Application of 2-Hydroxychalcone in preparation of anti-inflammatory drugs

Abstract

The invention discloses an application of 2-Hydroxychalcone in preparation of an anti-inflammatory drug, and belongs to the technical field of medicinal chemistry, and the drug comprises 2-Hydroxychalcone with a therapeutically effective amount or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier. Wherein the 2-Hydroxychalcone has an action target of TRPV1, the expression of the TRPV1 protein is inhibited through the 2-Hydroxychalcone, so that various inflammatory factors are down-regulated, the antibacterial effect is achieved, a new thought is provided for developing the TRPV1 and an inhibitor, and a new direction is provided for developing anti-inflammatory drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to the application of 2-Hydroxychalcone in the preparation of anti-inflammatory drugs. Background Art

[0002] The mechanism of inflammation is related to the pathophysiological changes of the body. As an important self-protection reaction of the body, inflammation not only clears the necrotic and malignant cells in the human body, but also has evolved a complete set of response procedures for invading foreign pathogens and any foreign substances that may cause cell damage in the body. However, when the inflammatory response is too intense and exceeds the range that the human body can bear, the risk factor of physical damage caused by it will greatly increase. Inflammation is generally divided into two major inflammatory signs: acute inflammation and chronic inflammation. Acute inflammation is self-limiting. It is caused by the overactivation of immune cells such as neutrophils and basophils. When the immune response is unbalanced and a large number of immune cells are mobilized to participate in the inflammatory response, the "inflammatory storm" reaction of the mononuclear macrophage system will occur, and then sepsis that endangers life and health will occur. Chronic inflammation is caused by the direct infiltration of mononuclear immune cells and the slow accumulation of inflammatory cytokines. For example, long-term chronic inflammation is a common feature of diabetes, cancer, obesity, cardiovascular disease, autoimmune diseases and other chronic diseases.

[0003] Traditional anti-inflammatory drugs, including non-steroidal drugs, have a history of decades and can no longer cover the occurrence and development of all inflammatory diseases. There is also a great demand for the development of new anti-inflammatory drugs in clinical practice. As a major country in the cultivation, production and sales of Chinese herbal medicine, my country can naturally play a key role in this.

[0004] Naturally occurring drugs are monomeric compounds with anti-inflammatory and antioxidant properties that can trigger protective pathways in the cell cascade, thus providing a promising strategy for therapeutic applications. Among the drugs that may meet these criteria are the phytochemicals chalcones, which have been shown to have antioxidant, oxygen free radical scavenging and anti-inflammatory properties in experiments. Chalcones are a class of compounds with mainly phenolic aromatic groups in the flavonoid family that are widely found in nature. It has been found that chalcones display a special chemical structure that allows them to participate in anti-cancer effects. In addition, studies have shown that rats fed a daily diet of dried broccoli sprouts can reduce oxidative stress, hypertension and inflammatory responses, thus providing evidence for the potency and diversity of the positive effects caused by this plant ingredient.

[0005] The anti-inflammatory effect of chalcone derivatives is related to the inhibition of the release of inflammatory mediators, such as nitric oxide (NO), tumor necrosis factor-(TNF-) and interleukin 1-β, which can be produced by macrophages stimulated by lipopolysaccharide (LPS). It may come from the simultaneous inhibition of the production of various inflammatory mediators or the activation of transcription factors (NFkB, AP-1) that regulate inflammatory responses by directly inhibiting them. Studies have shown that chalcone can also promote its anti-inflammatory effect by exerting its endogenous cytoprotective pathway. Studies have also reported that polyphenolic compounds, such as curcumin and caffeic acid phenethyl ester, also mediate cytoprotection through HO-1 induction, but they require the activation of the transcription factor Nrf2.

[0006] Currently, there is no literature report on the anti-inflammatory activity of 2-Hydroxychalcone. Summary of the invention

[0007] The purpose of the present invention is to provide the use of 2-Hydroxychalcone in the preparation of anti-inflammatory drugs, and to develop anti-inflammatory candidate drugs with development prospects by studying the anti-inflammatory ability and anti-inflammatory mechanism of 2-Hydroxychalcone.

[0008] In a first aspect, the present invention provides a use of 2-Hydroxychalcone in the preparation of an anti-inflammatory drug. The target of 2-Hydroxychalcone is TRPV1. The 2-Hydroxychalcone molecule inhibits the expression of TRPV1 channels, downregulates inflammatory factors, and achieves the anti-inflammatory purpose.

[0009] In a second aspect, the present invention further provides an anti-inflammatory drug comprising 2-Hydroxychalcone, wherein the drug comprises a therapeutically effective amount of 2-Hydroxychalcone or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.

[0010] In some embodiments, the medicament comprises a therapeutically effective amount of 2-Hydroxychalcone or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.

[0011] In some embodiments, the concentration of 2-Hydroxychalcone is 12.5 umol / ml.

[0012] In some embodiments, the pharmaceutically acceptable carrier includes any one or more of alumina, aluminum stearate, lecithin, serum protein, phosphate, glycerol, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax and lanolin.

[0013] In some embodiments, the drug is in the form of any one of tablets, capsules, powders, syrups, solutions, suspensions or aerosols.

[0014] In a third aspect, the present invention further provides a TRPV1 antagonist comprising 2-Hydroxychalcone, comprising contacting cells capable of expressing TRPV1 with a therapeutically effective amount of 2-Hydroxychalcone.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] 1. The present invention first discovered that 2-Hydroxychalcone down-regulates the expression of inflammatory factors by acting on the TRPV1 target to achieve the anti-inflammatory purpose.

[0017] 2. The present invention found that 2-Hydroxychalcone has a relatively obvious inhibitory effect on inflammatory factors under the condition of ... dosage. This type of compound is a potential new type of anti-inflammatory drug candidate and has further research and development value.

[0018] 3. 2-Hydroxychalcone can be used as a TRPV1 inhibitor and has certain development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 To establish the most suitable drug concentration for establishing different concentration gradient drug models; (a) Cumulative mortality of animals under different concentration gradient drug models; (b) Survival rate of animals under different concentration gradient drug models.

[0020] Figure 2 The behavioral changes of zebrafish in the experimental group and drug group models were measured; (a) the movement speed of zebrafish under different concentration gradient drug models; (b) the movement distance of zebrafish under different concentration gradient drug models.

[0021] Figure 3 Macrophage migration in zebrafish inflammation model; (a) 2'-Hydroxychalcone inhibits CuSO4-induced macrophage migration; (b) Comparison of macrophage migration.

[0022] Figure 4 Comparison of macrophage migration.

[0023] Figure 5 This is the change of ROS in zebrafish before and after drug action.

[0024] Figure 6 The changes of inflammatory factors before and after medication; (a) inflammatory factor IL-1β; (b) inflammatory factor TNF-a.

[0025] Figure 7 The cell viability under different concentration gradient drug models.

[0026] Figure 8 To screen the optimal LPS concentration range by measuring cell viability.

[0027] Fig. 9 The changes in gene expression levels of inflammatory factors before and after medication; (a) inflammatory factor IL-1β; b inflammatory factor TNF-a; (c) inflammatory factor NF-KB; (d) inflammatory factor NLRP3.

[0028] Fig.10 The changes of inflammatory pathway proteins before and after drug administration; (a) inflammatory factor NF-KB; (b) inflammatory factor IL-1β.

[0029] Fig.11 To explore the interaction between 2'-Hydroxychalcone and TRPV1 receptor, molecular docking experiments were carried out using MOE 2019 software.

[0030] Fig.12 To use CETSA technology to confirm the thermal migration phenomenon between 2-Hydroxychalcone and its target TRPV1; (a) CETSA binding state of 2'-Hydroxychalcone and TRPV1; (b) TRPV1 protein expression under temperature changes.

[0031] Fig.13 This is the protective mechanism of 2'-Hydroxychalcone against the inflammatory response induced by CuSO4 exposure. DETAILED DESCRIPTION

[0032] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0033] The process by which drugs play a role in diseases is to bind to target proteins in the body, change the physical and chemical properties of target proteins and downstream proteins, and ultimately play a role in treating diseases. Understanding the protein targets that bind to drugs has far-reaching significance for new drug development, clinical application of drugs, and the mechanism of action of drugs and the pathogenic mechanism of diseases.

[0034] At present, most methods for studying drug targets are based on chemical modification and related phenotypic analysis. However, the former method may lead to changes in drug activity to a certain extent and fail to verify the original pharmacodynamic characteristics of the drug. Therefore, phenotypic analysis methods are gaining more and more attention, and thermal proteome profiling (TPP) is one of them.

[0035] CuSO4 exposure can trigger mitochondrial oxidative stress, mitochondrial damage, and cause the massive release of ROS, which in turn causes the massive production of NLRP3, and ultimately triggers the massive release of pro-inflammatory factors, causing damage to the body.

[0036] Research Methods

[0037] 1. Phenomenon observation:

[0038] (1) A RAW264.7 cell model was constructed using LPS, and a blank control group and a 2-Hydroxychalcone concentration gradient treatment group were set up for 8 hours. The specific operation is as follows: ① Dissolve LPS powder in sterile water to make the mother solution concentration 1 mg / ml, and filter it with a 0.22um filter; similarly, use DMSO to dissolve 2-Hydroxychalcone to the recommended mother solution concentration of 200mmol / ml; ② Dilute the mother solution with a cell-specific antibiotic-free culture medium: LPS is diluted to 100 / 500 / 1000nmol / ml; 2-Hydroxychalcone concentrations are selected at 5 / 10 / 25 / 50umol / ml; ③ After 24 hours of cell plating, add the prepared LPS solution; ④ After 4 hours of LPS action, wash twice with sterile PBS and add the pre-prepared 2-Hydroxychalcone.

[0039] (2) Use 2-Hydroxychalcone to construct a zebrafish model, and set up a blank control group and a drug concentration gradient treatment group. Observe for 6 hours continuously and record the activity and survival rate of each group of zebrafish. The specific operation is as follows: ① Take 2-Hydroxychalcone powder and dissolve it in PBS to make its mother solution concentration 200mmol / ml; similarly, prepare anhydrous CuSO4 with PBS solution to make the mother solution concentration 80umol / ml; ② Dilute the mother solution with embryo culture water: dilute CuSO4 to 20nmol / ml; select 2-Hydroxychalcone concentrations of 100 / 50 / 25 / 12.5 / 6.25 / 3.13umol / ml; ③ The number of zebrafish in each group is 20, and the number and vitality of zebrafish surviving after soaking in drugs and CuSO4 of different concentrations for 6 hours are measured; ④ Extend the drug action time to 72h and measure the survival rate and vitality of zebrafish.

[0040] (3) qPCR was used to detect the mRNA levels of inflammatory factors in different groups.

[0041] (4) WB was used to detect the protein expression of inflammatory factors in different groups.

[0042] (5) Elisa was used to detect the levels of TNF-a and IL-1β in each group.

[0043] (6) The binding of 2-Hydroxychalcone to target proteins was analyzed using thermoproteomics combined with mass spectrometry.

[0044] 2. Functional study:

[0045] (1) ① The morpholino sequence of the corresponding possible target protein was constructed by molecular cloning technology; ② The morpholino sequence was injected into the zebrafish embryonic cleavage stage to establish a gene knockdown zebrafish model, and a negative control was set up; ③ qPCR was used to detect the mRNA level of the target in different groups; ④ WB was used to detect the expression of the target protein in different groups; ⑤ qPCR was used to detect the levels of TNF-a and IL-1β in each group.

[0046] (2) ① Cultivate RAW264.7 (mouse macrophage) cells; ② Transfect the target siRNA lentiviral vector to establish a RAW264.7 cell model with target gene knockdown, and set up a negative control; ③ qPCR to detect the level of target mRNA in different groups; ④ WB to detect the expression of target protein in different groups; ⑤ ELISA to detect the levels of TNF-a and IL-1β in each group; ⑥ qPCR to detect the expression level of corresponding inflammatory factors.

[0047] 3. Mechanism study:

[0048] ① Use the FDA molecular library (ZINC and DRUGBANK) to computer simulate the specific situation of 2-Hydroxychalcone docking with molecules; ② Use WB technology to explore the expression of inflammatory pathway proteins such as IL-1β, TNF-a, NF-KB, and NLRP3; ③ Clarify the expression of the target TRPV1 under inflammatory conditions and its interaction with the NF-KB pathway.

[0049] Experimental protocol:

[0050] Part I: Cell Experiments

[0051] 1. RAW264.7 cells were seeded into 6-well plates at a density of 5×106 and cultured in a complete medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator

[30] . RAW264.7 cells were randomly divided into an inflammation group, a 2-Hydroxychalcone immersion group, and a blank control group. LPS (100 ng / ml, 6 hours) was used to establish an inflammation model. After successful modeling, cells were collected for the following tests: qPCR to detect the mRNA levels of TNF-a and IL-1β; WB to detect the protein levels of TNF-a and IL-1β; ELISA to detect the levels of TNF-a and IL-1β in the supernatant.

[0052] 2. RAW264.7 cells were cultured and randomly divided into inflammation group, 2-Hydroxychalcone immersion group and blank control group. The corresponding cell supernatant after centrifugation was extracted using thermal proteomics technology and sent for mass spectrometry sequencing.

[0053] 3. RAW264.7 cells were cultured and randomly divided into inflammation group, 2-Hydroxychalcone immersion group and blank control group, and transfected with siRNA lentiviral vectors of corresponding targets. After 24 hours, LPS (100 ng / ml, 6 hours) was used to establish an inflammation model. After successful modeling, cells were collected for the following tests: qPCR to detect the mRNA level of the target; WB to detect the protein expression of the target; ELISA to detect the levels of TNF-a and IL-1β in the supernatant.

[0054] Part II: Animal Experiments

[0055] 1. Zebrafish were randomly divided into inflammation group, 2-Hydroxychalcone immersion group and blank control group at 72 hours after fertilization, with 20 fish in each group. The inflammation model was established by immersion (CuSO4 20umol / ml, 6 hours). The zebrafish were observed 4 times a day for 72 hours, and the activity and survival rate of the zebrafish were recorded.

[0056] 2. Take zebrafish 72 hours after fertilization and randomly divide them into inflammation group, 2-Hydroxychalcone immersion group and blank control group, with 10 in each group. The inflammation model was established by immersion (CuSO4 20umol / ml, 6 hours). After the model was successfully established, the zebrafish were killed and the fish bodies were collected for the following tests: qPCR detection of TNF-a and IL-1β mRNA levels; WB detection of TNF-a and IL-1β protein expression levels.

[0057] 3. Zebrafish were taken 3 days after fertilization and randomly divided into inflammation group, 2-Hydroxychalcone immersion group and blank control group, with 18 in each group. The embryos were injected with the morpholino sequence of the target, and the inflammation model was established by embryo immersion (CuSO4 20umol / ml, 6 hours) 24 hours later. After the modeling was successful, 6 zebrafish were killed in each group, and the fish bodies were collected for the following tests: qPCR detection of the mRNA level of the fish body target; WB detection of the protein expression of the target; qPCR detection of TNF-a and IL-1β levels in each group.

[0058] Experimental conclusion

[0059] Part I: Animal Experiments

[0060] like Figure 1 As shown, we evaluated the MTC of 2'-Hydroxychalcone at 25umol / mL after 6h. Exposure to 100 and 200umol / mL of 2'-Hydroxychalcone caused rapid death of all juveniles. Doses up to 25umol / mL had no significant effect on mortality at 6h. Thus, the concentration used in the experiment was controlled below 25umol / mL. And the mortality rate of the 2'-Hydroxychalcone and CuSO4 group at a concentration of 25umol / ml was about 90% at 48h, the mortality rate at 12.5umol / ml was about 30%, the mortality rate at 6.25 was about 50%, the mortality rate at 3.13 was about 70%, and the mortality rate of the CuSO4 modeling group was about 75%.

[0061] like Figure 2 As shown in the figure, the effect of 2'-Hydroxychalcone exposure on the swimming behavior of zebrafish larvae. The anti-inflammatory effect of the drug was confirmed by measuring the behavior of the zebrafish experimental group and drug group model. The results showed that the motor ability of the experimental group decreased significantly compared with the control group, while the motor ability recovered after drug administration.

[0062] like Figure 3As shown, 2'-Hydroxychalcone inhibited the migration of macrophages induced by CuSO4. The number of migrating macrophages in the experimental group increased compared with the control group, and the number of migrating macrophages after drug administration was improved compared with before.

[0063] like Figure 4 As shown, in the inflammation inhibition rate, as the concentration of the zebrafish drug group increased, the inflammation inhibition rate showed a gradient upward trend compared with the control group.

[0064] like Figure 5 As shown, 2'-Hydroxychalcone inhibited the production of ROS induced by CuSO4. The results showed that the ROS in the experimental group changed more significantly than that in the control group, and the index was adjusted back after medication.

[0065] like Figure 6 As shown in (a) and (b), the inflammatory factors IL-1β and TNF-a in the experimental group showed an upward trend compared with the control group; while under the action of the three drug gradients, their gene expression levels decreased.

[0066] Part II: Cell experiments

[0067] like Figure 7 As shown, 2-hydroxychalcone had little effect on cell viability at concentrations of 5umol / ml, 10umol / ml and 25umol / ml.

[0068] like Figure 8 As shown, cell viability was optimal when the LPS concentration reached 500 nmol / ml.

[0069] like Fig. 9 As shown in (a), (b), (c) and (d), the inflammatory pathway factors IL-1β, TNF-a, NF-KB, NLRP3, etc. were increased in the experimental group compared with the control group, while the gene expression decreased after medication.

[0070] like Fig.10 As shown in (a) and (b), the expression of two inflammatory pathway proteins, IL-1β and NF-KB, was downregulated before and after drug administration.

[0071] like Fig.11 As shown, the Asp738 residue on the TRPV1 receptor forms a hydrogen bond interaction with 2'-Hydroxychalcone, while the Lys325 residue participates in hydrophobic, electrostatic interactions, and carbon-hydrogen interactions with 2'-Hydroxychalcone. In addition, the Leu366 and Arg740 residues on the receptor establish hydrophobic interactions with 2'-Hydroxychalcone, while the Glu328 residue forms an electrostatic interaction with it.

[0072] like Fig.12 (a) and (b) show the binding state of 2'-Hydroxychalcone to TRPV1's CETSA. For cells treated at the same temperature, the control group reached the best stable state at 42°C, and then the curve showed a downward trend; while the cells treated with 2-Hydroxychalcone had a more stable structural configuration and stronger thermal stability.

[0073] result

[0074] like Fig.13 As shown in the results, 2'-Hydroxychalcone can inhibit the generation of ROS and the release of downstream NLRP3, IL-1β and TNF-a; and it can affect the functional state of the target TRPV1 by binding to it, causing changes in intracellular calcium ion levels, affecting the functional state of mitochondria and indirectly affecting cell vitality, thereby improving the inflammatory response of zebrafish induced by CuSO4.

[0075] 2-Hydroxychalcone has anti-inflammatory and antioxidant activities and reduces oxidative stress under the induction of CuSO4 and LPS. It can reduce the expression level of inflammatory factors and explore the possible binding state of 2-Hydroxychalcone with the target TRPV1 through CETSA technology; molecular docking technology shows that 2'-Hydroxychalcone has good binding activity with TRPV1. These results indicate that 2'-Hydroxychalcone can be used as a possible new inflammatory and immune regulator.

[0076] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

Application of 1.2-Hydroxychalcone in the preparation of drugs for improving CuSO4-induced inflammation in zebrafish.

2. An anti-inflammatory drug comprising 2-Hydroxychalcone, characterized in that: The medicine comprises a therapeutically effective amount of 2-Hydroxychalcone or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier.

3. An anti-inflammatory drug comprising 2-Hydroxychalcone according to claim 2, characterized in that: The concentration of 2-Hydroxychalcone is 12.5 umol / ml.

4. The anti-inflammatory drug comprising 2-Hydroxychalcone according to claim 2, characterized in that: The pharmaceutically acceptable carrier includes any one or more of aluminum oxide, aluminum stearate, lecithin, serum protein, phosphate, glycerol, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, beeswax and lanolin.

5. The anti-inflammatory drug comprising 2-Hydroxychalcone according to claim 2, characterized in that: The drug is in the form of tablets, capsules, powders, syrups, solutions, suspensions or aerosols.

6. A ROS inhibitor comprising 2-Hydroxychalcone, characterized in that The method comprises contacting cells capable of releasing ROS with a therapeutically effective amount of 2-Hydroxychalcone.

7. A TRPV1 antagonist comprising 2-Hydroxychalcone, characterized in that The method comprises contacting a cell capable of expressing TRPV1 with a therapeutically effective amount of 2-Hydroxychalcone.

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