Application of NEP protein as sepsis biomarker and therapeutic target
By discovering the elevation of NEP protein in the plasma of sepsis patients, using it as a biomarker for early diagnosis, and treating sepsis with NEP protein inhibitors, the problem of the lack of effective treatment methods in existing technologies has been solved, and personalized treatment effects have been achieved.
Patent Information
- Application Number
- CN202511888411.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-15
- Publication Date
- 2026-03-03
AI Technical Summary
Current technologies lack effective treatments for sepsis, and individualized treatment is limited by unclear pathogenesis and patient heterogeneity. The correlation between NEP protein and sepsis has not been verified.
NEP protein was found to be significantly elevated in the plasma of sepsis patients, and its concentration was correlated with the severity of the disease. NEP protein was used as a biomarker for early diagnosis of sepsis, and NEP protein inhibitors such as racecadotril were used for therapeutic intervention to reduce inflammatory factors and protect organ function.
NEP protein has high diagnostic efficacy as a biomarker of sepsis. Racecadotril significantly reduces mortality and organ damage in septic mice, improves systemic inflammatory response, and provides the possibility of personalized treatment.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to the application of NEP protein as a biomarker and therapeutic target for sepsis. Background Technology
[0002] Sepsis, a life-threatening organ dysfunction caused by a dysregulated host response to infection, is one of the major challenges facing global healthcare. With high morbidity and mortality rates, sepsis imposes a significant disease burden worldwide; in 2017, there were over 48.9 million sepsis patients globally, and approximately one-fifth of deaths were attributable to sepsis. Sepsis is also one of the leading causes of death among critically ill patients in intensive care units. Despite advancements in current treatments, effective therapies are lacking, and individualized treatment faces challenges due to unclear pathogenesis and patient heterogeneity.
[0003] Existing research indicates that sepsis is associated with immune dysregulation, and changes in plasma proteins may be a key factor. Neprilysin (NEP), also known as neutral endopeptidase, is encoded by the MME gene. It is a 750-amino acid transmembrane protein with intracellular, transmembrane, and extracellular segments, and is widely expressed in various tissues and cells. NEP exists in two different forms: membrane-bound and soluble. Soluble NEP (sNEP) is the extracellular segment of NEP, present in its active form in the peripheral circulation, urine, and cerebrospinal fluid. It plays a role in degrading enkephalins and cleaving substances such as substance P, angiotensin I, angiotensin II, ANP, and BNP. It is well known that diseases can induce corresponding pathophysiological changes in the body, manifested as abnormalities in a series of biochemical indicators. Previous studies have found elevated NEP levels in the plasma of sepsis patients, but elevated biochemical indicators do not necessarily indicate a link to sepsis; the elevated NEP levels may be indirectly caused by sepsis triggering other diseases.
[0004] To date, no experimental research results have shown that NEP protein is associated with sepsis, and there is no relevant experimental data to confirm that NEP protein can serve as a therapeutic target for sepsis. Summary of the Invention
[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to discover whether NEP protein is related to sepsis, and on the basis of verifying the correlation, to further discover whether NEP protein can serve as a therapeutic target for sepsis.
[0006] This invention is based on the following findings: Our researchers were the first to discover that NEP protein levels were significantly elevated in the plasma of sepsis patients, and that the NEP protein concentration in the plasma of a severe sepsis model was higher than that in a moderate sepsis model. Further investigation revealed that the elevation of plasma NEP protein occurred earlier than other biochemical indicators. Therefore, we investigated NEP protein as a potential therapeutic target for sepsis, and based on this, we completed this invention.
[0007] The first aspect of the present invention is to provide a sepsis biomarker and therapeutic target, wherein the sepsis biomarker and therapeutic target is the NEP protein.
[0008] The second aspect of this invention provides the application of NEP protein as a biomarker and therapeutic target for sepsis, for screening, diagnosis and treatment of pathological conditions in sepsis patients.
[0009] A third aspect of the invention provides the use of inhibitors of the NEP protein in the preparation of medicaments for the prevention and / or treatment of sepsis.
[0010] The drug comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an inhibitor of the NEP protein.
[0011] Preferably, the NEP protein inhibitor can significantly reduce inflammatory factors in plasma and reduce the levels of ALT, AST, serum creatinine, and serum lactate in plasma.
[0012] Preferably, the inflammatory factors include TNFα and IL-6, which are associated with sepsis.
[0013] Preferably, the inhibitor of the NEP protein includes racecadotril.
[0014] The term "effective amount" or "effective dose" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.
[0015] The term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.
[0016] The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.
[0017] Pharmaceutically acceptable carriers include, but are not limited to: water, saline, buffer solutions, glycerol, ethanol, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be appropriate for the route of administration, as is well known to those skilled in the art.
[0018] This invention, through research on the function and mechanism of NEP protein in sepsis, provides applications of NEP protein in the diagnosis and treatment of sepsis. From a diagnostic perspective, it offers NEP protein as a biological biomarker for sepsis diagnosis, suitable for rapid detection of NEP protein in plasma, facilitating early screening. From a therapeutic perspective, it provides NEP protein as a therapeutic target for sepsis, demonstrating the effectiveness of treatment using NEP protein inhibitors, and offering insights for researchers to develop more suitable drugs.
[0019] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All equivalent substitutions made in accordance with this disclosure are within the scope of protection of the present invention. Attached Figure Description
[0020] Figure 1 This study investigated the changes in NEP levels in the plasma of patients with sepsis.
[0021] Figure 2 ROC curve for diagnosing sepsis using plasma NEP protein concentration.
[0022] Figure 3 The changes in NEP protein concentration in the plasma of septic mice.
[0023] Figure 4 To investigate the changes in sepsis and biochemical parameters in mice after racecadotril treatment.
[0024] Figure 5 The results show the effects of LPS-induced organ damage in mice by racecadotril.
[0025] Figure 6 The results show the effects of racecadotril on LPS-induced inflammatory factors in various major organs of mice. Detailed Implementation
[0026] The technical solution of the present invention will be further explained below with reference to experiments.
[0027] Example 1 Changes in NEP levels in the plasma of sepsis patients and non-sepsis patients in human samples.
[0028] Researchers collected plasma from sepsis and non-sepsis patients who had previously been treated at the Intensive Care Unit of Zhujiang Hospital, Southern Medical University, and used ELISA to detect the concentration of NEP protein in the plasma of sepsis patients (n=92) and non-sepsis patients (n=34).
[0029] Figure 1A represents the NEP content in the plasma of sepsis patients and non-sepsis patients (logarithmic transformation); B represents the NEP content in the plasma of sepsis patients and non-sepsis patients after gender stratification (logarithmic transformation); C represents the NEP content in the plasma of sepsis patients and non-sepsis patients in young adults (less than 65 years old) and older adults (65 years old and above) (logarithmic transformation); D represents the NEP content in the plasma of sepsis patients and non-sepsis patients at different infection sites (logarithmic transformation). "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001. Figure 1 The results showed that the plasma NEP level in sepsis patients was significantly higher than that in non-sepsis patients. Figure 1 A). Further stratification by gender revealed that, regardless of whether the patients were male or female, the plasma NEP levels in sepsis patients were significantly higher than those in non-sepsis patients. Figure 1 B). Subsequently, we further stratified all patients by age and found that the plasma NEP levels in both young sepsis patients (under 65 years old) and elderly sepsis patients (65 years old and above) were significantly higher than those in young and elderly non-sepsis patients. Figure 1 C). Finally, stratifying sepsis patients according to the site of infection revealed that regardless of the site of infection, the plasma NEP levels in sepsis patients were significantly higher than those in non-sepsis patients. Figure 1 D). To further clarify whether the level of NEP in the plasma of sepsis patients is related to the severity of sepsis, we compared the NEP levels in the plasma of sepsis patients who died within 28 days with those who survived within 28 days. We found that the NEP levels in the plasma of sepsis patients who died within 28 days were significantly higher than those of sepsis patients who survived within 28 days. Figure 1 E). In summary, by detecting NEP levels in the plasma of sepsis patients, we confirmed that the NEP protein level in the plasma of sepsis patients was significantly higher than that in non-sepsis patients, and that the higher the NEP level, the more severe the sepsis. Furthermore, NEP levels were not affected by gender, age, or site of infection, strongly suggesting that plasma NEP protein can serve as a biomarker for sepsis.
[0030] Subsequently, to further evaluate whether the NEP protein could help differentiate between sepsis and non-sepsis, we plotted ROC curves from... Figure 2 The ROC curve area under the curve (AUC) was 0.82, the sensitivity was 0.78, and the specificity was 0.72, indicating that plasma NEP protein has good efficacy in distinguishing between sepsis and non-sepsis. The plasma NEP protein concentration threshold is 169.89 pg / ml; a plasma NEP protein concentration greater than 169.89 pg / ml strongly suggests the onset of sepsis.
[0031] Example 2 Changes in sepsis levels in animal experiments The cecal ligation and perforation (CLP) procedure was performed on mice using a standard experimental method. The changes in NEP levels in the mouse plasma after treatment were observed. Figure 3 The data represent the changes in NEP protein concentration in the plasma of septic mice. A shows the ELISA measurement results of NEP protein concentration in plasma of CLP-induced model mice at different time points; B shows the changes in NEP protein concentration in plasma of mice with moderate and severe sepsis models; C shows the changes in IL-6 concentration in plasma of mice at different time points after intraperitoneal injection of LPS; and D shows the changes in NEP concentration in plasma of mice at different time points after intraperitoneal injection of LPS. "ns" indicates no significant difference between the two groups, "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001.
[0032] The results showed that the NEP content in the plasma of mice was significantly increased after CLP surgery. Figure 3 A), and the concentration of NEP protein in the plasma of severe sepsis model mice was higher than that in moderate sepsis model mice ( Figure 3 B). Furthermore, the time point for the increase in plasma NEP protein is earlier than that for interleukin-6 (IL-6), showing a significant increase as early as 15 minutes after intraperitoneal injection of lipopolysaccharide (LPS). Figure 3 (C, 3D). Animal studies strongly suggest that plasma NEP protein has the potential for early diagnosis of sepsis and that it plays an important role in the pathogenesis of sepsis.
[0033] Example 3 Intervention experiment of racecadotril in septic mice To determine whether NEP protein could serve as a potential therapeutic target for sepsis, racecadotril was used to intervene in septic mice. Racecadotril is an enkephalinase inhibitor clinically used for acute diarrhea in infants and children over one month of age. Racecadotril selectively and reversibly inhibits enkephalinase, thereby protecting endogenous enkephalins from degradation, prolonging the physiological activity of endogenous enkephalins in the gastrointestinal tract, and reducing excessive secretion of water and electrolytes. Oral administration of racecadotril acts on peripheral enkephalinase without affecting the activity of enkephalinase in the central nervous system, and has no significant effect on gastrointestinal motility and basal intestinal secretion.
[0034] Mice were treated with racemic caldotril by gavage for 7 days, followed by intraperitoneal injection of LPS to induce sepsis. The plasma levels of TNFα, IL-6, lactate, ALT, AST, and Cr were measured 12 hours after LPS injection. At the same time, major organs (heart, lung, kidney, liver, and intestine) were collected for molecular and pathological examinations. Figure 4A represents the effect of racecadotril treatment on the survival curve of septicemia; BD represents the effect of racecadotril treatment on the plasma concentrations of TNFα (B), IL-6 (C), and L-lactate (D) in LPS-induced septic mice; EG represents the effect of racecadotril treatment on the plasma concentrations of ALT (E), AST (F), and Cr (G) in LPS-induced septic mice. "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001.
[0035] The results showed that mice treated with racecadotril had a significantly lower sepsis mortality rate than control mice. Figure 4 A), mice treated with racecadotril also had lower levels of inflammatory factors (TNFα, IL-6) in their plasma. Figure 4 B, 4C). Racecadotril can improve tissue perfusion and protect organ function in septic mice, as evidenced by a significant decrease in blood lactate levels in mice after racecadotril treatment. Figure 4 D), plasma ALT, AST, and serum creatinine (Cr) levels were significantly lower than in the control group. Figure 4 E, 4F, 4G).
[0036] Subsequently, HE staining was performed on the major organs (heart, lungs, kidneys, and liver) to assess pathological damage in each organ. Figure 5 A shows the HE staining results of the heart, lungs, liver, and kidneys; B shows the myocardial cell edema score; C shows the myocardial interstitial inflammatory cell infiltration score; D shows the lung tissue pathological score; and E shows the liver tissue pathological score. "ns" indicates no significant difference between the two groups, "*" indicates P < 0.05, and "***" indicates P < 0.001. Pathological examination results confirmed that under normal conditions, racecadotril treatment does not cause pathological damage in the heart, lungs, liver, and kidneys of mice that can be detected by HE staining. LPS treatment significantly induced inflammatory cell infiltration in various organs, accompanied by varying degrees of cell damage. In septic mice treated with racecadotril, the heart ( Figure 5 B, 5C), lungs ( Figure 5 D) Liver ( Figure 5 The pathological damage score of E) was significantly lower than that of the control mice.
[0037] Example 4 Does racecadotril inhibit LPS-induced overactivation of inflammatory factors in major organs?
[0038] RNA was further extracted from major organs (heart, lung, liver, and intestine) of mice, and the transcriptional levels of inflammatory factors (Tnfα, IL-1β, and IL-6) in these organs were further detected by qPCR. The qPCR results confirmed that racemic doxorubicin gavage treatment in normal mice did not cause excessive activation of inflammatory factors in these major organs, while racemic doxorubicin significantly reduced the transcription of inflammatory factors in these major organs in septic mice. Specifically, racemic doxorubicin significantly inhibited LPS-induced heart (…). Figure 6 A, 6B), lungs ( Figure 6 C-6E), liver ( Figure 6 F, 6G) and gut ( Figure 6 Excessive activation of inflammatory factors in H-6J.
[0039] In summary, by detecting NEP levels in the plasma of septic mice and patients, we confirmed that plasma NEP protein levels are significantly elevated in sepsis, regardless of sex, age, or site of infection. Higher NEP levels indicate more severe sepsis and a worse prognosis. The AUC for NEP protein in diagnosing sepsis was 0.82, with a sensitivity of 0.78 and a specificity of 0.72, indicating that plasma NEP protein has good efficacy in distinguishing between sepsis and non-septic conditions. A plasma NEP protein concentration greater than 169.89 pg / ml strongly suggests the onset of sepsis, and the time point of NEP protein elevation is earlier than that of IL-6. This result confirms that plasma NEP protein can serve as a new early biomarker for sepsis. Furthermore, mouse experiments demonstrated that the NEP protein inhibitor racecadotril significantly reduces mortality in septic mice, effectively improves sepsis-induced multi-organ damage, and alleviates systemic inflammatory responses associated with sepsis. In normal mice, the use of racecadotril does not cause organ dysfunction. In summary, the NEP protein content in sepsis plasma is significantly higher than that in non-septic plasma, demonstrating a strong ability to differentiate between sepsis and non-septic conditions. Furthermore, targeting the NEP protein may become a novel approach for treating sepsis.
[0040] The above description is for illustrative purposes and not for limitation. Many implementations and applications beyond the provided examples will become apparent to those skilled in the art upon reading the above description.
Claims
1. A biomarker and therapeutic target for sepsis, characterized in that, The biomarker and therapeutic target for sepsis is the NEP protein.
2. The application of NEP protein as a biomarker and therapeutic target for sepsis, characterized in that, NEP protein is used for screening, diagnosis, and treatment of pathological conditions in patients with sepsis.
3. Use of NEP protein inhibitors in the preparation of drugs for the prevention and / or treatment of sepsis.
4. The use according to claim 3, characterized in that, The drug comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient, wherein the active ingredient is an NEP protein inhibitor.
5. The use according to claim 4, characterized in that, The NEP protein inhibitor can significantly reduce inflammatory factors in plasma and decrease the levels of ALT, AST, serum creatinine, and serum lactate in plasma.
6. The use according to claim 5, characterized in that, The inflammatory factors include TNFα and IL-6, which are associated with sepsis.