blood scDNA in nucleic acid gels of sepsis patients and healthy controls.

[0040] The results showed that the expression level of scDNA in the peripheral blood of patients with high inflammatory response in sepsis was significantly higher than that in healthy people, and the difference was statistically significant (P<0.001). Therefore, scDNA can be used as a diagnostic indicator for clinical sepsis.

[0041] 2. Comparison of scDNA expression in peripheral blood between sepsis model mice and sham-operated mice Cecal ligation and puncture (CLP) was used to establish septic mice. The specific method is as follows: adult C57 mice were selected and anesthetized with 100 μL of 1.5% sodium pentobarbital injected intraperitoneally. The mice were fixed to an operating board. The abdominal hair of the mice was shaved with a special shaver. The skin was disinfected. A 1 cm long incision was made in the middle of the abdomen. The cecum was ligated and punctured with a 26-gauge syringe needle. Finally, the wound was sutured and the skin was disinfected. (For specific experimental methods, please refer to: Daniel Rittirsch, Peter A Ward, et al. Immunodesign of experimental sepsis by cecalligation and puncture. Nat Protoc. 2009; 4(1): 31-36). This modeling method is a classic modeling method for sepsis animals and is currently the standard animal model for studying sepsis.) Sixty male mice weighing 20-22 g were randomly divided into two groups: a sham-operated group and a CLP6h group. A sepsis model was established according to the above method. Cecal ligation and puncture were omitted in the sham-operated group, and all other procedures were the same. Cardiac blood was collected 6 hours after surgery in the sham-operated group and 6 hours after the CLP6h group. Peripheral blood mononuclear cells were collected using Percoll, and cytoplasmic DNA was isolated using the same procedures as above. scDNA expression was detected by gel electrophoresis, and statistical analysis was performed as above.

[0042] Figure 1 a and Figure 1 b shows a comparison of the expression levels of scDNA in the serum of sepsis model mice and sham operation group mice in nucleic acid gel.

[0043] The results showed that compared with the control group, the expression level of scDNA in mouse serum was significantly increased 6 hours after the sepsis model was established, and the difference was statistically significant, suggesting that scDNA can be used as a diagnostic indicator for the mouse sepsis model.

[0044] Example 2 Mouse survival rate experiment Twenty male mice weighing 20-22 g were selected and divided into two groups: a protein-treated group (n=10) and a control group (n=10). A sepsis model was then established according to the method of Example 1. The scDNA-treated group was pre-injected with scDNA (0.625 mg / kg) solvent via the tail vein, while the control group was injected with an equal volume of transfection reagent and ddH2O via the tail vein. The survival of the mice was observed twice daily for 14 consecutive days, and each experiment was repeated at least twice.

[0045] Figure 2Shown are statistical graphs of the survival rates of sepsis model mice in the scDNA protein treatment group and the PBS control group.

[0046] The results showed that after scDNA was replenished in wild-type mice, their survival rate was significantly higher than that of the blank control group after 14 days of observation after CLP modeling, suggesting that scDNA can improve the survival outcomes of septic mice.

[0047] Example 3 Bacterial load experiment Ten adult C57 male mice weighing 20-22 g were selected and modeled according to the method in Example 1. They were equally divided into scDNA-treated and control groups, and injected with scDNA and a blank control solvent according to the method in Example 2. After 24 hours, cardiac blood, peritoneal lavage fluid, kidneys, and livers were collected. Cardiac blood was diluted 10-100-fold, peritoneal lavage fluid was diluted 100-1000-fold, and kidneys and spleens were homogenized in 1 ml of sterile PBS and diluted 100-1000-fold. Ten microliters of each agar was then plated, and the number of colonies on the blood agar plates was counted 12 hours later.

[0048] Figure 3 a shows a comparison of bacterial loads in the blood of sepsis model mice in the scDNA treatment group and the blank control group. Figure 3 b shows the comparison of bacterial load in peritoneal lavage fluid of sepsis model mice in scDNA treatment group and blank control group. Figure 3 c shows the comparison of bacterial load in the liver of sepsis model mice in the scDNA treatment group and the blank control group. Figure 3 d shows a comparison of bacterial loads in the kidneys of sepsis model mice in the scDNA-treated group and the blank control group.

[0049] The results showed that compared with the control group, after scDNA treatment, the bacterial load in the heart blood, peritoneal lavage fluid, liver and kidney of sepsis model mice was significantly reduced, and the difference was statistically significant, indicating that scDNA enhanced the bacterial clearance ability of the mouse body.

[0050] Example 4 Liver and kidney function tests Twelve adult C57 male mice weighing 20-22 g were selected and modeled according to the method described in Example 1. The mice were equally divided into an scDNA-treated group and a control group, and injected with scDNA and a blank control solvent, respectively, according to the method described in Example 2. After 24 hours, cardiac blood was collected and centrifuged at 1400 × g for 7 minutes at 4°C to separate serum. Liver and kidney function biochemical markers were then measured.

[0051] Figure 4 a and Figure 4b shows the comparison of liver function between the scDNA treatment group and the blank control group in the sepsis model mice. Figure 4 c and Figure 4 d shows a comparison of renal function in sepsis model mice between the scDNA-treated group and the blank control group.

[0052] The results showed that compared with the control group, after scDNA treatment, the liver and kidney function damage indicators in the peripheral blood of sepsis model mice were significantly reduced, and the difference was statistically significant, indicating that scDNA enhanced the liver and kidney function recovery ability of mice.

[0053] Example 5 Flow cytometry was used to detect the ability of peritoneal macrophage recruitment Twelve adult C57 male mice weighing 20-22 g were selected and modeled according to the method in Example 1. They were equally divided into scDNA-treated and control groups, and injected with scDNA and blank control solvent according to the method in Example 2. After 24 h, the peritoneal lavage fluid of the CLP mice was collected, centrifuged and the supernatant discarded, and 1 ml / tube of red blood cell lysis buffer was added for 3 minutes, washed once with 1× PBS, centrifuged at 900g, 4°C for 8 minutes, and the supernatant discarded. The cells were resuspended in PBS. Flow cytometry antibody staining: 2 μl / tube of blocking antibody CD16 / 32 was added and incubated on ice in the dark for half an hour: 2 ul of mouse Ly6G-BV421Ab, mouse F4 / 80-PEAb, mouse CD11b-FITCAb, mouse CD206-APCAb, and mouse CD86-Cyaine7Ab were added to each tube, gently mixed, incubated in the dark for 30 minutes, centrifuged at 900g, 4°C for 8 minutes, and the supernatant discarded. Resuspend the cells in pre-cooled 1× PBS to wash away any unbound antibodies, then resuspend the cells in flow cytometry staining buffer for detection.

[0054] Figure 5 a shows a comparison of cell recruitment in the peritoneal lavage fluid of sepsis model mice in the scDNA treatment group and the blank control group. Figure 5 b shows the statistical graph of the percentage of macrophage recruitment in the peritoneal lavage fluid of sepsis model mice in the scDNA treatment group and the blank control group. Figure 5 c shows the statistical graph of neutrophil recruitment percentage in the peritoneal lavage fluid of sepsis model mice in the scDNA treatment group and the blank control group.

[0055] The results showed that compared with the control group, the proportion of macrophages in the peritoneal lavage fluid of sepsis model mice was significantly increased after scDNA treatment, and the difference was statistically significant, indicating that scDNA enhanced the ability of mice to recruit macrophages into the peritoneal cavity.

[0056] Example 6 Serum chemokines to detect the body's ability to recruit macrophages Ten adult C57 male mice weighing 20-22 g were selected and modeled according to the method in Example 1. They were equally divided into an scDNA-treated group and a control group, and injected with scDNA and a blank control solvent, respectively, according to the method in Example 2. After 24 hours, cardiac blood and peritoneal lavage fluid were collected from CLP mice, and serum CXCL1 and CCL2 expression was assayed using the CXCL1 and CCL2 ELISA kit procedures.

[0057] Figure 6 a shows the expression of CXCL1 in the heart blood serum of sepsis model mice in the scDNA treatment group and the blank control group. Figure 6 b shows the expression of CCL2 in the heart blood serum of sepsis model mice in the scDNA treatment group and the blank control group. Figure 6 c shows the expression of CXCL1 in the peritoneal lavage fluid of sepsis model mice in the scDNA treatment group and the blank control group. Figure 6 d shows the expression of CCL2 in the peritoneal lavage fluid of sepsis model mice in the scDNA treatment group and the blank control group.

[0058] The results showed that compared with the control group, the proportion of chemokine CCL2, which recruits macrophages, in the cardiac blood and peritoneal lavage fluid of sepsis model mice was significantly increased after scDNA treatment, and the difference was statistically significant, indicating that scDNA enhanced the ability of mice to recruit macrophages.

[0059] Example 7 Macrophage phagocytosis and bacterial killing function test (plate counting method) Paraffin was injected into the peritoneal cavity of mice 3 days in advance. After the mice were sacrificed, the peritoneal lavage fluid was collected and centrifuged to obtain primary macrophages. The macrophages were divided into two groups, with 3 replicates in each group. One group was transfected with scDNA for 24 hours, and the other group was added with an equal amount of blank transfection reagent as a control. Pseudomonas aeruginosa and Escherichia coli were collected, and a bacterial suspension was prepared. The macrophages were infected with an infection index MOI = 1:100. The cell culture plate was placed on a 37°C shaker. The macrophages were co-cultured with Pseudomonas aeruginosa or Escherichia coli for 30 minutes, washed three times with sterile PBS to wash away excess extracellular bacteria that were not phagocytosed. 200 μl of antibiotics (200 ug / ml) were added to each well and placed at 4°C for about 15 minutes to kill extracellular bacteria. The cells were washed three times with 1× PBS to wash away extracellular antibiotics. For phagocytic plates: Add ddH2O to lyse cells and collect the lysate. For killer plates: Aspirate DMEM, wash three times with 1× PBS, add DMEM, incubate at 37°C for 2 hours, then wash three times with 1× PBS. Add ddH2O to lyse cells and collect the lysate. Pipette the lysed bacterial solution into an EP tube and dilute 10x, 100x, and 1000x. Take 10 μl and inoculate it onto a blood agar plate. Incubate at 37°C for 24 hours and count the number of colonies.

[0060] Figure 7 a shows the number of E. coli phagocytosed by primary peritoneal macrophages in mice in the scDNA treatment group and the blank control group. Figure 7 b shows the percentage of Escherichia coli killed by primary peritoneal macrophages in the scDNA-treated group and the blank control group; Figure 7 c shows the number of Pseudomonas aeruginosa phagocytosed by primary macrophages in the peritoneal cavity of mice in the scDNA treatment group and the blank control group. Figure 7 d shows the percentage of primary peritoneal macrophages killing Pseudomonas aeruginosa in the scDNA-treated group and the blank control group.

[0061] The results showed that compared with the control group, the number of bacteria phagocytosed and killed by primary mouse peritoneal macrophages increased significantly after scDNA treatment, and the difference was statistically significant, indicating that scDNA enhanced the ability of primary mouse peritoneal macrophages to phagocytose and kill bacteria.

[0062] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical concepts disclosed in this application shall be covered by the claims of this application.

Claims

1. Application of small cytoplasmic double-stranded DNA as a biomarker in the preparation of sepsis diagnostic reagents, prognostic reagents or efficacy evaluation reagents.

2. The use according to claim 1, characterized in that: The reagent is used for determining the concentration of cytoplasmic small molecule double-stranded DNA in a blood sample and / or a body fluid sample.

3. The use according to claim 2, characterized in that: The method for the reagent to detect the concentration of cytoplasmic small molecule double-stranded DNA is selected from nucleic acid gel electrophoresis method or real-time fluorescence quantitative PCR.

4. The use according to claim 2, characterized in that: The concentration of cytoplasmic small molecule double-stranded DNA in the blood sample and / or body fluid sample is positively correlated with the severity of sepsis.

5. The use according to claim 2, characterized in that: The blood sample is selected from at least one of serum, plasma or whole blood; And / or, the body fluid sample is selected from at least one of urine, cerebrospinal fluid, pleural effusion or ascites.

6. Use of cytoplasmic small molecule double-stranded DNA in the preparation of drugs for improving and / or treating sepsis.

7. The use according to claim 6, characterized in that: The drug has at least one of the following effects (I) to (IV): (Ⅰ) Inhibit bacterial growth in vivo; (II) Slowing down liver and / or kidney damage; (III) Promote the recruitment of macrophages to the inflammatory site and increase the proportion of macrophages in the inflammatory site; (IV) Enhance the ability of macrophages to phagocytize and kill bacteria.

8. The use according to claim 7, characterized in that: The drug promotes the recruitment of macrophages to the inflammatory site by enhancing the expression of the chemokine CCL2, thereby increasing the proportion of macrophages in the inflammatory site.

9. The use according to claim 6, characterized in that: The active ingredients of the drug include cytoplasmic small molecule double-stranded DNA; And / or, the drug further includes a pharmaceutically acceptable carrier and / or excipient.

10. The use according to claim 6 or 9, characterized in that: When the drug is used, the effective dosage of the cytoplasmic small molecule double-stranded DNA is 0.1-1.25 mg / kg body weight.

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