WO2021073249A1 - β-NMN在制备脓毒症器官损伤的治疗、预防药物中的应用 - Google Patents

β-NMN在制备脓毒症器官损伤的治疗、预防药物中的应用 Download PDF

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WO2021073249A1
WO2021073249A1 PCT/CN2020/110120 CN2020110120W WO2021073249A1 WO 2021073249 A1 WO2021073249 A1 WO 2021073249A1 CN 2020110120 W CN2020110120 W CN 2020110120W WO 2021073249 A1 WO2021073249 A1 WO 2021073249A1
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sepsis
mice
nicotinamide mononucleotide
damage
tissue
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French (fr)
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彭天庆
曹婷
郑东
李会丽
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Suzhou University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the invention relates to the field of biomedicine, in particular to the application of ⁇ -NMN in the preparation of a medicine for the treatment and prevention of septic organ damage.
  • Sepsis is a life-threatening multiple organ dysfunction caused by the imbalance of the body response after infection, which can develop into septic shock and multiple organ dysfunction syndrome (MODS), which is clinical One of the leading causes of death in critically ill patients.
  • MODS multiple organ dysfunction syndrome
  • the research data of domestic and foreign scholars shows that despite the continuous progress of "clustered" treatment strategies such as capacity assessment and resuscitation, early anti-infection and organ support, the mortality rate of patients remains high. So far, there are no effective measures to prevent and treat sepsis. Therefore, it is urgent to find new targets and new treatment measures for the prevention and treatment of sepsis.
  • Fluid resuscitation Most patients with sepsis will have circulatory failure in the early stage. It is determined that patients with sepsis have persistent hypotension. Before being admitted to the intensive care unit, the initial resuscitation and volume expansion treatment will be carried out through intravenous rehydration. However, in patients with sepsis, the diastolic function of the heart is limited, and a large amount of crystalloid infusion within a short period of time may exceed the compensatory capacity of the patient’s heart. This will not only not increase cardiac output, but will cause pulmonary edema and high center. Serious hemodynamic consequences including liver and kidney damage caused by venous pressure. Therefore, this also puts forward higher requirements for clinicians in the evaluation of fluid type, measurement, and course of treatment.
  • vasoactive drugs In order to make the patient's average arterial pressure greater than or equal to 65mmHg, vasopressor drugs are needed. Generally, norepinephrine is the first choice, and vasopressin can be used to increase the average arterial pressure value. However, the actual application, dosage and choice of drugs for patients with septic shock are also controversial. In addition to norepinephrine and vasopressin, angiotensin II has gradually become more prominent in the role of vasoactive drugs. Studies have found that angiotensin II can effectively increase the arteries of patients with vasodilatory shock whose conventional vasoactive drugs are ineffective. blood pressure. As for the timing of the application of vasoactive drugs and the appropriate reduction or even discontinuation, there is still no unified conclusion.
  • vasoactive drugs and anti-infective drugs have always been the core strategy of the campaign to save sepsis, but there is a lack of specific treatment measures. Therefore, it is particularly urgent to continue pre-clinical basic research to provide effective and specific prevention and treatment programs.
  • Nicotinamide Mononucleotide is a nucleotide derived from ribose and nicotinamide. It can be synthesized in the body through two pathways: one is catalyzed by nicotinamide via nicotinamide phosphoribosyl transferase (NAMPT) Synthesis; the other way is by Nicotinamide riboside (Nicotinamide riboside) catalyzed synthesis by nicotinamide riboside kinase.
  • NAMPT nicotinamide phosphoribosyl transferase
  • ⁇ -Nicotinamide Mononucleotide ( ⁇ -Nicotinamide Mononucleotide, ⁇ -NMN) is the active form of nicotinamide mononucleotide, which acts as Nicotinamide adenine dinucleotide (NAD + ; also known as coenzyme)
  • NAD + is a coenzyme that transfers protons. It is an indispensable coenzyme in glycolysis, gluconeogenesis, tricarboxylic acid cycle, and respiratory chain. It plays an important role in providing energy to cells, repairing DNA, and anti-aging. The role of substitution.
  • NAD + Nicotinamide nucleotide adenylytransferase 1-3
  • NAD + accounts for 85% of the total body's NAD + and is the most important way to maintain the NAD+ content in the body.
  • studies have reported that the NAD + content in the body significantly decreases under sepsis conditions, and it may be closely related to the continued deterioration of the main organs in sepsis.
  • the purpose of the present invention is to provide an application of ⁇ -NMN in the preparation of drugs for the treatment and prevention of septic organ damage.
  • the present invention discloses a new type of treatment and/or prevention of septic organ damage. medicine.
  • the invention discloses the application of ⁇ -nicotinamide mononucleotide ( ⁇ -NMN) in the preparation of medicines for the treatment and/or prevention of organ damage caused by sepsis.
  • ⁇ -NMN ⁇ -nicotinamide mononucleotide
  • organ damage includes one or more of heart damage, lung damage, liver damage, and kidney damage.
  • the drug is administered by injection or oral administration.
  • injection administration includes intravenous and/or intraperitoneal injection administration.
  • the administration dose of the drug is 300-1000 mg/kg.
  • the dosage of the drug is 500 mg/kg.
  • the medicine is used to increase the reduction of coenzyme I (NAD + ) content caused by sepsis.
  • the medicine is used to reduce the activity of myeloperoxidase and the content of active oxygen and malondialdehyde in organs caused by sepsis.
  • the present invention has at least the following advantages:
  • the present invention discloses the new use of ⁇ -nicotinamide mononucleotide, which has therapeutic, preventive and protective effects on multiple organ damage caused by sepsis, thereby providing new treatment methods and approaches for diseases in related fields.
  • a single administration of ⁇ -nicotinamide mononucleotide can increase the level of NAD + in the body, improve the heart function of septic mice, and reduce the level of oxidative stress in sepsis mice. Reduce the damage of heart, liver, lung, kidney and other organs.
  • ⁇ -nicotinamide mononucleotide is simple: ⁇ -nicotinamide mononucleotide is easy to dissolve in water, no other special solvents are required, and it is very popular and maneuverable.
  • the route of administration can be diversified: In the present invention, both intravenous injection and intraperitoneal injection are effective. In addition, it can also be administered orally.
  • Figure 1 is the detection result of heart function, oxidative stress and inflammatory response in Example 2 of the present invention
  • Figure 2 is the detection results of lung histopathology, oxidative stress, inflammatory response and cell apoptosis in Example 2 of the present invention
  • Fig. 3 is the detection result of oxidative stress, cell damage and inflammatory reaction of liver tissue in Example 2 of the present invention
  • Figure 4 is the detection results of oxidative stress, cell damage and inflammatory reaction in kidney tissue in Example 2 of the present invention.
  • Figure 5 is the detection results of heart function, oxidative stress and inflammatory response in Example 3 of the present invention.
  • Figure 6 is the detection results of lung tissue oxidative stress, inflammatory response and cell apoptosis in Example 3 of the present invention.
  • Fig. 7 is the detection result of oxidative stress, cell damage and inflammatory reaction of liver tissue in Example 3 of the present invention.
  • Fig. 8 shows the detection results of oxidative stress, cell damage and inflammatory reaction in kidney tissue in Example 3 of the present invention.
  • the present invention mainly uses the following experimental methods to detect representative biochemical parameters and damage indicators of multiple organs in septic mice, which are briefly described as follows:
  • Serological testing Collect mouse peripheral serum, and use commercial kits to detect alanine aminotransferase (Alamine aminotransferase, also known as GPT) and aspartate aminotransferase (AST also known as GOT) that reflect liver function indicators; reaction Creatinine (Cr) and blood urea nitrogen (BUN), which are indicators of renal function.
  • GPT alanine aminotransferase
  • AST aspartate aminotransferase
  • BUN blood urea nitrogen
  • tissue reactive oxygen species Grind mouse heart, lung, liver and kidney tissues, add horseradish peroxidase and Amplex Red to detect the amount of reactive oxygen species produced in each tissue.
  • the final form of reactive oxygen species in tissue cells is hydrogen peroxide.
  • the product of hydrogen peroxide catalyzed by horseradish peroxidase can be labeled with a fluorescent signal carried by Amplex Red.
  • the strength of the fluorescence signal represents the production of reactive oxygen species in the tissue.
  • MDA Malondialdehyde
  • TBA Thiobarbituric acid
  • MDA is a natural product produced by organisms after lipid oxidation. When the cells of the body undergo oxidative stress, lipid oxidation also occurs. Some fatty acids will be decomposed into a series of complex compounds after oxidation, including MDA. The level of lipid oxidation in the body can be reflected by detecting the content of MDA, so the measurement of MDA is widely used as an indicator of lipid oxidation.
  • MDA can react with TBA to form a red MDA-TBA adduct. The latter has a maximum absorption at 535nm, so it can be detected by colorimetry.
  • TBA Thiobarbituric acid
  • Detection of protein carbonyl groups during oxidative stress, the effects of free radicals on proteins include protein peptide chain scission, cross-linking and polymerization between protein molecules, oxidative deamination of protein amino acids, and free radicals attacking protein reducing groups, The malondialdehyde produced by oxidative cleavage of lipids is cross-linked with molecules produced by amino groups on proteins.
  • protein oxidative damage there are two main detection indicators for protein oxidative damage, namely protein carbonyl production and nitrotyrosine production.
  • the protein carbonyl detection kit of Sigma-Aldrich is used to form a stable dinitrophenylhydrazine complex by the interaction between the protein carbonyl and 2,4-dinitrophenylhydrazine (DNPH), the latter can be detected at 375nm Maximum light absorption.
  • DNPH 2,4-dinitrophenylhydrazine
  • the commonly used detection methods for cell apoptosis mainly include: caspas-3 activity determination, caspase-3 cleavage fragment determination and TUNEL in situ staining. Among them, caspase-3 activity determination and TUNEL in situ staining are performed using commercial kits, and caspase-3 cleavage fragments are detected by western blot using related antibodies.
  • Evans blue staining method is mainly used in the present invention to evaluate the permeability of pulmonary capillary vessels. 30 minutes before euthanasia, the mice were injected with 0.4% Evans Blue solution (50 mg/kg) through the tail vein. After lavage with 10 ml of normal saline, the mouse lung tissue was weighed and homogenized with normal saline. Mix the tissue lysate with 2ml of formamide, incubate at 60°C for 16 hours and then centrifuge at 20000g at 4°C for 5 minutes. Aspirate the supernatant to determine the concentration of Evans Blue by spectrophotometry (620, 740nm), and evaluate the permeability of the capillaries by the leakage rate of Evans Blue in the lung tissue.
  • Myeloperoxidase is a heme protease containing heme prosthetic group secreted by neutrophils, monocytes and macrophages in certain tissues. It is a member of the heme peroxidase superfamily. Myeloperoxidase is a function and activation mark of neutrophils, and its level and activity changes represent the function and activity status of neutrophil polymorphonuclear leukocytes.
  • a commercial kit is used to reflect the level of enzyme activity by detecting the content of the myeloperoxidase catalytic substrate.
  • Hematoxylin-Eosin (HE) staining method is mainly used for pathological analysis of mouse lung tissue. After the mice were euthanized, the right lobe of the lung was collected, and an appropriate volume of 10% formalin tissue fixative was added. After being allowed to stand at room temperature for 24 hours, it was dehydrated by alcohol and embedded in paraffin. A paraffin section with a thickness of 0.5 mm was cut with a paraffin microtome, and hematoxylin-eosin staining was performed through deparaffinization, hydration, nuclear and cytoplasmic staining, and decolorization, and then the pathological changes of lung tissue structure were observed under a microscope.
  • HE Hematoxylin-Eosin
  • mice 7-8 weeks old C57 male mice (body weight around 25 grams) are placed in the animal turnover room for later use. Randomly select several mice as fecal donor mice for future use. Then the remaining mice were randomly divided into four groups: the control group, the control group plus ⁇ -nicotinamide mononucleotide group, the implanted stool group and the implanted stool plus ⁇ -nicotinamide mononucleotide group (the number of each group was greater than 5). only).
  • Implant feces to prepare a sepsis model take out the prepared fecal suspension, and implant feces into the abdominal cavity of experimental mice by intraperitoneal injection at a ratio of 50 ml/kg body weight. Subsequently, 1 ml of normal saline containing 4 micrograms of buprenorphine was subcutaneously injected into the mice to replenish electrolytes and relieve pain in the mice. The end of the experiment was 6 hours later, and the damage of the heart, liver, and kidney of mice in different experimental groups was analyzed.
  • the control group and the control group plus ⁇ -nicotinamide mononucleotide group did not undergo any operation, the stool implantation group and the stool implantation plus ⁇ -nicotinamide mononucleotide group were first prepared according to the above method to prepare the sepsis model.
  • Example 2 ⁇ -nicotinamide mononucleotide prevents multiple organ damage in septic mice
  • the administration method of intraperitoneal injection is adopted, and the specific steps are as follows:
  • ⁇ -nicotinamide mononucleotide solution weigh the ⁇ -nicotinamide mononucleotide required by the experimental mice at a ratio of 500 mg/kg body weight, and dissolve it in an appropriate amount of normal saline (each mouse The injection volume is 100 microliters). For example, when preparing the ⁇ -nicotinamide mononucleotide injection required for 10 mice weighing 25 grams, the weighed 125 mg of ⁇ -nicotinamide mononucleotide should be dissolved in 1 ml of normal saline.
  • ⁇ -nicotinamide mononucleotide For the implanted stool plus ⁇ -nicotinamide mononucleotide group constructed in Example 1, 100 microliters were injected into the mouse's abdominal cavity while the experimental mice were implanted in the stool The prepared ⁇ -nicotinamide mononucleotide solution, for the control group of Example 1 plus ⁇ -nicotinamide mononucleotide group, the mice were injected with the same amount of ⁇ -nicotinamide mononucleotide. The control group and the stool implantation group were injected with the same amount of normal saline.
  • mice The heart function of the four groups of mice was measured by ultra-high resolution echocardiography after implantation of feces for 6 hours. After euthanasia, peripheral serum and heart, liver, lung and kidney tissue samples were collected from all mice for analysis of different tissues.
  • FIG. 1 Vehicle represents control mice; Feces represents sepsis In the mouse group, Saline represents normal saline; NMN represents the application of ⁇ -nicotinamide mononucleotide at the onset of sepsis.
  • Fractional Shortening (%) is the short-axis shortening rate, which represents cardiac contractility; MPO represents myeloperoxidase activity; ROS represents the production of reactive oxygen species; MDA represents the content of malondialdehyde.
  • Figure 1 the cardiac contractile function of septic mice was significantly weaker than that of control mice, and ⁇ -nicotinamide mononucleotide improved the cardiac function of septic mice (Figure 1A). Grinding mouse myocardial tissues, using a commercial kit to detect the activity of myeloperoxidase and the production of reactive oxygen species and malondialdehyde in myocardial tissues. The results are shown in Figure 1B-D. Sepsis leads to myocardial tissue marrow.
  • ⁇ -nicotinamide mononucleotide significantly reduced myeloperoxidase activity and reactive oxygen species and malondialdehyde in myocardial tissue of septic mice content. The results show that ⁇ -nicotinamide mononucleotide can improve the heart function of septic mice.
  • FIG. 2 shows the relevant test results of lung tissue.
  • Caspase-3 activity represents the level of apoptosis.
  • the other letters and symbols have the same meaning as in Figure 1.
  • the lung tissue is embedded in formalin, dehydrated and embedded in paraffin. After sectioning, hematoxylin-eosin staining was performed.
  • the representative picture is shown in 2A: the lung tissue of septic mice showed inflammatory reactions such as alveolar wall thickening and cell nucleus aggregation.
  • ⁇ -nicotinamide mononucleotide effectively improved sepsis Inflammatory damage to lung tissue caused by disease.
  • FIG 3 shows the relevant test results of liver tissue.
  • Protein carbonyl represents the content of protein carbonyl group
  • AST represents aspartate aminotransferase that causes liver cell damage
  • other letters and symbols have the same meaning as Figure 1.
  • Protein in liver tissue of septic mice The content of carbonyl and malondialdehyde and the activity of myeloperoxidase were significantly higher than those of control mice, suggesting that the liver tissue of sepsis mice had oxidative stress and inflammatory damage.
  • ⁇ -nicotinamide mono Nucleotides can significantly improve the level of oxidative stress and inflammatory damage in liver tissue caused by sepsis (Figure 3A, B and D).
  • ⁇ -nicotinamide mononucleotide can improve liver tissue lesions in septic mice.
  • FIG 4 shows the related test results of kidney tissue.
  • BUN stands for urea nitrogen that reflects kidney cell damage.
  • Other letters and symbols have the same meaning as Figures 1 and 3.
  • the protein carbonyl and malondialdehyde in the kidney tissue of septic mice The content and the activity of myeloperoxidase were significantly higher than those of control mice, suggesting that oxidative stress and inflammatory damage occurred in the kidney tissue of sepsis mice.
  • ⁇ -nicotinamide mononucleotide can be significantly Improve the level of oxidative stress and inflammatory damage in the kidney tissue caused by sepsis ( Figure 4A, B and D).
  • the increase in serum urea nitrogen content indicates renal insufficiency in septic mice.
  • ⁇ -nicotinamide mononucleotide After the application of ⁇ -nicotinamide mononucleotide, the urea nitrogen content is down-regulated to protect renal function (Figure 4C).
  • ⁇ -nicotinamide mononucleotide can improve renal tissue lesions in septic mice.
  • Example 3 ⁇ -nicotinamide mononucleotide treatment of multiple organ damage in septic mice
  • the administration method of intraperitoneal injection is adopted, and the specific steps are as follows:
  • ⁇ -nicotinamide mononucleotide solution weigh the ⁇ -nicotinamide mononucleotide required by the experimental mice at a ratio of 500 mg/kg body weight, and dissolve it in an appropriate amount of normal saline (each mouse The injection volume is 100 microliters). For example, when preparing the ⁇ -nicotinamide mononucleotide injection required for 10 mice weighing 25 grams, the weighed 125 mg of ⁇ -nicotinamide mononucleotide should be dissolved in 1 ml of physiological saline.
  • mice were injected with 100 into the tail vein Microliters of the prepared ⁇ -nicotinamide mononucleotide solution, for the control group of Example 1 plus ⁇ -nicotinamide mononucleotide group, mice were injected with the same amount of ⁇ -nicotinamide mononucleotide.
  • the control group and the stool implantation group were injected with the same amount of normal saline.
  • mice The heart function of the four groups of mice was measured by ultra-high resolution echocardiography after implantation of feces for 6 hours. After euthanasia, peripheral serum and heart, liver, lung and kidney tissue samples were collected from all mice for analysis of different tissues.
  • mice were anesthetized with 1-2% isoflurane, and the contractile function of the mouse heart was detected by ultra-high resolution echocardiography.
  • the results are shown in Figure 5.
  • NMN-1H represents the application of ⁇ - when sepsis is onset for 1 hour. Nicotinamide mononucleotide, other letters and symbols have the same meaning as above.
  • the cardiac contractile function of sepsis mice was significantly weaker than that of control mice.
  • ⁇ -nicotinamide mononucleotide was used to improve the cardiac function of sepsis mice (Figure 5A).
  • the mouse myocardial tissue was ground, and a commercial kit was used to detect the activity of myeloperoxidase and the production of reactive oxygen species and malondialdehyde in the myocardial tissue.
  • the results are shown in Figure 5B-D.
  • Sepsis leads to myocardial tissue marrow. Increased peroxidase activity and excessive production of reactive oxygen species and malondialdehyde.
  • Application of ⁇ -nicotinamide mononucleotide 1 hour after the onset of sepsis significantly reduced myeloperoxidase activity in myocardial tissue of septic mice And the content of active oxygen and malondialdehyde.
  • the application of ⁇ -nicotinamide mononucleotide one hour after the onset of sepsis can improve the heart function of septic mice.
  • FIG. 6 illustrates the relevant test results of lung tissue.
  • NMN-1H represents the application of ⁇ -nicotinamide mononucleotide at the onset of sepsis one hour, and other letters and symbols have the same meaning as above.
  • Formalin-embedded lung tissues were dehydrated and paraffin-embedded in sections and then stained with hematoxylin and eosin.
  • a representative picture is shown in 6A: The lung tissue of septic mice showed inflammation such as alveolar wall thickening and cell nucleus aggregation.
  • the application of ⁇ -nicotinamide mononucleotide 1 hour after the onset of disease can effectively improve the inflammatory damage of lung tissue caused by sepsis.
  • FIG. 7 shows the results of the related tests of liver tissue.
  • NMN-1H represents the application of ⁇ -nicotinamide mononucleotide at the onset of sepsis one hour, and other letters and symbols have the same meaning as above.
  • FIG 8 illustrates the related test results of kidney tissue.
  • NMN-1H represents the application of ⁇ -nicotinamide mononucleotide at the onset of sepsis one hour, and other letters and symbols have the same meaning as above.
  • the content of protein carbonyl and malondialdehyde and the activity of myeloperoxidase in the kidney tissue of sepsis mice were significantly higher than those of control mice, suggesting that the kidney tissue of sepsis mice had oxidative stress and inflammatory damage
  • the application of ⁇ -nicotinamide mononucleotide 1 hour after the onset of disease can significantly improve the level of oxidative stress and inflammatory damage in the kidney tissue caused by sepsis ( Figure 8A, B and D).
  • ⁇ -nicotinamide mononucleotide was used to down-regulate the urea nitrogen content and protect renal function one hour after the onset of disease ( Figure 8C).
  • ⁇ -nicotinamide mononucleotide can be used to improve renal tissue lesions in septic mice one hour after the onset of sepsis.
  • the present invention proves through preliminary in vivo experiments that the intraperitoneal injection of ⁇ -nicotinamide mononucleotide at the same time or after the preparation of the mouse sepsis model effectively improves the damage of the main organs of the mouse sepsis, and proves that the ⁇ -nicotinamide mononuclear
  • the preventive and therapeutic protective effects of Glycolic acid on multiple organs of septic mice indicate that ⁇ -nicotinamide mononucleotide can be used to prepare drugs for the treatment and prevention of septic organ damage, thereby providing a good basis for diseases in related fields. New treatment methods and approaches.

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Abstract

β-烟酰胺单核苷酸在制备脓毒症引起的器官损伤的治疗和/或预防药物中的应用。β-烟酰胺单核苷酸的新用途，其对脓毒症引发的多器官损伤具有治疗和预防保护作用，而且β-烟酰胺单核苷酸易溶于水，普及性和可操作性强，给药途径多样。

Description

β-NMN在制备脓毒症器官损伤的治疗、预防药物中的应用 技术领域
本发明涉及生物医药领域，尤其涉及一种β-NMN在制备脓毒症器官损伤的治疗、预防药物中的应用。
背景技术
脓毒症(sepsis)是感染后机体反应失调所导致危及生命的多器官功能障碍，可发展为脓毒性休克(septic shock)和多器官功能障碍综合征(multiple organ dysfunction syndrome,MODS)，是临床危重患者的最主要死亡原因之一。据报道，全球每年有近1800万脓毒症患者，我国每年脓毒症患病人数估计在500万以上，且患者数目以近10％的速度逐年递增，造成严重的医疗负担。国内外学者的研究资料表明，尽管随着容量评估与复苏、早期抗感染及器官支持等“集束化”治疗策略的不断进步，患者死亡率仍然居高不下。到目前为止，尚无有效的预防和治疗脓毒症的措施。因此，寻找预防和治疗脓毒症的新靶点和新治疗措施迫在眉睫。
迄今为止，临床上没有任何针对脓毒症的特效治疗措施，目前临床上主要以支持疗法为主，简述如下：
1.液体复苏：绝大多数脓毒症患者早期都会出现循环衰竭，确定脓毒症患者存在持续性低血压，在入住重症监护病房之前，会通过静脉补液的方式进行初始复苏和扩容治疗。然而，脓毒症患者心脏舒张功能受限，短时间内给予大量晶体液弹丸式输注可能会超出患者心脏的代偿能力，这不仅不能增加心输出量，反而会引起包括肺水肿、高中心静脉压所致的肝肾功能损害在内的严重的血流动力学后果。因此，这也对临床医生在液体的类型、计量、疗程的评估提出了更高的要求。
2.血管活性药物的运用：为了使患者平均动脉压大于等于65mmHg，需使用血管升压药，一般首选去甲肾上腺素，配合使用血管加压素时以提高平均动脉压值。但是，脓毒症休克患者活血性药物的应用实际、剂量及药物选择也存在争议。除了去甲肾上腺素和血管加压素，血管紧张素II在血管活性药物在的地位也逐渐凸显，有研究发现血管紧张素II能有效升高常规血管活性药物无效的血管扩张性休克患者的动脉血压。至于血管活性药物的应用时机和合适减量甚至停用目前仍没有统一的定论。
3.抗生素的运用：较早的研究发现脓毒症休克患者每延迟1小时应用抗生素其病死率 评价增加7.6％。随后的研究并未发现在复苏治疗前的6小时从急诊分诊到抗生素应用的时间与住院期间病死率的相关性，但是延迟到出现脓毒症休克再应用抗生素则显著增加死亡风险。最近的临床研究发现与入院后应用抗生素相比，救护车上应用抗生素的脓毒症患者的28天或90天的病死率差异无统计学意义。目前尚有的证据并不支持所有的脓毒症患者在严格时间窗内应用抗生素，且抗生素的滥用还会引起耐药性的增加。
4.其他辅助治疗方式：严重脓毒症患者还需用高流量鼻导管或面罩吸氧等方式来保护通气以治疗呼吸窘迫和低血氧症；当患者血糖水平连续超过180mg/dl时，则需使用胰岛素，但是床旁指标检测的毛细血管的血糖水平不能充分地反映患者血浆和动脉的血糖情况。
综上所述，目前临床上通过液体复苏、使用血管活性药物和抗感染药物等集束化治疗方案一直是拯救脓毒症运动的核心策略，但是缺乏特效治疗措施。因此继续进行临床前的基础研究以提供有效和特异的防治方案就显得尤为迫切。
烟酰胺单核苷酸(Nicotinamide Mononucleotide，NMN)是衍生自核糖和烟酰胺的核苷酸，在体内它可以通过两条途径合成：一是由烟酰胺经烟酰胺磷酸核糖转移酶(NAMPT)催化合成；另一途径是由烟酰胺核苷(Nicotinamide riboside)经烟酰胺核苷激酶催化合成。β-烟酰胺单核苷酸(β–Nicotinamide Mononucleotide，β-NMN)是烟酰胺单核苷酸的活性形式，它作为烟酰胺腺嘌呤二核苷酸(Nicotinamide adenine dinucleotide,NAD +；又称辅酶I)的前体，其功能也主要通过NAD +来实现。NAD +是一种传递质子的辅酶，它是糖酵解、糖异生、三羧酸循环及呼吸链中必不可少的辅酶，在为细胞提供能量、DNA修复、抗衰老等方面发挥着不可替代的作用。因此，维持细胞内NAD +的含量就显得尤为重要。尽管体内有多种途径可以合成NAD +，但研究表明β-烟酰胺单核苷酸通过烟酰胺单核苷酸酶1-3(Nicotinamide nucleotide adenylytransferase 1-3,NMNAT1-3)催化作用下产生的NAD +占人体NAD +总量的85％，是维持体内NAD +含量最重要的途径。有意思的是，研究报道在脓毒症条件下体内NAD +的含量显著下降，并且可能与脓毒症主要脏器持续恶化的功能衰竭密切相关。发明人所在团队最近的研究证实应用烟酰胺核苷提高NAD +含量能够有效减轻脓毒症主要脏器损伤，但是它需要在脓毒症发生之前预防给药，如在脓毒症发生后给予烟酰胺核苷则没有任何保护作用，其原因是脓毒症条件严重降低烟酰胺核苷激酶的表达，从而导致烟酰胺核苷不能在体内转变为β-烟酰胺单核苷酸(Hong G,et al.Free Radiac Bio Med 2018；123:125-137)。因此，烟酰胺核苷不可能用于治疗脓毒症主要脏器损伤。
发明内容
为解决上述技术问题，本发明的目的是提供一种β-NMN在制备脓毒症器官损伤的治疗、 预防药物中的应用，本发明公开了一种新型治疗和/或预防脓毒症器官损伤的药物。
本发明的技术方案如下：
本发明公开了β-烟酰胺单核苷酸(β-NMN)在制备脓毒症引起的器官损伤的治疗和/或预防药物中的应用。
进一步地，器官损伤包括心损伤、肺损伤、肝损伤和肾损伤中的一种或几种。
进一步地，药物的给药方式为注射给药或口服给药。
进一步地，注射给药包括静脉和/或腹腔注射给药。
进一步地，药物的给药剂量为300-1000mg/kg。优选地，药物的给药剂量为500mg/kg。
进一步地，药物用于提高脓毒症引起的辅酶I(NAD +)的含量降低。
进一步地，药物用于减少脓毒症引起的器官中髓过氧化物酶的活性和活性氧、丙二醛的含量。
借由上述方案，本发明至少具有以下优点：
1、本发明公开了β-烟酰胺单核苷酸的新用途，其对脓毒症引发的多器官损伤具有治疗和预防保护作用，从而为相关领域的疾病提供了新的治疗手段和途径。在脓毒症发病前或后，单次给予β-烟酰胺单核苷酸可以提升体内NAD +水平，并改善脓毒症小鼠心功能、降低脓毒症小鼠体内的氧化应激水平和减轻心、肝、肺、肾等器官的损伤作用。
2、β-烟酰胺单核苷酸配制方法简单：β-烟酰胺单核苷酸易溶于水，无需准备其他特殊的溶剂，普及性和可操作性很强。
3、给药途径可以多样化：本发明中通过静脉注射和腹腔注射的给药方式均有效，此外，也可以通过口服的方式给药。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1是本发明实施例2中心脏功能、氧化应激和炎性反应的检测结果；
图2是本发明实施例2中肺组织病理学、氧化应激、炎性反应和细胞凋亡的检测结果；
图3是本发明实施例2中肝组织氧化应激、细胞损伤和炎性反应的检测结果；
图4是本发明实施例2中肾组织氧化应激、细胞损伤和炎性反应的检测结果；
图5是本发明实施例3中心脏功能、氧化应激和炎性反应的检测结果；
图6是本发明实施例3中肺组织氧化应激、炎性反应和细胞凋亡的检测结果；
图7是本发明实施例3中肝组织氧化应激、细胞损伤和炎性反应的检测结果；
图8是本发明实施例3中肾组织氧化应激、细胞损伤和炎性反应的检测结果。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
本发明主要运用了以下实验方法来检测脓毒症小鼠多个器官代表性的生化参数和损伤指标，简述如下：
1.超声评价心功能：1％异氟烷麻醉小鼠，应用高分辨率的心脏超声探头(35-MHz linear array transducer attached to Vevo 2100 ultrasound system)在二尖瓣乳头肌水平获取心室切面，在M模式图像测量心室前后壁厚度、心室腔直径大小及心室壁移动，然后分析收缩功能：短轴缩短率(FS％)和左室射血分数(EF％)。在心尖四腔心切面，利用脉冲多普勒组织超声心动图分析舒张功能：峰值心房血流速度A，峰值舒张早期血流速度E，和它们的比值(E/A)。
2.血清学检测：采集小鼠外周血清，利用商业化的试剂盒检测反应肝功能指标的谷丙转氨酶(Alamine aminotransferase,ALT又称GPT)和谷草转氨酶(Aspartate aminotransferase,AST又称GOT)；反应肾功能指标的肌酐(Creatinine,Cr)和尿素氮(Blood urea nitrogen,BUN)。
3.组织活性氧的检测：研磨小鼠心肺肝肾组织，加入辣根过氧化物酶和Amplex Red检测各组织活性氧的产生量。组织细胞内活性氧的最终形式是过氧化氢，辣根过氧化物酶催化过氧化氢的产物可以被Amplex Red携带的荧光信号标记。荧光信号的强弱代表组织活性氧产量的多少。
4.丙二醛(Malondialdehyde,MDA)的检测：该检测方法是基于MDA和硫代巴比妥酸(Thiobarbituric acid,TBA)产生红色产物的显色反应。MDA是生物体发生脂质氧化后产生的一种天然产物。当机体细胞发生氧化应激(oxidative stress)时，同时也会发生脂质氧化，一些脂肪酸氧化后会分解为一系列复杂的化合物，其中包括MDA。通过检测MDA的含量可以反应体内脂质氧化的水平，因此MDA的测定被广泛用作脂质氧化的指标。在酸性环境中处于较高的温度时MDA可以于TBA发生反应，形成红色的MDA-TBA加合物，后者在535nm处有最大吸收光，因此可以通过比色法进行检测。具体的反应原理如下所示：
Figure PCTCN2020110120-appb-000001
5.蛋白羰基的检测：氧化应激过程中，自由基对蛋白质的作用包括蛋白质肽链断裂、蛋白质分子之间相互交联聚合，蛋白质氨基酸发生氧化脱氨反应、自由基攻击蛋白质还原性集团、脂类氧化裂解所产生的丙二醛与蛋白质上的氨基产生的分子交联等。目前对蛋白质氧化损伤的检测指标主要有两个，分别是蛋白羰基生成和硝基酪氨酸的生成。本发明中使用Sigma-Aldrich的蛋白羰基检测试剂盒通过由蛋白质羰基与2，4-二硝基苯肼(DNPH)相互作用形成稳定的二硝基苯肼络合物，后者可在375nm有最大吸收光。
6.细胞凋亡的检测：细胞凋亡常用的检测方法主要包括：caspas-3活性测定、caspase-3的裂解片段测定和TUNEL原位染色等。其中caspase-3活性测定和TUNEL原位染色利用商业化的试剂盒进行，caspase-3裂解片段则通过western blot利用相关抗体检测。
7.肺毛细血管通透性的检测：本发明中主要运用伊文思蓝染色法评估肺毛细血管通透性。在安乐死前30分钟给小鼠通过尾静脉向小鼠注射0.4％伊文思蓝溶液(50mg/kg)。用10毫升生理盐水灌洗后小鼠肺组织后，称重并用生理盐水匀浆。将组织裂解液和2毫升甲酰胺混合，在60度下孵育16小时后20000g,4度离心5分钟。吸取上清液通过分光光度法(620，740nm处)测定伊文思蓝的浓度，通过肺组织中伊文思蓝的漏出率评估微血管的通透性。
8.髓过氧化物酶(myeloperoxidase,MPO)的检测：髓过氧化物酶是由中性粒细胞、单核细胞和某些组织的巨噬细胞分泌的含血红素辅基的血红素蛋白酶，是血红素过氧化物酶超家族成员之一。髓过氧化物酶是中性粒细胞的功能和激活标志，其水平及活性变化代表着嗜中性多形核白细胞的功能和活性状态。本发明中利用商业化的试剂盒通过检测髓过氧化物酶催化底物的含量来反应酶活性的高低。
9.组织病理学分析：本发明中主要运用苏木素-伊红(Hematoxylin-Eosin，HE)染色法进行小鼠肺组织的病理学分析。小鼠安乐死后采集肺右叶，加入适当体积的10％福尔马林组织固定液，室温静置24时后经酒精脱水、石蜡包埋。用石蜡切片机切取0.5毫米厚度的石蜡切片，经脱蜡、水化、细胞核和胞质着色、脱色等步骤进行苏木素-伊红染色，然后在显微镜下观察肺组织结构的病理变化。
实施例1:小鼠脓毒症模型的制备
本发明以下实施例运用的是腹腔注射粪便的方式制备脓毒症小鼠模型，具体方法步骤如下：
1、小鼠：7-8周龄C57雄性小鼠(体重在25克左右)，将其安置在动物周转房备用。随机挑选若干只作为粪便捐献者小鼠备用。然后将剩余小鼠随机分成四组：对照组、对照组加β-烟酰胺单核苷酸组、植入粪便组和植入粪便加β-烟酰胺单核苷酸组(每组数量大于5只)。
2、采集粪便：安乐死后解剖捐献者小鼠，采集其盲肠内粪便后称重，每75mg粪便溶于1毫升生理盐水中(植入粪便体积为50毫升/公斤体重)，充分混匀后4度静置24小时，待用。如，拟对10只体重为25克小鼠制备脓毒症模型则应采集938毫克粪便，溶于12.5毫升生理盐水中。
3、植入粪便制备脓毒症模型：取出准备好的粪便混悬液，按50毫升/公斤体重的比例采用腹腔注射的方式给实验小鼠腹腔植入粪便。随后给小鼠皮下注射1毫升含有4微克丁丙诺啡的生理盐水以补充电解质、减轻小鼠疼痛感。6小时后即为实验终点，分析不同实验组小鼠的心、肝、肾等的损伤。对照组和对照组加β-烟酰胺单核苷酸组不进行任何操作、植入粪便组和植入粪便加β-烟酰胺单核苷酸组均按照上述方法先制备脓毒症模型。
实施例2:β-烟酰胺单核苷酸预防脓毒症小鼠多器官的损伤
本发明中采用的是腹腔注射的给药方式，具体步骤如下：
1、配制β-烟酰胺单核苷酸溶液：按照500毫克/公斤体重的比例称量实验小鼠所需的β-烟酰胺单核苷酸，溶于适量的生理盐水中(每只小鼠的注射体积为100微升)。如，配置10只体重为25克的小鼠所需的β-烟酰胺单核苷酸注射液时应将称取的125毫克β-烟酰胺单核苷酸溶于1毫升生理盐水中。
2、腹腔注射β-烟酰胺单核苷酸：对于实施例1构建的植入粪便加β-烟酰胺单核苷酸组，在实验小鼠植入粪便的同时给小鼠腹腔注射100微升配置好的β-烟酰胺单核苷酸溶液，对于实施例1对照组加β-烟酰胺单核苷酸组，对小鼠注射等量的β-烟酰胺单核苷酸。对照组和植入粪便组注射等量生理盐水。
植入粪便6个小后利用超高分辨率心动超声仪检测四组小鼠心功能，安乐死后采集所有小鼠外周血清和心肝肺肾等组织样本，进行不同组织的分析。
用1-2％异氟烷麻醉小鼠，借助超高分辨率心动超声仪检测小鼠心脏的收缩功能，结果如图1所示，图1中，Vehicle代表对照组小鼠；Feces代表脓毒症小鼠组，Saline代表生理盐水；NMN代表脓毒症发病时应用β-烟酰胺单核苷酸。Fractional Shortening(％)为短轴缩短率，代表心脏收缩功能；MPO代表髓过氧化物酶活性；ROS代表活性氧的产量；MDA代表丙二醛的含量。图1中数据为平均值±标准差，采用单因素方差分析，n＝5，*P＜0.05vs Vehicle (Saline)，
Figure PCTCN2020110120-appb-000002
vs Feces(Saline)。图1中，脓毒症小鼠心脏收缩功能较对照组小鼠明显减弱，β-烟酰胺单核苷酸提高脓毒症小鼠的心功能(如图1A)。研磨小鼠心肌组织，利用商业化的试剂盒检测心肌组织中髓过氧化物酶的活性和活性氧、丙二醛的产生量，结果如图1B-D所示，脓毒症导致心肌组织髓过氧化物酶活性增加和活性氧和丙二醛的过量产生，β-烟酰胺单核苷酸显著减少脓毒症小鼠心肌组织中髓过氧化物酶的活性和活性氧、丙二醛的含量。结果表明，β-烟酰胺单核苷酸可提高脓毒症小鼠心功能。
图2图示了肺组织的相关检测结果，图中Caspase-3 activity代表细胞凋亡水平，其他字母和符号含义与图1相同，用福尔马林包埋肺组织，经脱水、石蜡包埋切片后进行苏木素-伊红染色，代表性图片如2A所示：脓毒症小鼠肺组织出现了肺泡壁增厚、细胞核聚集等炎症反应，β-烟酰胺单核苷酸有效地改善脓毒症造成的肺组织炎性损伤。这一结果和代表炎性损伤的髓过氧化物酶活性、代表氧化应激水平的活性氧和丙二醛以及细胞凋亡的检测结果也是一致的(如图2B-D)。脓毒症诱导肺组织细胞凋亡，β-烟酰胺单核苷酸降低脓毒症小鼠肺组织细胞凋亡水平(如图2E)。综上，β-烟酰胺单核苷酸可减轻脓毒症小鼠肺组织病变。
图3图示了肝组织的相关检测结果，图中Protein carbonyl代表蛋白羰基的含量，AST代表肝细胞损伤的谷草转氨酶，其他字母和符号含义与图1相同，脓毒症小鼠肝组织中蛋白羰基、丙二醛的含量和髓过氧化物酶的活性明显高于对照组小鼠，提示脓毒症小鼠肝组织出现了氧化应激反应和炎性损伤，类似的，β-烟酰胺单核苷酸可以显著改善脓毒症引起的肝组织氧化应激水平和炎性损伤程度(如图3A、B和D)。此外，代表肝细胞损伤的谷草转氨酶在脓毒症小鼠外周血清中显著增加，而β-烟酰胺单核苷酸能够降低脓毒症小鼠谷草转氨酶的表达量(如图3C)。综上，β-烟酰胺单核苷酸可改善脓毒症小鼠肝组织病变。
图4图示了肾组织的相关检测结果，图中BUN代表反映肾细胞损伤的尿素氮，其他字母和符号含义与图1和3相同，脓毒症小鼠肾组织中蛋白羰基、丙二醛的含量和髓过氧化物酶的活性明显高于对照组小鼠，提示脓毒症小鼠肾组织出现了氧化应激反应和炎性损伤，类似的，β-烟酰胺单核苷酸可以显著改善脓毒症引起的肾组织氧化应激水平和炎性损伤程度(如图4A、B和D)。血清尿素氮含量增加提示脓毒症小鼠肾功能不全，应用β-烟酰胺单核苷酸后尿素氮含量下调、保护肾功能(如图4C)。综上，β-烟酰胺单核苷酸可改善脓毒症小鼠肾组织病变。
实施例3:β-烟酰胺单核苷酸治疗脓毒症小鼠多器官的损伤
本发明中采用的是腹腔注射的给药方式，具体步骤如下：
1、配制β-烟酰胺单核苷酸溶液：按照500毫克/公斤体重的比例称量实验小鼠所需的β-烟酰胺单核苷酸，溶于适量的生理盐水中(每只小鼠的注射体积为100微升)。如，配置10 只体重为25克的小鼠所需的β-烟酰胺单核苷酸注射液时应将称取的125毫克β-烟酰胺单核苷酸溶于1毫升生理盐水中。
2、静脉注射β-烟酰胺单核苷酸：对于实施例1构建的植入粪便加β-烟酰胺单核苷酸组，在实验小鼠植入粪便1小时后给小鼠尾静脉注射100微升配置好的β-烟酰胺单核苷酸溶液，对于实施例1对照组加β-烟酰胺单核苷酸组，对小鼠注射等量的β-烟酰胺单核苷酸。对照组和植入粪便组注射等量生理盐水。
植入粪便6个小后利用超高分辨率心动超声仪检测四组小鼠心功能，安乐死后采集所有小鼠外周血清和心肝肺肾等组织样本，进行不同组织的分析。
用1-2％异氟烷麻醉小鼠，借助超高分辨率心动超声仪检测小鼠心脏的收缩功能，结果如图5所示，图中NMN-1H代表脓毒症发病1小时候应用β-烟酰胺单核苷酸，其他字母和符号与上文含义相同。脓毒症小鼠心脏收缩功能较对照组小鼠明显减弱，脓毒症发病1小时后应用β-烟酰胺单核苷酸提高脓毒症小鼠的心功能(如图5A)。研磨小鼠心肌组织，利用商业化的试剂盒检测心肌组织中髓过氧化物酶的活性和活性氧、丙二醛的产生量，结果如图5B-D所示，脓毒症导致心肌组织髓过氧化物酶活性增加和活性氧和丙二醛的过量产生，脓毒症发病1小时后应用β-烟酰胺单核苷酸显著减少脓毒症小鼠心肌组织中髓过氧化物酶的活性和活性氧、丙二醛的含量。综上，脓毒症发病1小时后应用β-烟酰胺单核苷酸可提高脓毒症小鼠心功能。
图6图示了肺组织的相关检测结果，图中NMN-1H代表脓毒症发病1小时候应用β-烟酰胺单核苷酸，其他字母和符号与上文含义相同。福尔马林包埋肺组织，经脱水、石蜡包埋切片后进行苏木素-伊红染色，代表性图片如6A所示：脓毒症小鼠肺组织出现了肺泡壁增厚、细胞核聚集等炎症反应，发病1小时后应用β-烟酰胺单核苷酸有效地改善脓毒症造成的肺组织炎性损伤。这一结果和代表炎性损伤的髓过氧化物酶活性、代表氧化应激水平的活性氧和丙二醛以及细胞凋亡的检测结果也是一致的(如图6B-D)。脓毒症诱导肺组织细胞凋亡，发病1小时后应用β-烟酰胺单核苷酸降低脓毒症小鼠肺组织细胞凋亡水平(如图6C)。综上，脓毒症发病1小时后可应用β-烟酰胺单核苷酸减轻脓毒症小鼠肺组织病变。
图7图示了肝组织的相关检测结果，图中NMN-1H代表脓毒症发病1小时候应用β-烟酰胺单核苷酸，其他字母和符号与上文含义相同。脓毒症小鼠肝组织中蛋白羰基、丙二醛的含量和髓过氧化物酶的活性明显高于对照组小鼠，提示脓毒症小鼠肝组织出现了氧化应激反应和炎性损伤，类似的，发病1小时后应用β-烟酰胺单核苷酸可以显著改善脓毒症引起的肝组织氧化应激水平和炎性损伤程度(如图7A、B和D)。此外，代表肝细胞损伤的谷草转氨酶在脓毒症小鼠外周血清中显著增加，而发病1小时后应用β-烟酰胺单核苷酸能够降低脓毒 症小鼠外周血清谷草转氨酶的表达量(如图7C)。综上，脓毒症发病1小时后应用β-烟酰胺单核苷酸可改善脓毒症小鼠肝组织病变。
图8图示了肾组织的相关检测结果，图中NMN-1H代表脓毒症发病1小时候应用β-烟酰胺单核苷酸，其他字母和符号与上文含义相同。脓毒症小鼠肾组织中蛋白羰基、丙二醛的含量和髓过氧化物酶的活性明显高于对照组小鼠，提示脓毒症小鼠肾组织出现了氧化应激反应和炎性损伤，类似的，发病1小时后应用β-烟酰胺单核苷酸可以显著改善脓毒症引起的肾组织氧化应激水平和炎性损伤程度(如图8A、B和D)。外周血清尿素氮含量增加提示脓毒症小鼠肾功能不全，发病1小时后应用β-烟酰胺单核苷酸下调尿素氮含量、保护肾功能(如图8C)。综上，脓毒症发病1小时后可应用β-烟酰胺单核苷酸改善脓毒症小鼠肾组织病变。
本发明通过前期体内实验证实在制备小鼠脓毒症模型的同时或之后腹腔注射β-烟酰胺单核苷酸有效改善小鼠脓毒症主要脏器的损伤，证明了β-烟酰胺单核苷酸对脓毒症小鼠多器官的预防保护作用和治疗保护作用，说明β-烟酰胺单核苷酸可用于制备脓毒症器官损伤的治疗、预防药物，从而为相关领域的疾病提供了新的治疗手段和途径。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (6)

  1. β-烟酰胺单核苷酸在制备脓毒症引起的器官损伤的治疗和/或预防药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于：所述器官损伤包括心损伤、肺损伤、肝损伤和肾损伤中的一种或几种。
  3. 根据权利要求1所述的应用，其特征在于：所述药物的给药方式为注射给药或口服给药。
  4. 根据权利要求3所述的应用，其特征在于：注射给药包括静脉和/或腹腔注射给药。
  5. 根据权利要求3所述的应用，其特征在于：所述药物的给药剂量为300-1000mg/kg。
  6. 根据权利要求1所述的应用，其特征在于：所述药物用于提高脓毒症引起的辅酶I的含量降低。
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