CN114306307A

CN114306307A - Application of DSF in preparing anti-inflammatory drugs or anti-oxidation drugs and drugs

Info

Publication number: CN114306307A
Application number: CN202210068119.7A
Authority: CN
Inventors: 田静; 何亚文; 朱宏杰; 张东伟
Original assignee: Xi'an Yingchuang Biotechnology Co ltd
Current assignee: Xi'an Yingchuang Biotechnology Co ltd
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2022-04-12

Abstract

The application relates to the technical field of biological medicines, in particular to application of DSF in preparation of anti-inflammatory drugs or anti-oxidation drugs and drugs. The LPS is utilized to induce the zebra fish embryo inflammation model, after the LPS inflammation model group is processed by the DSF, the ROS level in the zebra fish embryo is reduced, the expression level of relevant inflammation indexes is obviously reduced, and the DSF has good anti-inflammatory activity and has an application prospect of being developed into anti-inflammatory drugs or anti-oxidation drugs.

Description

Application of DSF in preparing anti-inflammatory drugs or anti-oxidation drugs and drugs

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of DSF in preparation of anti-inflammatory drugs or anti-oxidation drugs and drugs.

Background

DSF (differential signal factor) is discovered in 1997, is a small molecule signal substance secreted by Xanthomonas campestris (Xcc) wild strains, and the small molecule substance can diffuse in an agar culture medium, promote the expression of pathogenic related genes, inhibit the formation of a biological membrane, promote the metabolic regulation of flora and adapt to the environment with high population density. (He YW, Zhang LH. Quorum sensing and virology regulation in Xanthomonas campestris. FEMS Microbiol Rev.2008 Aug; 32(5):842-57.) it was found that DSF induces plant innate immunity, reduces disease severity in host plants and inhibits pathogenic bacteria growth (Kakkar A, Nizamptnam NR, Kondreddy A, Pradhan BB, Chatterjee S. Xanthomonas campestris l-cell signalling mole DSF (differential signalling) immunity in plants and is supplied by the exolytic reaction plasmid Bop Exp. Nov; 66: 6697). At present, DSF mainly has related application research in plant disease control, and no report that DSF has anti-inflammatory activity exists.

Bacterial endotoxins, also known as Lipopolysaccharides (LPS), are the main structural components of the outer membrane of all gram-negative bacterial cell walls, consist of polysaccharides and lipid A, and can cause fever, immune dysfunction, microcirculation disturbance, multiple organ inflammation and the like of animal organisms (Dong Z, mean Y. involved and inflammatory stress induced by LPS in acid solution in Jun, I inhibition by ST1926.int J Mol Med.2018 Jun; 41(6): 3405-) 3421). The mechanism of injury usually begins with the activation of the monocyte-macrophage system, the induction of secretion of a variety of inflammatory mediators, and the activation of inflammatory signaling pathways by these mediators, which trigger an inflammatory response in the local or other parts of the body, causing injury (Guha M, Mackman N.LPS indication of gene expression in human monocytes. cell Signal. 2001Feb; 13(2): 85-94.). In addition, LPS can also directly act on target cells such as endothelial cells, fibroblasts and the like, so that the expression of surface molecules is abnormal, the permeability of cell membranes is increased, and the damage is caused.

Zebra fish (Danio rerio) is a tropical freshwater teleost fish, and the membrane begins to hatch about 3 days after fertilization, and sexual maturity can be reached in about 3 months. The zebra fish has the advantages of small size, strong reproductive capacity, low breeding cost, transparent embryos, easiness in living body observation and the like, and has high homology with human genome. Zebrafish possess an immune system similar to that of humans, with various organs and tissues highly resembling humans at the anatomical, physiological, molecular level. Under the induction of LPS, inflammatory responses In the Zebrafish intestinal tract can be triggered, making it an ideal subject for the study of Inflammation and other metabolic diseases (Wang S, Ni L, Fu X, Duan D, Xu J, Gao X. asulfamed Polysaccharaide from Saccharina japonica super press LPS-Induced Inflammation Both In a macro Cell Model via Blocking MAPK/NF-. kappa.B Signal pathway In Vitro and a Zebraphish Model of emulsions and Lavae In vivo. Mar drugs 2020Nov 26; 18(12): 593.).

Disclosure of Invention

The invention aims to provide an application of DSF in preparing anti-inflammatory drugs or antioxidant drugs and a drug, wherein the DSF has good anti-inflammatory activity and an application prospect of being developed into the anti-inflammatory drugs or the antioxidant drugs.

In order to achieve the purpose, the invention provides the following technical scheme: in a first aspect, there is provided a medicament comprising, as an active ingredient, DSF having the formula cis 11-methyl-2-dodecenoic acid:

further, in the medicament, the DSF is the only active ingredient.

Further, in the medicament, the dose of the DSF is 20 μ M/L.

Further, the composition also comprises a pharmaceutically acceptable carrier or auxiliary material.

Furthermore, the dosage form of the medicine is one or more of liquid injection, powder injection, tablets, capsules, soft capsules, powder, pills, oral liquid, paste, granules or dressings.

In a second aspect, the invention also provides an application of DSF in preparing anti-inflammatory drugs or anti-oxidation drugs, wherein the structural formula of DSF is cis 11-methyl-2-dodecenoic acid:

further, the DSF is used for preparing an inflammation mediator inhibitor.

Further, the DSF inhibits LPS-induced inflammatory responses, or inhibits LPS-induced inflammatory responses and LPS-induced oxidative stress responses.

Further, said inhibiting LPS-induced inflammatory responses comprises reducing abnormally high expression of inflammatory mediators in LPS-induced cells.

Further, the inflammatory mediators include TNF alpha, IL-1 beta, IL-6, and IL-10.

In a third aspect, there is also provided the use of DSF in the manufacture of an inhibitor of inflammatory mediators, wherein said inflammatory mediators comprise at least one of TNF α, IL-1 β, IL-6 or IL-10.

Compared with the prior art, the invention has the beneficial effects that:

the LPS is utilized to induce the zebra fish embryo inflammation model, after the LPS inflammation model group is processed by the DSF, the ROS level in the zebra fish embryo is reduced, the expression level of relevant inflammation indexes is obviously reduced, and the DSF has good anti-inflammatory activity and has an application prospect of being developed into anti-inflammatory drugs or anti-oxidation drugs.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a graph showing the statistics of survival and teratogenicity of zebrafish embryos with DSF at various concentrations according to an embodiment of the present application;

FIG. 2 is a DCFH-DA staining pattern of three groups of zebrafish embryos, as shown in an embodiment of the present application;

FIG. 3 is a graph of the quantitative analysis of ROS levels of three groups of zebrafish embryos, according to an embodiment of the present application;

FIG. 4 is a graph showing the relative expression level of inflammatory mediators in three groups of zebra fish embryos according to an embodiment of the present application.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

It should be noted that: the examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The application provides that DSF can be used for preparing anti-inflammatory drugs or anti-oxidation drugs, the chemical formula of the DSF is cis-11-methyl-2-dodecenoic acid, and the structural formula is as follows:

in the experimental process, the DSF is taken as the only active ingredient by the inventor, and the DSF is found to have no obvious cytotoxicity to zebra fish embryos within a certain dosage range, so that the anti-inflammatory activity of the DSF is not realized through cytotoxicity.

Specifically, the inventor finds that DSF can remarkably inhibit the release of inflammatory mediators such as TNF alpha, IL-1 beta, IL-6, IL-10 and the like in zebra fish embryos induced by LPS, thereby playing an anti-inflammatory role.

Thus, the present application provides that DSF may be used in the preparation of anti-inflammatory drugs, in particular, in the preparation of inhibitors of inflammatory mediators. In alternative embodiments, the inflammatory mediator may be at least one of TNF α, IL-1 β, IL-6, and IL-10, and accordingly, DSF may be used to prepare a TNF α inhibitor, an IL-1 β inhibitor, an IL-6 inhibitor, or an IL-10 inhibitor.

In addition, the inventor also finds that the DSF can inhibit the generation of ROS in zebra fish embryos induced by LPS, thereby playing anti-inflammatory and antioxidant roles.

Therefore, the application also provides that the DSF can be used for preparing antioxidant medicines.

As mentioned above, DSF in zebra fish embryos not only does not cause a large amount of ROS, but also inhibits the ROS generation induced by LPS, and also significantly inhibits the expression of inflammatory mediators such as TNF alpha, IL-1 beta, IL-6, IL-10 and the like, and the results show that DSF has good anti-inflammatory activity and has application prospect in developing anti-inflammatory drugs.

In another aspect, the present application also proposes a medicament comprising DSF as an active ingredient, which may be an anti-inflammatory drug or an anti-oxidant drug.

Optionally, the medicine further comprises pharmaceutically acceptable carrier or adjuvant, such as filler, binder, and disintegrant. Specifically, lactose, starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, and the like can be included.

Alternatively, the dosage form of the drug can be an oral dosage form or an injection dosage form. Specifically, the injection can be one or more of liquid injection, powder injection, tablet, capsule, soft capsule, powder, pill, oral liquid, paste, granule or dressing.

In an alternative embodiment, DSF is the only active ingredient of the drug, at a dose of 20 μ M/L.

The following is a further description with reference to specific examples.

EXAMPLE first examination of the Effect of different concentrations of DSF on the survival and teratogenicity of Zebra fish embryos

1. Collecting fish eggs

Selecting adult fish with good state and not laying eggs in 7 days in the evening before the experiment, placing one male fish and one female fish in each mating box, separating the two fish by using a baffle plate, pulling the baffle plate on the morning the next day, obtaining fertilized eggs in 5 minutes, collecting the fish eggs, and transferring the fertilized eggs into a culture dish containing embryo culture solution.

2. Zebra fish embryos were treated with different concentrations of DSF

Eggs of 8hpf (8 hours post fertilization) were transferred to 6-well plates, 30 eggs per well, and treated as follows:

control group (0. mu.M/L DSF): 5ml of fresh embryo culture;

20 μ M/L DSF treatment group: changing to 5ml of embryo culture solution containing DSF with concentration of 20 μ M/L;

30 μ M/L DSF treatment group: changing to 5ml of embryo culture solution containing DSF with concentration of 30 μ M/L;

40 μ M/L DSF treatment group: changing to 5ml of embryo culture solution containing DSF with concentration of 40 μ M/L;

50 μ M/L DSF treatment group: changing to 5ml of embryo culture solution containing DSF with concentration of 50 μ M/L;

75 μ M/L DSF treatment group: changing to 5ml of embryo culture solution containing DSF with concentration of 75 μ M/L;

100 μ M/L DSF treatment group: the medium was replaced with 5ml of an embryo culture medium containing DSF at a concentration of 100. mu.M/L.

The culture was continued to 3dpf (3 days after fertilization) in an incubator at 28 ℃ and zebrafish embryos were collected ready for statistics of survival and teratogenicity.

3. Counting the survival rate and teratogenesis rate of zebra fish embryos

And collecting the zebra fish embryos of each group, and counting the survival number of the zebra fish embryos of each group and the number of the deformed zebra fish under a microscope. As shown in FIG. 1, no significant death and teratogenicity of zebrafish embryos occurred at the treatment concentration of 20. mu.M/L, and significant decrease in the survival rate and significant increase in the teratogenicity rate of zebrafish embryos occurred at the treatment concentration of 30. mu.M/L and thereafter. The result shows that the zebra fish embryos are in a relatively safe state at the DSF treatment concentration of 20 mu M/L and can be used as the treatment concentration of subsequent experiments.

EXAMPLE two measurements of reactive oxygen species ROS levels in Zebra fish embryos after LPS-induced inflammation and DSF treatment

1. Collecting fish eggs

2. Construction of zebra fish embryo inflammation model and DSF treatment

Eggs of 8hpf (8 hours post fertilization) were transferred to 24-well plates, 10 eggs per well, and treated as follows:

control group: 1ml of fresh embryo culture;

model group (LPS group): changing to 1ml of an embryo culture solution containing LPS with the concentration of 25 mug/ml;

experimental group (DSF group): the medium was replaced with 1ml of an embryo culture medium containing LPS at a concentration of 25. mu.g/ml and DSF at a concentration of 20. mu.M/L.

The culture was continued to 3dpf (3 days after fertilization) in an incubator at 28 ℃ and zebrafish embryos were harvested and prepared for detection of reactive oxygen species ROS levels.

3. Detection of zebra fish embryo Reactive Oxygen Species (ROS) levels

As shown in FIG. 2, each of the collected groups of zebrafish embryos were transferred to a 1.5ml EP tube and washed twice with fresh embryo culture medium, and the washed zebrafish embryos were treated with ROS fluorescence probe (DCFH-DA) at a concentration of 20. mu.g/ml and incubated in an incubator at 28 ℃ for 1 hour in the absence of light. After incubation was complete, the zebrafish embryos were washed with fresh embryo culture medium and the remaining portion of the fluorescent probe was removed. And adding a tricaine solution into the cleaned zebra fish embryos for anesthesia, observing and photographing fluorescence images of the zebra fish embryos of each group under a body type fluorescence microscope, and analyzing and counting the relative fluorescence intensity of the zebra fish embryos of each group by using Image J software.

The results are shown in fig. 3, and it can be seen that the reactive oxygen species ROS levels of zebrafish embryos increased significantly after LPS-induced inflammatory responses, with statistical differences (× p < 0.05, × p < 0.01, × p < 0.001). Compared with a control group, the LPS induces the zebra fish embryo inflammatory reaction to cause the ROS level to rise, and meanwhile, the expression of related inflammatory signal factors shows a rising trend, which indicates that the model building of the inflammation model is successful. After DSF treatment, the ROS level of the active oxygen is obviously reduced, and the difference has statistical significance (# p < 0.05, # p < 0.01, # p < 0.001), which indicates that the DSF has obvious anti-inflammatory activity and antioxidant effect.

Example three real-time fluorescent quantitative PCR detection of the expression level of inflammation-related factors in zebra fish embryos after LPS-induced inflammation and DSF treatment

1. Collecting fish eggs

2. Construction of zebra fish embryo inflammation model and DSF treatment

control group: 5ml of fresh embryo culture;

model group (LPS group): changing to 5ml of an embryo culture solution containing LPS with the concentration of 25 mug/ml;

experimental group (DSF group): the medium was replaced with 5ml of an embryo culture medium containing LPS at a concentration of 25. mu.g/ml and DSF at a concentration of 20. mu.M/L.

3. Extraction of zebra fish embryo total RNA

The Trizol method is adopted, and the specific steps are as follows:

1) three groups of zebra fish embryos are collected in RNase-free EP tubes respectively, 1ml of Trizol is added into the EP tubes, and the zebra fish embryos are blown evenly (on ice) repeatedly by using a 1ml syringe until the zebra fish embryos are fully lysed.

2) Add 200. mu.l chloroform, sample to chloroform ratio 5: 1, vigorously shaken for 20s, left on ice for 3min and then centrifuged at 12000rpm for 15min at 4 ℃.

3) Carefully sucking the supernatant of the uppermost layer in an EP tube, placing the supernatant in a new EP tube without RNase, adding isopropanol with the same volume as the volume in the tube, turning upside down to fully mix the supernatant, and then placing the mixture at-80 ℃ for 30 min.

4) Samples were removed from-80 ℃ and centrifuged at 12000rpm for 30min at 4 ℃.

5) Carefully discard the supernatant, then add 600. mu.l of 75% ethanol pre-chilled, mix well, centrifuge at 12000rpm for 5min at 4 ℃ and carefully remove the supernatant.

6) And (5) repeating the step.

7) The residual 75% ethanol was removed as clean as possible, left to stand for 5min with the lid open, and when the flaky white precipitate at the bottom of the EP tube was cooled to be transparent, 20. mu.l of nucleic Free Water was added to dissolve it sufficiently.

8) Mu.l of RNA was taken for concentration determination, and 1. mu.l of RNA was mixed with Loading Buffer and subjected to nucleic acid electrophoresis to detect the total RNA mass.

4. Synthesis of cDNA

Reverse transcription was performed using the Thermo Scientific reverse First Strand cDNA Synthesis Kit.

1) To the tube were added:

2) 5min at 65 ℃ (in a PCR instrument) and then immediately removed and inserted into ice for 5 min.

3) After the centrifugal of a miniature centrifugal machine, adding the following components in sequence:

4) mix gently.

5) Carrying out reverse transcription in a PCR instrument, wherein the reaction conditions are as follows: 5min at 25 ℃, 60min at 2 ℃, 5min at 70 ℃ and infinity at 4 ℃.

5. Real-time fluorescent quantitative PCR (qRT-PCR)

By KAPA

The FAST Universal fluorescent quantitative PCR Kit is used for qRT-PCR, and the reaction system is as follows:

wherein the qRT-PCR reaction conditions are as follows: 95 ℃ for 3min, 95 ℃ for 3s, 60 ℃ for 20s, 40cycles, 95 ℃ for 15s, 60 ℃ for 1min, 95 ℃ for 15 s.

Designing zebra fish related qRT-PCR primers by using qRT-PCR primer design software, wherein the primer sequences are as follows:

tnfa(TNFα)	Fw：5’-AGTCGGGTGTATGGAGGGTGTT-3’
		Rv：5’-GGATGGCAGCCTTGGAAGTGAA-3’
il1b(IL-1β)	Fw：5’-ATGGCGAACGTCATCCAAGAG-3’
		Rv：5’-TTCAAGTCGCTGCTTCCGGCT-3’
il6(IL-6)	Fw：5’-TGCTACACTGGCTACACTCTT-3’
		Rv：5’-CACATCCTGAACTTCGTCTCC-3’
il10(IL-10)	Fw：5’-GCTCTGCTCACGCTTCTTC-3’
		Rv：5’-CCAAGTCATCGTTGGACTCATA-3’
gapdah	Fw：5’-CCAAGGCTGTAGGCAAAGTAA-3’
		Rv：5’-AAGATGGATGAACGGCAATC-3’

the experimental results are shown in fig. 4, and compared with the control group, the levels of inflammation-related indicators in the model group are significantly increased, and the differences have statistical significance ([ p ] < 0.05, [ p ] < 0.01, [ p ] < 0.001); compared with a model group, the levels of inflammation-related indexes in cells of the DSF group are obviously reduced, and the differences have statistical significance (# p < 0.05, # p < 0.01, # p < 0.001), which indicates that the DSF has obvious anti-inflammatory effect and has the prospect of being developed into anti-inflammatory drugs and inflammation mediator inhibitors.

In summary, the following steps:

the LPS is utilized to induce the zebra fish embryo inflammation model, compared with a control group, the LPS induces the zebra fish embryo inflammation reaction to cause the ROS level to rise, and meanwhile, the expression of related inflammation signal factors shows a rising trend, so that the success of model building of the inflammation model is demonstrated. After the LPS inflammation model group is treated by the DSF, the ROS level in the zebra fish embryo is reduced, the expression level of related inflammation indexes is obviously reduced, and the DSF has good anti-inflammatory activity and has an application prospect of being developed into anti-inflammatory drugs.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A medicament comprising, as an active ingredient, DSF having the formula cis 11-methyl-2-dodecenoic acid:

2. the medicament of claim 1, wherein said DSF is the only active ingredient in said medicament.

3. The medicament of claim 1 or 2, wherein in the anti-inflammatory medicament the DSF dose is 20 μ Μ/L.

4. The medicament of claim 1 or 2, further comprising a pharmaceutically acceptable carrier or excipient.

5. The medicament of claim 1 or 2, wherein the medicament is an anti-inflammatory or anti-oxidant medicament.

The application of DSF in preparing anti-inflammatory drugs or anti-oxidation drugs is characterized in that the structural formula of the DSF is cis-11-methyl-2-dodecenoic acid:

7. use according to claim 6, wherein said DSF is used for the preparation of an inhibitor of inflammatory mediators.

8. The use of claim 7, wherein said DSF inhibits LPS-induced inflammatory responses or inhibits LPS-induced inflammatory responses and LPS-induced oxidative stress responses.

9. The use of claim 8, wherein the inhibition of LPS-induced inflammatory responses comprises a reduction in abnormally high expression of inflammatory mediators in LPS-induced cells.

Use of DSF in the preparation of an inhibitor of inflammatory mediators, wherein said inflammatory mediators comprise at least one of TNF α, IL-1 β, IL-6 or IL-10.