Puromycin-sensitive aminopeptidase activators and their compositions for the treatment and / or prevention of inflammatory diseases

Abstract

This application discloses the use of a puromycin-sensitive aminopeptidase activator in the preparation of medicaments for the treatment and / or prevention of inflammatory diseases. The puromycin-sensitive aminopeptidase activator may be selected as abscisic acid and / or andrographolide. This application is the first to discover that abscisic acid and andrographolide can exert therapeutic effects on acute pancreatitis, gouty arthritis, and cholestatic liver disease by activating puromycin-sensitive aminopeptidase, thus expanding the new uses of abscisic acid and andrographolide. A mouse model of acute pancreatitis induced by strychnine, a mouse model of gouty arthritis induced by MSU, and a mouse model of cholestatic liver disease induced by gallbladder ligation were constructed to verify the therapeutic effects. The results showed that intraperitoneal injection of abscisic acid effectively improved acute pancreatitis, gouty arthritis, and cholestatic liver disease in mice, while intraperitoneal injection of andrographolide effectively improved acute pancreatitis and gouty arthritis in mice.

Description

Technical Field

[0001] This application relates to the field of compound pharmaceutical technology, and specifically to puromycin-sensitive aminopeptidase activators and compositions thereof for the treatment and / or prevention of inflammatory diseases. Background Technology

[0002] Acute pancreatitis (AP) is a severe inflammatory disease characterized by sudden inflammation and damage to the pancreatic tissue. This disease typically has a sudden onset, and symptoms can worsen rapidly within hours, even becoming life-threatening. One of the main causes of AP is the early activation of pancreatic enzymes, leading to pancreatic tissue damage accompanied by infiltration of inflammatory cells (primarily macrophages and neutrophils) and activation of the NLRP3 inflammasome. Simultaneously, the NLRP3 inflammasome further exacerbates the inflammatory response in the pancreatic tissue by modulating the adaptive immune system (CARS). The most common treatment for AP is non-surgical (conservative) treatment, including fasting, gastrointestinal decompression, fluid and nutritional support, drug therapy, and traditional Chinese medicine. For some patients with severe AP, surgery may be a necessary option. Currently, treatment options for AP are quite limited, aiming only to alleviate pain and prevent complications; there is no effective strategy to completely cure AP.

[0003] Gout (GA) is a metabolic disease clinically characterized by joint swelling, redness, and pain. This is caused by the deposition of monosodium urate crystals in both joint and non-joint structures under persistently high serum uric acid levels. Monosodium urate crystals can stimulate the activation of the NLRP3 inflammasome, leading to the massive release of pro-inflammatory cytokines such as IL-1β. This results in neutrophils flooding the synovium and synovial fluid, causing joint cavity damage. Typical gout can cause a series of complex complications, including impaired joint function, metabolic syndrome, diabetes, cardiovascular disease, and kidney disease. During acute attacks, gouty arthritis is often treated with Western medicines such as nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, and glucocorticoids, but these often have significant adverse reactions. Traditional Chinese medicine also has some efficacy in treating gouty arthritis, but its onset of action is slow. Therefore, although these therapies are effective for most patients, new treatment options still need to be developed to overcome the shortcomings of existing treatments.

[0004] Cholestatic liver disease is a liver disorder caused by impaired bile secretion or excretion, often leading to hepatocellular damage and dysfunction. Clinical features include fatigue, pruritus, jaundice, and abdominal pain, primarily due to the accumulation of bile salts and bilirubin in the liver. Cholestasis can cause hepatocellular inflammation, necrosis, and fibrosis, potentially leading to cirrhosis and liver failure in severe cases. The causes of cholestatic liver disease are diverse, mainly including primary biliary cholangitis, primary sclerosing cholangitis, and secondary cholestasis caused by other diseases. These diseases are often accompanied by autoimmune reactions or hepatobiliary tract diseases, forming a complex pathophysiological mechanism. In the acute or chronic phase, traditional treatments include using medications to control liver function, improve bile excretion, and treat the underlying cause, such as immunosuppressants. In some cases, surgical intervention may be necessary to relieve biliary obstruction. However, these treatment options often have certain side effects and limitations. Traditional Chinese medicine and other adjunctive therapies have shown potential in improving symptoms and liver function, but the effects may be slower. Given the need for improvement in current treatment methods, the development of new therapies for cholestatic liver disease is of particular importance in order to improve patients' quality of life and reduce the occurrence of complications.

[0005] Abscisic acid (ABA) is a sesquiterpene plant hormone first discovered in plants in the 1960s. It can regulate plant responses to environmental stresses such as drought, cold, and nutrient deficiency. In the 1980s, researchers discovered that ABA can be endogenously synthesized in various human and animal cells (such as macrophages and granulocytes). Recent studies have shown that ABA exhibits good preventive and therapeutic effects in type II diabetes, inflammatory bowel disease, atherosclerosis, and neuroinflammation. Androlide is a natural product extracted and isolated from the plant Andrographis paniculata. It possesses various pharmacological activities, including antitumor, antibacterial, anti-inflammatory, antiviral, antifibrotic, anti-obesity, immunomodulatory, and hypoglycemic activities. However, there are no reports on its application as a PSAP activator in the preparation of anti-inflammatory drugs. Summary of the Invention

[0006] In view of the above-mentioned technical limitations, this application proposes a puromycin-sensitive aminopeptidase activator and its composition for the treatment and / or prevention of inflammatory diseases; which overcomes the deficiencies and defects mentioned in the background art.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] The inventive point of this application is to provide the use of a puromycin-sensitive aminopeptidase (PSAP) activator in the preparation of a medicament for the treatment and / or prevention of inflammatory diseases.

[0009] Optionally, in the above-described uses, the puromycin-sensitive aminopeptidase activator is selected from any one or more of abscisic acid and andrographolide.

[0010] The structural formula of abscisic acid is shown in the following formula (Ⅰ).

[0011]

[0012] The structural formula of andrographolide is shown in formula (II) below.

[0013]

[0014] Optionally, in the above-described uses, the puromycin-sensitive aminopeptidase activator is selected as abscisic acid and andrographolide.

[0015] Optionally, in the above-described uses, the inflammatory disease is selected from any one or more of acute pancreatitis, gouty arthritis, and cholestatic liver disease.

[0016] Optionally, for the above-described uses, the formulation type of the drug is selected from any one or more of tablets, capsules, granules, micro-pellets, droplets, oral liquids, soft capsules, water injections, powder injections, infusions, ointments, gels, and microemulsions.

[0017] The unit dose of tablets, capsules, granules, micro-pellets, drop pills, oral liquids, and soft capsules is 5-1000mg; the unit dose of water injections, powder injections, and infusions is 5-500mg.

[0018] Optionally, in the above-described uses, the dosage of the puromycin-sensitive aminopeptidase activator is 0.8-40 mg / kg, preferably 13 mg / kg.

[0019] Optionally, in the above-described uses, the dosage of abscisic acid in the puromycin-sensitive aminopeptidase activator is 0.3-30 mg / kg, preferably 3 mg / kg.

[0020] Optionally, in the above-described uses, the dosage of andrographolide in the puromycin-sensitive aminopeptidase activator is 0.5-10 mg / kg, preferably 10 mg / kg.

[0021] Compared with the prior art, this application has the following advantages:

[0022] This application is the first to discover that abscisic acid and andrographolide can exert therapeutic effects on acute pancreatitis, gouty arthritis, and cholestatic liver disease by activating puromycin-sensitive aminopeptidase. This expands the new applications of abscisic acid and andrographolide. A mouse model of acute pancreatitis induced by strychnine, a mouse model of gouty arthritis induced by MSU, and a mouse model of cholestatic liver disease induced by gallbladder ligation were constructed to verify the therapeutic effects. The results show that in the strychnine-induced mouse model of acute pancreatitis, intraperitoneal injection of abscisic acid (0.3 mg / kg, 3 mg / kg, 30 mg / kg) or andrographolide (0.5 mg / kg, 1 mg / kg, 2 mg / kg) can effectively improve pancreatic damage and inhibit the development of puromycin-sensitive aminopeptidase. Abnormally elevated lipase and amylase activities in mouse serum effectively inhibited the infiltration of inflammatory cells in pancreatic tissue. In an MSU-induced gouty arthritis model, intraperitoneal injection of abscisic acid (0.3 mg / kg, 3 mg / kg, 30 mg / kg) or andrographolide (5 mg / kg, 10 mg / kg) effectively alleviated paw tissue damage in mice. In a gallbladder ligation-induced cholestatic liver disease model, intraperitoneal injection of abscisic acid (1 mg / kg, 3 mg / kg, 10 mg / kg) effectively improved survival rate, alleviated inflammation and liver damage in mice, and effectively prevented and treated cholestatic liver disease. Intraperitoneal injection of abscisic acid or andrographolide provides a new therapeutic approach for acute pancreatitis, gouty arthritis, and cholestatic liver disease. Attached Figure Description

[0023] Figure 1 The results showed that abscisic acid, andrographolide, and PSAP were bound together; among them... Figure 1 A represents the detection of the binding of abscisic acid (ABA) and PSAP by isothermal titration calorimetry (ITC) (Kd = 23.9 μM); Figure 1 B represents the isothermal titration calorimetry (ITC) method for detecting the binding of andrographolide and PSAP (Kd = 120 nM). Figure 1 C indicates the presence of ABA and PSAP binding in protein quality control testing. Figure 1 D represents 293T cells incubated with 10 nM ABA for 3 h, and intracellular thermal migration analysis (CETSA) was performed to determine the thermal stability of PSAP at different temperatures; Figure 1 E represents the molecular docking simulation site for the binding of ABA and PSAP.

[0024] Figure 2 Abscisic acid and andrographolide are shown to be activators of PSAP; Figure 2 A represents the effect of ABA on PSAP oligomerization detected by co-incubation of ABA and PSAP proteins. Figure 2 B represents the effect of ABA on PSAP enzyme activity when ABA and PSAP protein are co-incubated. Figure 2C represents the enzyme kinetic curve detected by co-incubation of ABA and PSAP proteins; Figure 2 D represents the effect of ABA treatment on intracellular PSAP enzyme activity in THP1 cells. Figure 2 E represents the effect of ABA on PSAP enzyme activity detected using macrophage-conditioned knockout PSAP mice and WT mice via BMDM. Figure 2 F represents the effect of ABA on PSAP enzyme activity detected by BMDM stimulation with LPS and ATP / Nigericin / MSU. Figure 2 G represents the enzyme kinetic curve detected by co-incubation of Andro and PSAP proteins; Figure 2 H represents the effect of Andro on PSAP enzyme activity detected by BMDM using LPS and ATP / Nigericin / MSU stimulation.

[0025] Figure 3 The results showed that abscisic acid significantly alleviated taurine-induced acute pancreatitis in mice; Figure 3 A is a schematic diagram of an acute pancreatitis model induced by taurine; Figure 3 B represents the determination of serum amylase activity in mice; Figure 3 C represents the determination of serum lipase activity in mice; Figure 3 D represents the IL-1β content in mouse pancreatic tissue detected by ELISA; Figure 3 E represents the detection of myeloperoxidase activity in mouse pancreas; Figure 3 F represents the detection of myeloperoxidase activity in mouse lung tissue; Figure 3 G represents the flow cytometry detection of CD4 in mouse spleen. + CD69 + The proportion of T cells; Figure 3 H represents H&E staining of mouse pancreas and lung tissue (scale bar = 100 μm).

[0026] Figure 4 Andrographolide significantly alleviated taurine-induced acute pancreatitis in mice; Figure 4 A represents the determination of serum amylase activity in mice; Figure 4 B represents the determination of serum lipase activity in mice; Figure 4 C represents the IL-1β content in mouse pancreatic tissue detected by ELISA; Figure 4 D represents the TNF-α content in mouse pancreatic tissue detected by ELISA; Figure 4 E represents the flow cytometry detection of CD4 in mouse spleen. + CD69 + The proportion of T cells; Figure 4 F represents H&E staining of mouse pancreas and lung tissue (scale bar = 100 μm).

[0027] Figure 5Abscisic acid significantly alleviated MSU-induced gouty arthritis in mice; Figure 5 A is a schematic diagram of an MSU-induced gouty arthritis model; Figure 5 B represents the change in foot thickness at different time points after MSU injection; Figure 5 C represents the morphological changes in the mouse paws 6 hours after injection of MSU; Figure 5 D represents H&E staining of mouse pancreas and lung tissue;

[0028] Figure 6 Andrographolide showed that it significantly alleviated MSU-induced gouty arthritis in mice; Figure 6 A represents the change in foot thickness at different time points after MSU injection; Figure 6 B shows H&E staining of mouse pancreas and lung tissue;

[0029] Figure 7 The results showed that abscisic acid significantly alleviated cholestatic liver injury induced by gallbladder ligation; Figure 7 A represents the survival status of patients within seven days after BDL surgery, as recorded by medication. Figure 7 B represents the change in mouse body weight within seven days after administration of the drug following BDL surgery. Figure 7 C represents the serum AST level measured seven days after BDL surgery. Figure 7 D represents the serum ALT level measured seven days after BDL surgery. Figure 7 E represents the serum IL-1β level measured seven days after BDL surgery. Figure 7 F represents H&E staining of mouse liver tissue. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0032] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0033] Example 1

[0034] Use of puromycin-sensitive aminopeptidase (PSAP) activators in the preparation of medicaments for the treatment and / or prevention of inflammatory diseases.

[0035] Puromycin-sensitive aminopeptidase can also be written as PSA or NPEPPS (puromycin-sensitive aminopeptidase).

[0036] The puromycin-sensitive aminopeptidase activator is selected from any one or more of abscisic acid and andrographolide.

[0037] The structural formula of abscisic acid is shown in the following formula (Ⅰ).

[0038]

[0039] The structural formula of andrographolide is shown in formula (II) below.

[0040]

[0041] Inflammatory diseases are selected from any one or more of acute pancreatitis, gouty arthritis, and cholestatic liver disease.

[0042] The drug formulation type is selected from any one or more of the following: tablets, capsules, granules, micro-pellets, droplets, oral liquids, soft capsules, water injections, powder injections, infusions, ointments, gels, and microemulsions.

[0043] The unit dose of tablets, capsules, granules, micro-pellets, drop pills, oral liquids, and soft capsules is 5-1000mg; the unit dose of water injections, powder injections, and infusions is 5-500mg.

[0044] The dosage of puromycin-sensitive aminopeptidase activator is 0.8-40 mg / kg, preferably 13 mg / kg.

[0045] In puromycin-sensitive aminopeptidase activators, the dosage of abscisic acid is 0.3-30 mg / kg, preferably 3 mg / kg; the dosage of andrographolide is 0.5-10 mg / kg, preferably 10 mg / kg.

[0046] Example 2

[0047] Abscisic acid, andrographolide, and PSAP are bound together.

[0048] 1. Experimental cells:

[0049] 293T cells.

[0050] 2. Experimental methods:

[0051] 2.1 Intracellular thermal migration:

[0052] Prepare two dishes of healthy 293T cells and treat them with 10 nM ABA for 3 h (the control group was treated with the same volume of DMSO for 2 h). Digest the drug-treated cells and transfer them to 1.5 mL EP tubes, washing twice with PBS. Resuspend the cells in 300 μL PBS, then divide the cell suspension into 10 equal portions of 30 μL each, and transfer each portion to a 200 μL EP tube. Heat the cells using a PCR instrument (setting the temperature range to 43℃-70℃, with 10 temperature points every 3℃, corresponding to 10 samples). Ensure that the control and drug groups are heated simultaneously for 3 min at each temperature point. After heating, remove the samples, equilibrate to room temperature for 3 min, and then store on ice. After all samples have been heated, freeze the cells at -80℃ for 15 min, then remove them and thaw them in a 37℃ water bath, repeating this process 3 times to lyse the cells. Transfer the lysed samples to 1.5 mL EP tubes. Subsequently, the mixture was centrifuged at 13,000 rpm and 4°C for 10 min. The supernatant was collected and 5× loading buffer was added. The mixture was then boiled in water for 7 min. The expression of PSAP was detected by Western blotting. Figure 1 D).

[0053] 2.2 ITC protein interactions:

[0054] The PSAP protein was expressed and purified, and its composition was determined using an ITC200 microcalorimeter with ABA or Andro drugs. Figure 1 A, Figure 1 B) The initial injection volume is 0.4 μL, followed by the injection of 1 μL aliquots of abscisic acid or andrographolide solution into a 300 μL PSAP protein solution at a constant stirring rate of 200 × g. Baseline correction is required for the initial calorific value. The dissociation constant Kd = 1 / Ka is calculated using the binding constant.

[0055] 2.3 Protein quality control:

[0056] Prepare a solution containing 100 ng of protein and 10 nM ABA. Use a control containing 100 ng of protein and DMSO solution. Aspirate from the sample tube and perform analysis. Figure 1 C).

[0057] 3. Analysis of experimental results:

[0058] like Figure 1 The figure shows the molecular docking simulation results of the binding sites of abscisic acid, andrographolide and PSAP, and ABA and PSAP. Figure 1 As shown in E.

[0059] Example 3

[0060] Abscisic acid and andrographolide are activators of PSAP.

[0061] 1. Experimental cells:

[0062] BMDM and THP-1 cells.

[0063] 2. Experimental methods:

[0064] 2.1 PSAP Oligomerization Detection:

[0065] PSAP protein (0.14 mg / ml) and ABA drugs (0.03, 0.1, 0.3, 1, 3 nM) were diluted with HEPES solution containing 60 mM KCl. 70 μL of diluted PSAP solution, 10 μL of ABA solution, and 18 μL of HEPES solution containing 60 mM KCl were added to a 96-well plate. After incubation on ice for 30 min, 2 μL of dye was added, and the plate was quickly placed in a microplate reader to measure the absorbance (ex. / em. = 550 / 600 nm).

[0066] 2.2 Detection of the effects of ABA and Andro on PSAP enzyme activity in a protein system:

[0067] Add 100 ng PSAP to each well of a 96-well plate and incubate with 0.03, 0.1, 0.3, 1, or 3 nM ABA or 30 μM Andro on ice for 30 min, then add the substrate and measure the absorbance (ex. / em. = 380 / 460 nm) using a multi-mode microplate reader.

[0068] 2.3 Detection of the effects of ABA and Andro on PSAP enzyme activity in a cell system:

[0069] (1) THP-1 cells or BMDM cells were seeded into 96-well plates. The cells were treated with phenol red-free medium one day before the experiment, followed by treatment with different concentrations of ABA for one hour. The cells were then washed with Hank Buffer, and enzyme activity detection buffer containing 40 μM of the detection substrate was added to the wells. The absorbance (ex. / em. = 380 / 460 nm) was measured using a multi-functional microplate reader. Data were collected over 30 minutes, and the reaction rate was calculated every 2 minutes.

[0070] (2) BMDM cells were seeded into 96-well plates. The day before the experiment, phenol red-free medium was added for treatment. After LPS induction, ABA or Andro was added, followed by stimulation with ATP, MSU, and Nigericin. Cells were then washed with Hank Buffer, and enzyme activity detection buffer containing 40 μM of the detection substrate was added to the wells. The absorbance (ex. / em. = 380 / 460 nm) was measured using a multi-mode microplate reader. Data were collected over 30 minutes, and the reaction rate was calculated every 2 minutes.

[0071] 3. Analysis of experimental results:

[0072] like Figure 2 As shown, ABA enhances PSAP enzyme activity by promoting PSAP oligomerization. Figure 2 A), in extracellular purified protein ( Figure 2 B, C) and intracellular system ( Figure 2 D) Detection showed that ABA could enhance PSAP enzyme activity, and this conclusion was further verified in PSAP knockout cells. Figure 2 E). Secondly, stimulation of BMDM with LPS and ATP / MSU / Nigericin followed by ABA treatment can enhance PSAP enzyme activity. Figure 2 F). Similarly, Andro enhanced PSAP enzyme activity in both extracellular purified protein systems and intracellular stimulation with LPS and ATP / MSU / Nigericin. Figure 2 G, H).

[0073] Example 4:

[0074] Intraperitoneal injection of abscisic acid improves taurine-induced acute pancreatitis in mice:

[0075] 1. Laboratory animals:

[0076] C57 / B6J male mice, 6-8 weeks old, 18-22g.

[0077] 2. Experimental reagents:

[0078] Rain frog extract (1352KD), Nanjing Peptide Industry.

[0079] 3. Experimental methods:

[0080] Six- to eight-week-old normal C57BL / 6 male mice were randomly divided into a Control group and an acute pancreatitis model group. Figure 3A) Five groups of mice were established: abscisic acid (A) low-dose group (0.3 mg / kg), abscisic acid medium-dose group (3 mg / kg), and abscisic acid high-dose group (30 mg / kg), with six mice in each group. Mice were fasted but allowed free access to water the day before modeling. On the second day, mice were injected intraperitoneally with A at the above-mentioned doses. Subsequently, mice were injected intraperitoneally at a dose of 200 μg / kg of abscisic acid every hour for a total of eight injections. One hour after the injections, the mice were sacrificed, and their serum and pancreatic tissue were collected for subsequent testing.

[0081] 4. Analysis of pharmacological experimental results:

[0082] like Figure 3 Abscisic acid significantly alleviated taeniacin-induced acute pancreatitis in mice. In mice given taeniacin, acute pancreatitis symptoms were accompanied by significant pancreatic and lung damage. Intraperitoneal injection of abscisic acid significantly reduced serum amylase and lipase activities. Figure 3 B, C). Furthermore, compared to the acute pancreatitis model group, abscisic acid treatment reduced the level of IL-1β in pancreatic tissue (B, C). Figure 3 D). As an indicator of systemic inflammation, abscisic acid administration reduces myeloperoxidase activity in pancreatic and lung tissues. Figure 3 E and F indicate that abscisic acid effectively reduced the degree of inflammatory response. Flow cytometry revealed CD4+ in the spleen. + CD69 + The total number of T cells decreased after abscisic acid treatment, indicating that abscisic acid effectively inhibited T cell activation. Figure 3 G). H&E staining showed that abscisic acid treatment reduced pancreatic necrosis and lung injury (G). Figure 3 H).

[0083] Example 5:

[0084] Intraperitoneal injection of andrographolide ameliorate reduced lecithin-induced acute pancreatitis in mice.

[0085] 1. Laboratory animals:

[0086] C57 / B6J male mice, 68 weeks old, 18 22g.

[0087] 2. Experimental reagents:

[0088] Rain frog extract (1352KD), Nanjing Peptide Industry.

[0089] 3. Experimental methods:

[0090] Six- to eight-week-old normal C57BL / 6 male mice were randomly divided into a Control group and an acute pancreatitis model group. Figure 3A) Five groups of mice were established, including a low-dose group (0.5 mg / kg), a medium-dose group (1 mg / kg), and a high-dose group (2 mg / kg), with six mice in each group. Mice were fasted but allowed free access to water the day before modeling. On the second day, mice were injected intraperitoneally with the above-mentioned doses of andrographolide. Subsequently, mice were injected intraperitoneally with 200 μg / kg of andrographolide every hour for a total of eight injections. One hour after the injections, the mice were sacrificed, and their serum and pancreatic tissue were collected for subsequent testing.

[0091] 4. Analysis of pharmacological experimental results:

[0092] like Figure 4 Andrographolide significantly alleviated lecithin-induced acute pancreatitis in mice. In mice given lecithin, acute pancreatitis symptoms were accompanied by significant pancreatic and lung damage. Intraperitoneal injection of andrographolide significantly reduced serum amylase and lipase activities. Figure 4 A, B). Furthermore, compared to the acute pancreatitis model group, andrographolide treatment reduced the levels of IL-1β and TNF-α in pancreatic tissue (…). Figure 4 C, D). Flow cytometry revealed CD4 levels in the spleen. + CD69 + The total number of T cells decreased after abscisic acid treatment, indicating that andrographolide effectively inhibited T cell activation. Figure 4 E). This indicates that andrographolide effectively reduced the degree of inflammatory response. H&E staining showed that andrographolide treatment reduced pancreatic necrosis and lung injury (E). Figure 4 F).

[0093] Example 6:

[0094] Intraperitoneal injection of abscisic acid improves MSU-induced gouty arthritis in mice:

[0095] 1. Laboratory animals:

[0096] C57 / B6J male mice, 6-8 weeks old, 18-22g.

[0097] 2. Experimental reagents:

[0098] MSU (190KD) Sigma-Aldrich.

[0099] 3. Experimental methods:

[0100] Six- to eight-week-old normal C57BL / 6 male mice were randomly divided into a Control group and a gouty arthritis model group. Figure 5A) Six groups were established: abscisic acid (A) low-dose group (0.3 mg / kg), A) medium-dose group (3 mg / kg), A) high-dose group (30 mg / kg), and colchicine positive control group (1 mg / kg), with six mice in each group. Before modeling, mice were intraperitoneally injected with the above-mentioned doses of A) or colchicine, followed by an intraperitoneal injection of MSU suspension into the paw. Paw thickness was measured every 3 hours until 24 hours after modeling, at which point the mice were sacrificed. The hind paws were harvested, and the skin tissue was carefully removed. The paws were then immersed in a decalcification solution for two weeks for subsequent testing.

[0101] 4. Analysis of pharmacological experimental results:

[0102] like Figure 5 The results showed that abscisic acid significantly alleviated MSU-induced gouty arthritis in mice. MSU injection induced paw edema in mice, accompanied by severe tissue damage, while abscisic acid administration significantly improved paw edema in mice. Figure 5 B, C), H&E staining showed that abscisic acid treatment reduced tissue damage (B, C). Figure 5 D).

[0103] Example 7:

[0104] Intraperitoneal injection of andrographolide improves MSU-induced gouty arthritis in mice:

[0105] 1. Laboratory animals:

[0106] C57 / B6J male mice, 6-8 weeks old, 18-22g.

[0107] 2. Experimental reagents:

[0108] MSU (190KD) Sigma-Aldrich.

[0109] 3. Experimental methods:

[0110] Six- to eight-week-old normal C57BL / 6 male mice were randomly divided into a Control group and a gouty arthritis model group. Figure 5 A) Four groups of mice were established, including a low-dose andrographolide group (5 mg / kg) and a high-dose andrographolide group (10 mg / kg), with six mice in each group. Before modeling, mice were injected intraperitoneally with the above-mentioned doses of andrographolide, followed by an intraperitoneal injection of MSU suspension into the paw. Paw thickness was measured every 3 hours until 24 hours after modeling, at which point the mice were sacrificed. The hind paws were harvested, and the skin tissue was carefully removed. The paws were then immersed in a decalcification solution for two weeks for subsequent testing.

[0111] 4. Analysis of pharmacological experimental results:

[0112] like Figure 6Andrographolide significantly alleviated MSU-induced gouty arthritis in mice. MSU injection induced paw swelling in mice, accompanied by severe tissue damage, while andrographolide administration significantly improved paw swelling in mice. Figure 6 A), H&E staining showed that andrographolide treatment reduced tissue damage ( Figure 6 B).

[0113] Example 8:

[0114] Intraperitoneal injection of abscisic acid improves cholestatic liver disease induced by gallbladder ligation:

[0115] 1. Laboratory animals:

[0116] C57 / B6J male mice, 6-8 weeks old, 18-22g.

[0117] 2. Experimental methods:

[0118] Six- to eight-week-old normal male C57BL / 6 mice were randomly divided into six groups: a control group, a BDL model group, a low-dose abscisic acid (1 mg / kg) group, a medium-dose abscisic acid (3 mg / kg) group, a high-dose abscisic acid (10 mg / kg) group, and a ursodeoxycholic acid (UDCA) positive control group (15 mg / kg), with six mice in each group. After gallbladder ligation or sham surgery to establish the model, the mice were intraperitoneally injected with the above-mentioned doses of abscisic acid, ursodeoxycholic acid, or PBS. The daily survival and weight changes of the mice in each group were recorded until they were sacrificed 7 days after the model was established. Blood was collected from the liver and eyeballs of the mice, and the paws were fixed in tissue fixation solution for subsequent testing.

[0119] 3. Analysis of pharmacological experimental results:

[0120] like Figure 7 The results showed that abscisic acid significantly alleviated cholestatic liver injury induced by gallbladder ligation. In mice, gallbladder ligation resulted in severe liver damage, significant weight loss, and mortality; however, abscisic acid administration significantly improved survival and slowed weight loss. Figure 7 A, B), ALT, AST enzyme activity, and H&E staining showed that abscisic acid treatment reduced liver tissue damage. Figure 7 C, D, F) reduced inflammation ( Figure 7 E).

[0121] Example 9:

[0122] Intraperitoneal injection of abscisic acid and andrographolide improved lecithin-induced acute pancreatitis and MSU-induced gouty arthritis in mice.

[0123] 1. Laboratory animals:

[0124] C57 / B6J male mice, 6-8 weeks old, 18-22g.

[0125] 2. Experimental methods:

[0126] The mouse acute pancreatitis model was constructed as shown in Example 4 or Example 5, except that the dosage of abscisic acid was 3 mg / kg and the dosage of andrographolide was 2 mg / kg, with the two being administered at intervals of 1 hour; a total of 8 administrations were given, and the mice were sacrificed and their serum, pancreatic tissue, etc. were collected for testing.

[0127] The mouse model of gouty arthritis was constructed as shown in Example 6 or Example 7, except that the dosage of abscisic acid was 3 mg / kg and the dosage of andrographolide was 5 mg / kg, administered at intervals of 24 hours; a total of 4 administrations were performed. The hind paws of the mice were removed and the skin tissue was carefully removed. The paws were placed in a decalcification solution for two weeks for subsequent testing.

[0128] 3. Analysis of pharmacological experimental results:

[0129] The treatment regimen of alternating administration of abscisic acid and andrographolide can effectively reduce the levels of IL-1β in pancreatic tissue and serum amylase. The comparison data of IL-1β levels are shown in Table 1, and the comparison data of serum amylase levels are shown in Table 2.

[0130] Table 1

[0131]

[0132] Table 2

[0133]

[0134]

[0135] The treatment regimen of alternating administration of abscisic acid and andrographolide significantly improved paw swelling in mice. Table 3 shows the comparison data of paw thickness measured 6 hours after MSU injection.

[0136] Table 3

[0137]

[0138] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Use of puromycin-sensitive aminopeptidase activators in the preparation of medicaments for the treatment and / or prevention of inflammatory diseases.

2. The use according to claim 1, characterized in that, The puromycin-sensitive aminopeptidase activator is selected from any one or more of abscisic acid and andrographolide.

3. The use according to claim 2, characterized in that, The puromycin-sensitive aminopeptidase activator was selected as abscisic acid and andrographolide.

4. The use according to claim 3, characterized in that, The inflammatory disease is selected from any one or more of acute pancreatitis, gouty arthritis, and cholestatic liver disease.

5. The use according to claim 4, characterized in that, The formulation type of the drug is selected from any one or more of the following: tablets, capsules, granules, micro-pellets, droplets, oral liquids, soft capsules, water injections, powder injections, infusions, ointments, gels, and microemulsions.

6. The use according to any one of claims 1-5, characterized in that, The dosage of the puromycin-sensitive aminopeptidase activator is 0.8-40 mg / kg, preferably 13 mg / kg.

7. The use according to claim 6, characterized in that, In the puromycin-sensitive aminopeptidase activator, the dosage of abscisic acid is 0.3-30 mg / kg, preferably 3 mg / kg.

8. The use according to claim 6, characterized in that, In the puromycin-sensitive aminopeptidase activator, the dosage of andrographolide is 0.5-10 mg / kg, preferably 10 mg / kg.

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