Inhibitor of phospholipase A2 family and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an inhibitor of a phospholipase A2 family and application thereof, and the inhibitor comprises isoquinoline alkaloid. The phospholipase A2 family inhibitor disclosed by the invention has high safety and tolerance, and can be specifically combined with phospholipase A2 family members by adopting natural isoquinoline alkaloid as an active component, so that the activity level of the phospholipase A2 family members is remarkably inhibited, the high expression level of inflammatory cytokines caused by abnormal enzyme activity is reduced, and the activity of the phospholipase A2 family members is improved. The application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to inhibitors of the phospholipase A2 family and applications thereof. Background Art

[0002] The phospholipase A2 (PLA2) family is a ubiquitous enzyme that catalyzes the hydrolysis of phospholipids to produce free fatty acids and lysophospholipids. This family comprises numerous members, including secretory phospholipase A2 (sPLA2), cytosolic phospholipase A2 (cPLA2), and calcium-independent phospholipase A2 (iPLA2). These enzymes play important roles in physiological processes such as cell signaling, inflammatory responses, and lipid metabolism. However, abnormal expression of the phospholipase A2 family can lead to a variety of diseases. For example, overexpression of sPLA2 at inflammatory sites is closely associated with inflammatory diseases such as rheumatoid arthritis and atherosclerosis. Abnormal activation of cPLA2 may contribute to the development and progression of tumors. For example, the activity of cytosolic phospholipase A2α (cPLA2α) is low in normal tissues but is significantly elevated in tumor tissues of certain cancer types or in various inflammatory cells activated during inflammatory responses. Therefore, the development of enzyme activity inhibitors of the phospholipase A2 family is of great significance for the treatment of diseases caused by abnormal expression of the phospholipase A2 family.

[0003] Currently, common phospholipase A2 family inhibitors are typically synthetic small molecule compounds, such as aryl fluorophosphate inhibitors. These inhibitors bind to the active site of the phospholipase A2 family, inhibiting its catalytic activity and thus exerting their therapeutic effects. However, these synthetic inhibitors may cause some side effects in clinical applications, such as hepatotoxicity and gastrointestinal discomfort.

[0004] Therefore, it is of great significance to find a natural product as an inhibitor of the phospholipase A2 family, and thus provide a new option for the treatment of diseases caused by abnormal expression of the phospholipase A2 family. Summary of the Invention

[0005] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, the present invention provides inhibitors of the phospholipase A2 family and their uses. The phospholipase A2 family inhibitors of the present invention are highly safe and well-tolerated. By employing naturally derived isoquinoline alkaloids as active ingredients, they are able to specifically bind to phospholipase A2 family members, thereby significantly inhibiting their activity levels and reducing the elevated expression of inflammatory cytokines caused by abnormal enzyme activity. They have broad application prospects.

[0006] In its first aspect, the present invention provides an inhibitor of the phospholipase A2 family. According to embodiments of the present invention, the inhibitor comprises an isoquinoline alkaloid. The inhibitor according to embodiments of the present invention, comprising an isoquinoline alkaloid, has high safety and tolerability. It can specifically bind to phospholipase A2 family members, thereby significantly inhibiting the activity levels of phospholipase A2 family members and reducing the high expression levels of inflammatory cytokines caused by abnormal enzyme activity. The inhibitor of the present invention has high specificity. At the same time, because the isoquinoline alkaloid is derived from a natural product, it has higher safety and tolerability, further reducing the side effects and risks of traditional synthetic inhibitors, and provides a new option for treating various diseases caused by abnormal expression of the phospholipase A2 family.

[0007] According to an embodiment of the present invention, the above-mentioned inhibitor may also have the following additional technical features:

[0008] According to an embodiment of the present invention, the phospholipase A2 family includes one or more of secretory phospholipase A2, cytoplasmic phospholipase A2, calcium ion-independent phospholipase A2, lipoprotein-associated phospholipase A2, lysosomal phospholipase A2 and adipose tissue-specific phospholipase A2.

[0009] According to an embodiment of the present invention, the cytoplasmic phospholipase A2 is a cytoplasmic phospholipase A2α enzyme.

[0010] According to an embodiment of the present invention, the isoquinoline alkaloid is an aporphine-type alkaloid.

[0011] According to a preferred embodiment of the present invention, the aporphine-type alkaloid is nuciferine.

[0012] In a second aspect, the present invention provides a method for inhibiting phospholipase A2 family activity in a sample. According to an embodiment of the present invention, the method comprises contacting the sample with the inhibitor and / or isoquinoline alkaloid described in the first aspect. According to the method of an embodiment of the present invention, by contacting the sample with an inhibitor or isoquinoline alkaloid, the phospholipase A2 family activity in the sample is efficiently and specifically inhibited.

[0013] According to an embodiment of the present invention, the above method may also have the following additional technical features:

[0014] According to an embodiment of the present invention, the phospholipase A2 family includes one or more of secretory phospholipase A2, cytoplasmic phospholipase A2, calcium ion-independent phospholipase A2, lipoprotein-associated phospholipase A2, lysosomal phospholipase A2 and adipose tissue-specific phospholipase A2.

[0015] According to an embodiment of the present invention, the cytoplasmic phospholipase A2 is a cytoplasmic phospholipase A2α enzyme.

[0016] According to an embodiment of the present invention, the isoquinoline alkaloid is an aporphine-type alkaloid.

[0017] According to a preferred embodiment of the present invention, the aporphine-type alkaloid is nuciferine.

[0018] According to an embodiment of the present invention, the sample includes one or more of a blood sample, a tissue sample, a saliva sample, and a stool sample.

[0019] In the third aspect of the present invention, the present invention proposes the use of isoquinoline alkaloids and the inhibitors described in the first aspect in the preparation of drugs for preventing, alleviating, assisting in the treatment or treating diseases caused by abnormal expression of the phospholipase A2 family.

[0020] Those skilled in the art will appreciate that the features and advantages described above for isoquinoline alkaloids or inhibitors of the phospholipase A2 family are also applicable to this use and will not be described in detail here.

[0021] According to an embodiment of the present invention, the above-mentioned use may also have the following additional technical features:

[0022] According to an embodiment of the present invention, the isoquinoline alkaloid is an aporphine-type alkaloid.

[0023] According to a preferred embodiment of the present invention, the aporphine-type alkaloid is nuciferine.

[0024] According to an embodiment of the present invention, the phospholipase A2 family includes one or more of secretory phospholipase A2, cytoplasmic phospholipase A2, calcium ion-independent phospholipase A2, lipoprotein-associated phospholipase A2, lysosomal phospholipase A2 and adipose tissue-specific phospholipase A2.

[0025] According to an embodiment of the present invention, the cytoplasmic phospholipase A2 is a cytoplasmic phospholipase A2α enzyme.

[0026] According to an embodiment of the present invention, the diseases caused by abnormal expression of the phospholipase A2 family include one or more of cardiovascular disease, rheumatoid arthritis, ulcerative colitis, neonatal necrotizing enterocolitis, obesity and colorectal cancer.

[0027] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments with reference to the following drawings, in which:

[0029] Figure 1This is a diagram showing the dynamic process of binding and dissociation between the C2 domain of cPLA2α protein and solutions of nuciferine at different concentrations as determined by SPR in Example 1 of the present invention;

[0030] Figure 2 This is a graph showing the affinity level between the C2 domain of cPLA2α protein and solutions of nuciferine at different concentrations as determined by SPR in Example 1 of the present invention;

[0031] Figure 3 Figure 2 is a graph showing the cPLA2α enzyme activity test results of the plasma and liver tissue samples of each group of mice in Example 2 of the present invention, wherein A is the cPLA2α enzyme activity test results of the liver tissue samples of each group of mice, and B is the cPLA2α enzyme activity test results of the plasma samples of each group of mice;

[0032] Figure 4 This is a graph showing the experimental results of the effect of nuciferine treatment on the proteolytic stability of cPLA2α in Example 3 of the present invention;

[0033] Figure 5 4 is a graph showing the expression level of inflammatory cytokines in each group of HepG2 cells in Example 4 of the present invention, wherein A is a graph showing the expression level of inflammatory cytokine TNF-α in each group of HepG2 cells; B is a graph showing the expression level of inflammatory cytokine IL-6 in each group of HepG2 cells; C is a graph showing the expression level of inflammatory cytokine IL-1β in each group of HepG2 cells; and D is a graph showing the expression level of inflammatory cytokine IL-8 in each group of HepG2 cells. DETAILED DESCRIPTION

[0034] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0035] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0036] The endpoints of the ranges and any values ​​disclosed herein 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 endpoints of each range, the endpoints of each range and 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 to be specifically disclosed herein.

[0037] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0038] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0039] Terms and Definitions

[0040] In this article, the term "isoquinoline alkaloids" refers to a class of N-based heterocyclic compounds, mainly including various alkaloids from the plant kingdom, which have a wide range of biological activities. Their core structure usually contains a nitrogen atom and often has multiple substituted groups such as hydroxyl and methoxy groups. Among them, nuciferine in the specific embodiment of the present invention belongs to the isoquinoline alkaloids. The inventors selected nuciferine as a representative and conducted experimental verification of its specific binding to the cytoplasmic phospholipase A2α enzyme, a member of the phospholipase A2 family.

[0041] As used herein, the term "aporphine-type alkaloid" refers to a type of isoquinoline alkaloid, which is structurally characterized by a unique four-ring system (ring AD) and a nitrogen atom in the B ring. Nuciferine in the specific embodiments of the present invention belongs to the aporphine-type alkaloid. The inventors selected nuciferine as a representative to experimentally verify its specific binding to the cytosolic phospholipase A2α enzyme, a member of the phospholipase A2 family.

[0042] In this article, the term "nuciferine" refers to a naturally occurring alkaloid, which is mainly extracted from plants such as the lotus of the Nymphaeaceae family and has multiple pharmacological effects and biological activities.

[0043] In this article, the term "cPLA2α" refers to the cytosolic phospholipase A2α enzyme, which belongs to the phospholipase A2 family. It is widely expressed in cells and is mainly involved in the release of arachidonic acid and hemolytic phospholipids. Among them, cPLA2α protein and cPLA2α enzyme (cytosolic phospholipase A2α enzyme) actually refer to the same molecule, that is, cPLA2α protein exhibits enzymatic activity, namely cPLA2α protease activity, under specific conditions (such as increased calcium ion concentration, cell activation, etc.).

[0044] Inhibitors of the phospholipase A2 family

[0045] The present invention provides an inhibitor of the phospholipase A2 family. According to an embodiment of the present invention, the inhibitor includes an isoquinoline alkaloid. The inhibitor according to the embodiment of the present invention, comprising an isoquinoline alkaloid, has high safety and tolerability. It can specifically bind to phospholipase A2 family members, thereby significantly inhibiting the activity levels of phospholipase A2 family members and reducing the high expression levels of inflammatory cytokines caused by abnormal enzyme activity. The inhibitor of the present invention has high specificity. At the same time, because the isoquinoline alkaloid is derived from a natural product, it has higher safety and tolerability, further reducing the side effects and risks of traditional synthetic inhibitors, and provides a new option for treating various diseases caused by abnormal expression of the phospholipase A2 family.

[0046] It should be noted that the activity level of phospholipase A2 family members can abnormally lead to high expression of multiple inflammatory cytokines, not limited to TNF-α, IL-6, IL-1β and IL-8 specifically verified in the embodiments of the present invention. All cases of high expression of inflammatory cytokines caused by abnormal expression of the phospholipase A2 family are within the scope of the present invention.

[0047] According to an embodiment of the present invention, the phospholipase A2 family includes one or more of secretory phospholipase A2, cytoplasmic phospholipase A2, calcium-independent phospholipase A2, lipoprotein-associated phospholipase A2, lysosomal phospholipase A2, and adipose tissue-specific phospholipase A2. Thus, the inhibitor of the present invention has a wide range of applications, targeting a variety of phospholipase A2 families, including secretory, cytoplasmic, and calcium-independent types.

[0048] According to an embodiment of the present invention, the cytoplasmic phospholipase A2 is a cytosolic phospholipase A2α enzyme. Therefore, the inhibitor of the present invention is particularly suitable for inhibiting the activity level of cytosolic phospholipase A2α enzyme and reducing the high expression levels of inflammatory cytokines caused by abnormal cytosolic phospholipase A2α enzyme activity, thereby providing a new option for treating diseases caused by abnormal cytosolic phospholipase A2α enzyme expression.

[0049] According to an embodiment of the present invention, the isoquinoline alkaloid is an aporphine-type alkaloid. Therefore, when the inhibitor of the present invention comprises an aporphine-type alkaloid, it is more effective in inhibiting the activity level of phospholipase A2 family members and reducing the high expression level of inflammatory cytokines caused by abnormal enzyme activity.

[0050] According to a preferred embodiment of the present invention, the aporphine-type alkaloid is nuciferine. As a naturally occurring alkaloid, nuciferine has high safety and biocompatibility. It is selected as the active ingredient in the inhibitor of the present invention. It can specifically bind to members of the phospholipase A2 family, effectively inhibiting the activity levels of phospholipase A2 family members and reducing the high expression levels of inflammatory cytokines caused by abnormal enzyme activity, while also having excellent medicinal potential. Exemplarily, the concentration of nuciferine can be 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, etc.

[0051] Among them, nuciferine has a structure as shown in Formula 1:

[0052]

[0053] It should be noted that although the examples of the present invention only use nuciferine and cytosolic phospholipase A2α enzyme as an example for detailed verification, due to the high similarity in structure and function between members of the phospholipase A2 family and the structural characteristics of isoquinoline alkaloids including nuciferine, they can also be used to inhibit the activity of other members of the phospholipase A2 family. Furthermore, all isoquinoline alkaloids that can specifically bind to members of the phospholipase A2 family and inhibit their activity levels should fall within the scope of protection of the present invention.

[0054] Method for inhibiting the activity of phospholipase A2 family in a sample

[0055] The present invention provides a method for inhibiting phospholipase A2 family activity in a sample. According to an embodiment of the present invention, the method comprises contacting the sample with the aforementioned inhibitor and / or isoquinoline alkaloid. The method according to an embodiment of the present invention efficiently and specifically inhibits phospholipase A2 family activity in the sample by contacting the sample with an inhibitor or isoquinoline alkaloid.

[0056] According to an embodiment of the present invention, the phospholipase A2 family includes one or more of secretory phospholipase A2, cytoplasmic phospholipase A2, calcium-independent phospholipase A2, lipoprotein-associated phospholipase A2, lysosomal phospholipase A2, and adipose tissue-specific phospholipase A2. Thus, the method of the present invention has a wide range of applicability and is applicable to different types of phospholipase A2 family members.

[0057] According to an embodiment of the present invention, the cytoplasmic phospholipase A2 is a cytoplasmic phospholipase A2α enzyme. Therefore, the method of the present invention is particularly suitable for cytoplasmic phospholipase A2α enzyme.

[0058] According to an embodiment of the present invention, the isoquinoline alkaloid is an aporphine-type alkaloid. Therefore, when an aporphine-type alkaloid is used, the method of the present invention has a better effect in inhibiting the activity level of phospholipase A2 family members.

[0059] According to a preferred embodiment of the present invention, the aporphine-type alkaloid is nuciferine. Thus, when nuciferine is used, the method of the present invention is more effective in inhibiting the activity level of phospholipase A2 family members. Exemplarily, the concentration of nuciferine can be 1 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, etc.

[0060] According to an embodiment of the present invention, the sample comprises one or more of a blood sample, a tissue sample, a saliva sample, and a stool sample. Thus, the method of the present invention is applicable to various types of samples, enabling the method of the present invention to be used in different research types and clinical scenarios.

[0061] It should be noted that the "sample" of the present invention can come from a healthy individual, a patient, or a subject, including but not limited to blood, urine, saliva, feces, tissue samples, cell samples, cerebrospinal fluid, semen, amniotic fluid, or other body fluids such as joint cavity fluid, pleural effusion, etc.; it can also come from other biological materials, including but not limited to cells and microorganisms obtained by biotechnology means.

[0062] use

[0063] The present invention proposes the use of isoquinoline alkaloids and the aforementioned inhibitors in preparing drugs for preventing, alleviating, assisting in the treatment or treating diseases caused by abnormal expression of the phospholipase A2 family.

[0064] It should be noted that the abnormal expression of the phospholipase A2 family of the present invention refers to an expression level higher than or much higher than the normal level, that is, overexpression.

[0065] Those skilled in the art will appreciate that the features and advantages described above for isoquinoline alkaloids or inhibitors of the phospholipase A2 family are also applicable to this use and will not be described in detail here.

[0066] According to an embodiment of the present invention, the isoquinoline alkaloid is an aporphine-type alkaloid. Therefore, the use is applicable to aporphine-type alkaloids.

[0067] According to a preferred embodiment of the present invention, the aporphine-type alkaloid is nuciferine. Therefore, the use is particularly suitable for nuciferine.

[0068] According to an embodiment of the present invention, the phospholipase A2 family includes one or more of secretory phospholipase A2, cytoplasmic phospholipase A2, calcium-independent phospholipase A2, lipoprotein-associated phospholipase A2, lysosomal phospholipase A2, and adipose tissue-specific phospholipase A2. Therefore, the use is broad and applicable to different types of phospholipase A2 family members.

[0069] According to an embodiment of the present invention, the cytoplasmic phospholipase A2 is a cytoplasmic phospholipase A2α enzyme. Therefore, the use is particularly suitable for cytoplasmic phospholipase A2α enzyme.

[0070] According to an embodiment of the present invention, the diseases caused by abnormal expression of the phospholipase A2 family include one or more of cardiovascular disease, rheumatoid arthritis, ulcerative colitis, neonatal necrotizing enterocolitis, obesity, and colorectal cancer. Thus, the use has a wide range of applications and is applicable to various diseases caused by abnormal expression of the phospholipase A2 family. For example, diseases caused by abnormal expression of cytoplasmic phospholipase A2α enzymes primarily include nervous system diseases, tumor-related diseases, cardiovascular diseases, and various inflammatory diseases, specifically, Alzheimer's disease, solid tumors such as breast cancer, hematologic tumors such as acute myeloid leukemia, cardiovascular diseases such as atherosclerosis, rheumatoid arthritis, and atopic dermatitis.

[0071] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0072] Example 1: Binding of Nuciferine to Target Protein cPLA2α

[0073] 1. Preparation of Nuciferine Solution

[0074] Nuciferine (NF) solutions with concentrations of 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM were prepared respectively and set aside.

[0075] 2. Plasmon Surface Resonance

[0076] Surface plasmon resonance (SPR) technology was used to investigate the binding of different concentrations of nuciferine solutions to the target protein cPLA2α. The specific steps are as follows:

[0077] The C2 domain of cPLA2α protein was fixed on the dedicated chip surface of the SPR molecular interaction instrument. The binding and dissociation dynamics and affinity levels of the C2 domain of cPLA2α protein with solutions of different concentrations of nuciferine were measured using the machine program of the SPR molecular interaction instrument. The experimental data were recorded and the results were analyzed.

[0078] The dynamic process of binding and dissociation between cPLA2α protein C2 domain and different concentrations of nuciferine solution was determined by SPR. Figure 1The results of SPR determination of the affinity between cPLA2α protein C2 domain and nuciferine solutions of different concentrations are shown in Figure 2 .

[0079] The results showed that as time gradually increased, the C2 domain of cPLA2α protein began to bind to solutions of different concentrations of nuciferine. After the binding reached equilibrium, as the buffer flowed, the C2 domain of cPLA2α protein began to dissociate from solutions of different concentrations of nuciferine. The higher the concentration of nuciferine, the stronger the binding signal. Figure 1 ); At the same time, the C2 domain of cPLA2α protein has a high affinity for nuciferine, with a KD value of 14.62μM ( Figure 2 ).

[0080] Example 2: Effect of Nuciferine Treatment on cPLA2 Enzyme Activity in Plasma and Liver Tissue of Mice with Hepatocellular Carcinoma

[0081] The inventors used the C57BL / 6J liver cancer mouse model to evaluate the effect of nuciferine on cPLA2 enzyme activity in the plasma and liver tissue of liver cancer mice. The specific scheme is as follows:

[0082] 1. Establishment of a mouse liver cancer model

[0083] Eighteen 3-week-old C57BL / 6J mice were randomly divided into a control group (Con), a liver cancer model group (DEN / Ccl4), and a nuciferine-treated group (DEN / Ccl4+NF), with 6 mice in each group.

[0084] (1) Control group (Con): When mice were three weeks old, they were intraperitoneally injected with an equal volume of PBS. After six weeks of development, they were intraperitoneally injected with an equal volume of olive oil twice a week for 16 weeks. At the same time, they were continuously gavaged with an equal volume of sodium dimethylcellulose (CMC-Na) to obtain control group mice.

[0085] (2) Liver cancer model group (DEN / Ccl4): At three weeks of age, mice were intraperitoneally injected with diethylnitrosamine (25 mg / kg) once. Six weeks after development, carbon tetrachloride (0.5 mL / kg) was intraperitoneally injected twice a week for 16 weeks. Simultaneously, an equal volume of sodium dimethylcellulose (CMC-Na) was continuously gavaged to obtain liver cancer model mice.

[0086] (3) Nuciferine-treated group (DEN / Ccl4+NF): Based on the treatment of the liver cancer model group mice, 10 mg / kg of nuciferine solution was gavaged daily for 16 weeks to obtain nuciferine-treated + liver cancer model group mice.

[0087] Among them, the solvent for dissolving diethylnitrosamine is PBS, and the solvent for dissolving carbon tetrachloride is olive oil.

[0088] 2. Sampling and testing

[0089] 12 h after the last oral administration of nuciferine solution, blood was collected from mice in each group and liver tissue samples were taken. The cPLA2α enzyme activity in plasma and liver tissue samples was then detected using an enzyme activity detection kit (purchased from Cayman, catalog number CAY-765021).

[0090] The results of cPLA2α enzyme activity detection in plasma and liver tissue samples of each group of mice are shown in Figure 3 .

[0091] The results showed that compared with the control group mice, the cPLA2α enzyme activity in the plasma and liver tissue samples of the liver cancer model group mice was significantly increased; however, after treatment with nucifera pine, the cPLA2α enzyme activity in the plasma and liver tissue samples of the mice in the nucifera pine treatment + liver cancer model group was significantly decreased.

[0092] The above results indicate that under the action of nuciferine, the cPLA2α enzyme activity in plasma and liver tissue was significantly inhibited.

[0093] Example 3: Effect of nuciferine treatment on the proteolytic stability of cPLA2α

[0094] 1. Cell culture

[0095] RAW264.7 cells (purchased from Shanghai Kanglang Biotechnology) in the logarithmic growth phase were selected and cultured at 3×10 5 / well were inoculated into six-well plates and cultured in a 37°C, 5% CO2 incubator overnight; then M-PER TM RAW264.7 cells were lysed using mammalian protein extraction reagent (purchased from Thermofisher, catalog number 78501) and centrifuged at 18,000X for 10 min at 4°C. The supernatant (cell lysate) was collected and the protein concentration of the supernatant was determined using a BCA kit (purchased from Solebro, catalog number PC0020).

[0096] 2. Nuciferine treatment

[0097] Blank control group: 300 μL of the supernatant (cell lysate) obtained in step 1 was incubated with an equal volume of dimethyl sulfoxide at 37°C and shaken at 100 rpm for 2 h to obtain the blank control group incubation solution;

[0098] Nuciferine-treated group: 300 μL of the supernatant (cell lysate) obtained in step 1 was incubated with nuciferine (30 μM) at 37° C. and shaken at 100 rpm for 2 h to obtain the nuciferine-treated group incubation solution.

[0099] After the incubation, the incubation fluid of the blank control group and the incubation fluid of the nuciferine-treated group were digested with protease (pronase E, 12ug pronase E was added for every 100ug protein) for 15min, and after the digestion was completed, 20μg of samples were taken for electrophoresis, gel transfer, and blocking solution was used for one hour. Then, the primary antibody was used at 4°C overnight, the membrane was washed, and the secondary antibody was incubated at room temperature for 1 hour, the membrane was washed, and then ECL development was performed; among them, the primary antibody was cPLA2 (purchased from Abmart, product number PA3288S) and GAPDH (purchased from Beyotime, product number AF1186), and the secondary antibody was HRP-labeled goat anti-mouse IgG (purchased from Beyotime, product number P0948).

[0100] The experimental results of the effect of nuciferine treatment on the proteolytic stability of cPLA2α are shown in Figure 4 .

[0101] The results showed that 30 μM nuciferine could bind well to cPLA2 protein, protect cPLA2 protein from protease degradation, and improve the enzymatic stability of the protein.

[0102] Example 4: Effect of Nuciferine Treatment on the Expression Levels of Inflammatory Cytokines

[0103] 1. Cell culture

[0104] HepG2 cells (purchased from Shanghai Kanglang Biotechnology) in the logarithmic growth phase were selected and cultured at 3×10 5 Each well was seeded with 100 μg of HepG2 cells per well and incubated overnight in a 37°C, 5% CO2 incubator. The following day, the HepG2 cells were observed and selected for the experiment. The cells were divided into a blank control group (Ctrl), a model group (Model), and a nuciferine-treated group (NF). The HepG2 cells in each group were treated as follows:

[0105] (1) Blank control group (Ctrl): HepG2 cells in this group were cultured in an equal volume of culture medium for 36 h to obtain the blank control group HepG2 cells;

[0106] (2) Model group: After the HepG2 cells in this group were treated with an equal volume of culture medium for 12 h, 1 μg / mL lipopolysaccharide (LPS) was added to stimulate the HepG2 cells in this group for 24 h to obtain the model group HepG2 cells;

[0107] (3) Nuciferine-treated group (NF): After the HepG2 cells in this group were treated with culture medium containing nuciferine (10 μM) for 12 h, 1 μg / mL LPS was added to the culture medium containing nuciferine (10 μM) to stimulate the HepG2 cells in this group for 24 h to obtain the nuciferine-treated HepG2 cells.

[0108] 2. RT-PCR detection

[0109] The culture medium of HepG2 cells in the blank control group, model group, and nuciferine-treated group was discarded, and the cells were washed once with PBS. 1 mL of RNA isolater (purchased from Vazyme, catalog number R401-01) was added to each well of the six-well plate. The cells were blown down with a pipette, and the lysate was transferred to a 1.5 mL centrifuge tube. RNA in the precipitate of each group was extracted according to the trizol method, and cDNA was synthesized using a high-throughput cDNA reverse transcriptase kit (purchased from Tiangen, catalog number KR116-02) according to the manufacturer's instructions. At the same time, primers were designed using the NCBI Primer-BLAST tool, and RT-PCR reactions were performed using Tiangen SYBR Green Supermix (purchased from Tiangen, catalog number FP217-02) and ThermoFisher QuantStudio 1 to detect the expression levels of inflammatory cytokines TNF-α, IL-6, IL-1β, and IL-8.

[0110] The results of the expression levels of inflammatory cytokines in HepG2 cells in each group are shown in Figure 5 .

[0111] The results showed that compared with the blank control group HepG2 cells, the expression levels of inflammatory cytokines TNF-α, IL-6, IL-1β, and IL-8 in the model group HepG2 cells were significantly increased; while under 10 μM nucifera treatment, the expression levels of inflammatory cytokines TNF-α, IL-6, IL-1β, and IL-8 in the nucifera treatment group HepG2 cells were significantly decreased.

[0112] The above results show that nucifera alkaloids have significant anti-inflammatory effects and can effectively inhibit the overexpression of inflammatory cytokines, thereby alleviating inflammatory responses. This finding further confirms the potential application value of nucifera alkaloids as a cPLA2α enzyme inhibitor in anti-inflammatory treatment, and provides a strong experimental basis for the development of anti-inflammatory drugs based on nucifera alkaloids.

[0113] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0114] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An inhibitor of the phospholipase A2 family, characterized in that Such inhibitors include isoquinoline alkaloids.

2. The inhibitor according to claim 1, characterized in that The phospholipase A2 family includes one or more of secretory phospholipase A2, cytoplasmic phospholipase A2, calcium ion-independent phospholipase A2, lipoprotein-associated phospholipase A2, lysosomal phospholipase A2 and adipose tissue-specific phospholipase A2; Optionally, the cytoplasmic phospholipase A2 is a cytosolic phospholipase A2α enzyme.

3. The inhibitor according to claim 1, characterized in that The isoquinoline alkaloid is an aporphine-type alkaloid; Preferably, the aporphine-type alkaloid is nuciferine.

4. A method for inhibiting the activity of phospholipase A2 family in a sample, characterized in that: include: The sample is contacted with the inhibitor and / or isoquinoline alkaloid according to any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The phospholipase A2 family includes one or more of secretory phospholipase A2, cytoplasmic phospholipase A2, calcium ion-independent phospholipase A2, lipoprotein-associated phospholipase A2, lysosomal phospholipase A2 and adipose tissue-specific phospholipase A2; Optionally, the cytoplasmic phospholipase A2 is a cytosolic phospholipase A2α enzyme.

6. The method according to claim 4, characterized in that The isoquinoline alkaloid is an aporphine-type alkaloid; Preferably, the aporphine-type alkaloid is nuciferine.

7. The method according to claim 4, characterized in that The sample includes one or more of a blood sample, a tissue sample, a saliva sample, and a stool sample.

8. Use of an isoquinoline alkaloid or the inhibitor according to any one of claims 1 to 3 in the preparation of a medicament for preventing, alleviating, assisting in the treatment or curing of diseases caused by abnormal expression of the phospholipase A2 family.

9. The use according to claim 8, characterized in that The isoquinoline alkaloid is an aporphine-type alkaloid; Preferably, the aporphine-type alkaloid is nuciferine.

10. The use according to claim 8, characterized in that The phospholipase A2 family includes one or more of secretory phospholipase A2, cytoplasmic phospholipase A2, calcium ion-independent phospholipase A2, lipoprotein-associated phospholipase A2, lysosomal phospholipase A2 and adipose tissue-specific phospholipase A2; Optionally, the cytoplasmic phospholipase A2 is a cytosolic phospholipase A2α enzyme.

11. The use according to claim 8, characterized in that The diseases caused by abnormal expression of the phospholipase A2 family include one or more of cardiovascular disease, rheumatoid arthritis, ulcerative colitis, neonatal necrotizing enterocolitis, obesity and colorectal cancer.

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