NEW USE OF ANTIPLATELET THROMBOLYSIN

Abstract

A method of preparing a medicament for inhibiting an inflammation includes the step of using an antiplatelet thrombolysin. The antiplatelet thrombolysin consists of two peptide chains of α chain and β chain, and the α chain amino acid sequence is shown in SEQ ID NO:1, β chain the amino acid sequence is shown in SEQ ID NO: 2; or the antiplatelet thrombolysin is derived from the α chain amino acid sequence by substitution, deletion, or addition of one or more amino acids and has at least 95% identity with SEQ ID NO: 1, or the antiplatelet thrombolysin is derived from the β chain amino acid sequence by substitution, deletion, or addition of one or more amino acids and has at least 95% identity with SEQ ID NO: 2; and the antiplatelet thrombolysin has an activity of inhibiting the inflammation.

Claims

1. A method of preparing a medicament for inhibiting an inflammation, comprising the step of using an antiplatelet thrombolysin, wherein the antiplatelet thrombolysin consists of two peptide chains of α chain and β chain, wherein the amino acid sequence of the α chain is shown in SEQ ID NO:1, the amino acid sequence of the β chain is shown in SEQ ID NO: 2; or the antiplatelet thrombolysin is derived from the amino acid sequence of the α chain by substitution, deletion, or addition of one or more amino acids and has at least 95% identity with SEQ ID NO: 1, or the antiplatelet thrombolysin is derived from the amino acid sequence of the β chain by substitution, deletion, or addition of one or more amino acids and has at least 95% identity with SEQ ID NO: 2; and the antiplatelet thrombolysin has an activity of inhibiting the inflammation.

2. The method according to claim 1, wherein the inflammation is selected from the group consisting of acute inflammation and chronic inflammation.

3. The method according to claim 1, wherein the inflammation is selected from the group consisting of degenerative inflammation, exudative inflammation, proliferative inflammation and specific inflammation.

4. The method according to claim 1, wherein the medicament is a chemical drug or a biological preparation.

5. The method according to claim 1, wherein the medicament is an oral preparation or an injection.

6. The method according to claim 5, wherein the oral preparation is a tablet, a capsule, a pill, a granule, a dripping pill, a microcapsule or a pellet.

7. The method according to claim 1, wherein the medicament is an external preparation.

8. The method according to claim 7, wherein the external preparation is a tincture, an ointment, a cream, a lotion, a rinse, a liniment or a gel.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0019] FIG. 1 shows that antiplatelet thrombolysin inhibits the binding of mouse platelets to αMβ2 integrin;

[0020] FIG. 2 shows that antiplatelet thrombolysin inhibits the binding of human platelets to αMβ2 integrin;

[0021] FIG. 3 shows the effect of antiplatelet thrombolysin in reducing TNF-α-induced neutrophil cell aggregation in wt mice;

[0022] FIG. 4 shows that antiplatelet thrombolysin reduces TNF-α-induced platelet-neutrophil binding in wt mice;

[0023] FIG. 5 shows the effect of antiplatelet thrombolysin in reducing the aggregation of human neutrophils;

[0024] FIG. 6 shows the effect of antiplatelet thrombolysin in reducing the binding between human platelets and neutrophils;

[0025] FIG. 7 shows images of platelets and neutrophils at different time points. From left to right, they are BSA group, 25 ng/g anfibatide group, and 50 g/g anfibatide group. From top to bottom, they are 0 s, 30 s, and 60 s maps, arrow indicates the direction of blood flow, green is to mark platelets, and red is to mark neutrophils;

[0026] FIG. 8 shows the blood flow velocity comparison chart of the drug administration group, from left to right are the BSA group, the 25 ng/g anfibatide group, and the 50 g/g anfibatide group, respectively;

[0027] FIG. 9 shows the platelet-neutrophil count chart, from left to right are the control IgG group, anti PDI group, BSA group, anfibatide group, IgG+BSA group, and anti PD-1+anfibatide group;

[0028] FIG. 10 shows the brain tissue slices of mice with ischemia/reperfusion induced stroke. From left to right, they are sham operation group, BSA group, 5 ng/ganfibatide group, and 25 ng/g anfibatide group;

[0029] FIG. 11 shows the experimental results of infarct volume in mice with ischemia/reperfusion induced stroke. The ordinate is the infarct volume. From left to right, they are sham operation group, BSA group, 5 ng/ganfibatide group, and 25 ng/g anfibatide group;

[0030] FIG. 12 shows the Grip score of ischemia/reperfusion induced stroke mice. From left to right, they are sham operation group, BSA group, 5 ng/g Anfibatide group, and 25 ng/g Anfibatide group;

[0031] FIG. 13 shows the Bederson score of ischemia/reperfusion-induced stroke mice, from left to right are sham operation group, BSA group, 5 ng/g Anfibatide group, 25 ng/g Anfibatide group.

DETAILED DESCRIPTION

[0032] The present invention provides use of antiplatelet thrombolysin in the preparation of a medicament for inhibiting inflamation. Those skilled in the art can achieve it in view of this disclosure and appropriate improvement of the process parameters. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and use of the present invention have been described through the preferred examples. It is obvious that those skilled in the art can make changes or appropriate modifications and combinations to the methods and uses described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0033] The reagents used in the present invention are all common commercially available products, and all are available in the market.

[0034] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific examples.

EXAMPLE 1

Antiplatelet Thrombolysin Inhibits the Binding Effect of αMβ2 Integrin and Platelets

[0035] Mouse platelets and human platelets were pre-incubated at 37° C. respectively with control IgG (10 μg/ml), BD34 (anti-PD1 antibody, 10 μg/ml), BSA (bovine serum albumin, 0.2 μg/ml) and anfibatide (antiplatelet, thrombolysin 0.2 μg/ml) for 30 minutes, and then washed with HEPES-Tyrode buffer (20 mM HEPES, pH 7.4, 136 mM NaCl, 2.7 mM KCl, 12 mM NaHCO.sub.3, 2 mM MgCl.sub.2, and 5.5 mM glucose). The washed platelets were incubated with recombinant human αMβ2 integrin (10 μg/ml) treated with or without 0.5 mM MnCl.sub.2 for 30 min at 37° C. to wash away unbound αMβ2, and the remaining platelets were incubated with 5 μg/ml IgG-FITC or goat anti-human β2 antibody, and tested by flow cytometry.

[0036] αMβ2, also called Mac-1 or CD11b/CD18, is a member of the integrin family. It is the main factor that mediates the adhesion between cells and extracellular matrix and between cells. It can recognize and bind to the corresponding ligands in the extracellular matrix. It is mainly distributed in leukocytes, and is an important type of leukocyte adhesion factor that participates in the adhesion between leukocytes and endothelial cells, mediates the function of leukocytes, and thus plays a key role in inflammation and immune response.

[0037] As shown in FIG. 1-2, FIG. 1 shows the binding effect of mouse platelets and αMβ2 integrin, and FIG. 2 shows the binding effect of human platelets and αMβ2 integrin. The ordinate is the fluorescence intensity, and the abscissa corresponds to the experimental groups as follows: control IgG group, BSA group, anti PDI group, IgG+BSA group, anti PDI+anfibatide group, where anti-PDT is protein disulfide isomerase inhibitior. The bar on the left is the result of αMβ2 without MnCl.sub.2 treatment, and the bar on the right is the result of αMβ2 treated with MnCl.sub.2. The experimental data were statistically analyzed, n=3, *P<0.05, **P<0.01, or *** P<0.001.

[0038] The results showed that antiplatelet thrombolysin can inhibit the interaction between platelets and leukocytes mediated by αMβ2 integrin.

EXAMPLE 2

Antiplatelet Thrombolysin Reduces TNF-α-Induced Platelet-Neutrophil Aggregation in wt Mouse Model

[0039] Neutrophils are the first immune cells to reach the site of infection. They differentiate into mature granular material-carrying cells in the bone marrow and are released into the blood. In the absence of infection, the neutrophils remain in a resting state, and then exist in the peripheral blood, but once the bacteria invade, the body will quickly recruit neutrophils to the infection site, neutrophils through phagocytosis jointly produce a variety of bactericidal substances, release proteases from the particles and neutrophils, at the same time, neutrophils also secrete chemokines to recruit other immune cells to enter the infection site and jointly destroy the infectious agent.

[0040] However, over-activated neutrophils will be recruited to some important organs. In these organs, the bactericidal substances carried by activated neutrophils will be released locally, and their respective destructive effects will lead to tissue damage and further organ disorders. When neutrophils invade an organ, they will induce the acctunulation of neutrophils in other important organs, leading to multiple organ failure. Inhibitors targeting different neutrophil substances can alleviate tissue damage in acute inflammation. At the same time, after activation of platelet thrombin, platelet selective receptors will be quickly expressed on the cell surface and mediate the binding and adhesion of activated platelets to neutrophils, neutrophils aggregation, and lysosomal enzyme release. Platelet-derived products can also promote the chemotaxis of neutrophils, the release of enzymes and phagocytosis, and inhibit the explosion of oxidation. On the other hand, platelets adhere to neutrophils, promote platelet aggregation, mediate vascular occlusion, and aggravate inflammation.

[0041] Experiment Method:

[0042] Platelets (2×107 cells/ml) and neutrophils (1×106 cells/ml) from WT mice and PDI CKO mice (PDIflox/flox mice were hybridized with PF4-Cre mice to obtain PDI CK0 mice) were respectively labeled with DyLight488-Anti-CD42C and Alexa Fluor647-anti-Ly-6G antibody, human neutrophils and platelets were respectively labeled with Alexa Fluor 488-anti-CD41 and FITC-anti-L selectin antibodies. Neutrophils were induced with 20 ng/ml TNF-α for 5 minutes, the platelets were pre-incubated with control IgG (10 μg/ml), anti PDI antibody (10 μg/ml), BSA (0.2 μg/ml), antiplatelet thrombolysin (0.2 μg/ml) and antiplatelet thrombolysin+antiPDI at room temperature for 30 minutes, then incubated with 0.025 U/ml thrombin at 37° C. for 5 minutes, then incubated with 50 uM PPACK. The mixed platelets and neutrophils were stirred at 1000 rpm, then fixed after 5 minutes and analyzed by flow cytometry.

[0043] The experimental results are shown in FIG. 3-6. Antiplatelet thrombolysin reduced TNF-α-induced platelet-neutrophil aggregation in the wt mouse model. The ordinate is cell-to-cell/platelet-to-neutrophil aggregation, relative to the control group. The abscissa is the experimental group, the experimental groups from left to right are: control IgG group, anti PDI group, BSA group, anfibatide group, IgG+BSA group, anti PD-I+anfibatide group. n=4, the experimental data were statistically analyzed, *P <0.05, **P<0.01, or ***P<0.001.

[0044] The test results show that antiplatelet thrombolysin can reduce the aggregation of neutrophils, and can also reduce the binding between platelets and neutrophils, thereby inhibiting inflammation.

EXAMPLE 3

Inhibition of Antiplatelet Thrombolysin on TNF-α-Induced Inflammation

[0045] TNF-α is an important pro-inflammatory cytokine, which has physiological functions such as participation in immunity, anti-tumor, anti-virus, and regulation of specific gene expression. It plays a vital role in the coordination of the body's immune-inflammation coordination signal network. If TNFα is overexpressed, it will cause inflammatory disease and natural immune disease.

[0046] WT mice were taken and injected with TNF-α into the scrotum after anesthesia. After 3 hours, they were administered with BSA 50 ng/gBW and Anfibatide (25, 50 ng/gBW), and recorded by real-time intravital microscope. The platelets of WT mice were labeled with calcein AM, and were respectively pre-incubated with IgG (10 μg/ml), anti-PDI (10 μg/ml), BSA (0.2 μg/ml), anfibatide (0.2 μg/ml), anfibatide+anti-PDI, 100 μl per TNF-α mouse was injected according to the number of platelets 2×10.sup.9/ml.

[0047] The experimental data were statistically analyzed by ANOVA and Tukey's test, *P<0.05, **P<0.01, or ***P<0.001. The test results showed that the administration of Anfibatide 25-50 ng/g after TNF-α treatment had basically no effect on the rolling and adhesion of neutrophils in endothelial cells (see FIG. 7). Compared with the BSA control, Anfibatide dose-dependently increased blood flow rate (see FIG. 8). At the same time, the platelet transfer model was used in the intravital microscope to carry out the experiment. Treatment with 0.2 μg/ml Anfibatide inhibited 50-55% of platelet-neutrophil adhesion (FIG. 9), which further illustrates that antiplatelet thrombolysin reduces TNF-α-induced platelet-neutrophils aggregation in the wt mouse model.

EXAMPLE 4

Effect of Antiplatelet Thrombolysin on Brain Tissue Injury in Mice with Ischemia/Reperfusion Stroke

[0048] Female and male C57BL/6 mice and WT mice (22-25 g, 7-10 weeks old) were anesthetized with 2% pentobarbital and intraperitoneally injected buprenorphine (30 ng/g BW) during the operation a heating blanket was used to maintain body temperature at 37° C. The left common carotid artery was exposed, the external carotid artery was dissected and the internal carotid artery was separated, the left middle cerebral artery was sealed with fibrils (diameter 0.15 mm, tip diameter 0.22-0.25 mm) for 1 h. Laser Doppler perfusion monitoring system (PF5010, Perimed AB, Ardmore, PA) was used till the local cerebral blood flow dropped to 20% of the baseline level, which showed the embolization was successful. The fibrils were then removed and blood flow was restored to baseline. One hour after embolization, BSA or antiplatelet thrombolysin (5, 25 ng/g BW 100 μl saline) was administered, buprenorphine (25 ng/gBW) was injected again ip, and reperfusion was performed for 23 h. Bederson and grisp were used to score animal behavior. After scoring, the mice were sacrificed and a 2 mm brain tissue section was taken, stained with 2% 2,3,5-triphenyltetrazolium chloride solution at 37° C. for 10 min, fixed with 4% paraformaldehyde, scanned and sliced and analyzed for infarction volume using ImageJ.

[0049] The ischemia/reperfusion brain tissue injury model is a mature inflammation model, and its inflammation is related to thrombosis. The experimental data were statistically analyzed by ANOVA and Tukey's test, n=6-7/group, using ANOVA and Tukey test, ***P<0.001, ****P<0.0001 were significant differences.

[0050] The results show that antiplatelet thrombolysin has a protective effect on brain tissue damage in mice with stroke induced by ischemia/reperfusion. Anfibatide 5-25 ng/g.Math.BW was given 1 hour after MCAO-induced local ischemia. Anfibatide at 5 ng/g BW can reduce the infarct volume to 40% of the BSA control group. Compared to the BSA control, Anfibatide dose-dependently reduced infarct volume, improved nerve function, improved balance ability and exercise ability (FIG. 10-13).

[0051] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded in the protection scope of the present invention.