Application of ribonuclease A in the preparation of drugs for treating sepsis or sepsis complications

Abstract

The present invention relates to the use of ribonuclease A in preparing a drug for treating sepsis or sepsis complications. The present invention simulates a sepsis model in an animal, injects ribonuclease A into the tail vein for treatment, and ultimately determines the relevant pathways of ribonuclease A in treating sepsis by observing the survival rate of septic mice and combining methods such as tissue pathology and flow cytometry. The test results show that ribonuclease A can effectively treat sepsis. In addition, the test found that ribonuclease A can effectively reduce early neutrophil infiltration into tissues and reduce NETs formation to alleviate the degree of tissue damage. This shows that ribonuclease A can be used for the preparation of drugs for treating or preventing sepsis-related diseases, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of ribonuclease A in preparing a medicine for treating sepsis or sepsis complications. Background Art

[0002] Sepsis is a life-threatening organ dysfunction syndrome caused by a dysregulated response to infection, characterized by a disrupted host immune response. As one of the leading causes of mortality in intensive care units (ICUs), sepsis poses a significant public health challenge worldwide. Epidemiological data show that sepsis has become the leading cause of infection-related death worldwide, posing a significant threat to human health. Notably, the incidence of sepsis continues to rise annually with the aging population and the increasing proportion of immunocompromised individuals. Although widespread use of antibiotics, optimized fluid resuscitation strategies, and advances in organ support technologies significantly reduced sepsis mortality between the 1990s and 2010, clinical data over the past decade show that sepsis mortality has plateaued, suggesting that previous treatment strategies have reached a bottleneck.

[0003] Therefore, studying and elucidating the main pathological features and key regulatory mechanisms of sepsis and identifying novel interventional therapies will help improve the prognosis of patients with sepsis. One of the core pathological mechanisms of sepsis is the abnormal recruitment of neutrophils and the massive production of neutrophil extracellular traps (NETs). In sepsis, as core effector cells of innate immunity, neutrophils early on eliminate pathogens through phagocytosis and NET production. However, their overactivation can lead to abnormal deposition of NETs, ​​triggering alveolar-capillary barrier disruption and tissue oxidative stress, which is a key cause of mortality in patients with sepsis. Deepening the molecular mechanisms of NET development after sepsis, identifying new diagnostic and therapeutic targets, and developing corresponding intervention strategies have great theoretical and clinical value. However, there are currently no effective drugs that specifically target NETs to treat sepsis.

[0004] Ribonuclease A is an endoribonuclease, also known as RNase A and RNase I. It can specifically attack the 3' end of pyrimidine residues on RNA and cleave the phosphodiester bond formed with the adjacent nucleotide. The final reaction product is pyrimidine 3' phosphate and oligonucleotides with a pyrimidine 3' phosphate at the end. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the present invention proposes the use of ribonuclease A in the preparation of a drug for treating sepsis or sepsis complications.

[0006] The technical solution of the present invention is:

[0007] Application of ribonuclease A in the preparation of medicines for treating sepsis or sepsis complications.

[0008] Furthermore, the drug can reduce the early infiltration of neutrophils in lung tissue of patients with sepsis.

[0009] Furthermore, the drug can reduce the formation of NETS in lung tissue, serum and alveoli.

[0010] Furthermore, the septic complication is acute lung injury, or pneumonia, or severe sepsis, or septic shock.

[0011] Furthermore, the sequence of the ribonuclease A in the 5'→3' direction is MALEKSLVRLLLLVLILLVLGWVQPSLGKESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDST (SEQ ID NO. 1).

[0012] Furthermore, the effective dosage of the ribonuclease A is 250 μg / kg.

[0013] Furthermore, the drug also includes pharmaceutically acceptable excipients or carriers.

[0014] Further, the excipient includes a salt, and the salt includes a salt formed with an organic acid, and the organic acid includes a combination of one or more of benzenesulfonic acid, formic acid, tartaric acid, acetic acid, oxalic acid, glycerophosphoric acid, camphoric acid, pectinic acid, butyric acid, cyclopentanepropionic acid, maleic acid, nicotinic acid, succinic acid, adipic acid, alginic acid, aspartic acid, citric acid, digluconic acid, camphorsulfonic acid, dodecylsulfuric acid, glucoheptonic acid, ethanesulfonic acid, 2-naphthalenesulfonic acid, pivalic acid, hexanoic acid, heptanoic acid, fumaric acid, 2-hydroxyethanesulfonic acid, propionic acid, 3-phenylpropionic acid, lactic acid, pamoic acid, methanesulfonic acid, undecanoic acid and p-toluenesulfonic acid.

[0015] Furthermore, the auxiliary material includes an excipient, and the excipient includes a combination of one or more of lactose, mannitol, fructose and sucrose.

[0016] Furthermore, the dosage form of the drug includes but is not limited to injection.

[0017] Furthermore, the administration method of the drug includes but is not limited to injection.

[0018] Compared with the prior art, the present invention has at least the following advantages:

[0019] The present invention relates to the use of ribonuclease A in preparing a drug for treating sepsis or sepsis complications. The present invention simulates a sepsis model in an animal, injects ribonuclease A into the tail vein for treatment, and ultimately determines the relevant pathways of ribonuclease A in treating sepsis by observing the survival rate of septic mice and combining methods such as tissue pathology and flow cytometry. The test results show that ribonuclease A can effectively treat sepsis. In addition, the test found that ribonuclease A can effectively reduce early neutrophil infiltration into tissues and reduce NETs formation to alleviate the degree of tissue damage. This shows that ribonuclease A can be used for the preparation of drugs for treating or preventing sepsis-related diseases, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention, the following briefly introduces the drawings required for the specific embodiments or the description of the prior art.

[0021] Figure 1 In Example 1 of the present invention, ribonuclease A improves the survival rate of mice and screens drug concentrations in a cecal ligation and puncture (CLP) and intraperitoneal injection of lipopolysaccharide (LPS) sepsis model;

[0022] Figure 2 In Example 2 of the present invention, ribonuclease A can reduce organ damage;

[0023] Figure 3 In Example 3 of the present invention, ribonuclease A can reduce the early infiltration of neutrophils and the formation of NETs in organs;

[0024] Figure 4 This is the expression of ribonuclease A in the serum of clinical sepsis patients according to Example 4 of the present invention. DETAILED DESCRIPTION

[0025] The present invention is described in further detail below. It is necessary to point out that the following specific implementation methods are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0026] This document provides general and / or specific descriptions of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods. All reagents and instruments used, unless the manufacturer is specified, are commercially available, conventional products and were prepared or used using conventional methods.

[0027] Sources

[0028] RNase A: purchased from Roche.

[0029] Example 1 Grouping and treatment of mice (n=10 per group)

[0030] In this example, multiple groups of animal models were constructed and the survival rate of mice in each group was observed on the 7th day to screen the effective therapeutic concentration of RNase A.

[0031] Vehicle group: solvent control group based on the CLP-induced sepsis model; Sham group: sham-operated negative control group (only the cecum was freed after laparotomy); RNase A 10 / 50 / 250 μg / kg group: RNase A at concentrations of 10 μg / kg, 50 μg / kg, and 250 μg / kg was injected once through the tail vein 2 hours and 24 hours after the CLP-induced sepsis model was established; LPS 30 mg / kg group: a sepsis model was established by a single intraperitoneal administration of 30 mg / kg of LPS; LPS 30 mg / kg + RNase A 250 μg / kg group: a sepsis model was established by a single intraperitoneal administration of 30 mg / kg of LPS, and RNase A at a concentration of 250 μg / kg was injected through the tail vein 2 hours and 24 hours later.

[0032] The experimental results show that RNase A improves the survival rate of mice in the cecal ligation and puncture (CLP) and intraperitoneal injection of lipopolysaccharide (LPS) sepsis models. Figure 1 As shown, Figure 1 a is the survival rate of mice in the Vehicle, Sham, RNase A 10μg / kg, RNase A 50μg / kg, and RNase A 250μg / kg groups under the CLP model. Figure 1 As shown in a, the survival rates of the Vehicle, RNase A 10 μg / kg, and RNase A 50 μg / kg groups became 0 on day 2, day 2, and day 5, respectively. The survival rate of the RNase A 250 μg / kg group was 40% on day 7, which was significantly improved compared with the Vehicle group (p=0.0044); Figure 1 b shows the survival rate of mice in the LPS 30 mg / kg and LPS 30 mg / kg + RNase A 250 μg / kg groups under the LPS model. As can be seen from the figure, the survival rate of the LPS 30 mg / kg group on day 7 was 30%, while the survival rate of the drug-treated group on day 7 was 80%. The survival rate of the drug-treated group was significantly improved compared with the positive control group (p=0.0301); Figure 1 c, 1d show the clinical manifestations and clinical scores of CLP sepsis model mice after treatment with RNase A (250 μg / kg). Figure 1As shown in d, the score of the RNase A+CLP group was significantly higher than that of the Vehicle+CLP group, indicating that RNase A can significantly improve the survival rate of mice.

[0033] Example 2: Ribonuclease A can reduce lung tissue damage

[0034] Figure 2 a is the HE staining of lung tissues of the control group (Sham), CLP-induced sepsis model solvent control group (Vehicle+CLP), and 250 μg / kg drug treatment group (RNase A+CLP). Figure 2 aThe first column shows the visual field under a 4x microscope, and the second column shows the visual field under a 40x high-power microscope within the red frame in the first column. As can be seen from the figure, the lung tissue of Vehicle+CLP mice showed obvious injury characteristics one day after injury, including pulmonary edema, congestion and congestion of the alveolar interstitium, and infiltration of inflammatory cells around the lung tissue and pulmonary microvessels. When 250μg / kg RNase A was used early after injury, the lung tissue damage of CLP mice was significantly alleviated, indicating that the use of RNase A can effectively alleviate the degree of sepsis-induced injury. In addition, this example scored the degree of lung tissue damage (Lung Injury Score), and the scoring results are shown as follows: Figure 2 As shown in b, the lung tissue injury score of Vehicle+CLP group was the highest, and the score of RNase A+CLP group was significantly reduced. Figure 2 aThe results were consistent with those observed.

[0035] Example 3: RNase A reduces early neutrophil infiltration and NETs formation in the lung tissue of septic mice

[0036] Lung tissue samples were obtained from mice with CLP sepsis 1, 2, and 3 days after surgery, and single-cell suspensions were prepared. Flow cytometry was used to assess immune cell infiltration in lung tissue. Lung tissue, serum, and bronchoalveolar lavage fluid were obtained from mice 1 day after CLP surgery, and the content of MPO-DNA, a marker for NET formation, was assessed by ELISA. Lung tissue was obtained from mice 1 day after CLP surgery, and multi-channel fluorescence detection of NETs was performed sequentially, including sample repair, probe hybridization, fluorescent dye binding, DAPI nuclear staining, anti-quenching machine mounting, and laser confocal microscopy. Multi-channel fluorescence detection of NETs revealed the formation of NETs in lung tissue, with the staining appearing as scattered or clustered fluorescent dots.

[0037] The experimental results are as follows Figure 3 As shown, Figure 3 a Flow cytometry showed that RNase A could reduce the early infiltration of neutrophils in the lung tissue of CLP sepsis mice 1 and 2 days after surgery, and the early drug treatment group showed significant differences compared with the solvent control group; Figure 3b ELISA assays showed that RNase A could reduce the formation of NETs in lung tissue, serum, and bronchoalveolar lavage fluid of CLP-septic mice one day after surgery; Figure 3 c Confocal imaging of immunofluorescence staining showed that RNase A reduced the formation of NETs in the lung tissue of CLP-septic mice 1 day after surgery. Figure 3 These results indicate that RNase A can reduce the early infiltration of neutrophils in the lung tissue of septic mice and effectively reduce the formation of NETs.

[0038] Example 4 RNase A levels in sepsis patients and healthy controls are significantly different

[0039] Methods: Forty-seven serum samples from healthy volunteers aged 21-89 years and 161 serum samples from patients with sepsis aged 17-95 years were obtained from the Specialized Medical Center of the Chinese People's Liberation Army. Informed consent was obtained from all participants or their legal guardians. Serum RNase A levels in these samples were measured by ELISA, and the correlation between serum RNase A levels, disease severity, and prognosis was analyzed.

[0040] The experimental results are as follows Figure 4 As shown in Figure 2, the level of RNase A in the peripheral blood of patients with sepsis (n = 161) was significantly lower than that in healthy volunteers (n = 47) (p < 0.0001) ( Figure 4 a); RNase A levels in patients who died of sepsis (n=60) were significantly lower than those in patients who survived sepsis (n=101) (p=0.007) ( Figure 4 b); RNase A levels in patients with septic shock were also significantly lower than those in patients without septic shock (p=0.0292) ( Figure 4 c). We then analyzed the correlation between RNase A and the main clinical prognostic indicators of sepsis patients, namely neutrophil-lymphocyte ratio (NLR), C-reactive protein (CRP), white blood cells (WBC), procalcitonin (PCT), and interleukin-6 (IL-6). Figure 4 dh), and found that RNase A was negatively correlated with IL-6 and PCT indicators. To further evaluate the sensitivity and specificity of serum RNase A levels as a prognostic indicator for sepsis, we plotted a receiver operating characteristic (ROC) curve based on the patients' 28-day mortality rate and compared the areas under the curves of RNase A with those of PCT, CRP, WBC, IL-6, and NLR indicators. It was found that the area under the curve of RNase A was the largest at 0.6895, which was higher than that of other indicators, indicating that serum RNase A levels have a better ability to predict the 28-day mortality of patients with sepsis than other indicators.

[0041] This study, using animal models, discovered the therapeutic effects of RNase A on sepsis. Treatment with RNase A significantly reduced tissue damage, thereby improving clinical scores and survival in septic mice. Combining histopathology with flow cytometry significantly enhanced the reliability of the results. This study provides a novel sepsis treatment drug—RNase A—that directly inhibits early neutrophil infiltration in tissues and reduces NET formation, thereby alleviating tissue damage and improving survival.

[0042] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. Application of ribonuclease A in the preparation of drugs for the treatment of sepsis.

2. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients or carriers.

3. The use according to claim 2, characterized in that The dosage form of the medicine is injection.

4. The use according to claim 3, characterized in that The drug is administered by injection.

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