阅读说明：本技术 一种具有c5蛋白靶向性的硫酸软骨素寡糖的制备方法与应用 (Preparation method and application of chondroitin sulfate oligosaccharide with C5 protein targeting property ) 是由 李连 于宸 权爽 臧恒昌 李妍 于 2020-05-26 设计创作，主要内容包括：本发明提供一种具有C5蛋白靶向性的硫酸软骨素寡糖的制备方法与应用,属于生物医药技术领域。本发明以鱿鱼硫酸软骨素为原料,通过酶解法制备硫酸软骨素寡糖后,利用本方法对各种类型寡糖的活性进行筛选,得到活性最好的片段,方法科学性高、可行性强。本发明进一步利用筛选出的硫酸软骨素二糖,通过体外、体内实验探究并揭示了硫酸软骨素二糖通过调节补体系统治疗骨性关节炎的机制,最终为骨性关节炎的治疗提供一种新的途径,因此具有良好的实际应用之价值。(The invention provides a preparation method and application of chondroitin sulfate oligosaccharide with C5 protein targeting property, belonging to the technical field of biological medicines. According to the method, the squid chondroitin sulfate is used as a raw material, the chondroitin sulfate oligosaccharide is prepared by an enzymolysis method, and then the activities of various oligosaccharides are screened by the method to obtain the fragment with the best activity, so that the method is high in scientificity and strong in feasibility. The invention further utilizes the screened chondroitin sulfate disaccharide, researches and reveals a mechanism that the chondroitin sulfate disaccharide treats the osteoarthritis by regulating a complement system through in vitro and in vivo experiments, and finally provides a new way for treating the osteoarthritis, thereby having good practical application value.)

1. A method for preparing C5 protein-targeted chondroitin sulfate oligosaccharide, which is characterized by comprising the following steps:

performing enzymolysis treatment on chondroitin sulfate, and separating enzymolysis products to obtain different oligosaccharide components;

and (3) verifying each oligosaccharide component by using a hemolysis test and a mouse cell proliferation promoting test, and primarily inspecting and screening the chondroitin sulfate oligosaccharide.

2. The method for preparing chondroitin sulfate oligosaccharide having the C5 protein targeting property, as claimed in claim 1, wherein the chondroitin sulfate is squid-derived chondroitin sulfate.

3. The method for preparing chondroitin sulfate oligosaccharide having C5 protein targeting property of claim 1, wherein the enzymatic treatment specifically employs chondroitin sulfate ABC enzyme.

4. The method for preparing chondroitin sulfate oligosaccharide having C5 protein targeting property according to claim 1, wherein the enzymatic hydrolysate is separated by a column separation method, preferably a Bio-GelP10 column;

preferably, the method for separating the enzymatic hydrolysate specifically comprises the following steps: taking 1mol/L sodium chloride and 10% ethanol as mobile phases, separating the enzymolysis products by adopting a Bio-GelP10 column, wherein the flow rate is 2.0mL/10min, collecting components flowing out every 10min into one tube, detecting the wavelength to be 210nm, and combining the same components; centrifuging, concentrating, desalting with G25 column, and lyophilizing to obtain oligosaccharide components with different fragments.

5. The method of claim 1, wherein the method further comprises characterizing the oligosaccharide component by electrospray mass spectrometry to characterize the relative molecular mass.

6. The method of claim 1, wherein the hemolysis assay comprises: taking rabbit erythrocyte suspension, and using Mg²⁺-diluting rabbit erythrocytes with EGTA buffer; placing CS oligosaccharide solutions with different concentrations and NHS at low temperature for shaking incubation; and immediately adding the rabbit red blood cell suspension after the reaction is finished, uniformly mixing and incubating.

7. The method of claim 1, wherein the mouse cell proliferation-promoting assay comprises: extracting primary mouse chondrocytes, adding CS oligosaccharide solutions with different concentrations, culturing at constant temperature, measuring the number of cells by adopting a CCK-8 kit, and calculating the proliferation rate of the chondrocytes.

8. The method for preparing chondroitin sulfate oligosaccharide having C5 protein targeting property of claim 1, wherein the chondroitin sulfate oligosaccharide comprises chondroitin sulfate disaccharide, chondroitin sulfate tetrasaccharide, chondroitin sulfate hexasaccharide, chondroitin sulfate octasaccharide and chondroitin sulfate decasaccharide; chondroitin sulfate disaccharide is preferred.

9. A chondroitin sulfate oligosaccharide obtained by the production method according to any one of claims 1 to 8.

10. Use of chondroitin sulfate oligosaccharides as claimed in claim 9 for the preparation of a medicament for the treatment of osteoarthritis;

preferably, the chondroitin sulfate oligosaccharide comprises chondroitin sulfate disaccharide, chondroitin sulfate tetrasaccharide, chondroitin sulfate hexasaccharide, chondroitin sulfate octasaccharide and chondroitin sulfate decasaccharide; further preferably chondroitin sulfate disaccharide;

preferably, the osteoarthritis treatment drug is a drug for treating osteoarthritis by regulating a complement system.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method and application of chondroitin sulfate oligosaccharide with C5 protein targeting property.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Osteoarthritis (OA) is a degenerative joint disease caused by the mechanical and biological degradation of articular cartilage, especially for the elderly, and the incidence rate is increasing and the population of the disease is younger, which seriously affects the health and quality of life of the patient, so it is important to prevent and treat OA. The pathogenesis of OA is complex, OA is believed to be cartilage damage due to genetic, metabolic, biochemical, biomechanical factors, and mild to moderate inflammation, however, to date, humans have not found a complete cure for OA. Non-steroidal anti-inflammatory drugs (NSAIDs) are clinically used for relieving pain of patients, but the NSAIDs do not help to cure diseases, and some patients have strong adverse reactions such as gastrointestinal bleeding, cardiovascular diseases and the like after using the NSAIDs. With the progress of research, more and more results indicate that the immune system is involved in the pathogenesis of OA as a whole, and that the complement system plays a central role in this process. Studies have shown that during the onset of OA, initial cartilage damage results in the release of cartilage extracellular matrix components such as cartilage oligomeric matrix proteins, which activate the complement system. The complement system is mainly composed of the Classical Pathway (CP), the Alternative Pathway (AP) and the Lectin Pathway (LP). On the one hand, the three pathways converge when forming complement proteins C3 and C5 to generate C3 convertase and C5 convertase, respectively, which induce the production of C3a and C5a, respectively, thereby producing proinflammatory cytokines; on the other hand, after complement activation, Membrane Attack Complex (MAC) is formed on the surface of chondrocytes, and MAC can be inserted into cells to form cavities to promote chondrocyte lysis and death, and can also promote secretion of factors such as IL-1 beta and TNF-alpha, so that signal pathways such as NF-kappa B, MAPK and the like are activated, MMPs, ADAMTS and the like are released, cartilage damage is caused, and OA is formed. In addition, the activation of complement system occurs before the level of inflammatory factors such as IL-1 beta, TNF-alpha, etc. is increased, further indicating the importance of complement. These findings further demonstrate that activation of the complement system is a central factor in the pathogenesis of OA. In conclusion, the complement system may be a new drug target for treating OA, and the regulation of the complement system may provide a new strategy for treating OA.

Chondroitin Sulfate (CS) is a class of unbranched linear anionic glycosaminoglycans (GAGs) consisting of alternating linkages of disaccharide units consisting of D-glucuronic acid and N-acetyl-D-galactosamine. CS has many clinical applications, such as anticoagulant, antitumor, anti-inflammatory, and anticomplementary activities. In europe, CS is recommended by the European union of antirheumatics (EULAR) as a slow-acting drug for the treatment of OA. Most studies show that CS can realize the treatment of OA through the intervention of an inflammatory system, and is a good multi-target natural polysaccharide potential drug. However, the mechanism by which CS achieves OA treatment through modulation of the complement system has not been reported, and the mechanism by which CS interacts with a range of complement components is not yet clear. This is because the structure of CS is more complex than GAGs such as heparin, and the structure of CS is influenced by a series of factors such as cartilage origin and age. In addition, the inventor finds that the CS with the complete chain length can hardly penetrate through gastric and intestinal mucosa, Low Molecular Weight Chondroitin Sulfate (LMWCS) can penetrate through the intestinal mucosa, and the LMWCS has better bioavailability and the effect of treating II type collagen-induced rheumatoid arthritis compared with the complete CS, but the drug effects of LMWCS with different sizes are different, so that the screening of the CS fragment with specific anticomplementary activity and targeting and the mechanism research of the CS fragment and the complement system have important significance for the treatment of OA.

Disclosure of Invention

The invention provides a preparation method and application of chondroitin sulfate oligosaccharide with C5 protein targeting property, wherein chondroitin sulfate oligosaccharide from squid is used as a raw material, the chondroitin sulfate oligosaccharide is prepared by an enzymolysis method, and the activity and targeting property of each type of oligosaccharide are screened by using the method disclosed by the invention, so that a fragment with C5 protein targeting property and optimal activity is obtained, and the method is high in scientificity and strong in feasibility. The invention further utilizes the screened chondroitin sulfate disaccharide, researches and reveals that the squid-derived chondroitin sulfate disaccharide has stronger C5 protein targeting property through in vitro and in vivo experiments, and finally provides a new way for treating the osteoarthritis by regulating a mechanism of a complement system, thereby having good practical application value.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a method for preparing chondroitin sulfate oligosaccharide having C5 protein targeting property, the method comprising:

performing enzymolysis treatment on chondroitin sulfate, and separating enzymolysis products to obtain different oligosaccharide components;

and (3) verifying each oligosaccharide component by using a hemolysis test and a mouse cell proliferation promoting test, and primarily inspecting and screening the chondroitin sulfate oligosaccharide.

Specifically, the chondroitin sulfate is selected from squid chondroitin sulfate.

The enzymolysis treatment specifically adopts chondroitin sulfate ABC enzyme;

the enzymolysis product separation method can adopt a column separation mode, such as a Bio-Gel P10 column, and the oligosaccharide component separation effect is better.

Preferably, the chondroitin sulfate oligosaccharide includes, but is not limited to, chondroitin sulfate disaccharide, chondroitin sulfate tetrasaccharide, chondroitin sulfate hexasaccharide, chondroitin sulfate octasaccharide, and chondroitin sulfate decasaccharide; chondroitin sulfate disaccharide is preferred. Experiments prove that the chondroitin sulfate disaccharide obtained by the preparation and screening method has the optimal C5 protein targeting property.

In a second aspect of the present invention, there is provided a chondroitin sulfate oligosaccharide obtained by the above-mentioned production method.

In a third aspect of the present invention, there is provided a use of the chondroitin sulfate oligosaccharide described above in the preparation of a medicament for treating osteoarthritis.

The beneficial technical effects of one or more technical schemes are as follows:

1. the screening method of chondroitin sulfate oligosaccharide with C5 protein targeting provided by the technical scheme has the advantages of clear principle, simple and convenient experimental operation and high result reliability.

2. The chondroitin sulfate disaccharide provided by the technical scheme has the advantages of low molecular weight, good penetrability, high oral bioavailability, good C5 protein targeting property, and capability of promoting mouse chondrocyte proliferation and obviously inhibiting the formation of C3a, C4b and C5 complements and MAC in a complement pathway and the expression of related proteins induced by MAC, so that the chondroitin sulfate disaccharide has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a chromatographic separation result and a mass spectrum of each oligosaccharide component of chondroitin sulfate in example 1 of the present invention, wherein: a is a chondroitin sulfate chromatographic separation result diagram, B is a mass spectrum of the component A, C is a mass spectrum of the component B, D is a mass spectrum of the component C, E is a mass spectrum of the component D, and f is a mass spectrum of the component E.

FIG. 2 shows anticomplementary activities of chondroitin sulfate at different concentrations of disaccharide, tetrasaccharide, hexasaccharide and octasaccharide in example 2 of the present invention.

FIG. 3 shows the activity of different concentrations of chondroitin sulfate oligosaccharide in promoting the proliferation of chondrocytes in example 3 of the present invention, wherein a is the relative cell number of mouse chondrocytes cultured for 24 hours under the condition of different concentrations of chondroitin sulfate disaccharide. P <0.05, ns was not significantly different; b is the proliferation promoting rate curve of different concentrations of chondroitin sulfate disaccharide to mouse chondrocytes.

FIG. 4 is a graph showing the interaction of chondroitin sulfate disaccharide with different proteins in the complement pathway in example 4 of the present invention.

FIG. 5 is the inhibitory activity of chondroitin sulfate disaccharide on alternative pathways C3, C3a, C4b, C5 complement formation in example 5 of the present invention, wherein: a is the inhibitory activity of CS disaccharide on the formation of C3 in the complement alternative pathway, b is the inhibitory activity of CS disaccharide on the formation of C3a in the complement alternative pathway, C is the inhibitory activity of CS disaccharide on the formation of C4b in the complement alternative pathway, and d is the inhibitory activity of CS disaccharide on the formation of C5 in the complement alternative pathway. P <0.05, P <0.01, P <0.001, ns were not significantly different.

FIG. 6 is a graph showing the inhibitory activity of chondroitin sulfate disaccharide on the formation of the complement pathway MAC in example 6 of the present invention, in which: a is the effect of different concentrations of CS disaccharide on the level of lactate dehydrogenase in mouse chondrocytes after NHS stimulation<0.001,****P<0.0001；b₁Blank group MAC level, b₂At 10% NHS group MAC level, b₃At 10% NHS +0.4mg/mL chondroitin sulfate disaccharide MAC level, b₄At 10% NHS +0.8mg/mL chondroitin sulfate disaccharide MAC level, b₅Is 10% NHS +1.2mg/mL chondroitin sulfate disaccharide group MAC level.

FIG. 7 is a graph showing the inhibitory activity of chondroitin sulfate disaccharide on MAC-induced expression of related proteins in example 7 of the present invention, in which: a is the effect of different concentrations of CS disaccharide on mouse chondrocyte MMP-13 protein expression after NHS stimulation<0.05，**P<0.01; b is the effect of different concentrations of CS disaccharide on CCL2 protein expression in mouse chondrocytes after NHS stimulation<0.05，**P<0.01，***P<0.001；c₁Result of COX2 imaging of chondrocytes in blank group, c₂As a result of COX2 imaging of chondrocytes of a control group (10% NHS), c₃As a result of imaging chondrocytes of the administered group (10% NHS +0.8mg/mL CS disaccharide).

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. The experimental procedures in the following detailed description, if specific conditions are not indicated, are generally in accordance with conventional procedures and conditions within the skill of the art.

In a typical embodiment of the present invention, there is provided a method for preparing chondroitin sulfate oligosaccharide having C5 protein targeting property, the method comprising:

performing enzymolysis treatment on chondroitin sulfate, and separating enzymolysis products to obtain different oligosaccharide components;

and (3) verifying each oligosaccharide component by using a hemolysis test and a mouse cell proliferation promoting test, and primarily inspecting and screening the chondroitin sulfate oligosaccharide.

In another embodiment of the present invention, the chondroitin sulfate is squid-derived chondroitin sulfate.

In another embodiment of the present invention, the enzymatic treatment specifically employs chondroitin sulfate ABC enzyme;

in another embodiment of the present invention, the method for separating enzymatic hydrolysate can adopt a column separation method, such as a Bio-Gel P10 column, which has a better effect of separating oligosaccharide components;

in another embodiment of the present invention, the method for separating the enzymatic hydrolysate specifically comprises: separating the enzymolysis product by using 1M sodium chloride and 10% ethanol as mobile phases and a Bio-Gel P10 column at a flow rate of 2.0mL/10min, collecting the components flowing out every 10min into a tube, detecting the wavelength of 210nm, and combining the same components; centrifuging, concentrating, desalting with G25 column, and lyophilizing to obtain oligosaccharide components with different fragments.

In yet another embodiment of the present invention, the method further comprises characterizing the oligosaccharide component by electrospray mass spectrometry (ESI-MS).

In still another embodiment of the present invention, the hemolysis test and the mouse cell proliferation test are both performed by conventional experimental methods.

In yet another embodiment of the present invention, the hemolysis assay comprises: taking rabbit erythrocyte suspension, and using Mg²⁺-diluting rabbit erythrocytes with EGTA buffer; placing CS oligosaccharide solutions with different concentrations and NHS at low temperature for shaking incubation; and immediately adding the rabbit red blood cell suspension after the reaction is finished, uniformly mixing and incubating.

In yet another embodiment of the present invention, the mouse cell proliferation promotion assay comprises: extracting primary mouse chondrocytes, adding CS oligosaccharide solutions with different concentrations, culturing at constant temperature, measuring the number of cells by adopting a CCK-8 kit, and calculating the proliferation rate of the chondrocytes.

In still another embodiment of the present invention, the concentration of the CS oligosaccharide is 0.4-1.2 mg/mL, and more preferably 0.4mg/mL,0.8mg/mL and 1.2 mg/mL.

In yet another embodiment of the present invention, the chondroitin sulfate oligosaccharide includes, but is not limited to, chondroitin sulfate disaccharide, chondroitin sulfate tetrasaccharide, chondroitin sulfate hexasaccharide, chondroitin sulfate octasaccharide, and chondroitin sulfate decasaccharide; chondroitin sulfate disaccharide is preferred. Experiments prove that the chondroitin sulfate disaccharide obtained by the preparation and screening method has the optimal C5 protein targeting property, and can treat osteoarthritis by regulating a complement system.

In still another embodiment of the present invention, there is provided a chondroitin sulfate oligosaccharide obtained by the above-mentioned production method.

The chondroitin sulfate oligosaccharide includes, but is not limited to, chondroitin sulfate disaccharide, chondroitin sulfate tetrasaccharide, chondroitin sulfate hexasaccharide, chondroitin sulfate octasaccharide, and chondroitin sulfate decasaccharide; chondroitin sulfate disaccharide is preferred.

In another embodiment of the present invention, there is provided a use of the chondroitin sulfate oligosaccharide described above in the preparation of a medicament for treating osteoarthritis.

The chondroitin sulfate oligosaccharide includes, but is not limited to, chondroitin sulfate disaccharide, chondroitin sulfate tetrasaccharide, chondroitin sulfate hexasaccharide, chondroitin sulfate octasaccharide, and chondroitin sulfate decasaccharide; chondroitin sulfate disaccharide is preferred.

The osteoarthritis treatment drug is a drug for treating osteoarthritis by regulating a complement system.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.