Application of CALCOCO1 as biomarker and therapeutic target in detection and treatment of sepsis

Abstract

The invention provides application of CALCOCO1 as a biomarker and a therapeutic target in sepsis detection and treatment, and belongs to the technical field of sepsis detection and treatment. The invention discloses application of CALCOCO1 as a biomarker in preparation of a reagent or a kit for early prediction of sepsis. The invention relates to an application of CALCOCO1 as a therapeutic target in preparation of drugs for treating sepsis. According to the invention, the autophagy receptor protein CALCOCO1 is taken as a potential candidate target for sepsis detection and treatment, and the expression of the autophagy receptor protein CALCOCO1 is regulated to promote the autophagy of the Golgi apparatus and relieve the stress of the Golgi apparatus; the generation of excessive inflammatory factors is inhibited; death of dendritic cells and organ injury are reduced; therefore, the clinical prognosis of sepsis patients is improved, and the case fatality rate is remarkably reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of sepsis detection and treatment, and in particular to the application of CALCOCO1 as a biomarker and therapeutic target in sepsis detection and treatment. Background Art

[0002] Sepsis is a global public health problem in the field of critical illness, and has attracted much attention due to its high morbidity and mortality. Currently, sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection, replacing the earlier definition of "systemic inflammatory response syndrome." This indicates that people's understanding of the pathophysiological mechanisms of sepsis is constantly improving. Its clinical characteristics are manifested as a systemic inflammatory response, but there are very complex pathophysiological changes in its occurrence and development. Among them, immune dysfunction is an important factor leading to the poor prognosis of sepsis, such as secondary infection and increased mortality. The immune dysfunction caused by sepsis is mainly manifested in the imbalance of secretion of pro-inflammatory cytokines and anti-inflammatory cytokines, abnormal death of immune effector cells, and excessive proliferation of immunosuppressive cells.

[0003] Dendritic cells (DCs) are a type of innate immune cell with a high degree of immune surveillance. They are also the most powerful antigen-presenting cells in the body's immune system, acting as a bridge between the innate and acquired immune responses. After mature DCs are activated, the expression of MHC-II molecules on their surface is upregulated, and they efficiently and transiently present antigens to CD4+ and CD8+ T cells, thereby initiating acquired immune responses. In addition, mature and activated DCs can promote the maturation, proliferation, and differentiation of naive T cells by secreting cytokines. In sepsis, DCs often show a significant decrease in number and impaired function, which is closely related to the severity of the disease, poor prognosis, and increased mortality in patients with sepsis. Studies have shown that the reduction in DCs caused by sepsis is mainly due to the occurrence of cell death, including multiple types of programmed cell death, such as apoptosis, pyroptosis, necroptosis, and ferroptosis. Pyroptosis is a novel cell death mechanism centered on the activation of the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome. The activated inflammasome binds to apoptosis-associated speck-like protein containing a CARD (ASC), forming an active complex that recruits and activates the core executioner protein, caspase-1. Activated caspase-1 cleaves pro-interleukin (IL)-1β and IL-18 into their active forms and cleaves gas-induced protein D (GSDMD). The released N-terminal domain of GSDMD spontaneously aggregates and inserts into the plasma membrane, forming pores. This pathway promotes the extracellular release of IL-1β and IL-18, mediating a proinflammatory response and leading to cell swelling and eventual cell rupture and death. It can be seen that DCs pyroptosis is an important factor leading to immune dysfunction and stimulating excessive inflammatory response in sepsis, but its exact regulatory mechanism has not yet been elucidated.

[0004] The Golgi apparatus (GA) is a crucial intracellular membrane-bound organelle responsible for the processing, sorting, and transport of proteins and lipids within eukaryotic cells. The Golgi apparatus is a stacked or lamellar structure, typically located in close proximity to membrane-bound organelles such as the endoplasmic reticulum, mitochondria, endosomes, and lysosomes. As a highly dynamic organelle, the Golgi apparatus responds to various cellular needs by regulating the turnover of its own vesicles, providing a structural foundation for coordinating intracellular signal transduction. In terms of antigen presentation, the Golgi apparatus regulates the trafficking and surface expression of MHC I and MHC II molecules, promoting the efficient presentation of antigenic peptides and enhancing T cell recognition. Furthermore, the Golgi apparatus plays a central role in cytokine secretion. By regulating the secretory pathway and vesicle trafficking, it efficiently releases inflammatory mediators such as IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ into the extracellular environment, modulating the intensity and duration of immune responses. The latest research has found that the dynamic changes in the structure and function of the Golgi apparatus are closely related to the activation state of immune cells. Abnormal function of the Golgi apparatus may lead to dysregulation of the immune response and promote the development of chronic inflammatory diseases.

[0005] Under stressful conditions, such as hyperthermia, oxidative stress, and malnutrition, cells synthesize excessive proteins, leading to Golgi overload and dysfunction, thus inducing Golgi apparatus stress (GAS). Furthermore, some viruses, after infecting cells, alter the Golgi apparatus to promote their own replication, and these structural changes can also lead to Golgi stress. The mechanisms underlying Golgi stress response are currently understudied. While pathways such as TFE3, CREB3-ARF4, PG, and HSP47 have been proposed, the specific receptors, transduction pathways, and transcription factors involved in each pathway remain unclear. Sustained or severe Golgi stress can lead to impaired protein transport or fragmentation of the Golgi apparatus, triggering programmed cell death (PCD), which plays a significant role in the development and progression of numerous human diseases, including neurodegenerative diseases, infectious diseases, and cancer. Recent studies have reported that after influenza A virus infects cells, Golgi stress induces Golgi fragmentation, generating a large number of dispersed vesicle-like trans-Golgi networks (dTGNs). The dTGNs are loaded with a large negatively charged phosphatidylinositol-4-phosphate (PI4P), and binding to PI4P is essential for NLRP3 activation. This suggests that Golgi stress is involved in inducing pyroptosis.

[0006] During the adaptive process of cells responding to Golgi stress, Golgi-phagy helps remove structurally and functionally damaged Golgi structures and maintain Golgi homeostasis. CALCOCO1 is a newly discovered soluble selective autophagy receptor that can mediate the occurrence of Golgi-phagy under conditions of cellular starvation. Studies have found that CALCOCO1 binds to the Golgi membrane through its zDABM domain and interacts with ATG8 family proteins through its LIR domain to initiate Golgi autophagy. Thus, CALCOCO1 plays a key role in maintaining Golgi and cellular homeostasis. However, the role of CALCOCO1 in DCs in sepsis has not been reported.

[0007] Current clinical treatments for sepsis primarily rely on broad-spectrum antibiotics, fluid resuscitation, and supportive therapy, but lack specific molecular targeted approaches for regulating Golgi-phagy, leading to: 1. Uncontrolled systemic inflammatory response - excessive Golgi stress exacerbates the release of inflammatory mediators; 2. Pyroptosis and organ dysfunction: Golgi stress-induced pyroptosis accelerates multiple organ failure; 3. There is a lack of innovative targets for effectively preventing or reversing sepsis-related multi-organ damage.

[0008] Current treatment limitations include: 1. Lack of specific treatment: Existing treatment options are mainly supportive and cannot accurately block the pathological mechanisms of sepsis (such as the vicious cycle of inflammation-coagulation-immune imbalance).

[0009] 2. Drug resistance problem: The abuse of broad-spectrum antibiotics has led to an increase in multidrug-resistant bacteria (such as MRSA and CRE).

[0010] 3. Large individual differences: Genetic polymorphism affects drug response (such as differences in endotoxin clearance ability among individuals with TLR4 gene mutations).

[0011] Therefore, it is necessary to find more efficient and accurate targets to overcome the shortcomings of existing technologies. Summary of the Invention

[0012] The purpose of the present invention is to propose the application of CALCOCO1 as a biomarker and therapeutic target in the detection and treatment of sepsis, improve the clinical prognosis of sepsis patients, and significantly reduce the mortality rate.

[0013] The technical solution of the present invention is achieved as follows: The present invention provides a use of CALCOCO1 as a biomarker in preparing a reagent or a kit for early prediction of sepsis.

[0014] As a further improvement of the present invention, the CALCOCO1 is used as a biomarker to detect the protein expression level of CALCOCO1 in body fluids, or to detect the transcription level of CALCOCO1 in organ tissues.

[0015] As a further improvement of the present invention, the body fluid is at least one of serum, plasma, whole blood, urine, cerebrospinal fluid, pleural effusion, ascites, and joint fluid.

[0016] As a further improvement of the present invention, the organ tissue is liver, lung, kidney or spleen tissue.

[0017] The present invention further protects the use of CALCOCO1 in preparing a reagent or kit for early prediction of sepsis.

[0018] The present invention further protects the use of CALCOCO1 as a therapeutic target in the preparation of drugs for treating sepsis.

[0019] As a further improvement of the present invention, the active ingredient of the drug includes at least one of the following components: CALCOCO1, CALCOCO1 recombinant protein, a product obtained by subjecting CALCOCO1 to certain chemical modifications, and a product obtained by subjecting CALCOCO1 recombinant protein to certain chemical modifications.

[0020] As a further improvement of the present invention, the drug has at least one of the following effects (I) to (II): (Ⅰ) Inhibit the production of excessive inflammatory factors; (II) Reduce dendritic cell death and organ damage.

[0021] The present invention has the following beneficial effects: The present invention proposes to use the autophagy receptor protein CALCOCO1 as a potential candidate target for sepsis detection and treatment, and by regulating its expression, it can: promote Golgi autophagy and alleviate Golgi stress; inhibit the production of excessive inflammatory factors; reduce dendritic cell death and organ damage; thereby improving the clinical prognosis of sepsis patients and significantly reducing the mortality rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1The expression level of CALCOCO1 in the spleen of mice after CLP surgery; WT mice were randomly divided into SHAM group and CLP (24H, 72H) group. The spleens of mice were obtained 24 h and 72 h after CLP surgery, fixed and sliced, and then immunohistochemical staining of CALCOCO1 was performed. The expression level and localization of CALCOCO1 in spleen tissue were examined under an optical microscope.

[0024] Figure 2 Expression levels of Golgi autophagy-related proteins in DCs after CLP in mice. Splenic DCs were isolated from mice in the SHAM and CLP groups. CALCOCO1 and LC3B expression levels were assessed by Western blot. The grayscale values of each band were statistically analyzed. *: P < 0.05; **: P < 0.01.

[0025] Figure 3 The colocalization of CALCOCO1 and Golgi apparatus in LPS-stimulated DC2.4 cells was observed. DC2.4 cells were stimulated with 1 μg / mL LPS for 24 h, fixed, and immunofluorescence staining was performed for TGN46, GM130, and CALCOCO1. The expression levels and colocalization of TGN46, GM130, and CALCOCO1 were observed using LSCM.

[0026] Figure 4 The colocalization of Golgi apparatus and autophagy protein LC3B in DC2.4 cells after LPS stimulation; DC2.4 cells were stimulated with 1 μg / mL LPS for 24 h, fixed, and immunofluorescence staining of TGN46, GM130, and LC3B was performed. The expression levels and colocalization of TGN46, GM130, and LC3B were observed using LSCM.

[0027] Figure 5 The binding phenomenon of CALCOCO1 and Golgi-related proteins in LPS-stimulated DC2.4 cells was observed. DC2.4 cells were stimulated with LPS 1 μg / mL for 24 h, and then the cells were collected. The interaction of CALCOCO1 with GM130, MAN2A1, TGN46, and LC3B was detected by immunoprecipitation.

[0028] Figure 6 This figure shows the expression levels of autophagy-related proteins on the Golgi apparatus of DC2.4 cells after LPS stimulation. DC2.4 cells were stimulated with 1 μg / mL LPS for 24 hours, then harvested and isolated using a Golgi isolation kit. Western blot analysis was performed to determine the expression levels of GM130, TGN46, and CALCOCO1 in Golgi components in different groups. The grayscale values of each band were used for statistical analysis. **: P < 0.01; ***: P < 0.005.

[0029] Figure 7 Transmission electron microscopy was used to observe Golgi-related autophagy structures. Splenic DCs from WT male C57BL / 6J mice were cultured in vitro and stimulated with 1 μg / mL LPS for 24 hours. TEM analysis was used to analyze the number of autophagosomes produced by DCs and the changes in the number of autophagosomes containing Golgi structures. Arrows mark 1: normal Golgi apparatus, 2: autophagosome, and 3: Golgi components within autophagosomes.

[0030] Figure 8 To investigate the effects of CALCOCO1 expression intervention, lentiviral transfection was used to construct CALCOCO1-overexpressing (OE-CALCOCO1) and CALCOCO1-silencing (siRNA-CALCOCO1) DC2.4 cell lines. Western blot analysis was performed to determine CALCOCO1 expression, and the grayscale values of each band were statistically analyzed. *: P < 0.05.

[0031] Figure 9 To intervene in Golgi protein expression in DC2.4 cells after CALCOCO1 expression, CALCOCO1-overexpressing (OE-CALCOCO1) and silencing (siRNA-CALCOCO1) DC2.4 cell lines were randomly divided into a control group (CON) and a sepsis group (LPS). After 24 hours of LPS stimulation, CALCOCO1 expression was detected by Western blot, and the grayscale values of each band were statistically analyzed. *: P < 0.05.

[0032] Figure 10 This figure shows the expression levels of Golgi-phagy-related proteins in DCs during sepsis in CALCOCO1-deficient mice. WT male C57BL / 6J mice and CALCOCO1 knockout (CAL-KO) male C57BL / 6J mice were randomly divided into a SHAM group and a CLP group. 24 hours after CLP, splenic DCs were isolated from each group. Western blot analysis was performed to determine the expression levels of Golgi-phagy-related proteins GM130, TGN46, and LC3B. Statistical analysis was performed using the grayscale values of each band. *: P < 0.05; **: P < 0.01.

[0033] Figure 11To investigate the effects of BFA and MON on the expression of Golgi autophagy-related proteins in DCs under sepsis. DC2.4 cells were prestimulated with BFA (0.1 μg / mL) or MON (1 μg / mL) for 30 minutes and then incubated with LPS (1 μg / mL) for 24 hours. Western blot analysis was used to determine the expression levels of Golgi autophagy-related proteins CALCOCO1, GM130, TGN46, and LC3B in DCs in each group. The grayscale values of each band were statistically analyzed. *: P < 0.05; **: P < 0.01; ***: P < 0.005.

[0034] Figure 12 Effects of BFA and MON stimulation on the expression of Golgi autophagy-related proteins in DC2.4 cells. DC2.4 cells were stimulated with 0.1 μg / mL BFA or 1 μg / mL MON for 24 h, and the expression levels of Golgi autophagy-related proteins CALCOCO1, GM130, TGN46, and LC3B were assessed by Western blot. The grayscale values of each band were statistically analyzed. **: P < 0.01; ***: P < 0.005; ****: P < 0.001.

[0035] Figure 13 The Golgi-phagy phenomenon mediated by CALCOCO1 in different Golgi stress responses was investigated; the interaction between CALCOCO1 and GM130, TGN46 and LC3B in the above cells was detected by co-immunoprecipitation.

[0036] Figure 14 Western blot analysis of Golgi autophagy-related protein expression in ARF4-transfected DCs. ARF4 expression was inhibited in DC2.4 cells using lentiviral transfection to construct an ARF4-overexpressing cell line (OE-ARF4) and an ARF4-silencing cell line (siRNA-ARF4). These cells were randomly divided into a control group (CON) and a sepsis group (LPS). After 24 hours of LPS stimulation at 1 μg / mL, Western blot analysis of Golgi autophagy-related proteins CALCOCO1 and LC3B was performed. Statistical analysis was performed using the grayscale values of each band. *: P < 0.05; **: P < 0.01.

[0037] Figure 15Effects of CALCOCO1 expression intervention on the expression of proteins involved in the pyroptosis pathway in DC2.4 cells. CALCOCO1 expression was manipulated by lentiviral transfection to construct a CALCOCO1-overexpressing cell line (OE-CAL) and a CALCOCO1-silencing cell line (siRNA-CAL). The cells were randomly divided into a control group (CON) and a sepsis group (LPS). After 24 hours of LPS stimulation at 1 μg / mL, Western blot analysis was performed to examine the expression of pyroptosis-related proteins NLRP3, ASC, GSDMD, Caspase-1, IL-1, and IL-18. Statistical analysis was performed using the grayscale values of each band. *: P < 0.05; **: P < 0.01; P < 0.005.

[0038] Figure 16 To investigate the changes in LPS-induced pyroptosis in DC2.4 cells after intervening with CALCOCO1 expression, flow cytometry was used to measure the pyroptosis rates in each group. The results were statistically analyzed. ****: P < 0.001.

[0039] Figure 17 Figure 2: Expression of pyroptosis-related proteins in DCs during sepsis in CALCOCO1-deficient mice. WT male C57BL / 6J mice and CALCOCO1 knockout (CAL-KO) male C57BL / 6J mice were randomly divided into the SHAM group and the CLP group. 24 hours after CLP, splenic DCs were isolated from each group. Western blot analysis was used to examine changes in the expression levels of pyroptosis-related proteins NLRP3, ASC, GSDMD, and CASPASE-1. The grayscale values of each band were statistically analyzed. *: P < 0.05; **: P < 0.01; ***: P < 0.005; ****: P < 0.001.

[0040] Figure 18 The pyroptosis rate of DCs in sepsis-induced CALCOCO1 gene-deficient mice was observed by flow cytometry. The results were statistically analyzed as follows: ***: P < 0.005; ****: P < 0.001.

[0041] Figure 19The results represent the activation levels of DC surface molecules in CALCOCO1-deficient mice during sepsis. Sepsis models were established in WT and CAL-KO mice using CLP surgery. Splenic DCs were isolated 24 hours after surgery using the miniMACS CD11c+ magnetic bead method. Flow cytometry was used to measure the expression of functional molecules CD80, CD86, and MHC-II on the DC surface in each group. The results were statistically analyzed. *: P < 0.05; **: P < 0.01.

[0042] Figure 20 To evaluate the ability of DCs from CALCOCO1-deficient mice to induce CD4+ T cell proliferation after CLP surgery; DCs isolated from each group were co-cultured with CD4+ T cells from untreated WT mice at a cell ratio of 1:20. Flow cytometry was used to evaluate the ability of DCs to induce T cell proliferation and differentiation. The results were statistically analyzed: ****: P <0.001. DETAILED DESCRIPTION

[0043] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention. Example

[0044] 1. Mouse sepsis model SPF-free male C57BL / 6J mice, 6-8 weeks old and weighing approximately 20-25 g, were selected and acclimated in the animal laboratory for 2-3 days. Before the experiment, mice were randomly divided into a sham injury group (SHAM group) and a surgical group (CLP 24 h and CLP 72 h groups) and fasted for 12 h without water deprivation. On the day of the experiment, mice in each group received an intraperitoneal injection of sodium pentobarbital (50 mg / kg). After thorough anesthesia, the mice were secured in the supine position on an operating board. The surgical area of the abdomen was disinfected with iodine. A 1 cm longitudinal incision was made along the midline of the abdomen using ophthalmic scissors to fully expose the abdominal cavity, taking care to maintain a moderate depth to avoid damaging internal organs. The cecum was located and exposed, and ligated with a 3-0 suture 1.0-1.2 cm from the distal end of the cecum. A 5 mL syringe needle was then used to puncture the ligature and squeeze both ends to allow colonic contents to enter the peritoneal cavity. The cecum was then returned to its original position, and the abdominal incision was sutured and disinfected with iodine. Finally, mice were injected subcutaneously with 1 mL of 0.9% saline behind the neck and kept warm postoperatively. Mice in the sham-injury group were anesthetized with the same dosage, and the laparotomy and cecum were exposed at the same location. After the cecum was located and isolated, it was immediately returned to its original position without ligation or perforation. The incision was sutured and disinfected.

[0045] 2. Extraction of DCs and CD4+ T cells from mouse spleen Isolation of mouse spleen DCs cells by miniMACS CD11c+ magnetic beads method: Mice were sacrificed by cervical dislocation and placed in right lateral recumbency on an operating table. The left side of the abdomen was disinfected and the spleen was dissected. The spleen was rinsed with pre-chilled PBS to remove blood. After removing the fascia in a clean hood, the spleen was torn into 2-3 sections and transferred to a disposable 100 μm cell strainer for thorough grinding. The strainer was rinsed with PBS (no more than 10 mL) to collect the cell suspension. After centrifugation (1500 rpm, 5 min), the supernatant was discarded and the cell suspension was resuspended in PBS to 4 mL. In a 15 mL centrifuge tube, 4 mL of mouse organ lymphocyte separation buffer was added to the lower layer. 4 mL of cell suspension was gently added to the upper layer, ensuring complete separation. After centrifugation (3000 rpm, 15 min), the middle cloud layer cell suspension was aspirated with a Pasteur pipette into a new tube and diluted to 10 mL with PBS. After another centrifugation (1500 rpm, 5 min), the supernatant was discarded and the cell suspension was resuspended in 5 mL of PBS for counting. Subsequently, the cell suspension was centrifuged (1500 rpm, 5 min) and the cell suspension was counted at 1×10 7 Resuspend the cells in 40 μL PBS buffer to obtain mononuclear cell suspension.

[0046] According to the CD11c MicroBeads reagent instructions, each 1×10 7 10 μL of magnetic beads were added to the cells and incubated at 4°C in the dark for 15 min. The cells were washed with 5 mL of PBS and centrifuged (1500 rpm, 5 min). The supernatant was discarded and the cells were diluted to 1×10 7 Resuspend the cells in 50 μL of PBS. Load the MS / MACS column into a miniMACS Separator and pre-wet the column with MACS buffer. Gradually add the cell suspension to the MACS column. When the cell suspension is completely removed, wash the column 3-4 times with MACS buffer (500 μL each time). Remove the column and place it in a centrifuge tube. Rinse with MACS buffer and push with a plunger to collect bound CD11c⁺ cells. Count the cells by centrifugation and use for subsequent experiments.

[0047] Isolation of mouse spleen CD4+ T cells by positive selection using the miniMACS system: After obtaining mononuclear cell suspension using the same method, the cell concentration was adjusted to 1×10 7For each mL of 1 mL of CD4 MicroBeads, add 10 μL of CD4 MicroBeads, mix thoroughly, and incubate on ice for 15 min in the dark. Load the LS column onto a miniMACS Separator. Pre-wet the column with 1 mL of MACS buffer. Slowly add the labeled cell suspension to the column, ensuring that the liquid passes through the column dropwise. Finally, wash the column three times with 1 mL of MACS buffer. Remove the column and place it in a clean 15 mL centrifuge tube. Gently push with the plunger to collect bound CD4+ T cells. Count them by centrifugation and use them for subsequent experiments.

[0048] 3. DC2.4 grouping, culture and transfection Culture DC2.4 cells in a T25 flask with 4-6 mL of RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Incubate at 37°C, 5% CO2, and maintain humidity above 95%. Change the medium every 2-3 days and observe cell morphology regularly. The cells were divided into blank control group (CON), LPS (1 μg / mL) stimulation group (LPS 24 H, LPS 72 H), BFA (0.1 μg / mL) stimulation group (BFA 24 H), MON (1 μg / mL) stimulation group (MON 24 H), BFA (0.1 μg / mL) pretreatment LPS (1 μg / mL) stimulation group (BFA+LPS 24 H), MON (1 μg / mL) pretreatment LPS (1 μg / mL) stimulation group (MON+LPS 24 H), OE-ARF4-CON group, siRNA-ARF4-CON group, OE-ARF4-LPS 24 H group, siRNA-ARF4-LPS 24 H group, OE-CALCOCO1-CON group, siRNA-CALCOCO1-CON group, OE-CALCOCO1-LPS 24 H group, and siRNA-CALCOCO1-LPS 24H group.

[0049] DC2.4 cells were plated at 5×10 4Cells were seeded at a density of 100 μg / mL in 6-well plates and infected according to the instructions for use of the recombinant lentiviral vector. After 24 hours of infection at an MOI of 50, the culture medium was replaced with RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin and cultured until the desired number of cells was reached. Following lentiviral-mediated gene transduction, the cell lines were transfected with ARF4 expression and divided into an overexpression virus group (OE-ARF4-CON), a silencing virus group (siRNA-ARF4-CON), an overexpression virus group (OE-ARF4-LPS 24 hours), and a silencing virus group (siRNA-ARF4-LPS 24 hours).

[0050] 5. Total cell protein extraction Cool the reagents required for the experiment in advance and perform the entire operation in a low temperature environment (such as on ice). Collect cells from each group and use 50-200 μL lysis buffer / 1×10 6 Prepare protein lysate at a ratio of 1:50 and 1:100 for cells. Add protease and phosphatase inhibitors at ratios of 1:50 and 1:100, respectively, and mix thoroughly. Lyse the sample on ice for 30 minutes, gently shaking every 5 minutes, and then freeze-thaw three times in liquid nitrogen. After lysis, centrifuge at 12,000 rpm for 30 minutes at 4°C, and collect the supernatant (protein extract) while avoiding disturbing the precipitate. Prepare a standard curve using the BCA assay, measure absorbance, and calculate protein concentration. Add 5× protein loading buffer, mix thoroughly, and heat in a metal water bath at 95°C for 5 minutes. Cool rapidly and store frozen at -80°C to avoid repeated freeze-thaw cycles.

[0051] 6. Western blotting (WB) to detect protein expression Assemble the electrophoresis apparatus and perform a leak test. Prepare the required buffers in order: protein electrophoresis gel (8 mL each of Resolving Gel A and Resolving Gel B, 3 mL each of Stacking Gel A and Stacking Gel B), running buffer (50 mL of 10× running buffer, 450 mL of distilled water), and electrotransfer buffer (50 mL of 10× electrotransfer buffer, 100 mL of anhydrous methanol, 350 mL of distilled water). Let the gel stand for 25 minutes until it is completely solidified. Then add running buffer and load the sample. Run electrophoresis at a constant voltage of 170 mV for 40-50 minutes. Carefully remove the excess gel. Activate the PVDF membrane with anhydrous methanol for 10 minutes. Assemble the electrotransfer apparatus and electrotransfer at a constant current of 200 mA for 1 hour and 50 minutes. Block with 10% skim milk for 2 hours and then wash three times with TBST (5 minutes each). Add primary antibody at a dilution of 1:500-1:1000 and incubate overnight at 4°C on a shaker. Remove the membrane the next day and wash three times with TBST (5 minutes each). Depending on the primary antibody species, goat anti-rabbit or goat anti-mouse secondary antibodies were used at a 1:5000 dilution. Incubate at room temperature for 1 hour and wash three times with TBST (5 minutes each). A 1:1 mixture of hypersensitive chemiluminescent solutions A and B was used for development and visualization using a gel imaging system. The grayscale values of each histone band were analyzed using ImageJ software.

[0052] 7. Laser scanning confocal microscopy (LSCM) for immunofluorescence observation Place the 12-well slide into a 12-well plate and seed an appropriate amount of cells (1×10 4 -1×10 5 After adding culture medium, incubate in a 37°C, 5% CO2 incubator for 24 hours to allow cells to attach. Stimulate the cells as desired, aspirate the culture medium, rinse twice with ice-cold PBS, and fix with 4% paraformaldehyde for 10-15 minutes. After fixation, rinse three times with PBS for 5 minutes each, permeabilize with 0.3% Triton X-100 for 10 minutes, and rinse three times with PBS. Add 5% BSA in PBS blocking buffer and incubate at room temperature for 30 minutes to prevent nonspecific binding. Incubate overnight with LC3B Mouse mAb (Alexa Fluor® 647 Conjugate) at a concentration of 1:300. The following day, rinse three times with PBS, then add a 1:500 dilution of the corresponding species-specific fluorescent secondary antibody and incubate at room temperature for 1 hour. Rinse three times with PBS to complete staining. Stained slides were mounted with anti-fluorescence quenching mounting medium, covered with a coverslip, and gently squeezed to remove air bubbles before microscopic observation.

[0053] 8. Separation and extraction of cytoplasmic and nuclear proteins Cells from different groups were scraped off with a cell scraper, transferred to centrifuge tubes and centrifuged (1500 rpm, 5 min). 6 Add lysis buffer A to the remaining pellet and mix thoroughly on ice for 10 minutes, gently shaking or pipetting to promote lysis. Centrifuge the sample at 12,000 g for 5 minutes at 4°C, and collect the supernatant for cytoplasmic proteins. Add 100 μL of lysis buffer B to the remaining pellet and mix thoroughly on ice for 10 minutes, gently shaking or pipetting to lyse the nuclear membrane. Centrifuge at 12,000 g for 5 minutes at 4°C, and collect the supernatant for nuclear proteins. Determine protein concentration using the BCA assay, and calculate and record the cytoplasmic and nuclear protein content. Add 5× loading buffer, mix thoroughly, and heat at 95°C for 5 minutes to denature the pellet for subsequent Western blot analysis.

[0054] 9. Transmission electron microscopy (TEM) observation of subcellular organelle structure DCs were isolated and extracted, and stimulated according to experimental requirements. At least 2×107 cells were collected from each group. After collection, the cells were placed in a 2-mL EP tube, washed once with 1 mL of pre-cooled PBS, and centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. 1.8 mL of electron microscopy fixative was slowly added to the EP tube, and the tube was incubated at 4°C overnight and sent to Beijing Kaiyue Biotechnology Co., Ltd. for subsequent preparation and testing.

[0055] 10. Flow cytometry analysis of DCs pyroptosis ratio and immune function activation DCs were isolated and extracted using miniMACS CD11c+ magnetic beads. After stimulation according to experimental requirements, 3×10 5 Aliquot the cells into flow cytometry tubes, wash once with pre-cooled PBS, centrifuge at 1500 rpm for 5 min, and discard the supernatant.

[0056] Analysis of the proportion of pyroptotic cells: Follow the instructions for the FAM-FLICA® caspase-1 detection kit, protecting from light throughout the procedure. Dissolve FAM-YVAD-FMK in DMSO and dilute to 5x the volume in PBS as the working solution. Add 1 μL of the working solution to each tube, mix thoroughly, and incubate at 37°C for 1 h. After incubation, wash three times with pre-chilled PBS (1500 rpm, 5 min) and discard the supernatant. Resuspend the cells in 100 μL of pre-chilled PBS per tube, then add 5 μL of 7-AAD, mix thoroughly, and incubate at room temperature for 15 min. Wash again three times with pre-chilled PBS and discard the supernatant. Finally, resuspend the cells in 300 μL of pre-chilled PBS and analyze by flow cytometry.

[0057] Analysis of the expression level of functional molecules on the surface of DCs: Flow cytometry antibodies against DCs surface molecules (PE-CD80, PECy7-CD86, and FITC-M HC-II) were added at a ratio of 1:100 and mixed evenly. The cells were incubated at room temperature in the dark for 30 min, washed once with pre-cooled PBS, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cells were fixed with 300 μL of 1% paraformaldehyde and analyzed by flow cytometry.

[0058] 11. Co-culture of DCs and CD4+ T cells to detect DCs function On the first day, dilute the 5 μM CFSE stock solution 1000 times to prepare the working solution. Resuspend the mouse spleen CD4+ T cells with 1 mL of pre-chilled PBS, add 1 μL of CFSE working solution, and incubate at room temperature for 30 minutes in the dark. After staining, add 5 mL of RPMI 1640 + 10% FBS culture medium to stop the staining, then centrifuge at 1300 rpm for 5 minutes to wash the cells and prepare them for subsequent co-culture experiments. Use RPMI-1640 + 10% FBS culture medium at 2×10 6 The cells were resuspended at a ratio of 100 μg / mL and 5 μg / mL concanavalin A (Con A) was added as a stimulator. 100 μL per well was seeded into a 96-well plate and incubated at 37°C in 5% CO2 for 24 h.

[0059] The next day, splenic CD11c+ DCs and CD4+ T cells were added at a ratio of 1:20 and incubated in a 37°C, 5% CO2 incubator for 72 hours. Cell supernatants were collected after co-culture and cytokine expression (such as IL-2, IL-4, and IFN-γ) was assayed using ELISA. CFSE dye fluorescence decay was measured using flow cytometry.

[0060] 12. Extract Golgi apparatus and analyze related protein expression Collect at least 5×10 7 1×10 cells, centrifuge at 500 g for 5 min, wash twice with pre-cooled PBS, and discard the supernatant. 7 Add Reagent A to the cells in a 1:1 ratio and homogenize using a glass homogenizer, taking care to avoid over-mixing to prevent disruption of the Golgi apparatus. After homogenization, place on ice for 5 minutes. Centrifuge at 600 g for 10 minutes at 4°C and collect the supernatant (to remove nuclei and debris). Transfer to a fresh tube and add Reagent B in a 1:1 ratio by volume. Centrifuge at 12,000 g for 20 minutes at 4°C to collect the precipitate. Resuspend the precipitate in Reagent C and wash twice with Reagent D, centrifuging each time at 12,000 g for 10 minutes at 4°C. Finally, resuspend in an appropriate amount of Reagent D to obtain the extracted Golgi apparatus for subsequent experiments, such as protein analysis.

[0061] 13. Co-immunoprecipitation (Co-IP) assay to detect protein-protein interactions Remove the cell culture medium from each group and wash the cells twice with PBS. Scrape the cells with a scraper and collect them into 1.5 mL EP tubes. Add 200 μL of IP Lysis / Wash Buffer to each group, along with appropriate inhibitors such as PMSF. Mix thoroughly and incubate on ice for 30 minutes (vortexing every 5 minutes). Centrifuge at 12,000 g for 10 minutes at 4°C in a balanced, low-temperature high-speed centrifuge. Collect the supernatant and prepare a standard curve using the BCA assay. Measure absorbance and calculate protein concentration. Add the primary antibody at a ratio of 1000 μg protein / 5 μg immunoprecipitation antibody. Dilute the antibody and prepared sample to 1 mL in IP Lysis / Wash Buffer and incubate at room temperature for 4 hours with end-over-end to form the immune complex. Vortex thoroughly to mix the Biolinkedin® Protein A / G magnetic beads. Add 50 μL of Biolinkedin® Protein A / G magnetic beads to a 1.5 mL centrifuge tube and gently mix with 1 mL of pre-chilled PBS for 1 minute. Place the centrifuge tube in a magnetic rack to collect the magnetic beads to one side of the centrifuge tube and remove the supernatant. Add the antigen sample / antibody mixture to the centrifuge tube containing the magnetic beads, mix well, and incubate at room temperature for 4 hours. Collect the magnetic beads using a magnetic rack, remove the unbound sample, and add 1 mL of IP Lysis / Was h Buffer again, gently mix the magnetic beads, and wash for 5 minutes. Collect the magnetic beads, discard the supernatant, and repeat the wash twice. Add the same volume of 1× Loading Buffer as the magnetic beads and heat in a 95°C metal bath for 10 minutes. Separate the magnetic beads using a magnetic rack, retain the supernatant containing the target antigen, and store at -20°C for use.

[0062] 14. Immunohistochemistry (IHC) experiments to observe the distribution and expression of CALCOCO1 protein Spleens were obtained from mice in different groups after dissection and fixed in 4% tissue fixative for 24 hours. The tissues, along with the CALCOCO1 antibody, were sent to Beijing Kaiyue Biotechnology Co., Ltd. for subsequent preparation and microscopic observation.

[0063] Here are the results: 1. Activation of Golgi-phagy mediated by CALCOCO1 in DCs during sepsis Immunohistochemical staining results Figure 1CALCOCO1 expression was significantly upregulated in the spleens of mice 24 hours after CLP surgery, and CALCOCO1 protein expression in the spleens 72 hours after surgery was almost identical to that in the SHAM group. This suggests that the Golgi-phagy receptor CALCOCO1 is highly expressed in the spleen of mice during the early stages of sepsis, potentially mediating Golgi-phagy in DCs.

[0064] After further separation of spleen DCs, the expression of CALCOCO1 protein was verified by Western blot. Figure 2 The results showed that CALCOCO1 protein expression in DCs was significantly upregulated 24 hours after CLP, and the ratio of LC3B-II to LC3B-I protein expression was significantly increased, suggesting that Golgi-phagy may occur in splenic DCs in the early stage of sepsis in mice.

[0065] In cell experiments, LPS was used to stimulate DC2.4 to simulate sepsis conditions. Figure 3 Compared with the CON group, the fluorescence intensity of CALCOCO1 in the LPS-stimulated group was significantly enhanced, and showed significant overlap with the fluorescence of the Golgi structural proteins TGN46 (trans-Golgi) and GM130 (cis-Golgi). At the same time, the fluorescence of TGN46 and GM130 in the LPS-stimulated group significantly overlapped with the fluorescence of LC3B, further suggesting that CALCOCO1 expression in DCs increases in sepsis and induces extensive Golgi-phagy.

[0066] To further prove that CALCOCO1 is localized in the Golgi apparatus, the present invention uses Co-IP to verify the interaction between CALCOCO1 and Golgi structural proteins. Figure 4 Compared with the CON group, CALCOCO1 in the stimulated group showed significantly enhanced interactions with the Golgi membrane structural proteins TGN46 and GM130, but no interaction with the functional protein MAN2A1 in the Golgi lumen. These results further suggest that CALCOCO1 in DCs under sepsis conditions is involved in mediating the occurrence of Golgi-phagy.

[0067] like Figure 5 After 24 h of LPS (1 μg / mL) stimulation, the Golgi apparatus of DC2.4 cells was extracted and the expression of GM130 and TGN46 was significantly decreased, but the expression of CALCOCO1 was increased. Figure 6Combining Western blot analysis of DCs' overall protein content, LSCM observations, and Co-IP assays, the present study hypothesized that in the early stages of sepsis, the Golgi matrix is swollen but structurally dispersed, with cleaved Golgi fragments dispersed throughout the cytoplasm. These fragmented Golgi structures are difficult to extract using a Golgi isolation kit. However, increased CALCOCO1 expression in the extractable Golgi structures was confirmed, and CALCOCO1 participates in the induction of Golgi phagy, thereby restoring Golgi homeostasis.

[0068] like Figure 7 After primary DCs from mouse spleen were stimulated with LPS (1 μg / mL) for 24 h, transmission electron microscopy was used to observe the cell ultrastructure and found that the Golgi structure in DCs was severely fragmented and diffusely dispersed in the cytoplasm, and obvious Golgi-phagy occurred, which was manifested by the formation and significant increase in the number of autophagosomes around the Golgi apparatus of DCs, and the number of autophagosomes containing Golgi structures was significantly increased.

[0069] like Figure 8 To verify the key role of CALCOCO1, the present study used lentiviral transfection to intervene in CALCOCO1 expression and then observed the expression of Golgi matrix proteins in each group of DC2.4 cells after LPS stimulation. First, DC2.4 cells transfected with the corresponding lentiviruses successfully overexpressed or underexpressed CALCOCO1 protein under normal culture conditions.

[0070] like Figure 9 In DC2.4 cells overexpressing CALCOCO1, the expression of GM130 and TGN46 proteins decreased significantly after LPS stimulation. Conversely, in DC2.4 cells in which CALCOCO1 expression was disrupted, the expression of related proteins increased significantly after LPS stimulation, indicating that the expression level of CALCOCO1 is negatively correlated with Golgi proteins, which is consistent with the dynamic characteristics of CALCOCO1-mediated Golgi degradation.

[0071] In in vivo experiments, Figure 10 The present invention constructed CALCOCO1 knockout (CAL-KO) mice. Twenty-four hours after CLP surgery, GM130 and TGN46 expression in DCs of CAL-KO mice was significantly higher than that of WT mice, and LC3B protein activation was absent. The results suggest that Golgi-phagy in DCs of CALCOCO1-deficient mice is significantly inhibited during sepsis.

[0072] 2. CALCOCO1-mediated Golgi-phagy in DCs during sepsis is induced by the CREB3-ARF4 pathway like Figure 1124 h after LPS (1 μg / mL) stimulation of DC2.4 cells, the expression levels of CALCOCO1 and LC3B-II / -I were significantly increased. Prestimulation with BFA (0.1 μg / mL) did not alter these results. Conversely, prestimulation with MON (1 μg / mL) further upregulated the expression of CALCOCO1 and LC3B-II / -I and decreased the protein levels of GM130 and TGN46. These results suggest that BFA-induced Golgi stress in sepsis does not further activate Golgi phagy in DCs, but MON-activated Golgi stress contributes to synergistic activation of Golgi phagy in DCs, which may be closely related to the activation of the CREB3-ARF4 pathway in the MON-induced Golgi stress response.

[0073] like Figure 12 Stimulation of DC2.4 cells with BFA (0.1 μg / mL) alone did not increase the expression of CALCOCO1 and LC3B-II / -I, nor did it decrease the expression of GM130 and TGN46 proteins. In contrast, stimulation with MON (1 μg / mL) significantly upregulated the expression of CALCOCO1 and LC3B-II / -I, while significantly decreased the expression of GM130 and TGN46 proteins. Comparison of the results from the two groups revealed that MON-specific activation of the Golgi stress CREB3-ARF4 pathway is closely associated with the development of Golgi phagy.

[0074] In subsequent experiments, Figure 13 The present invention used Co-IP to verify the interactions of Golgi-phagy-related proteins. Compared with the CON group, no significant interactions were observed between CALCOCO1 and GM130, TGN46, or LC3B in the BFA (0.1 μg / mL) stimulation group. However, compared with the CON group, CALCOCO1 showed significant interactions with GM130, TGN46, and LC3B in the MON (1 μg / mL) stimulation group. These results further suggest that activation of the CREB3-ARF4 pathway in DC2.4 cells is involved in the induction of CALCOCO1-mediated Golgi-phagy.

[0075] like Figure 14Based on this, the present invention used lentiviral transfection to interfere with ARF4 expression. The results showed that DC2.4 cells overexpressing ARF4 showed significantly increased expression of CALCOCO1 and LC3B-II / -I after LPS stimulation. Conversely, after silencing ARF4 expression, LPS stimulation failed to induce increased expression of CALCOCO1 and LC3B-II / -I. This preliminary suggests that under sepsis conditions, the Golgi stress response CREB3-ARF4 pathway in DCs is involved in inducing CALCOCO1-mediated Golgi phagy.

[0076] 3. CALCOCO1-mediated Golgi-phagy in DCs alleviates DC pyroptosis and inflammatory factor secretion during sepsis like Figure 15 In DC2.4 cells overexpressing CALCOCO1, the expression levels of pyroptosis-related proteins such as NLRP3, GSDMD, Caspase-1, and IL-1β did not decrease significantly after LPS stimulation, indicating that overexpression of CALCOCO1 can significantly antagonize LPS-induced pyroptosis. Conversely, in DC2.4 cells with silenced CALCOCO1 expression, LPS stimulation still induced upregulation of the expression of related proteins, suggesting that CALCOCO1-mediated Golgi-phagy may be involved in inhibiting pyroptosis in DCs during sepsis.

[0077] Flow cytometry analysis results further demonstrated that Figure 16 Compared with normal cells, DC2.4 cells overexpressing CALCOCO1 had a lower rate of pyroptosis after LPS stimulation. Conversely, DC2.4 cells with silenced CALCOCO1 expression had a higher rate of pyroptosis.

[0078] like Figure 17 Compared with WT mice, the expression levels of NLRP3, GSDMD and Caspase-1 in DCs of CAL-KO mice were higher after CLP surgery, indicating that pyroptosis of DCs in mice with CALCOCO1 gene deletion was more significant during sepsis.

[0079] like Figure 18 Flow cytometry analysis revealed that pyroptosis of DCs in the spleen of CAL-KO mice was higher after CLP compared with WT mice. This suggests that CALCOCO1-mediated Golgi-phagy can effectively alleviate sepsis-induced pyroptosis.

[0080] 4. CALCOCO1-mediated Golgi-phagy in DCs improves DCs immune function activation during sepsis like Figure 1924 hours after CLP, the expression of functional molecules CD80, CD86, and MHC-II on the surface of DCs in the spleen of WT mice was significantly upregulated, but under the same conditions, the expression of these molecules on the surface of DCs in the spleen of CAL-KO mice did not increase significantly. This suggests that DCs cannot be effectively activated when CALCOCO1 is missing.

[0081] like Figure 20 The researchers then used CFSE-labeled CD4+ T cells and DCs to co-culture and examine their proliferative effects. The results showed that compared with DCs from WT mice after CLP surgery, DCs isolated from the spleens of CAL-KO mice significantly reduced their induction of CD4+ T cell proliferation, indicating that CALCOCO1 gene deficiency significantly inhibits DC function.

[0082] Discussion and Analysis: This study reveals for the first time the critical role of CALCOCO1-mediated Golgi-phagy in sepsis. The study first observed that CALCOCO1-mediated Golgi-phagy occurs in DCs during the early stages of sepsis, and that this process may play an important role in maintaining Golgi homeostasis in DCs and cellular homeostasis.

[0083] Further mechanism inventions show that under sepsis conditions, the CREB3-ARF4 signaling pathway in the Golgi stress response is specifically activated, participating in the induction of CALCOCO1-mediated Golgi-phagy activation. Different from traditional macroautophagy-related inventions, the results of the present invention emphasize the important role of the Golgi-specific autophagy process and the Golgi homeostasis regulated by it in inflammation and immune responses. Specifically, under sepsis conditions, overexpression of ARF4 protein can further promote CALCOCO1-mediated Golgi-phagy activation. This process not only helps to clear damaged Golgi apparatus and reduce the accumulation of abnormal proteins and lipids, but also effectively alleviates the occurrence of DCs pyroptosis and participates in improving the immune function of DCs. This finding is consistent with the current invention direction on the regulation of programmed cell death of immune cells by organelle-selective autophagy, and further supports the view that immune cell dysfunction in sepsis is closely related to organelle damage. For example, NUNF1P1 can alleviate the endoplasmic reticulum stress response by mediating ribosome selective autophagy, inhibit the apoptosis rate of T lymphocytes in septic mice, and improve the immune dysfunction of septic mice

[13] . In addition, it was found that mitochondrial selective autophagy can clear damaged mitochondria in DCs during sepsis, inhibit DC apoptosis and maintain their normal immune function activation

[14] .

[0084] In a further mechanism invention, the present invention confirmed that in the state of sepsis, overexpression of CALCOCO1 can effectively inhibit the activation of NLRP3 inflammasomes in cells and reduce the release of inflammatory factors IL-1β and IL-18, thereby alleviating excessive inflammatory responses. It is worth noting that CALCOCO1-mediated Golgi-phagy has a significant positive effect on the activation of DCs immune function. Moderate Golgi-phagy not only enhances the antigen presentation ability and T cell activation efficiency of DCs, but also promotes effective immune responses. Due to the lack of CALCOCO1 in CAL-KO mice, Golgi-phagy activation in DCs is impaired, resulting in immune response dysfunction. These results indicate that in the pathophysiological process of sepsis, the core role of Golgi-phagy in the immune response of DCs may become an important target for regulating immune responses.

[0085] Although the present invention shows that by regulating CALCOCO1-mediated Golgi-phagy, it is possible to reduce cell pyroptosis, alleviate excessive inflammatory responses, and protect DCs function, it provides a new strategy for the intervention treatment of sepsis. The next invention will further explore the specific mechanism by which CALCOCO1 alleviates the pyroptosis rate of DCs in septic mice, providing a clearer invention direction for the future development of intervention targets. In addition, future inventions will focus on in-depth exploration of the interaction between CALCOCO1 and autophagy receptors and organelle-related proteins in different immune cells to fully illustrate the immunoregulatory role of CALCOCO1 in sepsis, which will help to clarify the mechanism of action of Golgi-phagy in different pathological environments and its interaction with other organelle quality control systems. The mechanism exploration around Golgi-phagy will provide strong support for the development of new treatment strategies for Golgi stress-related diseases.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of CALCOCO1 as a biomarker in the preparation of reagents or kits for early prediction of sepsis.

2. The use according to claim 1, characterized in that The CALCOCO1 as a biomarker is to detect the protein expression level of CALCOCO1 in body fluids, or to detect the transcription level of CALCOCO1 in organ tissues.

3. The use according to claim 2, characterized in that The body fluid is at least one of serum, plasma, whole blood, urine, cerebrospinal fluid, pleural effusion, ascites, and joint fluid.

4. The use according to claim 2, characterized in that The organ tissue is liver, lung, kidney or spleen tissue.

5. Application of CALCOCO1 in the preparation of reagents or kits for early prediction of sepsis.

6. Application of CALCOCO1 as a therapeutic target in the preparation of drugs for the treatment of sepsis.

7. The use according to claim 6, characterized in that The active ingredient of the drug includes at least one of the following components: CALCOCO1, CALCOCO1 recombinant protein, a product obtained by subjecting CALCOCO1 to certain chemical modifications, and a product obtained by subjecting CALCOCO1 recombinant protein to certain chemical modifications.

8. The use according to claim 6, characterized in that The drug has at least one of the following effects (I) to (II): (Ⅰ) Inhibit the production of excessive inflammatory factors; (II) Reduce dendritic cell death and organ damage.

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