Application of cordycepin metabolite in preparation of medicine for treating acute pancreatitis

Abstract

The invention provides an application of a cordycepin metabolite in preparation of a medicine for treating acute pancreatitis, and the cordycepin metabolite is 2, 3-dihydroxybenzoic acid. The application of the cordycepin metabolite in preparation of the medicine for treating acute pancreatitis provides a new thought for treatment of pancreatitis, opens up a new way for global treatment of acute pancreatitis, and is expected to fundamentally improve the living quality and survival rate of patients; besides, 2, 3-dihydroxy benzoic acid is used as a metabolite of cordycepin, is derived from a natural substance, has relatively high safety and tolerance, possibly has fewer side effects compared with a traditional treatment method, and is more attractive in clinical application.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of cordycepin metabolites in the preparation of medicines for treating acute pancreatitis. Background Art

[0002] The annual incidence of acute pancreatitis (AP) is (13-45) / 100,000 people, and about 20% may develop into moderately severe acute pancreatitis (MSAP) or severe acute pancreatitis (SAP). The worldwide obesity epidemic may also lead to an increase in the global incidence of acute pancreatitis.

[0003] Major cellular changes in the pathogenesis of acute pancreatitis include pathological calcium signaling, mitochondrial dysfunction, premature activation of trypsinogen in acinar cells and macrophages, endoplasmic reticulum stress, impaired unfolded protein response, and impaired autophagy. These are triggered by common acinar cell toxins, such as alcohol, nicotine, and bile acids. These events can also be indirectly triggered by intraductal events, such as increased pressure caused by pancreatic duct obstruction, luminal acidification, and exposure of pancreatic duct cells to bile acids. Interplay between acinar cells and the immune system perpetuates the inflammatory response. At the locoregional level, the role of intra- and peripancreatic fat saponification and mesenteric lymph in modulating the severity of acute pancreatitis under ischemic conditions is well established. Despite the high global burden of disease, there is currently no effective drug to treat or prevent acute pancreatitis. Summary of the invention

[0004] In view of the defects in the prior art, the purpose of the present invention is to provide a use of a cordycepin metabolite in the preparation of a drug for treating acute pancreatitis, wherein the cordycepin metabolite is 2,3-dihydroxybenzoic acid.

[0005] The first aspect of the present invention provides the use of a cordycepin metabolite in the preparation of a medicament for treating acute pancreatitis, wherein the cordycepin metabolite is 2,3-dihydroxybenzoic acid.

[0006] In one embodiment, the drug has at least one of the effects (1) to (3):

[0007] (1) Inhibit serum pancreatic amylase activity;

[0008] (2) Inhibit lipase activity;

[0009] (3) Inhibit the expression of anti-inflammatory factors.

[0010] The second aspect of the present invention provides the use of a cordycepin metabolite in the preparation of a product for inhibiting serum pancreatic amylase activity, wherein the cordycepin metabolite is 2,3-dihydroxybenzoic acid.

[0011] The third aspect of the present invention provides the use of a cordycepin metabolite in the preparation of a product for inhibiting lipase activity, wherein the cordycepin metabolite is 2,3-dihydroxybenzoic acid.

[0012] A fourth aspect of the present invention provides the use of a cordycepin metabolite in the preparation of a product for inhibiting the expression of an anti-inflammatory factor, wherein the cordycepin metabolite is 2,3-dihydroxybenzoic acid.

[0013] Compared with the prior art, the embodiments of the present invention have the following beneficial effects: the use of the cordycepin metabolites provided by the present invention in the preparation of drugs for treating acute pancreatitis provides a new idea for the treatment of pancreatitis, opens up a new approach for the treatment of acute pancreatitis worldwide, and is expected to fundamentally improve the quality of life and survival rate of patients; in addition, 2,3-dihydroxybenzoic acid, as a metabolite of cordycepin, is derived from natural substances, has higher safety and tolerability, and may have fewer side effects than traditional treatment methods, making it more attractive in clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0015] Figure 1 This is the result of testing the amylase level in mice with caerulein-induced acute pancreatitis treated with cordycepin;

[0016] Figure 2 This is the result of testing the lipase level in mice with caerulein-induced acute pancreatitis treated with cordycepin;

[0017] Figure 3 This is the result of detecting tumor necrosis factor-α level in mice with caerulein-induced acute pancreatitis treated with cordycepin;

[0018] Figure 4 This is the result of testing the interleukin-6 level in mice with caerulein-induced acute pancreatitis treated with cordycepin;

[0019] Figure 5 This is the result of testing the interleukin-1β level in mice with caerulein-induced acute pancreatitis treated with cordycepin;

[0020] Figure 6This is the result of testing the interleukin-18 level in mice with caerulein-induced acute pancreatitis treated with cordycepin;

[0021] Figure 7 This is the result of HE staining of pancreatic tissue after different treatments in the mouse model of acute pancreatitis induced by caerulein;

[0022] Figure 8 The results of the detection of transforming growth factor-β levels in the supernatant of acinar cells after different treatments;

[0023] Fig. 9 This is the result of the detection of interferon-α level in the supernatant of acinar cells after different treatments;

[0024] Fig.10 This is the result of detecting the interferon-γ level in the supernatant of acinar cells after different treatments;

[0025] Fig.11 The results of the interleukin-6 level detection in the supernatant of acinar cells after different treatments;

[0026] Fig.12 The results of the interleukin-1β level test in the supernatant of acinar cells after different treatments;

[0027] Fig.13 The results of the interleukin-10 level detection in the supernatant of acinar cells after different treatments;

[0028] Fig.14 This is the result of testing the amylase level in mice with acute pancreatitis induced by sodium taurocholate treated with cordycepin;

[0029] Fig.15 This is the result of testing the lipase level in mice with acute pancreatitis induced by sodium taurocholate treated with cordycepin;

[0030] Fig.16 This is the result of detecting tumor necrosis factor α level in mice with acute pancreatitis induced by sodium taurocholate treated with cordycepin;

[0031] Fig.17 This is the result of testing the interleukin-6 level in mice with acute pancreatitis induced by sodium taurocholate treated with cordycepin;

[0032] Fig.18 This is the result of testing the interleukin-1β level in mice with acute pancreatitis induced by sodium taurocholate treated with cordycepin;

[0033] Fig.19 This is the result of testing the interleukin-18 level in mice with acute pancreatitis induced by sodium taurocholate treated with cordycepin;

[0034] Fig. 20 This is the result of metabolomics analysis of the caerulein-induced acute pancreatitis mouse model;

[0035] Fig.21 This is the result of differential gene expression detection of cordycepin and its metabolites;

[0036] Fig. 22 This is a graph showing the results of differential gene expression detection of hypotaurine, 2,3-dihydroxybenzoic acid and 2,6-dihydroxybenzoic acid;

[0037] Fig.23 This is the result of the interleukin-6 level test in acinar cells treated with sodium taurocholate for 24 hours after the treatment with cordycepin and its metabolites;

[0038] Fig.24 This is the result of detecting tumor necrosis factor α level in acinar cells treated with sodium taurocholate and cordycepin and its metabolites for 24 hours;

[0039] Fig.25 This is the result of interleukin-1β level detection in acinar cells treated with sodium taurocholate and cordycepin and its metabolites for 24 hours. DETAILED DESCRIPTION

[0040] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0041] Acute pancreatitis is a common acute abdomen characterized by acinar necrosis and local or systemic inflammatory response. Acute pancreatitis is closely associated with multi-organ damage and increased mortality, so preventing the progression of acute pancreatitis is the top priority in the treatment of acute pancreatitis. Although many treatments for acute pancreatitis have emerged in the past few decades, there are still no specific drugs and effective targets for the treatment of acute pancreatitis. Therefore, the development of AP treatment methods is a priority.

[0042] In 1950, Cunningham et al. first isolated the main active ingredient, the natural compound cordycepin, from Cordyceps militaris. Cordycepin is the first discovered natural derivative of adenosine, which is a needle-shaped or flaky white crystal with a structural formula similar to that of adenosine. The molecular formula of cordycepin is C 10 H 13 N5O3, molecular weight is 251.24. Unlike adenosine, cordycepin lacks the 3'-hydroxyl group in the ribose part, which is replaced by hydrogen to form a deoxy analogue of adenosine.

[0043] Cordycepin has multiple intracellular targets and can act on multiple signaling pathways. Cordycepin shows a wide range of therapeutic potential in anti-tumor, anti-inflammatory, anti-oxidative stress, hypoglycemic and hypolipidemic effects. At present, research on cordycepin focuses on its anti-cancer, anti-inflammatory, anti-oxidative and immune regulation. In 2019, the study by Fu Xiaobing et al. showed that in terms of anti-oxidation and anti-inflammation, on the one hand, cordycepin upregulates the expression of cellular antioxidant-related proteases, inhibits the generation of radiation-induced reactive oxygen species (ROS), and reduces the damage of oxidative stress to cells; on the other hand, cordycepin can effectively prevent and treat radiation ulcer damage, and inhibit inflammation and aging through the dual pathways of NRF2 and AMPK, which is of great clinical development value. Cordycepin also has a significant effect in the treatment of metabolic inflammation. Cordycepin alleviates metabolic inflammation in mice fed a Western diet by targeting the integrity of the intestinal barrier and intestinal flora, inhibits intestinal inflammation, oxidative stress damage, reduces intestinal epithelial cell apoptosis and pyroptosis, and significantly improves intestinal barrier damage caused by a Western diet. At the same time, cordycepin exerts a significant protective effect on hepatic steatosis, inflammation, liver damage and fibrosis in metabolic stress mice by activating the AMP-activated protein kinase signaling pathway, thereby improving non-alcoholic fatty liver disease.

[0044] Obviously, cordycepin is a very valuable natural medicinal ingredient. However, cordycepin itself still has certain application defects. Since the structure of cordycepin is similar to adenosine, it is easily metabolized by adenosine deaminase (ADA) into 3'-deoxyinosine after entering the body. 3'-deoxyinosine is an inactive metabolite of cordycepin. Studies have shown that when cordycepin is given to rats by oral and intravenous routes, its pharmacokinetic results show that the level of cordycepin in the blood decreases rapidly, and cordycepin has a short elimination half-life and a high clearance rate. The poor pharmacokinetic properties of cordycepin seriously limit its further application. Therefore, it is of great significance to study methods to slow down its deamination reaction in order to improve its bioavailability and efficacy and expand its scope of application.

[0045] The present invention constructs a cell model and an animal model of acute pancreatitis by using caerulein, and verifies the anti-inflammatory effect of cordycepin on acute pancreatitis. At the same time, we also verified the effect of cordycepin in inhibiting pancreatic necrosis in severe acute pancreatitis induced by sodium taurocholate. Based on the above, metabolomics was further used to analyze the mechanism of action and differential metabolite changes of acute pancreatitis after cordycepin treatment. The results of GSEA gene set enrichment analysis showed that hypotaurine, 2,3-dihydroxybenzoic acid and 2,6-dihydroxybenzoic acid were metabolites with significant differential expression of endogenous metabolites of cordycepin. Compared with the control group, the levels of the above three metabolites increased after cordycepin treatment. These data indicate that the metabolites of mice with acute pancreatitis changed before and after cordycepin treatment.

[0046] Furthermore, the present invention verifies the effects of cordycepin and its metabolites in a cell model of acute pancreatitis induced by caerulein. By detecting typical markers of acute pancreatitis, the results show that 2,3-dihydroxybenzoic acid has a more excellent therapeutic effect.

[0047] The first aspect of the present invention provides the use of a cordycepin metabolite in the preparation of a medicament for treating acute pancreatitis, wherein the cordycepin metabolite is 2,3-dihydroxybenzoic acid.

[0048] In one embodiment, the drug has at least one of the effects (1) to (2):

[0049] (1) Inhibit serum pancreatic amylase activity;

[0050] (2) Inhibit lipase activity;

[0051] (3) Inhibit the expression of anti-inflammatory factors.

[0052] The second aspect of the present invention provides the use of a cordycepin metabolite in the preparation of a product for inhibiting serum pancreatic amylase activity, wherein the cordycepin metabolite is 2,3-dihydroxybenzoic acid.

[0053] The third aspect of the present invention provides the use of a cordycepin metabolite in the preparation of a product for inhibiting lipase activity, wherein the cordycepin metabolite is 2,3-dihydroxybenzoic acid.

[0054] A fourth aspect of the present invention provides the use of a cordycepin metabolite in the preparation of a product for inhibiting the expression of an anti-inflammatory factor, wherein the cordycepin metabolite is 2,3-dihydroxybenzoic acid.

[0055] The present invention is further described below by way of examples.

[0056] Example

[0057] The mouse model of acute pancreatitis induced by cerulein was adopted. Cerulein (20-100μg / kg, commonly used concentration 50μg / kg) was intraperitoneally injected 7 times at 1 hour intervals. Different drug treatments were given after the last modeling. The cordycepin treatment group was intraperitoneally injected with cordycepin (200mg / kg) after the last modeling, and the modeling group was treated with an equal amount of normal saline after the last modeling. The sham operation group was not treated, and the cordycepin control group was intraperitoneally injected with cordycepin (200mg / kg).

[0058] After 12 hours, serum was collected for ELISA testing. In this example, ELISA testing was performed according to the ELISA kit of Dakoway Biotechnology Co., Ltd. and the instructions of Shanghai ELISA Biological. The levels of amylase ( Figure 1 ), lipase level ( Figure 2 ), inflammatory factor tumor necrosis factor α level ( Figure 3 ), interleukin-6 level ( Figure 4 ), interleukin-1β level ( Figure 5 ) and interleukin-18 levels ( Figure 6 ). Specific operation steps: Before use, equilibrate the kit at room temperature for 30 minutes; Blank well: do not add sample, only add color developer A, B and stop solution for zero adjustment; Standard well: add 100μL of diluted standard to each well, and then add 50μL of biotin antigen working solution; Zero well: add 100μL of standard / sample diluent. Then add 50μL of biotin antigen working solution; Sample well: add 100μL of sample, and then add 50μL of biotin antigen working solution; Shake gently, cover with sealing film, and incubate in a 37℃ incubator for 90min; Dilute the 50-fold concentrated washing solution with 50-fold distilled water for use; First wash: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 60 seconds and discard, repeat 4 times, and pat dry; Add 100μL TMB was added to the zero well, standard and sample wells, shaken gently, covered with a sealing film, and incubated in a 37°C incubator for 30 minutes; second wash: carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let it stand for 60 seconds and then discard, repeat this 4 times, and pat dry; color development: add 100μL of color developer to each well, shake gently to mix, and color at 37°C for 10 minutes; stop: add 100μL of stop solution to each well to stop the reaction; determination: adjust the blank well to zero, and measure the absorbance of each well at a wavelength of 450nm. In the acute edematous pancreatitis group, the increase in serum pancreatic amylase was positively correlated with the severity of pancreatitis. Intraperitoneal injection of caerulein significantly induced the increase of amylase, lipase and proinflammatory cytokines tumor necrosis factor α, interleukin 6, interleukin and interleukin 18 in mice, while cordycepin treatment significantly inhibited the increase in amylase, lipase and proinflammatory factors ( Figure 1-Figure 6 ).

[0059] The results showed that compared with mice in the group with acute pancreatitis induced by caerulein, cordycepin significantly reduced serum amylase and lipase levels, inhibited the increase of inflammatory factors, and reduced pancreatic edema. Cordycepin treatment had a significant protective effect on acute pancreatitis induced by caerulein. Hematoxylin-eosin staining of pancreatic tissue also proved that cordycepin could reduce pancreatic damage ( Figure 7 ).

[0060] In this example, the therapeutic effect of cordycepin was also verified at the cellular level ( Figure 8-Figure 13 ). The acute pancreatitis model of MPC-83 mouse pancreatic acinar cells was induced by caerulein. The cordycepin control group and cordycepin treatment group were pretreated with 50μM and 100μM cordycepin for 30 minutes, respectively. The blank control group and model group were added with the same volume of PBS solution. After 30 minutes of pretreatment, the cordycepin treatment group and model group were added with 10 -7Mol / L caerulein was treated for 24 hours, and an equal volume of PBS solution was added to the blank control group and the cordycepin control group. After 24 hours, the cell supernatant of each group was collected, and the particles and polymers were removed after centrifugation at 4°C and 3000rpm for 15 minutes, and the supernatant was collected for ELISA detection. The ELISA detection in this embodiment was performed according to the instructions of the ELISA kit of Dakoway Biotechnology Co., Ltd., and the specific operation was the same as above. The results showed that cordycepin significantly reduced the growth factor β ( Figure 8 ), interferon-α ( Fig. 9 ), interferon-γ ( Fig.10 ), interleukin-6( Fig.11 ), interleukin-1β level ( Fig.12 ) and increased the level of anti-inflammatory factor interleukin-10 ( Fig.13 ). Consistent with the results in animals, cordycepin reduced the damage of alveolar cells caused by caerulein.

[0061] At the same time, this example also tests the therapeutic effect of cordycepin in the mouse model of severe acute pancreatitis induced by sodium taurocholate. The modeling group and the cordycepin treatment group used a microinjection pump to retrogradely infuse 50ul of 3mM sodium taurocholate (TLCS) into the pancreatic duct. After modeling, the cordycepin treatment group gave mice an intraperitoneal injection of cordycepin (200mg / kg), and the modeling group was treated with an equal amount of normal saline. The sham operation group did not undergo any operation, and the cordycepin control group was intraperitoneally injected with cordycepin (200mg / kg). After 12 hours, mouse serum was collected for ELISA testing. In this example, the ELISA test was performed according to the ELISA kit of Dakoway Biotechnology Co., Ltd. and the instructions of Shanghai ELISA Biology, and the specific operation was the same as above. The results also show that cordycepin can improve pancreatic tissue necrosis caused by sodium taurocholate and reduce serum amylase ( Fig.14 ) and lipase ( Fig.15 ) levels, and significantly reduced the levels of pro-inflammatory factors ( Figure 16-Figure 19 ).

[0062] In this example, metabolomics analysis was performed on the endogenous metabolite composition of mice with severe acute pancreatitis induced by sodium taurocholate induced by cordycepin treatment. The modeling group and the cordycepin treatment group used a microinjection pump to retrogradely infuse 50ul of 3mM sodium taurocholate (TLCS) into the pancreatic duct. After modeling, the cordycepin treatment group was given an intraperitoneal injection of cordycepin (200mg / kg), and the modeling group was treated with an equal amount of normal saline. The sham operation group did not undergo any operation, and the cordycepin control group was given an intraperitoneal injection of cordycepin (200mg / kg). The analysis found that compared with the sham operation group, the levels of metabolites such as hypotaurine and 2,3-dihydroxybenzoic acid increased after cordycepin treatment ( Fig. 20). And through the differential gene expression of metabolites, three metabolites with significant differences were enriched: hypotaurine, 2,3-dihydroxybenzoic acid and 2,6-dihydroxybenzoic acid ( Fig.21 , Fig. 22 ).

[0063] Based on metabolomics analysis, it was found that the therapeutic effects of cordycepin, hypotaurine, 2,3-dihydroxybenzoic acid and 2,6-dihydroxybenzoic acid at the same concentration were detected by ELISA in the model of sodium taurocholate-induced acinar cell acute pancreatitis. In this embodiment, the ELISA test was performed according to the ELISA kit of Dakoway Biotechnology Co., Ltd. and the instructions of Shanghai ELISA Biology, and the specific operation was the same as above. Compared with cordycepin, hypotaurine and 2,6-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid more significantly reduced the inflammatory factor interleukin-6 (IL-6) at 24h. Fig.23 ), tumor necrosis factor α ( Fig.24 ) and interleukin-1β levels ( Fig.25 ) levels. The metabolite 2,3-dihydroxybenzoic acid showed a more excellent therapeutic effect on acute pancreatitis.

[0064] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. Use of cordycepin metabolites in the preparation of a drug for treating acute pancreatitis, characterized in that: The cordycepin metabolite is 2,3-dihydroxybenzoic acid.

2. The use of the cordycepin metabolite according to claim 1 in the preparation of a medicament for treating acute pancreatitis, characterized in that: The drug has at least one of the effects (1) to (3): (1) Inhibit serum pancreatic amylase activity; (2) Inhibit lipase activity; (3) Inhibit the expression of anti-inflammatory factors.

3. Use of cordycepin metabolites in the preparation of products for inhibiting serum pancreatic amylase activity, characterized in that: The cordycepin metabolite is 2,3-dihydroxybenzoic acid.

4. Use of cordycepin metabolites in the preparation of products for inhibiting lipase activity, characterized in that: The cordycepin metabolite is 2,3-dihydroxybenzoic acid.

5. Use of cordycepin metabolites in the preparation of products for inhibiting the expression of anti-inflammatory factors, characterized in that: The cordycepin metabolite is 2,3-dihydroxybenzoic acid.

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