Use of aegyptianus egg mass toxin-vi protein in the preparation of anti-inflammatory agents

Abstract

The application provides application of an intermediodorsal or spider ovigorous granule toxin-VI protein in preparation of an anti-inflammatory reagent, which can inhibit M1 type macrophage activation, promote M2 type macrophage cell transformation, inhibit macrophage and microglia cell activation and inhibit LPS-induced inflammation. The intermediodorsal or spider ovigorous granule toxin-VI protein disclosed by the application can significantly inhibit excessive activation of inflammation in an LPS-induced inflammation model, has good anti-inflammatory effects on peripheral inflammation and neural inflammation, has good anti-inflammatory effects in in-vivo and in-vitro inflammation models, and experiments show that the intermediodorsal or spider ovigorous granule toxin-VI protein has no obvious toxic side effects on mice at an inflammation inhibiting dose, and has good clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of a spider oocyte toxin-VI protein in the preparation of anti-inflammatory agents. Background Technology

[0002] Inflammation refers to the body's or tissue's normal defense against damaging factors. Under normal circumstances, inflammation activates macrophages to secrete a series of immune cytokines, which in turn kill pathogens, eliminate damaging factors, and clear and phagocytose necrotic cells. Therefore, as an immune response, a moderate inflammatory response is beneficial to the body; however, an excessive inflammatory response can affect the body's normal metabolic processes, causing septic shock, multiple organ dysfunction, and even endangering life.

[0003] Antibiotics are commonly used to treat inflammation, but they are only effective against infectious inflammation and not non-infectious inflammation. Furthermore, while existing glucocorticoids and nonsteroidal anti-inflammatory drugs (NSAIDs) can be used for anti-inflammatory treatment, achieving the desired anti-inflammatory effect requires long-term, high-dose use of these drugs. Long-term, high-dose use of these anti-inflammatory drugs can cause adverse reactions and significant side effects, resulting in considerable suffering for patients.

[0004] Latroeggtoxin-VI (LETX-VI) is a protein with an average molecular weight of 6199 kDa, found in the eggs of the black widow spider. Previous studies have shown that Latroeggtoxin-VI can enter pc12 cells and promote the synthesis and release of dopamine, but its anti-inflammatory effects have not yet been investigated. Summary of the Invention

[0005] To overcome a series of problems caused by the use of antibiotics to treat inflammation in existing technologies, this invention provides the application of *Sargassum fusiforme* oocyte toxin-VI protein in the preparation of anti-inflammatory agents. *Sargassum fusiforme* oocyte toxin-VI protein of this invention exhibits good anti-inflammatory effects in both in vivo and in vitro inflammation models. Furthermore, experiments show that at the dose that inhibits inflammation, it has no obvious toxic side effects on mice, and it significantly inhibits excessive activation of inflammation in LPS-induced inflammation models, demonstrating excellent anti-inflammatory activity.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] The first aspect of this invention provides the use of the oocyte toxin-VI protein from the spider spider in the preparation of anti-inflammatory agents.

[0008] As an optional implementation, the application provided by the present invention includes the use of spider oocyte toxin-VI in the preparation of an agent that inhibits the activation of M1 macrophages.

[0009] As an optional implementation, the application provided by the present invention includes the use of *Sargassum fusiforme* oocyte toxin-VI in the preparation of a reagent that promotes the transformation of M2 macrophages.

[0010] As an optional implementation, in the applications provided by the present invention, the application includes the use of spider oocyte toxin-VI in the preparation of a reagent to inhibit macrophage activation.

[0011] As an optional implementation, the application provided by the present invention includes the use of *Sargassum fusiforme* oocyte toxin-VI in the preparation of a reagent to inhibit microglia activation.

[0012] As an optional implementation, the application provided by the present invention includes the use of spider otoxin-VI in the preparation of an agent that inhibits LPS-induced inflammation.

[0013] As an optional implementation, in the application provided by the present invention, the anti-inflammatory agent further includes a drug that is compatible with mitochondrial oocyst toxin-VI.

[0014] As an optional implementation, in the application provided by the present invention, the anti-inflammatory agent includes an agent that inhibits peripheral inflammation or neuroinflammation.

[0015] As an optional implementation, in the application provided by the present invention, the anti-inflammatory agent is formulated as a solution, colloidal solution, emulsion, or suspension.

[0016] As an optional implementation, in the application provided by the present invention, the dosage form of the anti-inflammatory agent is an oral fast-dissolving film, oral liquid, capsule, injection, or transdermal absorption preparation.

[0017] A second aspect of the present invention provides an anti-inflammatory agent comprising an effective therapeutic dose of tarantula ovotoxin-VI, and a pharmaceutically acceptable carrier or excipient.

[0018] As an optional implementation, in the anti-inflammatory reagent provided by the present invention, the excipients include excipients or diluents.

[0019] The amino acid sequence of the intermediate spider otoxin-VI in this invention is as follows: EMTCADTQGQ CVAGNDCSCCGQYDKCDCTW NLGVRTCKCK RVAILSDWKK NLNCPQ. Even after chemical modification, substitution, deletion, or addition of one or more amino acids in the shown amino-terminal sequence, it still possesses the anti-inflammatory activity described in this application.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The Latroeggtoxin-VI (LETX-VI) protein of this invention has good anti-inflammatory effects. It can significantly inhibit the excessive activation of inflammation in LPS-induced inflammation models, and has good anti-inflammatory effects on both peripheral and neuroinflammation. It also has good anti-inflammatory effects in in vivo and in vitro inflammation models. Furthermore, experiments have shown that at the anti-inflammatory dose, there are no obvious toxic side effects in mice, indicating good prospects for clinical application. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The graph shows the inhibition results of LETX-VI protein on the pro-inflammatory mediator NO in Example 1;

[0024] Figure 2 The graph shows the inhibition results of LETX-VI protein on inflammatory factors IL-6 and TNF-α in Example 2;

[0025] Figure 3 This is a diagram showing the results of LETX-VI protein inhibiting macrophage M1 activation in Example 3;

[0026] Figure 4 This is a diagram showing the results of LETX-VI protein promoting the conversion of macrophages from M1 to M2 types in Example 4;

[0027] Figure 5 This is a diagram showing the results of LETX-VI protein inhibiting M1 type activation of microglia in brain tissue in Example 5;

[0028] Figure 6 This is a diagram showing the results of LETX-VI protein promoting the conversion of microglia from M1 to M2 types in brain tissue in Example 6;

[0029] Figure 7 This is a diagram showing the results of LETX-VI protein inhibiting the activation of microglia in the hippocampus and substantia nigra regions of the brain in Example 7.

[0030] Figure 8 The figure shows the safety test results of LETX-VI protein in mice in Example 8. Detailed Implementation

[0031] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0032] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0033] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0034] The oocyte toxin-VI protein of *Cyprinus intercalatus*, referred to as LETX-VI protein below, can be prepared by referring to the protein expression method in "Gene cloning, heterologous expression and activity identification of oocyte toxin-VI of *Cyprinus intercalatus*", Chinese Journal of Biotechnology, 2021, 37(02).

[0035] Example 1

[0036] The inhibitory effect of LETX-VI protein on the pro-inflammatory mediator NO.

[0037] Lipopolysaccharide (LPS) can be used to establish inflammation models both in vivo and in vitro. LPS injection is a widely used model for exploring the pathobiology of inflammation. If inflammation is not controlled, LPS injection in mice leads to systemic inflammation, neuronal damage, and memory loss. Peripheral injection of LPS derived from *E. coli* can induce cerebrovascular and central nervous system inflammation. This invention aims to investigate the inhibition or attenuation of excessive inflammation by using *Latroeggtoxin-VI* prior to LPS inflammatory stimulation.

[0038] Cultured macrophages were divided into a control group, an LPS treatment group, and a LETX-VI intervention group. The LPS treatment group was treated with LPS (1 μg / ml) for 12 h, while the LETX-VI intervention group was treated with LETX-VI (0.1 μM and 4 μM, respectively) and LPS (1 μg / ml) for 12 h each. After treatment, cell culture medium was collected, and the release of nitric oxide (NO) from macrophages into the culture medium was quantitatively measured using a nitric oxide (NO) assay kit (Beyotime.s0021). The detection method was as follows: First, a sodium nitrite standard curve was established. 50 μl of cell-free culture medium was mixed with 50 μl of Griess reagent I and 50 μl of Griess reagent II, and the absorbance was recorded at 540 nm using a microplate reader (Bio-Rad Laboratories, CA, USA). The detection results are as follows. Figure 1 As shown, LPS treatment alone increased NO levels (P<0.001). Intervention with 0.1 μM LETX-VI significantly inhibited LPS-induced NO release (P<0.05), and interventions with 1 μM and 4 μM LETX-VI showed even more significant inhibitory effects on LPS-induced NO release (P<0.01), indicating that LETX-VI can significantly inhibit the increase of the pro-inflammatory mediator NO and possesses significant anti-inflammatory activity.

[0039] Example 2

[0040] The inhibitory effect of LETX-VI protein on inflammatory factors IL-6 and TNF-α.

[0041] After treating cultured macrophages with LETX-VI or LPS, the treatment process was the same as in Example 1. The levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) released into the culture medium were measured using ELISA kits (IL-6 ELISA Kit, PI326 and TNF-α ELISA Kit, PT512). Cells were divided into a control group, an LPS treatment group, and a LETX-VI intervention group. Collected culture medium with cells removed and 100 μl of standard samples of different concentrations were added to 96-well ELISA plates and incubated for 2 hours. The liquid in the wells was discarded, and the plates were washed 5 times with washing buffer. 100 μl of biotinylated anti-IL-6 or TNF-α antibody was added and incubated for 1 hour. After washing 5 times, 100 μl of horseradish peroxidase-coupled avidin solution was added, and the plates were incubated in the dark at room temperature for 20 minutes. Washing was repeated, and 100 μl of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added. The reaction was continued for 20 minutes, then stopped with 50 μl of stop solution. The absorbance was recorded at 450 nm using a microplate reader (Bio-Rad Laboratories, CA, USA). The concentrations of IL-6 and TNF-α were calculated based on a standard curve of known cytokine concentrations, and the results are as follows: Figure 2 A, Figure 2 As shown in B.

[0042] from Figure 2 As shown in Figure A, LPS treatment of macrophages significantly increased the level of the inflammatory cytokine IL-6 compared to the control group (P<0.001), while LETX-VI intervention significantly reduced the increase in IL-6 inflammatory cytokine induced by LPS stimulation (P<0.05). Figure 2 B indicates that LPS treatment of macrophages significantly increased the level of the inflammatory factor TNF-α compared with the control group (P<0.001), while intervention with 0.1 μM LETX-VI significantly inhibited the increase of TNF-α inflammatory factor induced by LPS stimulation (P<0.01). The inhibitory effects of 1 μM and 4 μM LETX-VI intervention on TNF-α released induced by LPS treatment were even more significant (P<0.001). Figure 2 The results show that LETX-VI can inhibit the increase of inflammatory factors and has significant anti-inflammatory activity.

[0043] Example 3

[0044] The inhibitory effect of LETX-VI protein on M1 activation of macrophages.

[0045] Macrophages exhibit different functional polarization phenotypes: M1 and M2. Polarized M1 macrophages promote inflammatory responses by secreting pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα), interleukins (ILs), and interferon-γ; they can also release excessive amounts of inflammatory mediators such as nitric oxide and oxygen free radicals, directly leading to neurotoxicity. However, polarized M2 macrophages secrete large amounts of anti-inflammatory factors, thereby mitigating or inhibiting inflammatory responses. The pathogenesis of inflammation is related to the pro-inflammatory factors secreted by M1 macrophages. Many anti-inflammatory drugs can inhibit M1 macrophage polarization and promote the polarization of macrophages from M1 to M2.

[0046] Macrophages were divided into a control group, an LPS treatment group, and a LETX-VI pretreatment group. After treating cultured macrophages with LETX-VI or LPS, the treatment process was the same as in Example 1, and whole-cell protein samples were prepared. Western blotting was used to detect the expression level of macrophage M1 protein: inducible nitric oxide synthase (iNOS) protein. The detection method was as follows: The obtained whole-cell protein samples were first separated by SDS-PAGE gel electrophoresis. Then, the gel containing the target band was cut off, and the target protein was transferred to a PVDF membrane (PALL, USA) at a constant current of 100 mA. After the transfer, the PVDF membrane was blocked with 5% skim milk at room temperature for 2 h, and then incubated overnight with the target primary antibody (iNOS antibody, Cat No: 22226-1-AP). After incubation, the membrane was washed three times with TBST buffer, and then incubated for 1 h with a secondary antibody conjugated with horseradish peroxidase. After incubation, the immunoreactivity was detected by enhanced chemiluminescence (ECL), and the results were displayed in an imaging system. The results are shown below. Figure 3 As shown.

[0047] Depend on Figure 3 It was found that, compared with the internal control β-action, the M1 protein iNOS of macrophages was significantly increased in the LPS-induced inflammation model, indicating that LPS promoted the transformation of macrophages into the M1 type. However, intervention with LETX-VI could significantly decrease the LPS-induced M1 marker protein iNOS in a concentration-dependent manner, indicating that LETX-VI can inhibit the activation of macrophage M1 type to alleviate or suppress the inflammatory response.

[0048] Example 4

[0049] The effect of LETX-VI protein on promoting the conversion of macrophages from M1 to M2.

[0050] Cells were divided into a control group, an LPS treatment group, and a LETX-VI intervention group. Macrophage cells were cultured and treated with LETX-VI or LPS, following the same procedures as in Example 1, to prepare total cell protein samples. Western blotting was used to detect the expression level of M2-type protein arginase-1 (Arg-1) in macrophage cells. The obtained total cell protein samples were first separated by SDS-PAGE gel electrophoresis. The gel containing the target band was then cut off, and the target protein was transferred to a PVDF membrane (PALL, USA) at a constant current of 100 mA. After transfer, the PVDF membrane was blocked with 5% skim milk at room temperature for 2 hours, and then incubated overnight with the primary antibody (Arg-1 antibody, Cat No: 1600-1-AP). After incubation, the membrane was washed three times with TBST buffer, and then incubated for 1 hour with a secondary antibody conjugated with horseradish peroxidase. After incubation, immunoreactivity was detected by enhanced chemiluminescence (ECL), and the results were displayed on an imaging system. The results are shown below. Figure 4 As shown.

[0051] Depend on Figure 4 The results showed that LPS treatment significantly reduced the protein expression level of Arg-1, a marker protein for M2 macrophages, while LETX-VI showed the opposite effect, demonstrating that LETX-VI inhibited LPS-induced M1 macrophages and polarized them into M2 macrophages. Similarly, the M2 protein Arg-1 in macrophages was significantly increased in the LETX-VI intervention group compared to the LPS group, indicating that LETX-VI intervention can inhibit LPS-induced activation of M1 macrophages to a certain extent and transform LPS-induced M1 macrophages into M2 macrophages in a concentration-dependent manner. This suggests that LETX-VI can transform M1 macrophages into M2 macrophages with anti-inflammatory effects, thereby exerting an anti-inflammatory effect.

[0052] Example 5

[0053] The inhibitory effect of LETX-VI protein on M1 activation of microglia in brain tissue.

[0054] Male C57BL / 6j mice (purchased from Hunan Silek Jingda Experimental Animal Co., Ltd.) were divided into a control group, an LPS group, and a LETX-VI intervention group, with 10 mice in each group. Mice in the control group were injected with saline; mice in the LPS model group were injected intraperitoneally with 0.83 mg / kg LPS; mice in the LETX-VI intervention group were injected with 3 mg / kg LETX-VI and 0.83 mg / kg LPS. Twenty-four hours after injection, mouse brain tissue homogenates were collected to prepare protein samples for Western blotting.

[0055] Western blotting was used to detect the expression level of M1 type protein: inducible nitric oxide synthase (iNOS) in microglia of brain tissue. The method was as follows: The obtained whole-cell protein samples were first separated by SDS-PAGE gel electrophoresis. The gel containing the target band was then cut off, and the target protein was transferred to a PVDF membrane (PALL, USA) at a constant current of 100 mA. After transfer, the PVDF membrane was blocked with 5% skim milk at room temperature for 2 hours, followed by incubation overnight with the target primary antibody (iNOS antibody Cat No: 22226-1-AP diluted 1:2000 with skim milk). After incubation, the membrane was washed three times with TBST buffer, and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour. Immunoreactivity was detected by enhanced chemiluminescence (ECL) and displayed on an imaging system. The results are shown below. Figure 5 As shown.

[0056] Depend on Figure 5 The results showed that the expression level of the M1 protein iNOS in microglia of brain tissue was significantly increased in the LPS-induced in vivo inflammation model, indicating that LPS promoted the transformation of microglia in brain tissue into the M1 type in the in vivo inflammation model. Intervention with LETX-VI significantly decreased the LPS-induced M1 marker protein iNOS in a concentration-dependent manner. This suggests that in the in vivo inflammation model, LETX-VI can inhibit the activation of the M1 type of microglia in brain tissue, thereby alleviating or suppressing the inflammatory response.

[0057] Example 6

[0058] The effect of LETX-VI protein on promoting the conversion of microglia from M1 to M2 in brain tissue.

[0059] Male C57BL / 6j mice (purchased from Hunan Silek Jingda Experimental Animal Co., Ltd.) were divided into a control group, an LPS group, and a LETX-VI intervention group, with 10 mice in each group. Mice in the control group were injected with saline; mice in the LPS model group were injected intraperitoneally with 0.83 mg / kg LPS; mice in the LETX-VI intervention group were injected with 3 mg / kg LETX-VI and 0.83 mg / kg LPS. Twenty-four hours after injection, mouse brain tissue homogenates were collected to prepare protein samples for Western blotting.

[0060] Western blotting was used to detect the expression level of arginase-1 (Arg-1) protein, an M2-type protein in microglia of brain tissue. The obtained whole-cell protein samples were first separated by SDS-PAGE gel electrophoresis. The gel containing the target band was then excised, and the target protein was transferred to a PVDF membrane (PALL, USA) at a constant current of 100 mA. After transfer, the PVDF membrane was blocked with 5% skim milk at room temperature for 2 hours, followed by incubation overnight with the target primary antibody (Arg-1 antibody Cat No: 1600-1-AP diluted 1:2000 with skim milk). After incubation, the membrane was washed three times with TBST buffer, and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour. Immunoreactivity was detected by enhanced chemiluminescence (ECL) and displayed on an imaging system. The results are shown below. Figure 6 As shown.

[0061] Depend on Figure 6 The results showed that the LPS treatment group significantly reduced the level of Arg-1, a marker protein for M2 microglia in brain tissue, while LETX-VI showed the opposite effect. This demonstrates that in an in vivo inflammation model, LETX-VI inhibits LPS-induced M1 microglia and polarizes them into M2 microglia. Similarly, the M2 protein Arg-1 level in microglia was significantly higher in the LETX-VI intervention group than in the LPS group, indicating that LETX-VI intervention can, to some extent, inhibit the activation of LPS-induced M1 microglia and convert LPS-induced M1 microglia into M2 microglia. This suggests that in an in vivo inflammation model, LETX-VI can convert M1 microglia in brain tissue into M2 microglia with anti-inflammatory effects, thereby exerting an anti-inflammatory effect.

[0062] Example 7

[0063] The inhibitory effect of LETX-VI protein on the activation of microglia in the hippocampus and substantia nigra of the brain.

[0064] Microglia are macrophages in the brain and are inherent immune effector cells in the central nervous system. Microglia are rapidly activated during brain inflammation, releasing pro-inflammatory factors to induce the spread of inflammation and subsequently affect neuronal function. Therefore, microglia activation is considered a standard indicator of neuroinflammation. IBA (Ionized calcium-binding adapter molecule 1) is a calcium-binding protein of approximately 17 kDa specifically expressed in microglia of the central nervous system. Normally, its expression level is low, but it significantly increases after microglia activation. Therefore, IBA-1 is used as a marker of microglia activation.

[0065] Male C57BL / 6j mice (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.) were divided into a control group, an LPS group, and a LETX-VI intervention group, with 10 mice in each group. Mice in the control group were injected with saline; mice in the LPS model group were injected intraperitoneally with 0.83 mg / kg LPS; and mice in the LETX-VI intervention group were injected with 3 mg / kg LETX-VI and 0.83 mg / kg LPS. Inflammatory cell activation was assessed in the mice 24 hours after injection.

[0066] After mouse euthanasia, brain tissue was rapidly dissected and fixed at 4°C with 4% paraformaldehyde for 24 hours. Samples were taken from the hippocampus and substantia nigra regions of the brain tissue and washed three times with PBS buffer (pH 7.4). Samples were dehydrated using a gradient of 75-100% ethanol, followed by treatment with ethanol, benzene, xylene, and paraffin melted at 65°C. The samples were then embedded in paraffin and sectioned using a paraffin microtome. Sections were dewaxed in xylene, rehydrated in diluted ethanol, washed in distilled water, and then subjected to antigen retrieval via microwave heating and blocked in 3% BSA. The sections were then incubated overnight at 4°C with primary antibody (IBA-1 antibody). After incubation, the sections were washed three times with PBS buffer and then incubated with a fluorescently conjugated secondary antibody at room temperature in the dark. DAPI staining was performed, and immunofluorescence images were recorded using a fluorescence microscope (Nikon Eclipse C1, Japan). Immunofluorescence staining was performed on microglia in the hippocampus and substantia nigra of mouse brain tissue. Microglia activation was marked using the 17 kDa calcium-binding protein IBA-1, a marker protein of microglia. The extent of IBA-1 positive staining was used to indicate the degree of microglia activation. Results are shown in [Figure number missing]. Figure 7 .

[0067] Depend on Figure 7 The results showed that LPS significantly increased the inflammatory activation of microglia in different brain regions of mice, while LETX-VI had a significant inhibitory effect on LPS-induced microglia activation. LETX-VI also showed a good inhibitory effect on excessive inflammatory activation in the brain in an in vivo inflammatory model, indicating that LETX-VI can also exert a good anti-inflammatory effect in vivo.

[0068] Example 8

[0069] Safety testing of LETX-VI protein in mice.

[0070] Male C57BL / 6J mice were anesthetized, and blood was collected from the eyeballs. The blood was collected in anticoagulant tubes, centrifuged at 800g for 5 min, and the plasma was removed. Red blood cells were washed four times with isotonic PBS buffer. Red blood cells were used for the hemolysis test when the final concentration was 1%. Negative control: added isotonic PBS buffer; positive control: added 0.1% Triton X-100. LETX-VI was serially diluted twofold with PBS, from 250 μM to 1.95 μM. Each group of drugs was incubated with red blood cells at 37°C for 30 min, and then centrifuged at 12000g for 3 min at 4°C to remove unlysed red blood cells, retaining the supernatant. The obtained supernatant was transferred to the wells of a 96-well plate, and the absorbance (A) was measured at 490 nm. The hemolytic activity (%) was calculated as follows: (sample - negative control / positive control - negative control) × 100. The results are shown in the figure. Figure 8 A.

[0071] To determine the effects of LETX-VI on blood biochemical parameters in mice, 16 male C57BL / 6J mice were randomly divided into one control group and three experimental groups, with four mice in each group. Mice were injected intraperitoneally with 3 mg / kg LETX-VI, and blood samples were collected at 0 min, 30 min, 60 min, and 90 min. The control group mice were injected with sterile saline. Blood biochemical parameters, including serum total protein (TP), albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), cholesterol (CHO), high-density lipoprotein (HDL), and creatinine (Cr), which are closely related to liver and kidney function, were measured using a fully automated biochemical analyzer (DXC800, Beckman, USA). The results are shown below. Figure 8 B-8H.

[0072] Depend on Figure 8 As can be seen from A, when measuring the hemolytic activity of LETX-VI on mouse erythrocytes, in this invention, LETX-VI showed no significant hemolytic activity on mammalian erythrocytes. After LETX-VI injection, a time gradient was set, and the levels of total protein (TP), albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), cholesterol (CHO), high-density lipoprotein (HDL), and creatinine (Cr) in mouse serum were quantitatively measured. The results showed that, compared with the control group, LETX-VI treatment did not significantly change the levels of the above blood biochemical indicators, and the degree of fluctuation was not significantly different. Figure 8 (BH). Given that most of the blood indicators detected are closely related to the physiological functions of the liver, kidneys, bile ducts, etc., and that these indicators are not significantly affected by LETX-VI, LETX-VI is non-toxic to mammals in this invention, indicating that the application of LETX-VI at anti-inflammatory doses is safe.

[0073] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. However, it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. The application of *Scalycanthus oocystis* vesicle toxin-VI protein in the preparation of anti-inflammatory agents, characterized in that, The amino acid sequence of the *Sargassum fusiforme* oocyte venom-VI protein is as follows: EMTCADTQGQCVAGNDCSCCGQYDKCDCTWNLGVRTCKCKRVAILSDWKKNLNCPQ. Anti-inflammatory agents are used to treat LPS-induced inflammation.

2. The application of the *Scalycanthus ovatus* oocyte venom-VI protein according to claim 1 in the preparation of anti-inflammatory agents, characterized in that, The application includes at least one of the following: The application of *Scalys scalyssum* oocyst toxin-VI protein in the preparation of a reagent to inhibit LPS-induced M1 activation of macrophages; or, Application of *Sargassum fusiforme* oocyst toxin-VI protein in the preparation of a reagent that promotes LPS-induced conversion of macrophages from M1 to M2 types; or, Application of the oocyte toxin-VI protein from the spider spider in the preparation of a reagent to inhibit LPS-induced microglial M1 activation.

3. The application of the *Scalycanthus ovatus* oocyte venom-VI protein according to claim 1 or 2 in the preparation of anti-inflammatory agents, characterized in that, The anti-inflammatory agents include those that inhibit LPS-induced peripheral inflammation or LPS-induced neuroinflammation.

4. The application of the *Scalycanthus ovatus* oocyte venom-VI protein according to claim 1 or 2 in the preparation of anti-inflammatory agents, characterized in that, The anti-inflammatory reagent is formulated as a solution, colloidal solution, emulsion, or suspension.

5. The use of the *Scalycanthus ovatus* oocyte venom-VI protein according to claim 1 or 2 in the preparation of anti-inflammatory agents, characterized in that, The anti-inflammatory agent is administered in the form of an oral fast-dissolving film, oral liquid, capsule, injection, or transdermal absorption preparation.

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