Use of midbrain astrocyte-derived neurotrophic factor as a biomarker and therapeutic target in sepsis

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses a use of mesencephalic astrocyte-derived neurotrophic factor (MANF) as a biomarker and a therapeutic target in sepsis. The application finds that MANF can be used as a new biological marker for predicting sepsis, and the expression level thereof is negatively correlated with the severity of the disease, so it is proposed that the MANF can be used as a diagnostic, prognostic and curative effect evaluation index of sepsis, for the diagnosis of sepsis and the reflection of the severity of the disease. Meanwhile, it is found that the administration of exogenous MANF recombinant protein to a sepsis mouse model can significantly improve the survival rate, so it is proposed that the MANF can be used as a potential therapeutic target for sepsis. The application provides a new technical means for the diagnosis, prognosis, curative effect evaluation and treatment of sepsis and related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of midbrain astrocyte-derived neurotrophic factor as a biomarker and therapeutic target in sepsis. Background Technology

[0002] Sepsis is a complex, heterogeneous, and highly fatal syndrome that is difficult to identify and treat. It is estimated that more than 30 million people are diagnosed with sepsis globally each year, resulting in 5 million deaths. A multicenter epidemiological survey in 2020 showed that the incidence of severe sepsis in ICUs in my country was as high as 20.6%, with a 90-day mortality rate of 35.5%. Currently, sepsis is defined as life-threatening organ dysfunction caused by an abnormal host response to infection, and it is one of the most important and urgent public health challenges worldwide.

[0003] The pathogenesis of sepsis involves a complex systemic inflammatory network, immune dysfunction, coagulation abnormalities, and abnormal host responses to various pathogens and toxins, closely related to pathophysiological changes in multiple systems and organs. The initiation event of sepsis is the recognition of pathogen-associated molecular patterns (PAMPs) or endogenous damage-associated molecular patterns (DAMPs) from microbial sources, mediated by a series of pattern recognition receptors (PRRs) located on the cell membrane or within the cell. This recognition leads to the activation of intracellular signaling pathways. PAMPs and DAMPs include microbial products, host glycoproteins, lipoproteins, and nucleic acids, which can bind to and interact with pattern recognition receptors—Toll-like receptors (TLRs), dectin 1 (a member of the C-type lectin domain family), and dectin 2 (a member of the C-type lectin domain family). This interaction gradually transitions the body from a pro-inflammatory state to an abnormal inflammatory dysregulation characterized by chronic immunosuppression. The release of pro-inflammatory mediators leads to tissue ischemia and abnormal cell apoptosis, which in turn causes diffuse endothelial damage and microvascular thrombosis. Ultimately, this results in organ damage in multiple systems, including the cardiovascular, respiratory, urinary, nervous, hematologic, and hepatic systems, making sepsis a life-threatening heterogeneous syndrome.

[0004] While sepsis research has deepened in recent years, its laboratory biomarkers have not been widely used clinically for disease diagnosis and prognosis. Furthermore, clinical trials of sepsis treatments have failed to yield satisfactory results; current treatment remains largely supportive, including controlling the source of infection, timely antibiotic use, and restorative therapies for organ dysfunction. The resolution of inflammation following severe infection is not merely a passive process of ebb and flow, but involves a series of cellular processes and molecular signaling. Therefore, identifying biomarkers applicable to clinical diagnosis, treatment, and prognosis, and thus controlling the progression of sepsis, has become a critical and urgent problem for the medical community. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide the use of mesencephalic astrocyte-derived neurotrophic factor (MANF) as a biomarker and therapeutic target in sepsis, to provide a new biomarker for the diagnosis, prognosis and efficacy evaluation of sepsis, and to provide a new technical means for the treatment of sepsis.

[0006] To achieve the above and other related objectives, the first aspect of the present invention provides the use of midbrain astrocyte-derived neurotrophic factor as a biomarker in the preparation of diagnostic reagents, prognostic reagents or efficacy assessment reagents for sepsis.

[0007] Furthermore, the reagent is used to determine the expression level of midbrain astrocyte-derived neurotrophic factor in body fluid samples.

[0008] Furthermore, the methods for detecting the expression level of astrocyte-derived neurotrophic factor in the brain using the reagents include, but are not limited to, chemiluminescence, ELISA, immunoturbidimetry, immunofluorescence, and colloidal gold methods.

[0009] Furthermore, the expression level of midbrain astrocyte-derived neurotrophic factor in the body fluid samples was negatively correlated with the severity of sepsis.

[0010] Furthermore, the body fluid sample is selected from at least one of serum, plasma, whole blood, urine, cerebrospinal fluid, pleural effusion, ascites, and synovial fluid.

[0011] Furthermore, the reagent is used to diagnose or monitor the presence and / or process and / or severity and / or prognosis of sepsis.

[0012] A second aspect of the present invention provides the use of midbrain astrocyte-derived neurotrophic factor in the preparation of medicaments for improving and / or treating sepsis.

[0013] Furthermore, the active ingredient of the drug includes at least one of the following components: midbrain astrocyte-derived neurotrophic factor (MDNF), recombinant MDNF protein, a product obtained by chemically modifying MDNF, and a product obtained by chemically modifying recombinant MDNF protein. The chemical modifications alter the protein structure, thereby affecting its stability, biological activity, and function, but do not affect its primary function. Furthermore, the purpose of these chemical modifications is to further effectively control and improve the quality and safety of the drug; for example, oxidative modification, glycosylation, phosphorylation, and acetylation.

[0014] Furthermore, the drug has at least one of the following effects (I) to (III):

[0015] (I) Enhance the body's ability to eliminate bacteria;

[0016] (II) Reduce liver damage;

[0017] (III) Inhibit inflammation and reduce the expression of pro-inflammatory inflammatory factors.

[0018] Furthermore, the pro-inflammatory inflammatory factor is selected from at least one of IL6, CXCL1, and IL1β.

[0019] Furthermore, when the drug is used, the effective dose of the active ingredient is 50-2000 μg / kg body weight, preferably 100-1000 μg / kg body weight, more preferably 100-500 μg / kg body weight, and most preferably 200-300 μg / kg body weight, such as 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, and 300 μg / kg body weight.

[0020] Furthermore, the drug can be a single-component substance or a multi-component substance.

[0021] Furthermore, the drug also includes pharmaceutically acceptable carriers and / or excipients.

[0022] Furthermore, the form of the drug is not particularly limited and can be in various forms such as solid, liquid, gel, semi-liquid, or aerosol.

[0023] Furthermore, the drug is primarily targeted at mammals, preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc., with monkeys, apes, or humans being the preferred primates.

[0024] Furthermore, the drug can be administered in various ways, including via the gastrointestinal tract or non-gastrointestinal route. Generally, known routes of administration for each chemical drug are recommended, such as intravenous injection or intraperitoneal injection.

[0025] As described above, the use of the midbrain astrocytocyte-derived neurotrophic factor of the present invention as a biomarker and therapeutic target in sepsis has the following beneficial effects:

[0026] On the one hand, this invention found that the content of midbrain astrocytogenetic factor in the serum of patients with clinical sepsis was significantly higher than that in healthy individuals, and that the content of midbrain astrocytogenetic factor in the serum of patients with clinical sepsis was negatively correlated with the severity of the disease. Therefore, it is proposed that midbrain astrocytogenetic factor can be used as an indicator for the diagnosis, prognosis and efficacy evaluation of sepsis, and can also predict the severity of sepsis.

[0027] On the other hand, this invention found that administration of exogenous midbrain astrocyte-derived recombinant neurotrophic factor protein to a mouse model of sepsis can significantly improve its survival rate, and therefore proposes that it can be a potential therapeutic target for the clinical treatment of sepsis.

[0028] In summary, this invention provides midbrain astrocyte-derived neurotrophic factor as a novel biomarker for sepsis, used in the diagnosis, prognosis, and efficacy assessment of sepsis, reflecting the severity of the disease. Furthermore, it can serve as a potential therapeutic target for sepsis, providing new technical means for the treatment of sepsis and related diseases. Attached Figure Description

[0029] Figure 1 Figure a shows the results of MANAF content detection in the serum of patients with sepsis and healthy individuals in Example 1 of this invention. Figure 1 b shows the results of the detection of MANF content in the serum of patients who died from sepsis and those who did not die in Example 1 of this invention.

[0030] Figure 2 The figure shown is a correlation analysis result of serum MANAF expression level and SOFA score in patients with sepsis in Example 2 of this invention.

[0031] Figure 3 The diagram shows a comparative analysis of ROC curves in Example 3 of this invention to illustrate the predictive effects of MANF, PCT, and CRP on 28-day mortality in sepsis.

[0032] Figure 4 a shows the survival rate statistics of mice in the MANF protein treatment group and control group in Example 4 of this invention. Figure 4 b shows the survival rate statistics of mice in the MANF antibody treatment group and control group in Example 4 of this invention.

[0033] Figure 5 a shows the bacterial load statistics in the peritoneal lavage fluid of the MANF protein group and the PBS control group in Example 5 of this invention. Figure 5 b shows the bacterial load statistics in cardiac blood of the MANF proteome and the PBS control group in Example 5 of this invention. Figure 5 c shows the bacterial load statistics in the liver of the MANF proteome and the PBS control group in Example 5 of this invention. Figure 5 d shows the bacterial load statistics in the spleen of the MANF proteome and the PBS control group in Example 5 of this invention.

[0034] Figure 6 a shows the statistical graph of alanine transaminase in the serum of the MANF protein group and the PBS control group in Example 6 of this invention. Figure 6 b shows the statistical graph of aspartate transaminase in the serum of the MANF protein group and the PBS control group in Example 6 of this invention.

[0035] Figure 7 a shows the serum IL6 statistics of the MANF protein group and the PBS control group after 6 hours in Example 7 of this invention. Figure 7 b shows the serum IL6 statistics of the MANF protein group and the PBS control group after 24 hours in Example 7 of this invention; Figure 7 c shows the serum CXCL1 levels in the MANF protein group and the PBS control group 6 hours after the event in Example 7 of this invention. Figure 7 d shows the statistical graph of CXCL1 in serum of the MANF protein group and PBS control group after 24 hours in Example 7 of this invention; Figure 7 e shows the statistical graph of serum IL1β levels in the MANF protein group and PBS control group 6 hours after the event in Example 7 of this invention. Figure 7 f shows the statistical graph of IL1β in serum of the MANF protein group and PBS control group 24 h after the experiment in Example 7 of this invention. Detailed Implementation

[0036] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. This application 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 this application.

[0037] This invention focuses on exploring the application value of mesencephalic astrocyte-derived neurotrophic factor (MANF) in sepsis. The MANF gene, encoded on human chromosome 325, is 4.3 kb in length, contains 158 amino acids, and has a molecular weight of 18 kDa. Primarily located in the endoplasmic reticulum lumen, it is an endoplasmic reticulum stress protein that is upregulated and secreted into the bloodstream under endoplasmic reticulum stress induced by ischemia, hypoxia, or trauma. The MANF sequence is highly conserved evolutionarily, with the human MANF amino acid sequence sharing 98% homology with that of mouse MANF. Outside of non-neuronal cells, MANF is highly expressed in adult organs such as the liver, spleen, testes, and salivary glands. Current research shows that MANF can exert protective effects in various in vitro and in vivo inflammation models and is a key regulatory element in metabolic control in the human body. The sap-like domain at the C-terminus of MANF interacts with the DNA-binding domain of the NF-κB p65 subunit, negatively regulating NF-κB signaling transduction and inhibiting the p38 mitogen-activated protein kinase (MAPK) pathway. Furthermore, MANF has been shown to play a crucial role in macrophage differentiation, inhibiting the release of various pro-inflammatory factors and promoting tissue repair. However, research on MANF in sepsis has not been reported. Therefore, this invention, through a series of investigations, reveals that MANF, as an immunomodulatory factor, participates in the diagnosis and prognosis of sepsis, providing a new diagnostic method for clinical practice. Simultaneously, it can also serve as a potential therapeutic target for sepsis, offering new technical means for its treatment.

[0038] The inventors of this application used ELISA to detect serum levels in 50 adult patients with sepsis and 40 healthy individuals. They found that the MANF level in sepsis patients was significantly higher than that in healthy individuals, with a statistically significant difference. Further analysis revealed that the MANF level in deceased patients was significantly lower than that in non-deceased patients, with a statistically significant difference. Simultaneously, correlation analysis showed a negative correlation between MANF level and the Sequential Organ Failure Assessment (SOFA) score. Subsequently, ROC curve analysis was used to analyze the diagnostic value and predictive ability of MANF, PCT, and CRP in sepsis regarding 28-day mortality. The results showed that the area under the curve for MANF was significantly higher than that for CRP and PCT, indicating that MANF can serve as a diagnostic indicator for sepsis and can reflect the severity of the disease, with superior diagnostic efficacy compared to CRP and PCT.

[0039] To further confirm the above results, the inventors of the present application constructed a mouse model of sepsis induced by cecal ligation and puncture (CLP), injected murine MANF recombinant protein into the mice in the protein treatment group, injected exogenous MANF antibody into the mice in the antibody treatment group, and intraperitoneally injected the same volume of PBS / antibody solvent into the mice in the control group. It was found that the survival rate of the mice in the protein treatment group was significantly higher than that of the control group, and the survival rate of the mice in the antibody treatment group was significantly decreased, with statistical differences. In addition, the inventors of the present application also found that the bacterial loads in the peritoneal lavage fluid, cardiac blood, liver, and spleen of the septic mice given exogenous MANF protein were significantly lower than those in the control group, indicating that MANF protein enhanced the bacterial clearance function of the septic mice; at the same time, the liver function of the mice was detected again, and it was found that the liver function of the mice in the MANF protein group was significantly better than that of the control group.

[0040] The implementation content of the present application will be further described in detail through specific examples below.

[0041] Regarding the experiments involved in the following examples, the description is as follows:

[0042] Study population: The blood of sepsis patients diagnosed and treated at the First Affiliated Hospital of Chongqing Medical University from September 2021 to September 2023 was collected as the experimental group, and healthy subjects undergoing physical examinations during the same period were used as the healthy control group. The samples were centrifuged at 4000 rpm at 4 °C for 7 min, and then the serum was frozen at -80 °C. The expression level of MANF protein in the serum was detected by ELISA kit. All patients met the diagnostic criteria for the disease Sepsis 3.0 in the International Sepsis Conference, excluding those with malignant tumors, HIV infections, hemoglobin lower than 7 g / mL, or active bleeding, or those requiring more than two units of red blood cells. All studies on the subjects were approved by the Science and Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.

[0043] Experimental animals: Wild-type C57BL / 6J mice (Wild type, WT), male mice, weighing 18 - 22 g, about 6 - 8 weeks old, were purchased from Vital River Laboratory Animal Technology Co., Ltd. in Beijing and were housed in the SPF (Specific Pathogen Free) - level laboratory of the Experimental Animal Center of Chongqing Medical University. All mice used in this experiment were SPF - level experimental animals. Animal experiments were strictly carried out in accordance with the guidelines for the care and use of laboratory animals and complied with the management regulations of the Experimental Animal Ethics Committee of Chongqing Medical University. Experimental animal license number: SYXK(Yu)2022 - 0016.

[0044] Example 1

[0045] Determination of the expression level of MANF in the serum of sepsis patients

[0046] Blood samples were collected from healthy individuals and patients with sepsis. Statistical analysis was performed on gender, age, and lesion location (results are shown in Table 1). After centrifugation, the expression level of MANAF was measured using an ELISA kit (purchased from Biorbyt, Catalog#orb776948). The procedure was strictly followed according to the kit's instructions. The results were then statistically analyzed using SPSS 20.0 software, and graphs were plotted using GraphPad Prism 8.0.2 software.

[0047] Table 1. Statistical results of sepsis patients and healthy individuals undergoing physical examinations

[0048]

[0049] Figure 1 Figure a shows the serum MANAF expression levels in patients with sepsis and healthy controls. Figure 1 Figure b shows the results of MANF expression levels in the serum of patients who died from sepsis and those who did not.

[0050] The results showed that the expression level of MANF in the serum of patients with sepsis was significantly higher than that in healthy individuals, and the difference was statistically significant (P<0.0001). The expression level of MANF in patients who died from sepsis was significantly lower than that in patients who survived sepsis, and the difference was statistically significant (P<0.001). Therefore, MANF can be used as a diagnostic indicator for clinical sepsis.

[0051] Example 2

[0052] Serum MANAF expression levels in patients with sepsis were negatively correlated with SOFA scores.

[0053] 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.

[0054] Figure 2 The figure shows the results of the correlation analysis between serum MANAF expression and SOFA score in patients with sepsis.

[0055] The results showed that the expression level of MANF in the serum of patients with sepsis was negatively correlated with the SOFA score, therefore MANF indicates the severity of sepsis.

[0056] Example 3

[0057] ROC curve analysis compares the predictive value of MANF, CRP, and PCT for 28-day mortality.

[0058] The laboratory indicators CRP and PCT values ​​of sepsis patients were collected using the laboratory LIS system. The ROC curves were plotted and the statistical differences in the area under the ROC curves were compared using GraphPadPrism 8.0.2 software.

[0059] Figure 3 The graph shows the effect of ROC curve comparison analysis on the prediction of 28-day mortality in sepsis by MANF, CRP, and PCT.

[0060] Table 2. ROC curve analysis comparing the correlation between MANF / CRP / PCT in predicting sepsis-related deaths.

[0061] Correlation coefficient of ROC curve for predicting death of septicpatients

[0062]

[0063] like Figure 3 As shown in Table 2, by calculating the area under the ROC curves for predicting 28-day mortality using MANF, CRP, and PCT, we found that the AUC for MANF on the day of ICU admission was 0.8700 ([95% CI] 0.7578 - 0.9822, p = 0.0003), which was higher than that for PCT (AUC = 0.7250, [95% CI] 0.5645 - 0.8855, p = 0.0290) and CRP (AUC = 0.6475, [95% CI] 0.4190 - 0.8760, p = 0.1524). This indicates that MANF has certain diagnostic value in predicting 28-day mortality in sepsis.

[0064] Example 4

[0065] Mouse survival experiment

[0066] Sepsis mice were constructed using cecal ligation and puncture (CLP). The specific method is as follows: Adult C57 mice were selected and anesthetized by intraperitoneal injection of 100 μL of 1.5% sodium pentobarbital. The mice were fixed to the operating board, and the abdomen was shaved with pet electric clippers. The skin was disinfected, and 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. Nat Protoc. 2009; 4(1): 31-36). This modeling method is a classic modeling method for sepsis animal models, and this model is currently the standard animal model for studying sepsis.

[0067] Sixty male mice weighing 20-21g were selected and randomly divided into three groups: a CLP+protein treatment group, a CLP+PBS control group, and a sham surgery group. The sham surgery group underwent only laparotomy. The other two groups developed a sepsis model using the method described above. The protein treatment group received an intraperitoneal injection of 100ul of 500ng murine MANAF recombinant protein (purchased from Biobyt, Catalog #orb251682); the control group received an equal volume of sterile PBS intraperitoneally. Another 30 male mice weighing 20-21g were also randomly divided into three groups: a CLP+antibody treatment group, a CLP+PBS control group, and a sham surgery group. The sham surgery group underwent only laparotomy. The other two groups established sepsis models using the method described above. The antibody treatment group received an intraperitoneal injection of 100 μL of MANF antibody (purchased from Thermo Fisher Scientific, Catalog #PA5-96542), which was at a dose of 2 μg. The control group received an intraperitoneal injection of an equal volume of sterile antibody solvent. The survival of the mice was observed and recorded twice daily for two weeks until the mice stopped dying. Each experiment was repeated at least twice.

[0068] Figure 4 Figure a shows the survival rate statistics of mice in the MANF protein treatment group and mice in the PBS control group. Figure 4 b shows the survival rate statistics of mice in the MANF antibody treatment group and the control group.

[0069] The results show:

[0070] After wild-type mice were supplemented with exogenous recombinant mouse-derived MANF, their survival rate was 80% after 14 days of CLP modeling, while the survival rate of the PBS control group after CLP modeling was only 40%, suggesting that exogenous MANF can play a protective role in septicemia mice.

[0071] In another model, wild-type mice were supplemented with MANDF antibodies. After CLP modeling, the mice were observed for 14 days. It was found that compared with mice that were injected with antibody solvents intraperitoneally, the survival rate of mice injected with MANDF antibodies intraperitoneally was significantly reduced to 10%, while the survival rate of the antibody solvent group was 60%. This reversely verified the protective effect of MANDF and the potential of exogenous MANDF protein as a therapeutic target for this disease.

[0072] Example 5

[0073] Bacterial load experiment

[0074] Ten adult male C57 rats weighing 21-22g were selected and modeled according to the CLP method in Example 4. They were randomly divided into a protein treatment group (n=5) and a control group (n=5). The control group received injections of mouse-derived MANF recombinant protein (purchased from Biorbyt, Catalog #orb251682) and sterile PBS, respectively, as described in Example 4. Forty-eight hours later, peritoneal lavage fluid, cardiac blood, liver, and spleen were collected. The peritoneal lavage fluid was diluted 10... 3 -10 5 The heart blood was diluted 10-100 times, and the liver and spleen were homogenized with 1 ml of sterile PBS and then diluted 100-1000 times. 15 μL of each was then plated and incubated in an incubator for 12 hours before counting the number of colonies on the blood agar plates.

[0075] Figure 5 Figure a shows the bacterial load statistics in peritoneal lavage fluid of the MANF proteomic group and the PBS control group. Figure 5 b shows the bacterial load statistics in cardiac blood of the MANF protein group and the PBS control group. Figure 5 c shows the bacterial load statistics in the liver of the MANF protein group and the PBS control group. Figure 5 d shows the bacterial load statistics in the spleen of the MANF protease group and the PBS control group.

[0076] The results showed that, compared with the control group, the bacterial load in peritoneal lavage fluid, cardiac blood, liver and spleen was significantly reduced after administration of exogenous recombinant MANF, and the difference was statistically significant, indicating that MANF enhanced the body's ability to clear bacteria.

[0077] Example 6

[0078] Mouse liver function index detection

[0079] To further understand organ damage in mice after modeling, this example selected 10 adult male C57 mice weighing 21-22g and established a sepsis model according to the CLP method in Example 4. The mice were randomly divided into a control group (n=5) and a protein treatment group (n=5). The control group received an intraperitoneal injection of 100μL sterile PBS buffer after modeling, while each mouse in the protein treatment group received an intraperitoneal injection of 500ng / 100μL of mouse-derived MANF recombinant protein (purchased from Biobyt, Catalog #orb251682). Forty-eight hours later, the mice were anesthetized, and cardiac blood was collected. After centrifugation, the supernatant was collected, and liver function indicators, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were measured.

[0080] Figure 6 a shows the statistical graph of alanine transaminase levels in the serum of the MANF protein group and the PBS control group. Figure 6 b shows the statistical graph of aspartate aminotransferase in the serum of the MANF protein group and the PBS control group.

[0081] The results showed that the liver function damage in mice injected with recombinant MANDF protein was significantly reduced compared with that in the control group, suggesting that injection of MANDF enhanced liver function in mice.

[0082] Example 7

[0083] Mouse inflammatory factor expression detection

[0084] Fifteen adult male C57 mice weighing 21-22g were selected and modeled according to the CLP method in Example 4. They were evenly divided into protein treatment group 1 (n=5 mice sampled at 6h), protein treatment group 2 (n=5 mice sampled at 24h), and control group (n=5 mice). The mice were injected with mouse-derived recombinant MANF protein (purchased from Biorbyt, Catalog #orb251682) and sterile PBS, respectively, according to the method in Example 4. After 6h, the mice were anesthetized, and blood was collected from the heart. After centrifugation, the supernatant was collected, and the expression of IL6, CXCL1, and IL1β was detected.

[0085] Figure 7 Figure a shows the serum IL6 levels in the MANF protein group and the PBS control group after 6 hours. Figure 7 b shows the serum IL6 statistics of the MANF protein group and the PBS control group after 24 hours; Figure 7 c shows the serum CXCL1 statistics of the MANF protein group and the PBS control group after 6 hours. Figure 7 d shows the serum CXCL1 statistics of the MANF protein group and the PBS control group 24 h later; Figure 7 e shows the statistical graph of serum IL1β levels in the MANF protein group and the PBS control group 6 hours later. Figure 7f shows the statistical graph of serum IL1β levels in the MANF protein group and the PBS control group 24 hours later.

[0086] The results showed that the expression of inflammatory factors in mice injected with recombinant MANDF protein was significantly lower than that in the control group, suggesting the anti-inflammatory effect of MANDF injection.

[0087] In summary, this invention found that serum MANF levels in patients with sepsis were significantly higher than in healthy individuals, and were negatively correlated with SOFA scores. ROC curve analysis revealed that MANF could serve as a novel biomarker for the diagnosis of sepsis. Further animal experiments showed that supplementation with exogenous recombinant MANF protein significantly increased the survival rate of sepsis-affected mice, while MANF antibodies significantly decreased their survival rate, suggesting that MANF could be a potential therapeutic target for sepsis in clinical practice. Further animal experiments also showed that MANF protein enhanced bacterial clearance in mice, reduced liver damage, and decreased the expression of pro-inflammatory cytokines. Therefore, this invention provides MANF as an indicator for the diagnosis, prognosis, and severity assessment of sepsis, and also offers a potential therapeutic target for clinical use.

[0088] 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. Application of midbrain astrocyte-derived neurotrophic factor in the preparation of drugs for the treatment of sepsis.

2. The application according to claim 1, characterized in that: The active ingredient of the drug includes at least one of the following components: midbrain astrocyte-derived neurotrophic factor and recombinant midbrain astrocyte-derived neurotrophic factor protein.

3. The application according to claim 1, characterized in that: The drug has at least one of the following effects (I) to (III): (I) Enhance the body's ability to eliminate bacteria; (II) Reduce liver damage; (III) Inhibit inflammation and reduce the expression of pro-inflammatory inflammatory factors.

4. The application according to claim 2, characterized in that: When the drug is used, the effective dose of the active ingredient is 50-2000 μg / kg body weight.

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