Natural NLRP3 inflammasome inhibitor and application of natural NLRP3 inflammasome inhibitor in preparation of medicine for treating inflammatory intestinal diseases

Abstract

The invention discloses a natural NLRP3 inflammasome inhibitor and application thereof in preparation of a medicine for treating inflammatory intestinal diseases, and relates to the technical field of biological medicines. According to the invention, a natural compound 2 ', 4'-dihydroxy chalcone with an effect of inhibiting NLRP3 inflammasome activation is screened from abrus cantoniensis hance, and the natural compound 2 ', 4'-dihydroxy chalcone can inhibit the activation of the NLRP3 inflammasome by inhibiting the activation of Caspase-1 and the expression of NLRP3 and can reduce the secretion level of anti-inflammatory factors. The natural compound plays an important role in regulating intestinal homeostasis and inhibiting colitis and colon cancer. The NLRP3 inflammasome inhibitor provided by the invention can be used for effectively inhibiting the activation of the NLRP3 inflammasome in vivo and in vitro, and can be used for effectively treating diseases related to the NLRP3 inflammasome. The invention provides technical support for treating diseases related to the NLRP3 inflammasome.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a natural NLRP3 inflammasome inhibitor and an application thereof in the preparation of a drug for treating inflammatory bowel diseases. Background Art

[0002] Acanthopanax odoratus is the dried whole plant of Guangzhou Abrus preparata, a traditional Chinese medicinal herb in Lingnan region of China. It has a long history of use and has the effects of calming the liver, clearing away heat and detoxifying. It can be made into cold tea or cold soup for consumption. Since the 1960s, Chinese scholars have begun to study the chemical components of Acanthopanax odoratus. Modern pharmacological studies have shown that Acanthopanax odoratus has multiple biological activities such as liver protection, anti-inflammatory, immunomodulatory, antioxidant, and anti-tumor, which are closely related to its rich chemical components. Combining phytochemical and pharmacological activity studies, it was found that its main active ingredients are alkaloids, flavonoids, triterpenes and others. So far, most of the studies on the activity of the total extract have not been analyzed for chemical components, and its main active ingredients are unknown, making it difficult to gain a deep understanding of its pharmacodynamic mechanism, targets, pathways, and possible applications in other diseases.

[0003] Inflammasome is a complex protein structure in the body that has the function of regulating inflammatory responses. The general inflammasome structure contains NLRP (Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing), ASC protein (Apoptosis-associated Speck-like protein containing a CARD) and Caspase-1. Inflammasome is considered to be the inflammatory switch in the body. The NLRP3 inflammasome is the most studied inflammasome. It is a multiprotein complex that plays a key role in innate immunity and is also the first line of defense for the human body against pathogens. It belongs to the NLR protein family. The NLRP3 inflammasome plays a vital role in a variety of inflammation-driven human diseases. Its dysregulation can lead to excessive inflammation and is associated with various diseases, including autoinflammatory and autoimmune diseases, metabolic diseases and cancer.

[0004] The present invention intends to separate and identify components that have an inhibitory effect on NLRP3 inflammasome from the natural product of Acanthopanax gracilis, thereby providing technical support for the development of NLRP3 inflammasome inhibitors and the treatment of diseases related to NLRP3 inflammasome. Summary of the invention

[0005] The purpose of the present invention is to provide a natural NLRP3 inflammasome inhibitor and its use in the preparation of a drug for treating inflammatory bowel diseases to solve the problems of the above-mentioned prior art. The natural NLRP3 inflammasome inhibitor can effectively inhibit the activation of NLRP3 inflammasomes and can effectively treat diseases related to NLRP3 inflammasomes.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of 2',4'-dihydroxychalcone in preparing an NLRP3 inflammasome inhibitor.

[0008] The present invention also provides an NLRP3 inflammasome inhibitor, wherein the active ingredient includes 2',4'-dihydroxychalcone.

[0009] The present invention also provides the use of 2',4'-dihydroxychalcone in preparing a drug for treating diseases associated with NLRP3 inflammasome.

[0010] Furthermore, the disease associated with NLRP3 inflammasome is inflammatory bowel disease.

[0011] Furthermore, the inflammatory bowel disease is ulcerative colitis or colon cancer.

[0012] The present invention also provides a drug for treating diseases associated with NLRP3 inflammasome, wherein the active ingredient includes 2',4'-dihydroxychalcone.

[0013] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the dosage form of the drug is an oral dosage form.

[0015] The present invention discloses the following technical effects:

[0016] The present invention screened out a natural compound, 2',4'-dihydroxychalcone, from Glechoma longituba, which has the effect of inhibiting the activation of NLRP3 inflammasomes. It can inhibit the activation of Caspase-1 and the expression of NLRP3, inhibit the activation of NLRP3 inflammasomes, and reduce the secretion level of pro-inflammatory inflammatory factors. The natural compound plays an important role in regulating intestinal homeostasis and inhibiting colitis and colon cancer.

[0017] The NLRP3 inflammasome inhibitor provided by the present invention can effectively inhibit the activation of NLRP3 inflammasome both in vivo and in vitro, and can effectively treat diseases related to NLRP3 inflammasome. The present invention provides technical support for treating diseases related to NLRP3 inflammasome.

[0018] The 2',4'-dihydroxychalcone of the invention is used as an effective ingredient in the herb. Not only is the raw material easily available and cheap, but it is also helpful to expand the application research of the herb in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 Statistical graphs of cell activity and IL-1β secretion expression in different treatment groups; A is a statistical graph of THP-1 cell activity in different treatment groups; B is a statistical graph of J774A.1 cell activity in different treatment groups; C is a statistical graph of IL-1β secretion expression in THP-1 cells in different treatment groups; D is a statistical graph of IL-1β secretion expression in J774A.1 cells in different treatment groups; *** P<0.001; ns means no significant difference;

[0021] Figure 2 The statistical graphs of TNF-α secretion expression in J774A.1 cells in different treatment groups; ns means no significant difference;

[0022] Figure 3 Western blotting results of J774A.1 cells in different treatment groups;

[0023] Figure 4 Western blotting results of HT29 (A) and CT26 (B) cells in different treatment groups;

[0024] Figure 5 The weight change (A), DAI index (B), colon length (C) and colon representative image (D) of mice in different experimental groups in Example 2; n = 10; compared with the DSS group, *** P < 0.001;

[0025] Figure 6 The colon tissues of mice in different experimental groups in Example 2 are stained with hematoxylin-eosin;

[0026] Figure 7 The statistical diagram of the content of colon inflammatory factors IL-1β (A), IL-6 (B) and TNF-α (C) in mice of different experimental groups in Example 2; compared with the DSS group, *** P < 0.001;

[0027] Figure 8 The Western blotting results of mice in different experimental groups in Example 2;

[0028] Fig. 9 The tumor growth curve (A), mouse body weight change (B) and tumor weight statistics (C) of mice in different experimental groups in Example 3; wherein n=10; * P<0.05, ** P<0.01, *** P < 0.001;

[0029] Fig.10 The staining images of Ki67 in HT29 tumor tissues of mice in different experimental groups in Example 3;

[0030] Fig.11 The staining images of NLRP3 in HT29 tumor tissues of mice in different experimental groups in Example 3;

[0031] Fig.12 The Western blotting results of mice in different experimental groups in Example 3;

[0032] Fig.13 Statistical graphs of liver index (A) and spleen index (B) of mice in different experimental groups in Example 3; wherein n=10; * P<0.05, ** P<0.01, *** P<0.001. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Terminology Note:

[0039] PMA refers to phorbol 12-myristate 13-acetate;

[0040] LPS refers to lipopolysaccharide;

[0041] Nigericin refers to nigericin;

[0042] DSS refers to dextran sulfate sodium.

[0043] The present invention isolates a natural compound having an inhibitory effect on NLRP3 inflammasome from the herb of Glechoma longituba, which is identified as 2',4'-dihydroxychalcone, and has the following structural formula:

[0044]

[0045] Example 1 Study on the inhibitory activity of natural compounds on NLRP3 inflammasome in macrophages

[0046] 1. Experimental Methods

[0047] (1) Cytotoxicity studies

[0048] THP-1 and J774A.1 cells induced by LPS were cultured for 2e 4 / well were inoculated in 96-well plates and incubated in a 37°C constant temperature incubator at 5% carbon dioxide concentration. After they adhered to the wall, different concentrations of 2',4'-dihydroxychalcone (5, 10, 20 μM) were added and incubated for 2 hours, and the cell activity was measured by CCK-8 method.

[0049] (2) Study on inhibition of NLRP3 inflammasome activity in macrophages

[0050] 1.5e 6 THP-1 cells were seeded in 6-well plates, treated with PMA (10 ng / mL) overnight, then treated with LPS (500 ng / mL) for 3 h, and then treated with different concentrations of 2',4'-dihydroxychalcone (5, 10, 20 μM) for 1 h, and then incubated with Nigericin (10 μM, 1 h).

[0051] 1.0e 6 / well J774A.1 cells were seeded in 6-well plates and incubated with LPS (1 μg / mL) overnight after 24 h, then incubated with different concentrations of 2',4'-dihydroxychalcone (5, 10, 20 μM) for 1 h, and then co-incubated with Nigericin (10 μM, 1 h).

[0052] The supernatant of each treatment group was collected and tested by ELISA, and the cells were collected for protein Western blotting analysis.

[0053] (3) Study on inhibition of NLRP3 inflammasome activity in colon cancer cell lines

[0054] HT29 and CT26 cells were cultured at 3e 5 / well were inoculated in 6-well plates, and treated with different doses of 2',4'-dihydroxychalcone (5, 10, 20μM) for 24 hours after adhesion. BCA quantitative analysis was performed after cell lysis, and the lysate was mixed with 5× loading buffer, separated by 8% SDS-PAGE, and transferred to PVDF membrane. After blocking at room temperature for 1 hour, the PVDF membrane was incubated with specific primary antibodies at 4°C overnight. TBST buffer was washed 5 times, and secondary antibodies were added and incubated at room temperature for 1 hour. Washed 5 times, ECL reagent was used to illuminate the protein bands.

[0055] 2. Experimental results

[0056] Depend on Figure 1 As shown in Figures A and B, when the concentration of 2',4'-dihydroxychalcone was 20 μM, the relative viability of THP-1 and J774A.1 cells was close to 100%, indicating that the compound has good safety. Figure 1 As shown in Figures C and D, 2',4'-dihydroxychalcone inhibited the secretion of IL-1β in a dose-dependent manner; Figure 2 As shown in Figure 2, 2',4'-dihydroxychalcone does not inhibit TNF-α secretion independent of inflammasome activation. Figure 3As shown, 2',4'-dihydroxychalcone can inhibit the activation of Caspase-1 in J774A.1 macrophages. This indicates that 2',4'-dihydroxychalcone can inhibit the activation of NLRP3 inflammasome by inhibiting the cleavage of Caspase-1.

[0057] Depend on Figure 4 As shown in A and B, 2',4'-dihydroxychalcone can directly inhibit the expression of NLRP3, Caspase-1 and ASC in the NLRP3 inflammasome in colon cancer cells, and can inhibit the activation of Caspase-1, thereby directly inhibiting the activation of NLRP3 inflammasome.

[0058] The following uses colitis and colon cancer as examples to illustrate the effects of 2',4'-dihydroxychalcone in treating diseases associated with NLRP3 inflammasome.

[0059] Example 2 Study on the efficacy of natural compounds in improving DSS-induced ulcerative colitis

[0060] 1. Experimental Methods

[0061] (1) Construction of DSS-induced acute colitis model in mice and pharmacodynamic evaluation

[0062] Forty SPF-grade 6-8-week-old male C57BL / 6J mice were adaptively fed for 7 days and randomly divided into 4 groups (10 mice in each group): control group (Control group), model group (DSS group) and two treatment groups, of which the two treatment groups were low-dose group (10 mg / kg) and high-dose group (30 mg / kg). The DSS group was given 2.5% DSS (w / v) aqueous solution orally for 7 days (days 4-10). The low-dose and high-dose groups of the compound were gavaged with different doses of 2',4'-dihydroxychalcone (solvent was 0.4% sodium carboxymethyl cellulose) on days 1-10, and 2.5% DSS (w / v) aqueous solution was given orally for 7 days (days 4-10). The control group was not given 2',4'-dihydroxychalcone and DSS, and was replaced by an equal amount of solvent.

[0063] The weight of mice was recorded daily, and the disease activity index (DAI) was scored based on weight loss, stool consistency, and fecal blood. The score for each index ranged from 0 to 4. The mice were sacrificed on day 11, and colon tissues were collected for analysis.

[0064] (2) Histopathological analysis

[0065] Colon tissue was stained with hematoxylin and eosin (H&E) for histological analysis. The fixed colon was embedded in paraffin and sliced ​​for use. After hematoxylin-eosin staining, the sections were dehydrated and dried, and then sealed with neutral gum. They were then observed under a microscope.

[0066] (3) Determination of inflammatory factors in colon tissue

[0067] After the colon tissue was ground, the supernatant was centrifuged and used for ELISA and BCA protein quantification. According to the instructions of the ELISA kit, the inflammatory factors TNF-α, IL-6, and IL-1β were detected and analyzed.

[0068] (4) Analysis of NLRP3 inflammasome protein expression

[0069] Colon tissue was lysed using RAPI lysis buffer. After BCA quantitative analysis, the lysate was mixed with 5× loading buffer, separated by 8% SDS-PAGE, and transferred to a PVDF membrane. After blocking at room temperature for 1 hour, the PVDF membrane was incubated with specific primary antibodies at 4°C overnight. The membrane was washed 5 times with TBST buffer, and the secondary antibody was added and incubated at room temperature for 1 hour. After washing 5 times, the protein bands were displayed by ECL reagent luminescence.

[0070] 2. Experimental results

[0071] The results showed that compared with the mice in the Control group, the body weight of the mice in the model group and the two treatment groups gradually decreased after DSS-induced ulcerative colitis ( Figure 5 A), and the disease activity index DAI score increased ( Figure 5 Middle B), the colon length was significantly shortened ( Figure 5 Middle C), Figure 5 D is a representative colon diagram. The 2',4'-dihydroxychalcone group can increase the colon length of the DSS group mice in a dose-dependent manner and reduce the DAI score. The colon tissue was pathologically evaluated by H&E staining. The staining results are shown in Figure 6 The results showed that the overall structure of the intestinal tissue of mice with DSS-induced colitis was abnormal, local mucosal necrosis was obvious, the normal structure of the intestinal tissue basically disappeared, a large number of inflammatory cells were present in the whole layer of tissue, and there were dense areas of inflammatory cell aggregation. The histological microstructure of the H&E stained sections of the treatment group was improved, the infiltration of inflammatory cells was reduced, and there was no area of ​​inflammatory cell aggregation. The changes in the colon crypt morphology of mice in the DSS group were alleviated and goblet cells were protected. It can be seen that the natural NLRP3 inflammasome inhibitor of the present invention, 2',4'-dihydroxychalcone, can effectively improve the colon pathological damage of mice.

[0072] In addition, if Figure 7 As shown in the figure, the levels of pro-inflammatory factors IL-6, IL-1β and TNF-α in colon tissue can be significantly reduced after treatment with the natural NLRP3 inflammasome inhibitor of the present invention, 2',4'-dihydroxychalcone. Western blotting analysis found that 2',4'-dihydroxychalcone can inhibit the expression of NLRP3 protein and the activation of Caspase-1 in the colon ( Figure 8 ).

[0073] In summary, the natural NLRP3 inflammasome inhibitor of the present invention, 2',4'-dihydroxychalcone, can alleviate intestinal inflammation of DSS-induced ulcerative colitis in mice by significantly reducing the level of pro-inflammatory factors and inhibiting the activation of NLRP3 inflammasome.

[0074] Example 3 Study on the efficacy of natural compounds in inhibiting HT29 tumor growth in vivo

[0075] 1. Experimental Methods

[0076] (1) Construction of HT29 human colon cancer model and pharmacodynamic evaluation

[0077] Thirty SPF-grade BALB / c-Nude female mice aged 6-8 weeks were adaptively bred for 7 days. HT29 cells (8×10 6 Cells / 100 μL were injected subcutaneously into the right axilla of BALB / c nude mice. After obvious tumors were observed, the mice were divided into 3 groups (10 mice in each group): model group, low-dose group (30 mg / kg), and high-dose group (60 mg / kg). After grouping, the low-dose group and the high-dose group were gavaged with 2',4'-dihydroxychalcone every day according to their respective doses until the end of the experiment. After 13 days of administration, the mice were killed, the tumors were weighed and photographed, a part of the tumors were stored at -80℃ for Western blotting and other analyses, and the other part of the tumors were stored in 4% paraformaldehyde for IHC and other tests.

[0078] Tumor growth inhibition rate: Monitor tumor size and mouse weight every 2 days. Measure tumor volume with a vernier caliper. Take the long side and short side of the tumor as length (a) and width (b), volume = a × b × b / 2, and draw a tumor growth curve. Tumor inhibition rate (%) = (average tumor weight of model group - average tumor weight of drug administration group) / average tumor weight of model group × 100%.

[0079] (2) Histopathological analysis

[0080] The fixed tumor tissue was embedded in paraffin and sliced ​​before use. For IHC experiments, tissue sections were incubated with anti-NLRP3 antibody (GB114320, Servicebio), anti-Ki67 antibody (GB111141, Servicebio) and HRP-labeled anti-rabbit IgG (AB205718, Abcam), and stained with hematoxylin and DAB. The final stained sections were observed under a microscope system (Nikon EclipseCI, Nikon).

[0081] (3) Analysis of NLRP3 inflammasome protein expression

[0082] Tumor tissue was lysed using RIPA lysis buffer. After BCA quantitative analysis, the lysate was mixed with 5× loading buffer, separated by 8% SDS-PAGE, and transferred to a PVDF membrane. After blocking at room temperature for 1 hour, the PVDF membrane was incubated with specific primary antibodies at 4°C overnight. The membrane was washed 5 times with TBST buffer, and the secondary antibody was added and incubated at room temperature for 1 hour. After washing 5 times, the protein bands were displayed by luminescence with ECL reagent.

[0083] (4) Organ index analysis

[0084] After the mice were dissected, the liver and spleen, the main immune organs, were weighed and recorded. Organ index = organ weight / body weight × 100%.

[0085] 2. Experimental results

[0086] like Fig. 9 As shown, 2',4'-dihydroxychalcone can significantly inhibit the growth of HT29 transplanted tumors ( Fig. 9 A) and did not affect the body weight of mice ( Fig. 9 In B), tumor weight ( Fig. 9 Based on (C), the tumor inhibition rates of the low-dose group and the high-dose group were 49.2% and 61.0%, respectively.

[0087] 2',4'-dihydroxychalcone can significantly reduce Ki67 in HT29 tumor tissues ( Fig.10 ) and NLRP3( Fig.11 ) expression level, indicating that it can inhibit the proliferation of tumor cells and the expression of NLRP3 in the tumor. Western-blotting results also found that 2',4'-dihydroxychalcone can significantly inhibit the expression of NLRP3 and ASC in the tumor and the activation of Caspase-1 ( Fig.12 ). In addition, 2',4'-dihydroxychalcone can also reduce the liver index of tumor-bearing mice ( Fig.13 A) and spleen index ( Fig.13 (B) to normal levels, indicating that it can reduce the tumor burden in BALB / c-Nude mice.

[0088] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of 2',4'-dihydroxychalcone in the preparation of NLRP3 inflammasome inhibitors.

2. A NLRP3 inflammasome inhibitor, characterized in that The active ingredient includes 2',4'-dihydroxychalcone.

3. Use of 2',4'-dihydroxychalcone in the preparation of drugs for treating diseases associated with NLRP3 inflammasome.

4. The use according to claim 3, characterized in that: The disease associated with NLRP3 inflammasome is inflammatory bowel disease.

5. The use according to claim 4, characterized in that: The inflammatory bowel disease is ulcerative colitis or colon cancer.

6. A drug for treating a disease associated with NLRP3 inflammasome, characterized in that: The active ingredient includes 2',4'-dihydroxychalcone.

7. The drug according to claim 6, characterized in that The drug also includes pharmaceutically acceptable excipients.

8. The drug according to claim 7, characterized in that The dosage form of the drug is an oral dosage form.

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