Use of insulin-like growth factor binding protein 6 as a biomarker in the preparation of sepsis diagnosis and treatment reagents

Abstract

The application belongs to the technical field of biological medicine, and particularly discloses an application of insulin-like growth factors binding protein 6 (IGFBP6) as a biomarker in preparation of a sepsis diagnosis and treatment reagent. It is found by the application that IGFBP6 can be used as a new biological marker for assisting in diagnosis of sepsis, and can be used not only for diagnosis of diseases and reflecting severity of diseases, but also as a potential sepsis treatment target, so that a new technical means is provided for diagnosis, prognosis and treatment of sepsis diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of insulin-like growth factor binding protein 6 as a biomarker in the preparation of reagents for the diagnosis and treatment of sepsis. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulation of the host's anti-infective response. It is a serious complication of acute and critical illnesses such as trauma, burns, shock, and infection, and a leading cause of death in infected patients. Sepsis is usually caused by bacterial infection, but can also be caused by fungal or parasitic infections. The progression of sepsis mainly consists of four parts: general infection, systemic inflammatory response syndrome, sepsis, and severe sepsis. If not controlled promptly and effectively, it may progress to shock and organ failure. Due to the lack of specific symptoms and signs in sepsis patients, clinical diagnosis faces many challenges. A survey report showed that 86% of physicians believed that the symptoms of sepsis were atypical, leading to delays in diagnosis and treatment. Sometimes, the progression from sepsis to septic shock can occur in as little as 24 hours. A retrospective cohort study investigated 2,731 patients with septic shock from 14 intensive care units and 10 hospitals in Canada and the United States. The results showed that if patients did not receive effective anti-infective treatment within the first 6 hours, the risk of death increased, with survival decreasing by 7.6% (range 3.6%–9.9%) for each hour of delay. Only 50% of septic shock patients received antibiotics within 6 hours. This highlights the significant challenges clinicians face in improving early clinical identification, accurately assessing the condition, and implementing appropriate treatment strategies promptly.

[0003] The pathogenesis of sepsis is highly complex, involving a series of physiological and pathological processes such as infection, inflammation, immunity, coagulation, and tissue damage. Inflammatory factors released during infection involve multiple cells and systems throughout the body via the neuroendocrine-immune regulatory system, forming a vast and complex network with amplified cascade effects and mutual constraints. The instability of systemic inflammatory response syndrome (SIRS) and compensatory anti-inflammatory response syndrome (CARS) caused by the dysregulation of inflammatory and anti-inflammatory factors jointly contributes to sepsis-related immune dysfunction. The multi-organ inflammatory damage mediated by the SIRS cytokine storm and the CARS-related immunosuppression create a complex instability, making sepsis treatment difficult to achieve with simple anti-infective and anti-inflammatory drugs. Therefore, in recent years, novel immunomodulatory therapies have attracted increasing attention, with the potential immunomodulatory roles of some key cytokines in sepsis becoming a focus of research. Insulin-like growth factor binding protein-6 (IGFBP6) is one of the key factors proposed for the first time in this study that can intervene in the progression of sepsis, and it has significant implications for new strategies for the diagnosis and treatment of sepsis. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide the use of insulin-like growth factor binding protein 6 as a biomarker in the preparation of reagents for the diagnosis and treatment of sepsis. This invention is the first to discover that IGFBP6 can be used as a new biological marker for predicting and diagnosing sepsis. It can not only be used for the diagnosis of the disease and to reflect the severity of the disease, but it can also serve as a potential therapeutic target for sepsis, providing a new technical means for the diagnosis, prognosis and treatment of sepsis.

[0005] To achieve the above and other related objectives, the first aspect of the present invention provides the use of insulin-like growth factor binding protein 6 as a biomarker in the preparation or screening of diagnostic reagents and / or therapeutic drugs for sepsis.

[0006] In some embodiments of the present invention, the reagent is used to determine the expression level of insulin-like growth factor binding protein 6 in body fluid samples.

[0007] In some embodiments of the present invention, the expression level of insulin-like growth factor binding protein 6 in the body fluid sample is positively correlated with the severity of sepsis.

[0008] In some embodiments of the present invention, the detection method used with the reagent is selected from at least one of chemiluminescence, ELISA, immunoturbidimetry, immunofluorescence, and colloidal gold, but is not limited thereto.

[0009] In some embodiments of the present invention, the body fluid sample is selected from at least one of serum, plasma, whole blood, urine, cerebrospinal fluid, pleural fluid, and ascites, but is not limited thereto.

[0010] A second aspect of this invention provides the use of an insulin-like growth factor binding protein 6 (IGFBP6) blocking agent in the preparation of medicaments for the treatment and / or prevention of sepsis, said blocking agent being used to reduce the level of IGFBP6 protein. IGFBP6 blockade can reduce the level of IGFBP6 protein in the body, increase the body's ability to clear bacteria, significantly improve the condition of sepsis, and increase survival rate.

[0011] In some embodiments of the invention, the blocking agent contains an effective dose of an inhibitor, neutralizing antibody, and / or transcriptional regulation-related molecule associated with insulin-like growth factor binding protein 6, but is not limited thereto.

[0012] A third aspect of this invention provides the use of a STAT1 agonist in the preparation of a therapeutic and / or preventive medicament for sepsis caused by high expression of insulin-like growth factor binding protein 6 (IGFBP6). The STAT1 agonist increases the phosphorylation level of STAT1, and the medicament restores the macrophage recruitment capacity impaired by elevated IGFBP6 in vivo. The STAT1 agonist, i.e., a chemoagonist of STAT1, can restore macrophage recruitment impaired by increased IGFBP6, restore the bacterial clearance capacity of sepsis-affected mice impaired by increased IGFBP6, significantly improve sepsis symptoms, and increase survival rate.

[0013] In some embodiments of the present invention, the STAT1 agonist is selected from 2-NP.

[0014] The fourth aspect of the present invention provides the use of macrophage chemokines in the preparation of a therapeutic and / or preventive medicament for sepsis caused by high expression of insulin-like growth factor binding protein 6, said medicament restoring the macrophage recruitment capacity in vivo impaired by elevated insulin-like growth factor binding protein 6 through macrophage chemokines.

[0015] In some embodiments of the present invention, the macrophage chemokine is selected from at least one of CCL2 and its recombinant protein, preferably recombinant CCL2 protein. IGFBP6 promotes the development and progression of sepsis and is related to its inhibition of CCL2 expression; the therapeutic effect of blocking IGFBP6 on sepsis is also related to relieving its inhibitory effect on CCL2. Therefore, a CCL2-based dosing regimen has a therapeutic effect on sepsis. Recombinant CCL2 protein is used to increase CCL2 levels in septic mice, restore macrophage recruitment and bacterial clearance capabilities damaged by increased IGFBP6, significantly improve sepsis symptoms, and increase survival rates.

[0016] As described above, the use of insulin-like growth factor binding protein 6 as a biomarker in the preparation of reagents for the diagnosis and treatment of sepsis, according to the present invention, has the following beneficial effects:

[0017] This invention discovers that IGFBP6 can serve as a novel biomarker for diagnosing sepsis, and high expression of serum IGFBP6 in patients is positively correlated with disease severity. Furthermore, knocking out IGFBP6 in vivo increases macrophage recruitment in septic mice, enhances bacterial clearance, and improves survival. Further, macrophage chemokines (e.g., recombinant CCL2 protein) and STAT1 agonists (e.g., 2-NP) can restore macrophage recruitment impairment induced by IGFBP6, reduce bacterial load in animals, and increase survival in septic mice. Therefore, this invention provides IGFBP6 as a diagnostic and prognostic indicator for sepsis, as well as a potential therapeutic target. Attached Figure Description

[0018] Figure 1 The graph shows the expression levels of IGFBP6 in the serum of patients with sepsis, control group patients, and other infected patients in Example 1.

[0019] Figure 2 The image shows the correlation analysis between serum IGFBP6 expression and SOFA score in sepsis patients in Example 2.

[0020] Figure 3 The graph shown is a statistical chart of ROC curve analysis in Example 3, illustrating the positive diagnosis of serum marker IGFBP6 in sepsis and its correlation with CRP and PCT in the diagnosis of death.

[0021] Figure 4 The graph shows the expression changes of IGFBP6 in the sepsis mouse model in Example 4 and the survival rates of mice in the IGFBP6 protein treatment group and the control group.

[0022] Figure 5 The figures shown are statistical graphs of bacterial load in various tissues of septic mice in the IGFBP6 protein group and the control group, as well as statistical graphs of the proportion of macrophages in peritoneal lavage fluid (PLF) and lung tissue in Example 5.

[0023] Figure 6 The figures shown are survival curves, bacterial load statistics, and macrophage ratio statistics for IGFBP6 gene-deficient mice and wild-type sepsis mice in Example 6.

[0024] Figure 7 The graphs shown are the survival curves, bacterial load statistics, and macrophage ratio statistics of septic mice in the CCL2 treatment group, protein group, and control group in Example 7.

[0025] Figure 8 The graphs shown are the survival curves, bacterial load statistics, and macrophage ratio statistics of septic mice in the 2-NP treatment group, protein group, and control group in Example 8. Detailed Implementation

[0026] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0027] This invention focuses on exploring the application value of IGFBP6 in the diagnosis and treatment of sepsis. Insulin-like growth factor binding protein (IGFBP) is a family of seven highly homologous proteins with high affinity for IGF. The effects of IGFBPs in vivo mainly involve two aspects: IGF-dependent and IGF-independent. IGFBPs can aggregate free IGF molecules in body fluids and fix IGF using their binding ability to extracellular matrix proteins, thereby reducing IGF concentration and inhibiting IGF binding to IGF and related effects. In addition to the above classic pathways, IGFBPs, as exocrine proteins, have their own cellular receptors and can exert effects independent of the IGF molecular network.

[0028] IGFBP6 is distributed in various tissues and exhibits broad biological activity in vivo. IGFBP6 has an N-terminal and a C-terminal domain; the N-terminus binds IGFs, while the C-terminus assists in IGF binding and exerts IGF-independent effects. Unlike other IGFBPs, IGFBP6 binds to IGF-II much more readily than IGF-I, and it can regulate cell proliferation, differentiation, migration, survival, and angiogenesis by inhibiting IGF-II. Furthermore, IGFBP6 has been shown to regulate inflammatory cell activation, chemotaxis, apoptosis, and oxidative stress in IGF-dependent or IGF-independent ways. This suggests that IGFBP6 may play an immune role in sepsis, but no related research has been reported yet.

[0029] The inventors of this application used ELISA to detect IGFBP6 levels in the serum of 163 adult sepsis patients and 92 healthy individuals. They found that IGFBP6 levels in sepsis patients were significantly higher than in healthy controls, with a statistically significant difference. The IGFBP6 levels in deceased sepsis patients were significantly higher than in survivors, and the IGFBP6 levels in patients with septic shock were significantly higher than in those without septic shock. Subsequently, they also detected IGFBP6 levels in the serum of 143 pediatric sepsis patients and 98 healthy children. The results showed that the expression level of IGFBP6 in pediatric sepsis patients was significantly higher than in healthy individuals, with a statistically significant difference. Furthermore, the IGFBP6 levels in patients with septic shock were significantly higher than in those without septic shock. In addition, they conducted a correlation analysis between serum IGFBP6 levels in sepsis patients and their Sequential Organ Failure Assessment (SOFA) score, finding a positive correlation between IGFBP6 levels and SOFA scores, indicating that IGFBP6 can indicate the severity of sepsis. Subsequently, the diagnostic efficacy of IGFBP6 for sepsis was further evaluated using ROC curves, and its diagnostic efficacy for 28-day mortality was compared with that of PCT and CRP. The results showed that IGFBP6 has excellent diagnostic efficacy for sepsis, and the area under the curve for IGFBP6 in the deceased patient group was significantly higher than that for CRP and PCT. This indicates that IGFBP6 can be used as an auxiliary indicator for the diagnosis of sepsis and the prediction of patient mortality, and its diagnostic efficacy is superior to that of CRP and PCT.

[0030] To confirm our results, we further constructed a cecal ligation and puncture (CLP)-induced sepsis mouse model. The study found that after modeling, the levels of IGFBP6 in the blood, peritoneal lavage fluid (PLF), and various organs of the mice were significantly higher than those in the control group (Sham group), and the differences were statistically significant. Elevated IGFBP6 levels were associated with the development and progression of sepsis, manifested in septic mice injected intraperitoneally with exogenous recombinant IGFBP6 protein having higher bacterial loads and significantly lower survival rates. Further investigation revealed that recombinant IGFBP6 significantly reduced the proportion of macrophages in the peritoneal lavage fluid and lung tissue of septic mice. Macrophages are key immune cells involved in bacterial clearance during sepsis. These results indicate that high expression of IGFBP6 inhibits the recruitment of macrophages in septic mice, leading to impaired host clearance of pathogens and increased mortality.

[0031] Furthermore, in this embodiment of the invention, wild-type mice and IGFBP6 gene-deficient mice were used to model sepsis. Experimental results showed that the survival of IGFBP6 gene-deficient mice was significantly improved compared with wild-type mice, and the difference was statistically significant. Simultaneously, knocking out IGFBP6 enhanced the bacterial clearance ability of sepsis-affected mice and increased the recruitment of macrophages.

[0032] Subsequently, in the embodiments of this invention, it was found that IGFBP6 inhibits macrophage recruitment and is related to its inhibition of epithelial cell secretion of the macrophage chemokine CCL2. Further data showed that IGFBP6 inhibits CCL2 expression by inhibiting STAT1 phosphorylation in epithelial cells. Using 2-NP to activate STAT1 phosphorylation reversed the IGFBP6-induced decrease in CCL2 levels and sepsis-related damage. Therefore, high expression of IGFBP6 is an indicator of sepsis severity and can serve as a potential therapeutic target.

[0033] The following specific examples illustrate the present invention in detail. It should also be understood that the following examples are only for specific illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are all within the scope of protection of the present invention. The specific experimental parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below. The experiments involved in the following examples are described as follows:

[0034] Study population: Blood samples from children with sepsis treated at the Children's Hospital Affiliated to Chongqing Medical University from November 2020 to July 2022 were collected as the pediatric sepsis experimental group; blood samples from children with sepsis treated at the First Affiliated Hospital of Chongqing Medical University from July 2020 to September 2022 were collected as the adult sepsis experimental group; and healthy individuals who underwent physical examinations at their respective hospitals during the same period were collected as the healthy control group. Samples were centrifuged at 1400×g for 7 min at 4℃ and stored at -80℃. Serum IGFBP6 protein expression levels were detected using an ELISA kit. All patients met the diagnostic criteria for sepsis 3.0 in the international sepsis treatment guidelines and were excluded if they had malignant tumors, HIV infection, hemoglobin levels below 7 g / mL, active bleeding, or required more than two units of red blood cells.

[0035] Laboratory animals: Wild-type male C57BL / 6J mice, weighing 18-22g, approximately 6-8 weeks old, purchased from Beijing SPAF Biotechnology Co., Ltd., and housed in the SPF (Specific Pathogen Free) laboratory of the Laboratory Animal Center of Chongqing Medical University. All mice used in this experiment were SPF-grade laboratory animals. Animal experiments were strictly conducted in accordance with the Guidelines for Laboratory Animal Care and Use issued by the Ministry of Science and Technology of the People's Republic of China, and complied with the management regulations of the Laboratory Animal Ethics Committee of Chongqing Medical University.

[0036] Construction of a sepsis animal model: Sepsis mice were constructed using cecal ligation and puncture (CLP). The specific method is as follows: 6-8 week old male mice were selected, anesthetized, and fixed to a control board. The abdomen was shaved with pet electric clippers, the skin was disinfected, a 1 cm incision was made in the midline 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, refer to: Daniel Rittirsch, Peter A Ward, et al. Immunodesign of experimental sepsis by cecal ligation and puncture. NatProtoc. 2009; 4(1): 31-36). This modeling method is a classic modeling method for sepsis animal models and is currently the standard animal model for studying sepsis.

[0037] The nucleotide sequence of IGFBP6 involved in this invention is as follows:

[0038] >NC_000081.7:102052621-102057947Mus musculus strain C57BL / 6Jchromosome 15,GRCm39

[0039]

[0040]

[0041]

[0042]

[0043] The amino acid sequence of IGFBP6 involved in this invention is as follows:

[0044] >sp|P47880.2|IBP6_MOUSE RecName:Full=Insulin-like growth factor-binding protein 6; Short=IBP-6; Short=IGF-binding protein 6; Short=IGFBP-6; Flags: PrecursorMTWDGLPTQPLLMLLMLLFAAGSGSALAGCPGCGAGMQTGCRGGCVEEEDAGSPADGCTEAGGCLRREGQPCGVYSPKCAPGLQCQPRENEEAPLRALLIGQGR CQRARGPSEETTKESKPQGGASRSRDTNHRDRQKNPRTSAAPIRPNPVQDSEMGPCRRHLDSVLQQLQTEVFRGGARGLYVPNCDLRGFYRKQQCRSSQGNRRGPCWCVDPMGQPLPVSPDGQGSTQCSARSSG.

[0045] The following specific examples illustrate the present invention in detail. It should also be understood that the following examples are only for specific illustrative purposes and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0046] Example 1

[0047] Detection of IGFBP6 expression level in serum of sepsis patients

[0048] Serum samples were collected from healthy individuals undergoing physical examinations and sepsis patients (statistical information is shown in Tables 1 and 2) on the day of admission. IGFBP6 expression was detected using an ELISA kit (purchased from R&D Systems, catalog number DY876), with strict adherence to the kit instructions. Statistical analysis was performed using SPSS 20.0 software, and graphs were generated using GraphPad Prism 8.0 software.

[0049] Table 1. Statistical results of data from adult sepsis patients and healthy individuals undergoing physical examinations.

[0050]

[0051]

[0052] Table 2. Statistical results of data from children with sepsis and healthy individuals undergoing physical examinations.

[0053]

[0054] Figure 1 The graph shows the expression levels of IGFBP6 in the serum of patients with sepsis, control group patients, and other infected patients in this embodiment.

[0055] The results are as follows Figure 1 As shown in Figure A, IGFBP6 expression was significantly increased in adult patients with sepsis, and the increase was higher than that in patients with severe pneumonia and ordinary infections; Figure 1 As shown in Figure B, the IGFBP6 level in septic patients with shock was significantly higher than that in non-septic shock patients. Figure 1 C showed that the IGFBP6 levels in sepsis patients who eventually died were significantly higher than those in survivors. For example... Figure 1 As shown in D, further testing revealed that the expression level of IGFBP6 in children with sepsis was significantly higher than that in normal children, and also higher than that in children with severe pneumonia and common infections. Figure 1 E showed that the IGFBP6 level in the shock group was significantly higher than that in the non-shock group. Figure 1 F showed that the IGFBP6 levels in patients who died were significantly higher than those in survivors.

[0056] The results showed that IGFBP6 expression was significantly elevated in sepsis, suggesting it may play a role in the disease; IGFBP6 levels were closely related to disease severity and could be used to differentiate sepsis from other infectious diseases.

[0057] Example 2

[0058] Serum IGFBP6 expression levels in sepsis patients were positively correlated with SOFA scores.

[0059] In the Sepsis 3.0 diagnostic criteria, the SOFA score is an important indicator for diagnosing sepsis. Extensive research data shows that for ICU patients with infection or suspected infection, a SOFA score ≥2 is sufficient for a diagnosis of sepsis. Furthermore, the SOFA score reflects the degree of dysfunction of multiple organs in patients with multiple organ dysfunction syndrome (MODS) and is closely related to the in-hospital mortality rate.

[0060] Figure 2 The image shows the correlation analysis between serum IGFBP6 expression levels and SOFA scores in sepsis patients in this embodiment.

[0061] like Figure 2 As shown in Figure A, the correlation coefficient between serum IGFBP6 expression and SOFA score in adult sepsis patients was r = 0.4974, P < 0.0001. Figure 2 B-correlation analysis revealed that the correlation coefficient between IGFBP6 expression and SOFA score in children with sepsis was r = 0.6455, P < 0.0001, which was statistically significant.

[0062] The results showed that the expression level of IGFBP6 in the serum of both adult and pediatric sepsis patients was significantly positively correlated with the SOFA score, indicating that IGFBP6 can indicate the severity of sepsis.

[0063] Example 3

[0064] ROC curve analysis compared the diagnostic efficacy of serum IGFBP6 for sepsis infection and death.

[0065] Laboratory parameters, including CRP and PCT, were collected from patients in the sepsis group and the control group using the laboratory's LIS system. ROC curve analysis was used to assess the diagnostic efficacy of IGFBP6 in sepsis-positive patients, and its diagnostic efficacy in sepsis-related deaths was compared with that of CRP and PCT. ROC curves were generated using SPSS 20.0 software, and the statistical differences in the area under the ROC curves were compared using MedCalc 15.8 software.

[0066] Figure 3 This example shows the ROC curve analysis of the positive diagnosis of serum marker IGFBP6 in sepsis and its correlation with CRP and PCT in the diagnosis of death.

[0067] like Figure 3 As shown in Table A and Table 3, the ROC curve analysis showed that IGFBP6 had a diagnostic value in adult and pediatric sepsis-positive patients, with areas under the curve of 0.8837 and 0.8994, respectively.

[0068] The statistical results above show that the area under the curve for IGFBP6 in the diagnosis of sepsis in both adults and children is >0.85, indicating good diagnostic efficacy and statistical significance. This suggests that IGFBP6 has certain application value in the positive diagnosis of sepsis.

[0069] Table 3. ROC curve analysis of the correlation between IGFBP6 and sepsis-positive patients in adults and children.

[0070]

[0071] like Figure 3 As shown in B and Table 4, the diagnostic value of different indicators in patients who died from sepsis was analyzed by ROC curve analysis. The areas under the curve for serum IGFBP6, CRP, and PCT were 0.7899, 0.5712, and 0.6732, respectively.

[0072] The statistical results above show that the area under the curve for IGFBP6 is significantly larger than that for CRP and PCT, and this is statistically significant. This indicates that IGFBP6 has certain diagnostic value for patients who die from sepsis, and is the optimal one.

[0073] Table 4. ROC curve analysis comparing the correlation between IGFBP6 / CRP / PCT and the prediction of sepsis mortality.

[0074]

[0075]

[0076] Example 4

[0077] Animal models of sepsis further demonstrate that IGFBP6 exacerbates the severity of the disease.

[0078] After establishing a mouse sepsis model, samples of heart blood, peritoneal lavage fluid, lungs, and spleen were collected at four time points: 6 hours, 24 hours, 48 ​​hours, and 72 hours. The expression level of IGFBP6 was measured using an ELISA kit (purchased from R&D Systems, catalog number DY776).

[0079] To further verify the damaging role of IGFBP6 in sepsis, 10 wild-type mice of similar age and weight were randomly divided into two groups. After sepsis modeling, the mice were injected with PBS and 500 ng of IGFBP6 protein (purchased from R&D Systems, catalog number 776-B6) for three consecutive days. The survival rate of the mice was observed and recorded daily. The experiment was repeated three times. Kaplan-Meier survival curves were plotted and analyzed using GraphPad Prism 8.0.1 software.

[0080] Figure 4 The graph shows the expression changes of IGFBP6 in the sepsis mouse model in this embodiment and the survival rate statistics of mice in the IGFBP6 protein treatment group and the control group.

[0081] The result is as follows Figure 4 As shown in AD, IGFBP6 expression was significantly elevated in animals with sepsis. Figure 4 As shown in Figure E, the survival rate of mice in the control group reached 86.7%, while the survival rate of mice in the protein group was only 40%, showing a significant statistical difference. This further illustrates that the increase of IGFBP6 is a risk factor for the aggravation of sepsis, which promotes the occurrence and development of sepsis and increases the mortality rate of sepsis.

[0082] Example 5

[0083] IGFBP6 inhibits the recruitment capacity of macrophages, thereby impairing the ability of septic mice to clear bacteria.

[0084] The preceding data in this invention have confirmed that IGFBP6 plays a damaging role in sepsis, increasing the severity and mortality of sepsis. To further explore its pathogenic mechanism and potential therapeutic target for this disease, five 6-8 week old wild-type male C57BL / 6 mice were randomly selected from each group and injected intraperitoneally with IGFBP6 protein and PBS, respectively. The bacterial load assay was used to determine the bacterial clearance capacity of the sepsis mice, and flow cytometry was used to detect the recruitment capacity of macrophages in the peritoneal cavity and lung tissue.

[0085] Figure 5 The figures shown are statistical graphs of bacterial load in various tissues of septic mice in the IGFBP6 protein group and control group, as well as statistical graphs of the proportion of macrophages in peritoneal lavage fluid and lung tissue in this embodiment.

[0086] The results are as follows Figure 5 A showed that 24 and 48 hours after sepsis modeling, the bacterial load in the blood, peritoneal lavage fluid, lungs, and spleen of the proteomic mice was higher than that in the control group, suggesting that IGFBP6 impaired the bacterial clearance capacity of septic mice; flow cytometry results were as follows. Figure 5 BC showed that IGFBP6 treatment led to a decrease in the proportion of macrophages in peritoneal lavage fluid and lung tissue, suggesting that IGFBP6 impairs the ability to clear bacteria by affecting the number of macrophages, thereby increasing the mortality rate of sepsis and making it a therapeutic target for the development and progression of sepsis.

[0087] Example 6

[0088] IGFBP6 knockout mice exhibited increased recruitment of macrophages, enhanced bacterial clearance capabilities, and improved survival rates.

[0089] To further clarify the role of IGFBP6 in sepsis and evaluate its therapeutic value, we knocked out IGFBP6 at the gene level to reduce its protein level. We then modeled the disease using wild-type male mice of the same weight and age. We assessed the potential of IGFBP6 as a therapeutic target by detecting macrophage numbers, measuring bacterial load, and analyzing survival rates. Each experiment was repeated three times.

[0090] Figure 6 The figures shown are the survival curves, bacterial load statistics, and macrophage ratio statistics of IGFBP6 gene-deficient and wild-type sepsis mice in this embodiment.

[0091] The results are as follows Figure 6A showed that the survival rate of IGFBP6 gene-deficient mice was 60%, while the survival rate of wild mice was only 26.7%. The survival rate of sepsis mice was significantly improved after IGFBP6 knockout. Figure 6 B showed that 24 and 48 hours after sepsis modeling, the bacterial load in the blood, peritoneal lavage fluid, lungs, and spleen of defective mice was lower than that in the control group, and IGFBP6 deficiency significantly enhanced the ability to clear bacteria. Figure 6 C showed that the number of macrophages in IGFBP6-deficient mice was significantly higher than that in wild-type mice. This suggests that knocking out IGFBP6 has the potential to treat sepsis, and that IGFBP6 is a therapeutic target for the development and progression of sepsis.

[0092] Example 7

[0093] Macrophage chemokine CCL2 can reverse the damaging effects of IGFBP6 in sepsis.

[0094] In some embodiments of this invention, it was found that high expression of IGFBP6 inhibited the expression of CCL2 in septic mice, thereby impairing the body's ability to recruit macrophages. Therefore, this invention provides a recombinant mouse CCL2 protein (purchased from R&D Systems, catalog number 479-JE) for overexpressing CCL2 protein levels and assesses whether the damaging effect of IGFBP6 in sepsis depends on the inhibition of CCL2.

[0095] To clarify whether CCL2 overexpression under high IGFBP6 expression state has a protective effect against sepsis, we randomly divided C57BL / 6 male mice into three groups for sepsis modeling: control group (CLP+PBS), protein group (CLP+IGFBP6), and CCL2 treatment group (CLP+IGFBP6+CCL2), with 5 mice in each group, and the experiment was repeated three times.

[0096] Figure 7 The graphs shown here are the survival curves, bacterial load statistics, and macrophage ratio statistics for the CCL2 treatment group, protein group, and control group of septic mice in this embodiment.

[0097] Survival curve results as follows Figure 7 As shown in Figure A, CCL2 overexpression significantly restored the survival rate of septic mice. Figure 7 Bacterial load assays showed that CCL2 treatment reduced bacterial load levels in septic mice, reversing the impaired bacterial clearance capacity induced by IGFBP6. Figure 7As shown in Figure C, compared with the proteome, CCL2 treatment significantly increased the number of peritoneal macrophages recruited. This example demonstrates that CCL2, by enhancing the body's ability to recruit macrophages, restored the bacterial clearance capacity of septic mice and improved their survival rate. This proves that the damaging effect of IGFBP6 in sepsis is related to the inhibition of CCL2 expression, and that CCL2 administration can target this therapeutic point to address sepsis with high IGFBP6 levels.

[0098] Example 8

[0099] STAT1 agonist 2-NP can reverse the damaging effects of IGFBP6 in sepsis.

[0100] In some embodiments of this invention, it was found that high expression of IGFBP6 inhibited phosphorylation of STAT1 in intestinal and lung epithelial cells, thereby inhibiting CCL2 expression and ultimately impairing macrophage recruitment. Therefore, this invention provides a STAT1 chemokineant, 2-NP (purchased from MCE, catalog number HY-W013523), which can effectively increase the phosphorylation level of STAT1.

[0101] To clarify whether 2-NP in the state of high IGFBP6 expression has a protective effect against sepsis, we randomly divided C57BL / 6 male mice into three groups for sepsis modeling: control group (CLP+PBS+DMSO), protein group (CLP+IGFBP6+DMSO), and 2-NP treatment group (CLP+IGFBP6+2-NP), with 5 mice in each group, and the experiment was repeated three times.

[0102] Figure 8 The graphs shown here are the survival curves, bacterial load statistics, and macrophage ratio statistics for the 2-NP treatment group, protein group, and control group of septic mice in this embodiment.

[0103] Survival curve results as follows Figure 8 As shown in Figure A, 2-NP stimulation significantly restored the survival rate of septic mice, and was superior to that of the control group. Figure 8 Bacterial load assays showed that 2-NP treatment reduced bacterial load levels in the tissues of septic mice, reversing the impaired bacterial clearance capacity induced by IGFBP6. Figure 8 As shown in Figure C, compared with the protein control group, 2-NP treatment significantly increased the number of peritoneal macrophages recruited. This example demonstrates that 2-NP enhances the body's ability to recruit macrophages by activating the STAT1 pathway, restoring the ability of septic mice to clear bacteria and improving survival rate. This proves that the damaging effect of IGFBP6 in sepsis is related to the inhibition of the STAT1 signaling pathway, and that 2-NP activation of the STAT1 pathway can exert a protective effect by targeting this therapeutic point.

[0104] In summary, the inventors first determined that IGFBP6 expression was significantly elevated in sepsis patients, with the degree of elevation correlated with patient severity. Furthermore, the elevation of IGFBP6 in sepsis was significantly higher than in severe pneumonia and other common infections. Secondly, analysis of patient outcomes and survival revealed that IGFBP6 expression was higher in deceased patients than in surviving patients, and this high expression promoted mortality in sepsis patients. Moreover, IGFBP6 showed superior diagnostic efficacy for sepsis-related deaths compared to other indicators. Further in vivo animal experiments showed that IGFBP6 expression levels increased in various tissues after sepsis modeling, and high IGFBP6 expression was positively correlated with disease severity and damage. Modeling with wild-type and IGFBP6-deficient mice revealed enhanced bacterial clearance and significantly improved survival rates after IGFBP6 gene deficiency. On the other hand, the damaging effects of high IGFBP6 expression can be eliminated and reversed by recombinant CCL2 protein and the STAT1 agonist 2-NP. Therefore, this invention provides IGFBP6 as a diagnostic indicator for sepsis infection and mortality, as well as a potential therapeutic target.

[0105] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. The use of STAT1 agonists and / or macrophage chemokines in the preparation of therapeutic and / or preventive drugs for sepsis caused by high expression of insulin-like growth factor binding protein 6, characterized in that: The STAT1 agonist is used to increase the phosphorylation level of STAT1, and the drug restores the macrophage recruitment capacity in vivo that is impaired by elevated insulin-like growth factor binding protein 6 through STAT1 agonists and / or macrophage chemokines. The STAT1 agonist is selected from 2-NP; The macrophage chemokine is selected from at least one of CCL2 and its recombinant protein.

Images