Pharmaceutical composition and application thereof
Targeting PGAM5 by a composition of amphilin or nicotinamide mononucleotide NMN, the treatment problem of macrophage-related inflammation is solved, significantly reduces the expression of inflammatory factors, and improves sepsis and inflammatory bowel disease pathological conditions provide better therapeutic effects.
Patent Information
- Application Number
- CN202311296970.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-09
- Publication Date
- 2024-12-24
AI Technical Summary
The prior art is difficult to effectively prevent and treat macrophage-related inflammatory diseases, such as sepsis and its induced acute lung injury and inflammatory bowel disease, especially due to the fewer drugs developed for PGAM5 targets, resulting in therapeutic effects. limited.
Using a composition of Punicalagin or Punicalin and nicotinamide single nucleotide NMN/NAD+, it inhibits the expression and inflammatory response of macrophages by targeting PGAM5, providing a pharmaceutical composition. To prevent and treat sepsis, acute lung injury and inflammatory bowel disease.
It significantly reduced the expression of inflammatory factors such as TNF-α, IL-1β, IL-6, COX-2, etc., alleviated the pathological conditions of sepsis and inflammatory bowel disease. It has better compared to Punicalagin or NMN alone. The therapeutic effect is not obvious toxic.
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Figure CN119174774A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of medical technology, and particularly relates to a method for preparing a punicalagin or punicalin and nicotinamide mononucleotide NMN / NAD + The combination comprises a pharmaceutical composition, and its use in preventing and / or treating macrophage-related inflammation including inflammatory bowel disease, sepsis and inflammatory lung injury induced therein, and the therapeutic effect is derived from Punicalagin / Punicalin targeting the PGAM5 protein. Background Art
[0002] Nicotinamide mononucleotide (β-Nicotinamide Mononucleotide, NMN; CAS registration number: 1094-61-7) is a substance inherent in the human body. It is a derivative of vitamin B and is commonly found in daily foods such as broccoli, tomatoes, and raw beef. Its structural formula is shown in the figure below. NMN is used as NAD in the human body. + Precursor, whose biological activity is mainly through NAD + Implementation, NAD + Nicotinamide adenine dinucleotide, or coenzyme I, is a proton-transferring coenzyme that participates in thousands of biological catalytic reactions in the human body, such as glycolysis, gluconeogenesis, respiratory chain, and tricarboxylic acid cycle, covering energy supply, DNA repair (PARPs-mediated), and NADP (H) synthesis. It has antioxidant and anti-aging effects. There are also literature reports that NMN also plays a role in the treatment of diabetes, heart protection, and anti-Alzheimer's disease.
[0003] Punicalagin (CAS registration number: 65995-63-3) is a polyphenol component isolated from the leaves of Punica granatum L. or Terminalia catappa L. of the Punicaceae family. It is composed of the precursors Punicalin and Urolithin. Its molecular structure is shown in the figure below. Pomegranate fruit, peel, and leaves can be used as medicine. According to literature reports, in Chinese folk prescriptions, pomegranate medicinal materials have insecticidal, astringent, astringent, and anti-dysentery effects. Modern biochemical research has found that pomegranate extracts have antibacterial and antiviral pharmacological activities. However, so far, there are few reports on the anti-inflammatory biological activity of Punicalagin.
[0004] Phosphoglycerate mutase 5 (PGAM5) is a special enzyme that acts as an atypical SER / THR phosphatase in the body and is a regulator of oxidative stress and necrosis. According to the published literature, PGAM5 is mainly involved in inflammatory response, mitosis, cell apoptosis, lipid metabolism and other processes, and is one of the important proteins that regulate the normal functions of the human body. At present, there are still few drugs developed with PGAM5 as the target, which has broad research prospects.
[0005] Sepsis is a critical illness caused by pathogenic microorganisms such as bacteria invading the body. Clinical manifestations include systemic inflammatory response syndrome, accompanied by complications such as sepsis and insufficient organ blood perfusion. Sepsis can lead to septic shock, acute lung injury (ALI) / acute respiratory distress syndrome (ARDS), multiple organ failure, and even death. Epidemiological surveys have found that 18 million patients suffer from sepsis every year, and the number of patients continues to increase at a rate of 1.5% to 8% per year. It is currently believed that the uncontrolled inflammatory response and immune disorder caused by pathogenic microorganisms infecting the body are the main causes of sepsis. During sepsis infection, endotoxins and other substances stimulate immune cells to produce a large number of inflammatory mediators, prompting inflammatory response cells, especially macrophages, to be recruited and activated in tissues, further producing cytokines, chemokines, oxygen free radicals, etc., forming a waterfall-like cascade reaction, leading to organ damage or even failure, and ultimately causing the patient's death.
[0006] Acute lung injury (ALI) is a common and serious clinical disease. ALI often develops into acute respiratory distress syndrome (ARDS), which is characterized by pulmonary edema, decreased lung compliance, acute hypoxic respiratory failure, and ultimately death. According to statistics, there are about 150,000 ARDS patients in North America each year. Although the mortality rate has dropped significantly due to auxiliary medical equipment such as mechanical ventilation, 40%-70% of patients still die from ARDS. If accompanied by complications such as sepsis, the rate is as high as 90%, and sepsis is the most common cause of ALI / ARDS. The pathophysiological mechanism of ALI is often considered to be related to pulmonary inflammatory disorders. Its pathological characteristics are increased production of pro-inflammatory factors, increased inflammatory cell infiltration, and apoptosis of alveolar epithelial cells. Therefore, regulating the inflammatory response and improving the pathological state are important directions for the treatment of sepsis-induced acute lung injury and are also hot spots for drug development.
[0007] Inflammatory bowel disease (IBD) mainly includes Crohn's disease and ulcerative colitis, both of which are characterized by chronic inflammation in different parts of the gastrointestinal tract, and these inflammations can cause symptoms such as diarrhea, abdominal pain, and blood in the stool. Although the cause of inflammatory bowel disease is still not very clear, abnormalities in the immune system are considered to be an important factor in the progression of IBD. It is currently believed that its pathogenesis is an excessive intestinal mucosal immune response induced by a large number of intestinal bacteria, which causes intestinal mucosal damage in people with genetic susceptibility and further develops into chronic inflammation.
[0008] Summary of the invention
[0009] The present invention aims to provide a pharmaceutical composition comprising punicalagin or punicalin in combination with nicotinamide mononucleotide NMN / NAD + Or its pharmaceutically acceptable salt, use in drugs for treating macrophage-related inflammation, especially use in preparing drugs for preventing and / or treating sepsis and sepsis-induced acute lung injury.
[0010] The technical solution of the present invention is:
[0011] In a first aspect, a pharmaceutical composition is provided, wherein the pharmaceutical composition is composed of punicalagin / punicalin and nicotinamide mononucleotide NMN / nicotinamide adenine dinucleotide NAD + or a pharmaceutically acceptable salt thereof; the Punicalagin / Punicalin and NMN / NAD + The molar ratio of Punicalagin to NMN / NAD+ or a pharmaceutically acceptable salt thereof is 1:20 to 1:100. The target of the Punicalagin / Punicalin is PGAM5; the molar ratio of the Punicalagin to NMN / NAD+ or a pharmaceutically acceptable salt thereof is 1:20 to 1:100.
[0012] In some embodiments, the Punicalagin / Punicalin and NMN / NAD + or a pharmaceutically acceptable salt thereof in a molar ratio of 1:70 to 1:90, preferably 1:80 to 1:85, and more preferably 1:81.
[0013] In some embodiments, the Punicalagin / Punicalin and NMN / NAD +Typical but non-limiting mass ratios of the molar ratios of or pharmaceutically acceptable salts thereof are 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:90, etc., but are not limited to the listed values, and other unlisted values within the numerical range are also applicable.
[0014] In some embodiments, the concentration of Punicalagin / Punicalin is in the range of 15-25 mg / kg, and the concentration of Nicotinamide Mononucleotide NMN / Nicotinamide Adenine Dinucleotide NAD + The dosage concentration range is 400-600 mg / kg.
[0015] Preferably, the dosage range of Punicalagin / Punicalin is 20 mg / kg, and the dosage range of nicotinamide mononucleotide NMN / nicotinamide adenine dinucleotide NAD + The dosage concentration range is 500 mg / kg.
[0016] Wherein, the chemical structural formulas of Punicalagin and NMN are as follows:
[0017]
[0018] Punicalagin is a combination of the precursors Punicalin and Urolithin, and has a structural formula of C48H28O30.
[0019] In a second aspect, a pharmaceutical preparation is provided, which contains a therapeutically effective amount of the above-mentioned pharmaceutical composition and a pharmaceutically acceptable carrier, adjuvant or vehicle.
[0020] In some embodiments, the pharmaceutical preparation is in the form of a pulmonary or nasal inhalation nebulizer, a metered dose aerosol or a dry powder inhaler.
[0021] In a third aspect, the invention provides use of the pharmaceutical composition and the pharmaceutical preparation in the preparation of a drug for preventing and / or treating macrophage-related inflammation.
[0022] In some embodiments, the macrophage-related inflammation includes sepsis caused by peripheral inflammation, inflammatory lung injury, bronchial asthma, respiratory system-related diseases, and inflammatory bowel disease.
[0023] Fourthly, provide effective evidence that the core ingredients of the drug act on their specific targets and exert their effects.
[0024] The compounds Punicalagin / Punicalin and NMN / NAD+ Or the combination of their pharmaceutically acceptable salts has a synergistic effect, which can significantly enhance the inhibition of the expression and release of TNF-α, an inflammatory factor in macrophages, by Punicalagin / Punicalin, and enhance the effect of Punicalagin / Punicalin in down-regulating the gene expression levels of inflammatory factors in macrophages, such as TNF-α, IL-1β, IL-6, and COX-2.
[0025] The compounds Punicalagin / Punicalin and NMN / NAD + Or their pharmaceutically acceptable salts can relieve acute lung injury induced by sepsis caused by lipopolysaccharide, and the combination can better improve the pathological condition and reduce the expression and release of TNF-α in peripheral and lung tissues and the gene expression levels of related inflammatory factors compared with Punicalagin / Punicalin or NMN alone.
[0026] After knocking out PGAM5, the potential target of the compound Punicalagin / Punicalin, the acute lung injury induced by sepsis caused by lipopolysaccharide can be significantly alleviated, and the disease symptoms are significantly improved compared to when PGAM5 is not knocked out.
[0027] The compounds Punicalagin / Punicalin and NMN / NAD + The combination of punicalagin / punicalin or NMN can alleviate inflammatory bowel inflammation induced by sodium dextran sulfate, and the combination can better improve the pathological condition than Punicalagin / Punicalin or NMN alone. When the potential target PGAM5 is knocked out, it can produce similar effects to the use of Punicalagin / Punicalin.
[0028] The compound Punicalagin / Punicalin can strongly inhibit the phosphatase activity of the protein PGAM5, and PGAM5 is a key target that causes the macrophage-related inflammation.
[0029] In the present invention, when administering the above compounds and their pharmaceutically acceptable salts, and solvates of these compounds (collectively referred to herein as "therapeutic drugs") to mammals, they can be used alone, or preferably in combination with a pharmaceutically suitable carrier or diluent according to a standard pharmaceutical method. The administration method can be through various routes, including oral, intragastric, parenteral or topical administration. Parenteral administration referred to herein includes but is not limited to intravenous injection, intramuscular injection, subcutaneous injection and transdermal administration.
[0030] Beneficial effects: The advantages of the present invention are:
[0031] 1. The present invention discovered the natural product Punicalagin / Punicalin combined with nicotinamide mononucleotide NMN / NAD + Or its pharmaceutical use for administration of a pharmaceutically acceptable salt thereof to relieve sepsis and its induced acute lung injury;
[0032] 2. The present invention discovered that the natural product Punicalagin / Punicalin acts by targeting the protein PGAM5 in vivo;
[0033] 3. The present invention first discovered Punicalagin / Punicalin combined with nicotinamide mononucleotide NMN / NAD + or a pharmaceutically acceptable salt thereof can enhance Punicalagin or NMN / NAD + or a pharmaceutically acceptable salt thereof used alone to inhibit macrophage inflammation and has no obvious toxicity at an effective dose;
[0034] 4. The present invention first discovered Punicalagin / Punicalin combined with NMN / NAD + Or the administration of its pharmaceutically acceptable salt has the effect of alleviating inflammatory bowel disease, sepsis and the lung damage caused by it in mice, and has better therapeutic effect than Punicalagin / Punicalin or NMN used alone, indicating that the combination of Punicalagin / Punicalin and NMN has good clinical application prospects for the treatment of inflammatory bowel disease, sepsis and other macrophage inflammation-related diseases.
[0035] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is an example of the present invention with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 This is a schematic diagram of the effects of Punicalagin, NMN and their combination on lung tissue pathology in septic mice in the examples.
[0037] Figure 2 This is a schematic diagram of the effect of PGAM5 knockout on lung tissue pathology in mice with acute lung injury induced by tracheal instillation of LPS in the example.
[0038] Figure 3 This is a schematic diagram of the effects of Punicalagin, NMN and their combination on intestinal tissue pathology in mice with DSS-induced inflammatory bowel disease.
[0039] Figure 4 This is a schematic diagram of the effects of Punicalagin, NMN and their combination on the intestinal length of mice with DSS-induced inflammatory bowel disease in the examples.
[0040] Figure 5 Schematic diagram of the effect of Punicalagin on intestinal tissue pathology in wild-type and PGAM5 knockout mice with inflammatory bowel disease in the examples.
[0041] Figure 6 This is a schematic diagram of the effect of Punicalagin on intestinal length in wild-type and PGAM5 knockout mice with inflammatory bowel disease in the examples.
[0042] Figure 7 Schematic diagram of the effect of Punicalagin and its metabolites on PGAM5 phosphatase activity in vitro in the examples. DETAILED DESCRIPTION
[0043] In order to further illustrate the present invention, a series of examples are given below. These examples are purely illustrative and are only used to specifically describe the present invention, and should not be understood as limiting the present invention.
[0044] The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.
[0045] For the purpose of this specification and the appended claims, unless otherwise stated, all numbers expressing amounts, percentages or ratios and other numerical values used in this specification and the appended claims are understood to be modified in all cases by the term "about". In addition, all ranges disclosed herein are inclusive and independently combinable.
[0046] The term "and / or" in this article is only a description of the association relationship of the associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.
[0047] The present application is described in detail below in conjunction with specific embodiments. NMN / NAD + Or a pharmaceutically acceptable salt thereof, NMN is taken as an example in the embodiments.
[0048] Drugs: Punicalagin / Punicalin and NMN used in this experiment are easily soluble in physiological saline, with strong popularity and operability, and can be administered in various ways, including but not limited to intravenous, intraperitoneal, oral, nasal inhalation, nebulization and other methods. Reagents: Lipopolysaccharide (LPS) was purchased from Biotech Co., Ltd., and other reagents were commercially available analytical reagents. Cells: RAW264.7 macrophage cell line was purchased from China Center for Type Culture Collection. Kits: TNF-α ELISA kits were purchased from Shenzhen Dakoway Biotechnology Co., Ltd., RNA extraction kits were purchased from Novozyme Biotechnology Co., Ltd., and primers were purchased from Jinweizhi Biotechnology Co., Ltd.
[0049] Example 1
[0050] A pharmaceutical composition, consisting of punicalagin or punicalin and nicotinamide mononucleotide NMN; the molar ratio of punicalagin / punicalin to NMN is 1:20.
[0051] Example 2
[0052] A pharmaceutical composition, consisting of punicalagin or punicalin and nicotinamide mononucleotide NMN; the molar ratio of punicalagin / punicalin to NMN is 1:50.
[0053] Example 3
[0054] A pharmaceutical composition, consisting of punicalagin or punicalin and nicotinamide mononucleotide NMN; the molar ratio of punicalagin / punicalin to NMN is 1:100.
[0055] Example 4
[0056] A pharmaceutical preparation, consisting of punicalagin or punicalin, nicotinamide mononucleotide NMN and a carrier; the molar ratio of Punicalagin / Punicalin to NMN is 1:81.
[0057] Example 5
[0058] Determination of inflammatory factor expression in macrophages: Punicalagin (5, 10, 25, 6.25, 6.17, 5.88 μM), Punicalin (5, 10, 25, 6.25, 6.17, 5.88 μM), NMN (500 μM), and Punicalagin or Punicalin combined with NMN were used to pretreat RAW264.7 macrophages. After 3 hours, lipopolysaccharide LPS (100 ng / mL) was added for 2 hours. The expression and release of inflammatory factor TNF-α in the cell supernatant was detected by ELISA technology according to the operating instructions of the commercial kit. The doses of Punicalagin and Punicalin (6.25, 6.17, 5.88 μM) were obtained based on the molar ratios of 1:80, 1:81, and 1:85 with NMN. The experimental results are shown in Table 1. *** P<0.001, vs Control; ### P<0.001, vs Model (GraphPad Prism 8.0, Bonferroni's test). It can be seen that lipopolysaccharide significantly caused the upregulation of the expression of inflammatory factor TNF-α in macrophages RAW264.7. The combined use of Punicalagin and NMN can increase the anti-inflammatory activity of Punicalagin and better inhibit the expression and release of inflammatory factor TNF-α in macrophages. The effective doses are (Punicalagin 5μM+NMN 500μM, Punicalagin 10μM+NMN 500μM, Punicalagin 25μM+NMN 500μM, Punicalagin 6.25μM+NMN 500μM, Punicalagin 6.17μM+NMN 500μM, Punicalagin 5.88μM+NMN 500μM). The experimental results show that the combination of Punicalin and NMN can also improve the anti-inflammatory effect of Punicalin to a certain extent. The effective dose is (Punicalin 25μM+NMN 500μM)
[0059] Table 1
[0060] Group TNF-α percentage in the Model group (%) CCK-8 percentage in the Control group (%) Control 16.57±0.47 103.99±5.16 Model <![CDATA[100±0.76 *** ]]> 100±7.56 NMN (500 μM) 101.05±3.2 101.24±7.75 Punicalagin (5 μM) 106.1±1.4 98.24±1.34 Punicalagin (10 μM) <![CDATA[75.26±1.04 ### ]]> 96.7±1.18 Punicalagin (25 μM) <![CDATA[27.68±0.81 ### ]]> 96.78±3.69 Punicalagin (6.25 μM) 94.18±1.84 97±1.72 Punicalagin (6.17 μM) 90.78±0.18 101.59±2.9 Punicalagin (5.88 μM) 95.59±2.43 100.94±3.79 Punicalin (5 μM) 103.33±0.71 100.9±4.57 Punicalin (10 μM) 96.6±0.85 98.97±4.48 Punicalin (25 μM) <![CDATA[77.51±0.91 ### ]]> 95.5±3.23 Punicalin (6.25 μM) 99.68±1.36 99.66±3.66 Punicalin (6.17 μM) 102.56±1.3 97.6±3.97 Punicalin (5.88 μM) 97.93±2 97.25±4.12 Punicalagin (5μM) + NMN (500μM) <![CDATA[78.8±1.03 ### ]]> 98.03±1.2 Punicalagin(10μM)+NMN(500μM) <![CDATA[43.87±1.42 ### ]]> 97.43±1.3 Punicalagin(25μM)+NMN(500μM) <![CDATA[15.49±1.3 ### ]]> 102.53±2.33 Punicalagin(6.25μM)+NMN(500μM) <![CDATA[70.85±0.88 ### ]]> 102.02±3.94 Punicalagin(6.17μM)+NMN(500μM) <![CDATA[74.46±1.16 ### ]]> 102.23±5.49 Punicalagin(5.88μM)+NMN(500μM) <![CDATA[79.36±1.51 ### ]]> 100.3±4.86 Punicalin (5μM) + NMN (500μM) 104.59±2.34 102.87±3.26 Punicalin (10 μM) + NMN (500 μM) 93.27±3.37 103.52±4.37 Punicalin (25 μM) + NMN (500 μM) <![CDATA[64.44±2.61 ### ]]> 100.39±2.2 Punicalin(6.25μM)+NMN(500μM) 102.24±3.78 97.64±2.68 Punicalin(6.17μM)+NMN(500μM) 103.15±2.11 98.76±4.65 Punicalin(5.88μM)+NMN(500μM) 105.64±1.16 101.16±2.39
[0061] Example 6
[0062] Determination of the expression level of inflammation-related genes in macrophages: Punicalagin (5, 10 μM), NMN (500 μM) and the combination of the two were used to pretreat RAW264.7. After 3 hours, lipopolysaccharide LPS (100 ng / mL) was added for 2 hours. According to the instructions of the RNA extraction kit, the expression of inflammation-related genes in the cells was detected by RT-PCR technology. The experimental results are shown in Table 2, where *** P<0.001, vs Control; # P<0.05, ## P<0.01, ### P < 0.001, vs LPS; $ P<0.05, $$ P<0.01, $$$ P<0.001, vs single-dose group (Graphpad 8.0, Bonferroni's test). It can be seen that lipopolysaccharide significantly caused the upregulation of the expression of inflammatory-related genes TNF-α, IL-1β, IL-6, and Cox-2 in macrophages RAW264.7. The combined use of Punicalagin and NMN downregulated the inflammatory genes to varying degrees compared with single-dose administration. That is, the combined use of Punicalagin and NMN can increase the anti-inflammatory activity of Punicalagin, and the effective dose is (Punicalagin 5μM+NMN 500μM or Punicalagin 10μM+NMN 500μM).
[0063] Table 2
[0064] Group TNF-α fold change IL-1β fold change IL-6 fold change COX-2 fold change Control 1±0.11 1±0.05 1±0.03 1±0.03 Model <![CDATA[12.59±0.72 *** ]]> <![CDATA[47.09±7.42 *** ]]> <![CDATA[20.33±1.11 *** ]]> <![CDATA[9.49±1.8 *** ]]> Punicalagin (5 μM) <![CDATA[5.48±0.27 ### ]]> <![CDATA[29.31±2.26 # ]]> 20.46±1.01 6.97±0.86 Punicalagin (10 μM) <![CDATA[6.35±0.17 ### ]]> <![CDATA[15.03±3.48 ### ]]> 19.17±1.63 <![CDATA[3.68±0.77 ## ]]> NMN (500 μM) <![CDATA[5.83±1.31 ### ]]> <![CDATA[25.26±3.35 ## ]]> <![CDATA[10.72±1.01 ### ]]> <![CDATA[5.46±0.62 # ]]> Punicalagin (5μM) + NMN (500μM) <![CDATA[3.9±0.94 ### ]]> <![CDATA[11.26±0.54 ###,$$ ]]> <![CDATA[10.42±0.43 ###,$$$ ]]> <![CDATA[4.11±0.42 ##,$ ]]> Punicalagin(10μM)+NMN(500μM) <![CDATA[3.42±0.15 ###,$$ ]]> <![CDATA[4.99±0.47 ###,$ ]]> <![CDATA[8.9±0.62 ###,$$$ ]]> <![CDATA[2.7±0.17 ###,$$ ]]>
[0065] Example 7
[0066] Animal dosing grouping: The experimental animals were ICR♂ mice, and the experimental mice were randomly divided into control group, model group, and treatment group according to body weight; the control group and model group were intraperitoneally injected with normal saline, and the treatment group (500 mg / kg NMN, 20 mg / kg Punicalagin, 20 mg / kg Punicalagin + 500 mg / kg NMN, all dissolved in normal saline) was intraperitoneally injected. Each group was treated continuously for 3 days. 2 hours after the last administration, except for the control group, which was intraperitoneally injected with an equal volume of normal saline, the other groups were intraperitoneally injected with LPS (10 mg / kg).
[0067] Results Statistics:
[0068] Body weight and lung organ coefficient: The mice were randomly divided into groups by weighing on the first day and weighed before administration for 3 consecutive days. The weight on the first day was taken as 100% to calculate the growth status of the mice. LPS was injected intraperitoneally 2 hours after administration on the third day. The lung organs of the mice were dissected and weighed 6 hours later, as shown in Table 3. *** P<0.001, vs Control; # P<0.05, ## P<0.01, ### P < 0.001, vs LPS; $ P<0.05, $$$ P<0.01, vs single-dose group (GraphPad Prism 8.0, Bonferroni's test).
[0069] Lung pathology, serum and lung tissue inflammatory factor determination: 6 hours after intraperitoneal injection of LPS on the 3rd day, whole blood was collected from the eye sockets, centrifuged to prepare serum, and frozen at -80°C. Some mice were perfused with PBS and the right lobe of the lung was fixed with paraformaldehyde, while the lungs of other mice were removed after dissection and stored in liquid nitrogen. The fixed lungs were dehydrated, paraffin-embedded, and sectioned, and then stained with hematoxylin-eosin (H&E). The histopathological results are shown in Figure 1 The statistical results are shown in Table 3. *** P < 0.001, vs Control; # P<0.05, ### P < 0.001, vs LPS; $ P<0.05, $$ P<0.01, vs single-dose group (GraphPad Prism 8.0, Bonferroni's test).
[0070] Table 3
[0071] Group Weight(Day1) Weight(Day2) Weight(Day3) Lung coefficient (%) Alveolar cell area (%) G1:Control 100±0 101.89±0.86 108.69±1.46 0.607±0.014 27.08±1.69 G2:Model 100±0 103.46±1.05 109.8±1.56 <![CDATA[0.678±0.011 *** ]]> <![CDATA[45.53±1.27 *** ]]> G3: Dexamethasone 5mg / kg 100±0 103±0.98 107.01±1.61 <![CDATA[0.624±0.01 ## ]]> <![CDATA[33.59±1.4 ### ]]> G4: NMN 500mg / kg 100±0 102.59±1.09 106.78±1.75 0.657±0.009 <![CDATA[38.62±2.61 # ]]> G5: Punicalagin 20mg / kg 100±0 102.88±0.99 105.9±1.75 0.644±0.009 <![CDATA[35.05±1.53 ### ]]> G6:Punicalagin 20mg / kg+NMN 500m g100±0 103.11±0.99 105.85±1.55 <![CDATA[0.633±0.008 # ]]> <![CDATA[29.23±1.21 ###,$,$$ ]]>
[0072] Part of the lung tissue stored in liquid nitrogen was cut on ice, the homogenate was centrifuged and the supernatant was taken, the protein was determined and the total protein concentration was adjusted, and the serum and lung tissue homogenate supernatant were tested using a commercial TNF-α ELISA kit. The results of the effects of serum and lung tissue inflammatory factor TNF-α are shown in Table 4, where *** P<0.001, vs Control; # P<0.05, ## P<0.01, ### P < 0.001, vs LPS; $ P<0.05, $$ P<0.01, $$$P<0.001, vs single-dose group (GraphPad Prism 8.0, Bonferroni's test).
[0073] Table 4
[0074] Group TNF-α content in serum (% of Model) TNF-α content in lung tissue homogenate (% of Model) G1:Control 24.88±0.46 37.66±5.24 G2:Model <![CDATA[100±13.14 *** ]]> <![CDATA[100±5.64 *** ]]> G3: NMN 500mg / kg <![CDATA[55.56±6.64 ## ]]> 93.31±5.71 G4: Punicalagin 20mg / kg <![CDATA[46.15±7.09 ## ]]> <![CDATA[84.08±4.07 # ]]> G5:Punicalagin 20mg / kg+NMN 500mg / kg <![CDATA[36.94±2.86 ###,$,$$ ]]> <![CDATA[69.27±4.16 ###,$,$$ ]]>
[0075] Determination of the expression level of inflammation-related genes in lung tissue: Take a small amount of frozen lung tissue, grind it thoroughly, and use RT-PCR technology to detect the expression of inflammation-related genes in lung tissue according to the instructions of the RNA extraction kit. The experimental results are shown in Table 5, where *** P<0.001, vs Control; # P<0.05, ## P<0.01, ### P < 0.001, vs LPS; $ P<0.05, $$ P<0.01, $$$ P<0.001, vs single-dose group (Graphpad 8.0, Bonferroni's test).
[0076] Table 5
[0077] Group TNF-α fold change IL-1β fold change IL-6 fold change Fold change of iNOS G1:Control 1±0.29 1±0.19 1±0.35 1±0.21 G2:Model <![CDATA[7.4±0.82 *** ]]> <![CDATA[22.23±2.39 *** ]]> <![CDATA[13.93±1.76 *** ]]> <![CDATA[52.47±5.5 *** ]]> G3: NMN 500mg / kg <![CDATA[5.52±0.56 # ]]> 21.63±0.73 <![CDATA[21.63±1.06 ### ]]> 45.53±7.47 G4: Punicalagin 20mg / kg <![CDATA[3.41±0.42 ### ]]> <![CDATA[13.5±1.83 ## ]]> <![CDATA[6.27±0.73 ### ]]> <![CDATA[19.19±1.4 ### ]]> G5:Punicalagin 20mg / kg+NMN 500mg / kg <![CDATA[2.17±0.18 ###,$,$$ ]]> <![CDATA[12.23±1.91 ###,$$ ]]> <![CDATA[3.4±0.44 ###,$,$$ ]]> <![CDATA[22.25±3.72 ###,$ ]]>
[0078] In summary, there was no significant change in the weight of mice after administration of each drug group. 6 hours after intraperitoneal injection of lipopolysaccharide, the lung organ coefficient of mice was significantly upregulated, TNF-α in serum and lung tissue increased significantly, lung tissue was significantly damaged, and the expression level of related inflammatory genes in lung tissue increased significantly. Compared with the Punicalagin or NMN alone group, the Punicalagin combined with NMN administration group can significantly downregulate the lung coefficient, significantly reduce the expression and release of TNF-α in serum and lung tissue, reduce the expression of related inflammatory genes in lung tissue, and have a more significant improvement effect on lung pathological damage. These experimental results show that Punicalagin combined with NMN administration has a better preventive or therapeutic effect on sepsis and its caused acute lung injury than Punicalagin or NMN alone.
[0079] Example 8
[0080] Animal experiment groups: The experimental animals were C57♂ mice and C57♂PGAM5 - / - The experimental mice were randomly divided into WT control group, WT model group, PGAM5 - / - In the model group, LPS (20 mg / kg) was instilled into the trachea of each model group, and the control group was instilled into the trachea with an equal volume of normal saline.
[0081] Results Statistics:
[0082] Determination of lung organ coefficient and pathological condition: 6 hours after tracheal instillation of LPS, the lungs of mice were dissected and weighed. The right lobe of the lung was perfused with PBS and fixed with paraformaldehyde, and the left lobe of the lung was stored in liquid nitrogen. The fixed lungs were dehydrated, paraffin-embedded, sectioned, and stained with hematoxylin-eosin (H&E). The histopathological results are shown in Figure 2 The statistical results are shown in Table 6, where *** P<0.001, vs Control; ### P<0.001, vs WT-ALI (GraphPad Prism 8.0, Bonferroni's test).
[0083] Table 6
[0084] Group Lung coefficient (%) Alveolar cell area (%) G1:WT-CN 0.61±0.02 28.05±2.31 G2:WT-ALI <![CDATA[0.75±0.01 *** ]]> <![CDATA[43.89±1.62 *** <!-- 8 -->]]> <![CDATA[G3:PGAM5 - / - -BUT]]> <![CDATA[0.65±0.01 ### ]]> <![CDATA[28.7±1.45 ### ]]>
[0085] Part of the lung tissue stored in liquid nitrogen was cut on ice, the homogenate was centrifuged and the supernatant was taken, the protein was determined and the total protein concentration was adjusted, and the serum and lung tissue homogenate supernatant were tested using a commercial TNF-α ELISA kit. The results are shown in Table 7, where *** P<0.001, vs Control; ### P<0.001, vs WT-ALI (GraphPad Prism 8.0, Bonferroni's test).
[0086] Table 7
[0087] Group TNF-α content in lung tissue homogenate (% of WT-ALI) G1:WT-CN 25.12±4.63 G2:WT-ALI <![CDATA[100±5.71 *** ]]> <![CDATA[G3:PGAM5 - / - -BUT]]> <![CDATA[71.11±3.63 ### ]]>
[0088] Determination of the expression level of inflammation-related genes in lung tissue: Take a small amount of frozen lung tissue, grind it thoroughly, and use RT-PCR technology to detect the expression of inflammation-related genes in lung tissue according to the instructions of the RNA extraction kit. The experimental results are shown in Table 8, where ** P<0.01, *** P<0.001, vs Control; # P<0.05, ## P<0.01, ### P<0.001, vs WT-ALI (GraphPad Prism 8.0, Bonferroni's test).
[0089] Table 8
[0090] Group TNF-α fold change IL-1β fold change IL-6 fold change COX-2 fold change Fold change of iNOS G1:WT-CN 1±0.21 1±0.17 1±0.25 1±0.15 1±0.09 G2:WT-ALI <![CDATA[7.58±0.87 ** ]]> <![CDATA[19.59±4.55 *** ]]> <![CDATA[24.56±3.98 *** ]]> <![CDATA[4.75±1.02 ** ]]> <![CDATA[10.92±0.97 *** ]]> <![CDATA[G3:PGAM5 - / - -BUT]]> <![CDATA[5.61±0.36 # ]]> <![CDATA[9.89±1.69 # ]]> <![CDATA[11.28±1.67 ## ]]> <![CDATA[2.56±0.35 # ]]> <![CDATA[5.8±0.39 ### ]]>
[0091] In summary, 6 hours after tracheal instillation of LPS, the lung organ coefficient of mice was significantly upregulated, TNF-α in lung tissue increased significantly, lung tissue was significantly damaged, and the expression level of related inflammatory genes in lung tissue increased significantly; while PGAM5 - / - Compared with the WT model group, the group can significantly downregulate the lung coefficient, significantly reduce the expression and release of TNF-α in lung tissue, reduce the expression of related inflammatory genes in lung tissue, and have a more significant improvement on lung pathological damage. These experimental results show that PGAM5 plays a key role in acute lung injury induced by tracheal instillation of LPS, and inhibiting its activity may have a positive effect on the treatment or prevention of the disease.
[0092] Example 9
[0093] Animal dosing grouping: The experimental animals were C57♂ mice, which were randomly divided into control group, model group and treatment group according to body weight; the control group and model group were given normal saline by gavage, and the treatment group (500 mg / kg NMN, 20 mg / kg Punicalagin, 20 mg / kg Punicalagin + 500 mg / kg NMN, all dissolved in normal saline) was given by gavage, and each group was given the drug continuously for 7 days; from the beginning to the end of the experiment, the model group and the treatment group were given 3% DSS solution to drink freely, and the control group was given distilled water to drink freely.
[0094] Results Statistics:
[0095] Body weight and disease activity index score: The mice were weighed and randomly divided into groups on the first day, and fed with 3% DSS water and administered for 7 consecutive days. They were weighed and observed for diarrhea and bloody stools before administration. The weight on the first day was taken as 100%, and the growth status of the mice was counted. The results are shown in Tables 9 and 10. *** P<0.001, vs Control; # P<0.05, ## P<0.01, ### P<0.001, vs Vehicle (GraphPad Prism 8.0, Bonferroni's test).
[0096] Table 9
[0097]
[0098] Table 10
[0099]
[0100] Intestinal and histopathological scoring: On the 7th day, the mice were killed and their abdominal cavities were opened. The intestinal segment from the cecum to the anus was taken. The intestinal segment was measured and the end 0.5 cm was cut off. The intestinal segment was washed with PBS and fixed with paraformaldehyde. The fixed intestinal segment was dehydrated, embedded in paraffin, and then stained with hematoxylin-eosin (H&E). The histopathological results are shown in Tables 11 and Figure 3 ,in *** P<0.001, vs Control; # P<0.05, ### P < 0.001, vs Vehicle; & P<0.05, &&& P<0.01, vs single-dose group (GraphPad Prism 8.0, Bonferroni's test).
[0101] The results of colon length are shown in Tables 11 and Figure 4 ,in *** P<0.001, vs Control; ## P<0.01, ### P<0.001, vs Vehicle (GraphPad Prism 8.0, Bonferroni's test).
[0102] Table 11
[0103] Group Histopathological scoring Colon length G1:Ctrl 0±0 7.28±0.17 G2: Vehicle <![CDATA[13.67±0.88 *** ]]> <![CDATA[4.95±0.15 *** ]]> G3: Punicalagin (20 mg / kg) <![CDATA[6±0.58 ### ]]> <![CDATA[5.93±0.21 ## ]]> G4: NMN (500 mg / kg) <![CDATA[10.33±0.67 # ]]> 5.6±0.24 G5:Punicalagin(20mg / kg)+NMN(500mg / kg) <![CDATA[3±0.58 ###,&,&&& ]]> <![CDATA[6.32±0.14 ### ]]>
[0104] In summary, 7 days after DSS water-fed modeling in each drug group, the weight of mice decreased to varying degrees. The weight loss of the Punicalagin combined with NMN group was significantly reduced compared to the Punicalagin or NMN alone group, and it can significantly reduce IBD symptoms such as diarrhea and blood in the stool, effectively improve the disease activity index score and pathological score of the mouse intestine, reduce the shortening of the colon after IBD modeling in mice, and have a very significant improvement effect on intestinal tissue pathological damage. These experimental results show that Punicalagin combined with NMN administration has a better preventive or therapeutic effect on inflammatory bowel disease than Punicalagin or NMN alone.
[0105] Example 10
[0106] Animal dosing group: The experimental animals were C57♂ mice and C57♂PGAM5 - / - The experimental mice were randomly divided into control group (Control), WT model group (Vehicle-1), WT drug group (Punicalagin-1), PGAM5 - / -Model group (Vehicle-2), PGAM5 - / - The drug-treated group (Punicalagin-2) and the model group were intragastrically injected with normal saline, and the drug-treated group (50 mg / kg Punicalagin dissolved in normal saline) was intragastrically administered. Each group was given the drug continuously for 7 days. From the beginning to the end of the experiment, the model group and the drug-treated group were given 3% DSS solution to drink freely, and the control group was given distilled water to drink freely.
[0107] Results Statistics:
[0108] Body weight and disease activity index score: The mice were weighed and randomly divided into groups on the first day, and fed with 3% DSS water and administered for 7 consecutive days. They were weighed before administration and their diarrhea and bloody stools were observed. The weight on the first day was taken as 100%, and the growth status of the mice was counted. The results are shown in Tables 12 and 13, where * P<0.05, *** P<0.001, vs Control; ## P<0.01, ### P<0.001, vs Vehicle-1 (GraphPad Prism 8.0, Bonferroni's test).
[0109] Table 12
[0110] Group-Weight Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 G1:Control 100±0 100.11±0.56 100.98±1.01 101.07±0.94 100.84±1.2 102.35±1.59 102.59±2.61 G2: Vehicle-1 100±0 97.87±0.32 <![CDATA[96.05±0.02 * ]]> <![CDATA[93.26±0.89 *** ]]> <![CDATA[88.5±2.11 *** ]]> <![CDATA[82.74±2.99 *** ]]> <![CDATA[79.61±2.93 *** ]]> G3: Punicalagin-1 100±0 99.64±0.9 99.12±1.64 97.1±1.2 92.72±0.77 <![CDATA[89±1.61 ## ]]> <![CDATA[84.28±0.85 # ]]> G4: Vehicle-2 100±0 100.12±0.52 99.82±0.42 <![CDATA[98.62±0.85 ## ]]> <![CDATA[95.08±1.16 ### ]]> <![CDATA[91.06±1.04 ### ]]> <![CDATA[86.38±1.4 ### ]]> G5: Punicalagin-2 100±0 98.85±0.67 97.95±1.02 96.6±1.68 <![CDATA[93.88±0.89 ## ]]> <![CDATA[90.97±0.41 ### ]]> <![CDATA[88.6±0.92 ### ]]>
[0111] Table 13
[0112] Group-DAI Score Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 G1:Control 0±0 0±0 0±0 0±0 0±0 0±0 0±0 G2: Vehicle-1 0±0 0.67±0.33 <![CDATA[1.67±0.67 *** ]]> <![CDATA[6±1 *** ]]> <![CDATA[8±1 *** ]]> <![CDATA[10.67±0.33 *** ]]> <![CDATA[11.67±0.33 *** ]]> G3: Punicalagin-1 0±0 0±0 0.33±0.33 <![CDATA[2±0.58 ## ]]> <![CDATA[2.67±0.88 ### ]]> <![CDATA[5±0.58 ### ]]> <![CDATA[6.33±0.67 ### ]]> G4: Vehicle-2 0±0 0±0 0±0 <![CDATA[0.67±0.33 ### ]]> <![CDATA[2.5±0.99 ### ]]> <![CDATA[4.17±0.95 ### ]]> <![CDATA[6±1.32 ### ]]> G5: Punicalagin-2 0±0 0.17±0.17 0.33±0.21 <![CDATA[1±0.45 ### ]]> <![CDATA[1.67±0.67 ### ]]> <![CDATA[3.5±0.62 ### ]]> <![CDATA[4.67±0.95 ### ]]>
[0113] Intestinal and histopathological scoring: On the 7th day, the mice were killed and their abdominal cavities were opened. The intestinal segment from the cecum to the anus was taken. The intestinal segment was measured and the end 0.5 cm was cut off. The intestinal segment was washed with PBS and fixed with paraformaldehyde. The fixed intestinal segment was dehydrated, embedded in paraffin, and then stained with hematoxylin-eosin (H&E). The histopathological results are shown in Tables 14 and Figure 5 ,in *** P<0.001, vs Control; # P<0.05, ### P<0.001, vs Vehicle-1 (GraphPad Prism 8.0, Bonferroni's test).
[0114] The results of colon length are shown in Tables 14 and Figure 6 ,in *** P<0.001, vs Control; # P<0.05, ## P<0.01,### P<0.001, vs Vehicle-1 (GraphPad Prism 8.0, Bonferroni's test).
[0115] Table 14
[0116] Group Histopathological scoring Colon length G1:Control 0±0 7.35±0.13 G2: Vehicle-1 <![CDATA[12.83±0.6 *** ]]> <![CDATA[5.12±0.12 *** ]]> G3: Punicalagin-1 <![CDATA[5.67±0.49 ### ]]> <![CDATA[5.92±0.16 # ]]> G4: Vehicle-2 <![CDATA[5.17±0.75 # ]]> <![CDATA[6.18±0.22 ## ]]> G5: Punicalagin-2 <![CDATA[4±1.06 ### ]]> <![CDATA[6.55±0.24 ### ]]>
[0117] In summary, 7 days after DSS water feeding, the body weight of mice in each group decreased to varying degrees, especially in the WT group, PGAM5 group, - / - Model group and PGAM5 - / - Compared with the WT model group, the weight loss of the drug-treated group was reduced to varying degrees, and the IBD symptoms such as diarrhea and blood in the stool were significantly alleviated, the disease activity index score and pathological score of the mouse intestine were effectively improved, the shortening of the colon after IBD in mice was alleviated, and the pathological damage of intestinal tissue was significantly improved. These experimental results show that the preventive or therapeutic effect of Punicalagin administration on improving inflammatory bowel disease is very similar to the effect achieved by knocking out PGAM5, and Punicalagin may work by inhibiting the activity of PGAM5.
[0118] Embodiment 11
[0119] Determination of the effect of punicalagin, punicalin and urolithin on PGAM5 phosphatase activity: Take 10 μL of compound or solvent (compound diluted with reaction buffer) and 10 μL of ΔN21 PGAM5 (purified protein diluted with protein storage solution) and mix them evenly in a 384-well plate with a pipette. Then add 5 μL of 0.5 mM ASK1Ser-1029:NFEDH{pSER}APPSP (prepared with Phosphate-Free Water) to the wells of the 384-well plate reaction, mix evenly and place in an MB100-4A microplate constant temperature oscillator at 25°C for 10-30 minutes. Then add 20 μL of 1% hydroxylamine to the wells of the 384-well plate reaction. GREEN, mix well and place in MB100-4A microplate constant temperature oscillator at 25℃ for 10-40min. Detect the OD value at 620nm, and characterize the strength of PGAM5 phosphatase activity by the relative OD value. The statistical results of PGAM5 phosphatase activity are shown in Table 15 and Figure 7 .
[0120] Table 15
[0121] Concentration (μM) log(μM) Punicalagin Punicalin Urolithin 0.0005 -3.30103 100.06±0.6 98.9±0.62 N / A 0.005 -2.30103 99.3±0.53 98.5±0.62 N / A 0.05 -1.30103 99.23±0.68 96.35±0.84 109.54±1.99 0.5 -0.30103 77.71±2.22 83.36±1.55 104.19±2.62 1 0 65.9±1.65 71.78±2.78 119.33±4.53 2.5 0.39794 59.9±2.43 58.32±1.94 112.15±4.16 5 0.69897 44.54±2.12 37.83±1.5 109.05±3.62 10 1 32.68±2 20.53±1.43 100.34±4.4 25 1.39794 7.55±0.98 2.25±0.55 113.05±3.77 50 1.69897 8.14±0.5 2.49±0.62 109.16±4.19 100 2 10.55±0.37 4.18±0.77 108.05±2.08 200 2.30103 12.31±0.34 7.74±0.64 105.81±3.19
[0122] In summary, the IC values of Punicalagin and its metabolites in inhibiting PGAM5 phosphatase activity were determined by the PGAM5 phosphatase activity assay system. 50 The experimental results showed that Punicalagin and Punicalin had strong ability to inhibit PGAM5 phosphatase activity, and their IC 50 The results showed that Punicalagin may play a role by targeting PGAM5 protein in other embodiments.
[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A pharmaceutical composition, characterized in that The pharmaceutical composition is composed of punicalagin / punicalin and nicotinamide mononucleotide NMN / nicotinamide adenine dinucleotide NAD + or a pharmaceutically acceptable salt thereof; the Punicalagin / Punicalin and NMN / NAD + or a pharmaceutically acceptable salt thereof in a molar ratio of 1:20 to 1:
100.
2. The pharmaceutical composition according to claim 1, characterized in that The Punicalagin / Punicalin and NMN / NAD + or a pharmaceutically acceptable salt thereof in a molar ratio of 1:70 to 1:90, preferably 1:80 to 1:85, and more preferably 1:
81.
3. The pharmaceutical composition according to claim 1, characterized in that The dosage range of Punicalagin / Punicalin is 15-25 mg / kg, and the dosage range of nicotinamide mononucleotide NMN / nicotinamide adenine dinucleotide NAD + The dosage concentration range is 400-600 mg / kg.
4. The pharmaceutical composition according to claim 3, characterized in that The dosing concentration range of Punicalagin / Punicalin is 20mg / kg, nicotinamide mononucleotide NMN / nicotinamide adenine dinucleotide NAD + The dosage concentration range is 500 mg / kg.
5. A pharmaceutical preparation comprising a therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier, adjuvant or vehicle.
6. The pharmaceutical preparation according to claim 5, characterized in that The dosage form of the pharmaceutical preparation is a nebulizer for inhalation through the lungs or nose, a metered-dose aerosol or a dry powder inhaler.
7. Use of the pharmaceutical composition according to any one of claims 1 to 4 or the pharmaceutical preparation according to claim 5 or 6 in the preparation of a medicament for preventing and / or treating macrophage-related inflammation.
8. The use according to claim 7, characterized in that: The macrophage-related inflammation includes sepsis caused by peripheral inflammation, inflammatory lung injury, bronchial asthma, respiratory system-related diseases, and inflammatory bowel disease.
9. The use according to claim 7 or 8, characterized in that: Punicalagin / Punicalin exerts its effect by targeting PGAM5 protein.