Application of epigallocatechin gallate in preparation of preparation for treating oral ulcer

Abstract

The invention relates to the technical field of medicine, in particular to application of epigallocatechin gallate in preparation of a preparation for treating oral ulcers, the epigallocatechin gallate is used for inhibiting TLR4, MyD88 and NF-kappa B signal channels to treat oral inflammations, a new way and a new target spot are provided for treatment of the oral ulcers, and the application has the advantages that the application of the epigallocatechin gallate in preparation of the preparation for treating the oral ulcers can be realized by the application of the epigallocatechin gallate in preparation of the preparation for treating the oral ulcers; and a new drug for preventing and treating oral ulcer is expected to be developed.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of epigallocatechin gallate in preparing a preparation for treating oral ulcers. Background Art

[0002] RAU (recurrent aphthous ulcer) is the disease with the highest incidence among oral mucosal diseases. RAU is caused by systemic diseases or trauma, such as vitamin deficiency, oral microbial system disorders, hematological factors, stress, genetic diversity, oxidation-antioxidation imbalance, etc. Its incidence rate is as high as 20%, ranking first among oral mucosal diseases. Patients often have frequent attacks, manifested as severe pain in the ulcer site, which seriously affects eating and drinking, and even emotions, causing great distress to patients' daily lives. However, to date, its pathogenesis is not fully understood, and there is no satisfactory treatment. The main purpose of RAU treatment is to reduce the inflammatory process of ulcers. Most commonly used drugs can only relieve symptoms to a limited extent, and frequent use will also bring certain side effects.

[0003] Currently, there are very few studies on the use of natural extracts to treat RAU. Natural products have little toxicity and side effects, and may be very promising in the treatment of RAU, and can also be used to prevent oral ulcers. Green tea has a variety of ingredients and has multiple effects. The latest research shows that green tea is rich in natural active substances, especially EGCG (epigallocatechin gallate), which accounts for 50% to 70% of the total catechins and is the most important active ingredient in green tea, contributing greatly to its pharmacological effects.

[0004] EGCG exhibits important physiological and pharmacological properties such as anti-tumor, anti-infection, anti-radiation, improvement of lipid metabolism, anti-oxidation, and prevention of cardiovascular disease. However, no relevant studies have been reported on the relationship between EGCG and RAU. Summary of the invention

[0005] In view of this, the object of the present invention is to provide the use of epigallocatechin gallate in the preparation of a preparation for treating oral ulcers.

[0006] The present invention prepares EGCG oral lozenges, establishes a chemically induced RAU mouse model, and evaluates the pharmacological effect of EGCG on RAU. Experiments have found that the application of EGCG can improve the symptoms of RAU mice, reduce inflammation, promote the healing of oral ulcers, and improve feeding. Using an experimental RAU model induced by chemical damage, it was found that EGCG can significantly reduce weight changes, reduce the severity of ulcers, significantly reduce the ulcer area, and accelerate the wound healing of chemically corrosive ulcers. Through HE staining and MASSON staining, it was found that the pathological morphology of ulcer tissue tended to be complete after EGCG administration, and the cells and collagen fibers were arranged regularly. This shows that EGCG has a significant therapeutic effect on mouse RAU.

[0007] The pathogenic factors and pathogenesis of RAU are complex. The present invention uses disease databases and target databases to collect oral ulcer disease targets and EGCG drug targets. The results of GO biological enrichment analysis and KEGG enrichment pathways show that the process of EGCG improving RAU is closely related to inflammation. Studies have shown that IL-6 is a pleiotropic cytokine that promotes IFN-γ production, T cell differentiation, and B cell immunoglobulin secretion. As a bridge between innate and adaptive immunity, it is crucial in host defense. Elevated IL-6 may inhibit angiogenesis and delay wound healing. In the Toll-like receptor signaling pathway, TLR4 acts as a membrane-bound receptor, and IL-6 participates in proinflammatory responses. In the NF-κB pathway, IL-1β and IL-6 act as proinflammatory factors and have cell chemotactic effects. TNF-α is a key proinflammatory cytokine, usually secreted by activated T cells. Its level reflects the severity of inflammation, promotes the release of inflammatory factors, and leads to immune damage. The present invention detected the levels of inflammatory factors in ulcer tissues by ELISA and immunohistochemistry, and found that EGCG can reduce the expression of inflammatory factors such as TNF-α and IL-6 in a dose-dependent manner, indicating that the mechanism by which EGCG promotes ulcer healing may involve its inhibition of the expression of inflammatory factors.

[0008] On the basis of the above results, the potential protective mechanism of EGCG against RAU was further explored. Inflammatory response plays an important role in the pathogenesis of oral ulcers, and the TLR4 / MyD88 / NF-κB signaling pathway is the main pathway mediating inflammatory response. As an immune mediator, TLR can non-specifically bind to pathogen molecules, initiate signal transduction, activate NF-κB, and induce inflammation. TLR is expressed in a variety of cells, and its high expression is a sign of inflammation. Studies have shown that the expression of TLRs in oral epithelial cells of RAU patients is higher than that of the healthy group. TLR4 can recognize molecular signals from pathogens or damage, then dimerize, and interact with MyD1 through the Toll / IL-88 receptor homology region to form a receptor-associated kinase that interacts with MyD88 and IL-1, activate TRAF6 and release it into the cytoplasm to trigger the IKK complex, leading to the activation and nuclear translocation of NF-κB, promoting the release of inflammatory factors, forming a positive feedback loop, and exacerbating inflammation. The present invention uses Western blot experiments and RT-qPCR to find out the effect of EGCG on the expression of proteins and mRNA related to the TLR4 / MyD88 / NF-κB pathway in the ulcer site of mice, indicating that EGCG exerts anti-inflammatory effects by inhibiting this pathway.

[0009] As the main active ingredient in tea polyphenols, EGCG exhibits strong antioxidant capacity due to its unique stereochemical structure, and has significant anti-inflammatory and immunomodulatory effects. It reduces oral inflammation in mice by inhibiting TLR4, MyD88 and NF-κB signaling pathways. In order to further study the binding characteristics of EGCG with key targets, molecular docking and CETSA experiments found that EGCG has stable binding with core targets. EGCG has good binding activity with two important target proteins (NF-κB p65 and IKB), and the main forms of interaction between the components and the targets are hydrogen bonds and van der Waals forces. Through CETSA experiments, it was found that EGCG can bind to and stabilize TLR4, MyD88, and NF-κB P65 target proteins in cells.

[0010] In summary, EGCG has a significant effect in treating RAU in ICR mice, and its mechanism of action involves the inhibition of TLR4, MyD88 and NF-κB signaling pathways.

[0011] The beneficial effects of the present invention are as follows: the present invention provides a new approach and a new target for the treatment of oral ulcers, and is expected to develop new drugs for preventing and treating oral ulcers. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : The therapeutic effect of EGCG on ulcers, (A) images of ulcers in each group of mice; (B) ulcer healing areas in each group from D1 to D7, data are expressed as X±S, unit: mm 2 , compared with the RAU group, **p<0.01; (C) Body weight changes of mice in each group before membrane formation (0 day), 3, 5, and 7 days after membrane formation; (D) Control: normal control group; RAU: ulcer model group; EGCG-H: epigallocatechin gallate 150 mg / kg group; EGCG-L: epigallocatechin gallate 75 mg / kg group; Positive: vitamin positive control group.

[0013] Figure 2 : Histopathological examination of oral mucosa, (A) HE staining and Masson staining of oral ulcer tissues of RAU mice, a: NC (normal group); b: RAU-DAY1 (film formation 24H); c: RAU group; d: EGCG high-dose (EGCG-H) group; e: EGCG low-dose (EGCG-L) group; f: positive (V C +V B ) group (×100 / ×200 scale bar = 100 μm / 50 μm); (B) Masson staining of collagen fiber volume fraction in ulcer tissue compared with the normal group, * p<0.05; compared with RAU group, #p<0.05.

[0014] Figure 3 :Network pharmacology analysis screened the potential core targets of EGCG for RAU disease, (A) gene distribution volcano map; (B) clustered UMA map; (C) data set sample standardized box plot; (D) Venn diagram of EGCG and RAU disease intersection genes; (E) EGCG-RAU disease target protein PPI interaction network diagram; (F) EGCG-RAU disease common target intersection core network diagram.

[0015] Figure 4 :GO analysis and KEGG enrichment results, (A) GO functional enrichment analysis; (B) KEGG enrichment pathway network diagram; (C) TOLL-LIKE receptor signaling pathway pathway diagram.

[0016] Figure 5 :Effect of EGCG on the expression levels of TNF-ɑ and IL-6 in the ulcer sites of mice. (AB) ELISA was used to determine the expression levels of TNF-ɑ and IL-6 in the extracts of each group: X±S, unit: pg / ml; (C) Immunohistochemistry was used to detect the positive rates of TNF-ɑ and IL-6 in the ulcer sites of each group of mice. a: NC, b: RAU-DAY1, c: RAU, d: EGCG-H group, e: EGCG-L group, f: positive (V C +V B ) group (scale bar = 20 / 10 μm), compared with the NC group, ** p<0.01, **** p<0.0001; compared with RAU group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001; n=3.

[0017] Figure 6 :Effect of EGCG on TLR4 / MyD88 / NF-κB signaling pathway. (AB) Western blot was used to detect the expression levels of TLR4, MyD88, NF-κB p65, IKB-α and their phosphorylated proteins in mouse ulcer tissues; (C) RT-qPCR was used to detect the mRNA expression levels of TLR4, MyD88, NF-κB1, and NF-κB p65 genes in mouse ulcer tissues before and after modeling; (D) RT-qPCR was used to detect the effects of different drugs on the mRNA expression levels of TLR4, MyD88, NF-κB1, and NF-κB p65 genes in mouse ulcer tissues; compared with the NC group, * p<0.05, **p<0.01, *** p<0.001, **** p<0.0001; compared with RAU group, # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001; n=3.

[0018] Figure 7 :Verification of EGCG binding to core targets, (A) Epigallocatechin Gallate (EGCG) 2D structure image; (B) CCK-8 assay of the effect of drugs on cell activity; (C) Molecular docking model of EGCG with TLR4, NF-κB p65, and IKB; (D) CETSA experiment. DETAILED DESCRIPTION

[0019] The present invention discloses the use of epigallocatechin gallate in the preparation of a composition for treating oral ulcers. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the purpose. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The application of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0020] Example

[0021] 1 Materials and methods

[0022] 1.1 Materials

[0023] 1.1.1 Drugs and reagents

[0024] EGCG lozenge prescription: EGCG (purity 98%, Shanghai McLean Biochemical Technology Co., Ltd.) 15%, microcrystalline cellulose 15% (microcrystalline cellulose and EGCG account for about 30%), mannitol 34%, lactose 34%, sodium carboxymethyl starch 0.3%, magnesium stearate 0.2%, lemon flavor 0.1%, and an appropriate amount of 80% ethanol solution of povidone K30 (about 1%-2%).

[0025] Preparation method of EGCG lozenges: weigh microcrystalline cellulose, mannitol, lactose, and sodium carboxymethyl starch, pass them through a 120-mesh sieve respectively, and mix them evenly; dissolve povidone K30 in an appropriate amount of 80% ethanol as a binder, wet granulate, dry, add magnesium stearate, mix evenly, and press into tablets.

[0026] Other reagents: NaOH crystals (Biofroxx, Germany); isoflurane; sodium chloride injection; vitamin C tablets; vitamin B 2 slices; 4% paraformaldehyde (Wabcan Biotechnology Co., Ltd.); HE staining kit, Masson staining kit, Tween20, Trizol (Solarbio); TNF-α, IL-6 enzyme-linked immunosorbent assay kits (Wuhan Purity Biotechnology Co., Ltd.); chloroform (Tianjin Tianli Chemical Reagent Co., Ltd.); reverse transcription kit (Shanghai Yishen Biotechnology Co., Ltd.); fluorescence quantification kit, BCA protein quantification analysis kit (Nanjing Novogene Biotechnology Co., Ltd.); 5× protein loading buffer (Biosharp); RIPA lysis buffer (Biosharp Biotechnology Co., Ltd.); PMSF, protein marker, HRP-goat anti-rabbit secondary antibody, GAPDH antibody (Servicebio); TLR4 primary antibody, MYD88 primary antibody, NF-κBp65 primary antibody, p-NF-κBp65 primary antibody (Wuhan Sanying Biotechnology Co., Ltd.); IKB-α primary antibody, p-IKB-α primary antibody (Abmart Biotechnology Co., Ltd.).

[0027] 1.1.2 Main instruments and equipment

[0028] Fluorescence quantitative PCR instrument, electrophoresis instrument, vertical electrophoresis tank, protein transfer tank, microplate reader, gel imaging analyzer (BIO-RAD, USA); fully automatic microplate reader (Thermo, USA); inverted optical microscope (Olympus, Japan); high-speed low-temperature refrigerated centrifuge (Eppendorf, USA).

[0029] 1.2 Test methods

[0030] 1.2.1 Establishment of oral ulcer model

[0031] 50 SPF ICR mice, 6-8 weeks old, weighing about 20-30 g, half male and half female, were purchased from the Animal Center of Hubei Medical College, license number: SCXK (E) 2019-0008. Rearing conditions: room temperature 22-25 ° C, humidity 50%-60%, 12 h light and dark alternation, unlimited food and water. Animal experiments were carried out in accordance with the laws and institutional guidelines, and the experimental plan was approved by the ethics review number of Hubei Medical College (Fujian) No. 2024-Shi 082.

[0032] The mouse ulcer model was established by chemical burning method: After the mouse was anesthetized, a saturated NaOH crystal with a diameter of 2 mm was picked up with tweezers and burned for 5 seconds on the mucosa of the left lower lip near the corner of the mouth of the mouse. After burning, the burned area was rinsed with 0.9% sodium chloride injection to remove residual NaOH, and the immediate reaction was recorded. After anesthesia for 24 hours, the modeling was observed, and food and water were not allowed during the period. There were obvious traces of chemical burning in the treatment area, and the surface mucosa had red and white lesions with a diameter of 2 mm. The center of the wound was sunken and covered with a white pseudomembrane. Three mice were randomly selected before and after modeling and killed for histopathological examination.

[0033] 1.2.2 Grouping and Dosing

[0034] Ten rats were randomly selected as the normal control NC group, and the remaining 40 rats were modeled. After successful modeling (24h), they were randomly divided into four groups, each with 10 rats, divided into negative control (RAU) group, vitamin positive control (positive) group, 150mg / kg EGCG (EGCG-H) treatment group and 75mg / kg EGCG (EGCG-L) treatment group. The positive control group was given 0.01mL / g positive reagent by gavage every day, and the normal control group and negative control group were given 0.01mL / g distilled water (containing EGCG lozenge excipients) by gavage every day, once a day, for 7 consecutive days.

[0035] 1.2.3 Visually observe the healing of the ulcer surface and measure the healing area

[0036] The ulcer healing status was recorded by taking photos, and the average level of ulcer healing area in each group was calculated. The ulcer area was measured using ImageJ software in mm 2 , to determine the healing speed.

[0037] 1.2.4 Pharmacological network construction and analysis

[0038] The TCMSP platform was used to obtain the names of its target proteins, and the species "Homo sapiens" was selected in the Uniprot database to obtain the potential target genes of EGCG. In the GEO database of NCBI, "oral ulcer" was used as the keyword to search, and the datasets with species "Homo sapiens" and data type "DataSets" were screened out, and the datasets with large sample size and high data quality were selected for research. The intersection target genes of EGCG and RAU were obtained using the Venny2.1.0 platform (https: / / bioinfogp.cnb.csic.es / tools / venny / ). The human protein-protein interaction (PPI) network was established using the STRING database, and the species was set to "human". Cytoscape 3.9.1 was used for topological analysis and visualization. DAVID was used to perform GO and KEGG enrichment analysis on potential therapeutic targets, with a threshold of P<0.05. The microbial information platform integrates and displays metabolic pathways. Finally, AutoDock software was used to perform molecular docking of EGCG and core targets. The smaller the binding energy value, the stronger the binding ability. Targets with good binding activity were screened according to affinity and further visualized using PyMOL 2.4.0.

[0039] 1.2.5 Enzyme-linked immunosorbent assay

[0040] The levels of inflammatory factors in the oral ulcers of mice were detected by enzyme-linked immunosorbent assay. After 7 days of medication, the ulcer mucosal tissue was taken, weighed, and PBS (PH = 7.4) was added at 5 times the weight of the tissue. The specimen was homogenized thoroughly with a homogenizer. Centrifuged for about 20 minutes (3000 rpm). The supernatant was carefully collected and the levels of IL-6 and TNF-α were detected according to the instructions of the enzyme-linked immunosorbent assay kit.

[0041] 1.2.6 Pathological examination

[0042] After 7 days of administration, 3 mice were randomly selected from each group, anesthetized and killed by cervical dislocation, and the ulcer tissues were collected. The mice were fixed with 4% formaldehyde, dehydrated with ethanol, and transparentized with xylene, and then embedded in paraffin. The slices were cut into 5 μm slices, dewaxed, and stained with HE and Masson to observe the healing condition, and IL-6 and TNF-α were detected by immunohistochemistry. After staining, the slices were sealed, observed under a microscope, and photographed. The distribution of collagen fibers was counted by Image J. SP immunohistochemical staining showed brown nuclei as positive. Image J was used to count the number of positive cells / field of view, and 3 high-power fields of view were randomly selected from each group.

[0043] 1.2.7 Western blot detection

[0044] After tissue grinding, RIPA lysis buffer containing protein phosphatase inhibitors was added and lysed on ice for 20 minutes. Centrifuge at 12,000 rpm for 15 minutes at 4°C, take the supernatant and aliquot it, and quantify the protein using a BCA kit. 10% SDS-PAGE electrophoresis (concentration gel voltage 80V, separation gel voltage 120V), 350mA constant flow transfer for 40min, add fast blocking solution and block at room temperature for 15min, add appropriate amount of anti-TLR4 (1:2000), MyD88 (1:2000), NF-κBP65 (1:3000), p-NF-κBP65 (1:4000), IKB-α (1:1500), P-IKB-α (1:1500), GAPDH (1:3000) primary antibody dilution solution, shake gently at 4°C overnight, wash the membrane, incubate with secondary antibody at room temperature for 90min, wash the membrane with TBST, add ECL chemiluminescent solution for exposure and development.

[0045] 1.2.8 Real-time fluorescence quantitative PCR

[0046] Total RNA from ulcer tissue was extracted using Trizon reagent, and mRNA was extracted using RNA ultrapure extraction kit. After reverse transcription of mRNA into cDNA, gene expression was analyzed using fluorescent quantitative PCR. The program was set to pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 10 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds, for a total of 40 cycles. GAPDH was used as an internal reference, and 2- △△ The relative expression level was calculated by Ct method.

[0047] Table 1 Primer sequences

[0048]

[0049] 1.2.9 Cell proliferation assay

[0050] About 2000 oral squamous cell carcinoma (CAL27) cells were plated in a 96-well plate with 100 μL per well, and 100 μL PBS solution was added to the outer edge of the 96-well plate. After completion, the 96-well plate was placed in a cell culture incubator overnight. The next day, the cell growth was observed, and the culture medium containing different concentrations of EGCG (0, 0.2mM, 0.3mM, 0.6mM, 1.2mM) was replaced for 5 hours, and the CCK-8 method was used to evaluate the cell survival rate.

[0051] 1.2.10 Cellular thermal shift assay (CETSA) technique

[0052] CETSA validated the binding of EGCG to potential protein targets. CAL27 cells were treated with different concentrations of EGCG for 5 hours, and the pelleted cells were resuspended in PBS and distributed to 8 different PCR tubes, with 100 μL of cell suspension in each tube. The cells were then heated, and the cells were frozen and thawed twice using liquid nitrogen and a 25°C thermal cycle to lyse the cells and extract soluble proteins. Finally, Western blotting was performed using antibodies against the target protein to quantify the soluble proteins.

[0053] 1.2.11 Data Statistical Analysis

[0054] Statistical analysis was performed using Graphpad Prim 8, and data were expressed as mean ± standard deviation. Student's t-test was used for comparison between two groups, and ANOVA was used for comparison between multiple groups. P < 0.05 indicated a significant difference.

[0055] 2 Results

[0056] 2.1EGCG promotes RAU healing in ICR mice

[0057] On the first day after modeling, all mice developed RAU. Figure 1 As shown in A, the oral mucosa of mice in the normal control group was pink, intact, smooth, and without ulcers. However, in the other groups, obvious ulcers appeared on the mucosa of the left lower lip near the corner of the mouth 24 hours after NaOH cauterization, and there was no difference in the ulcer area between the groups (p>0.05). After 4 days of administration of low-dose and high-dose EGCG, the ulcer area of ​​mice decreased to varying degrees, the ulcer became shallower, and the congestion and edema decreased; after 7 days of treatment, the ulcers of mice in the RAU group were still visible, the congestion and edema of the mucosa around the ulcer decreased, and some pseudomembranes fell off the surface, but in the EGCG treatment group, the mucosa in the injured area was similar to the normal mucosa, without obvious congestion and edema; compared with the RAU group on D1-D7 days, the ulcer area of ​​the EGCG-H group, EGCG-L group and the positive control group was significantly reduced, and the wound healing rate was significantly improved (p>0.05). There was no significant difference in ulcer healing between the positive control group and the EGCG group, but the higher dose of EGCG showed a more favorable ulcer healing trend ( Figure 1 B); The weight of mice in each group was detected at the same time. The weight of the normal group was stable, and the average weight of mice in all modeling groups decreased. The weight of the RAU group decreased most significantly, and the weight of the EGCG-H group was slightly higher than that of the EGCG-L group ( Figure 1 C).

[0058] 2.2EGCG improves the pathological morphology of oral mucosa in mice

[0059] HE staining showed that EGCG and the positive control group had better ulcer healing effects. Figure 2As shown in A. The normal group mice had intact oral mucosal epithelial tissue and clear structure; 24 hours after cauterization in other groups, obvious circular ulcers appeared in the burned area; the mucosal epithelial layer and lamina propria in the ulcer area of ​​the model group were severely damaged and broken, the cells were arranged in disorder, the inflammatory cells in the submucosal layer were extensively infiltrated, and the red blood cell extravasation was obvious. The treatment group showed a relatively tight and orderly arrangement of the connective tissue of the lamina propria of the epithelial tissue, only the keratinized layer was slightly damaged, the rest of the epithelial layer had a clear structure, the red blood cell extravasation was significantly reduced, the amount of inflammatory cell infiltration was greatly reduced, and there was only a small gap between the submucosal layer and the lamina propria, indicating that EGCG effectively improved the mucosal barrier function.

[0060] like Figure 2 As shown in A, Masson staining of collagen fibers in the buccal mucosal ulcer tissue of mice showed that the collagen fibers in the mucosa of the model group were disorderly arranged and small and sparse, while the collagen fibers in the ulcer tissue of the treatment group were significantly more uniform and orderly than those in the model group, and the collagen fibers in the ulcer tissue of the EGCG-H group were significantly more than those in the EGCG-L and positive control groups, in a dose-dependent manner. Figure 2 B is the volume fraction of collagen fibers in ulcer tissue, compared with the normal group. * p<0.05; compared with RAU group, # p<0.05.

[0061] 2.3 Predicting targets and pathways using network pharmacology technology

[0062] First, after searching EGCG using the TCMSP database, the results showed that its oral availability was 55.09% (OB ≥ 30%) and its drug-like property was 0.77 (DL ≥ 0.18), which met the conventional requirements. At the same time, 124 drug targets corresponding to active compounds were integrated from TCMSP. Secondly, 8705 OU-related differential targets were collected from the GEO database ( Figure 3 AC). Obtain 71 intersection target genes of EGCG and RAU ( Figure 3 D). After the obtained intersection target genes were imported into the STRING database, the PPI protein selected 18 target genes that were not connected in the hidden network, and the PPI graph of EGCG against RAU disease and the genes that interacted with each other were obtained ( Figure 3 E). The 53 genes screened were then subjected to topological analysis and visualization analysis. The darker the color, the greater the correlation. IL-6, TLR4, IKB, etc. were found to be potential therapeutic targets ( Figure 3 F).

[0063] GO and KEGG analysis: GO reveals the core gene functions in three aspects. In the biological process (BP), the main genes are enriched in the negative regulation of apoptosis, the response of cells to mechanical stimulation, and I-κB kinase / NF-κB signal transduction; the cellular components (CC) mainly involve RNA polymerase II transcription factor complex, transcription factor complex, and interleukin-6 receptor complex; the molecular functions (MF) mainly include identical protein binding, protein serine / threonine / tyrosine kinase activity, enzyme binding, etc. The results of GO analysis are visualized as follows ( Figure 4 A). KEGG enrichment analysis showed that the pathways associated with the core target genes mainly involved multiple cancer-related pathways, tumor necrosis factor signaling pathway, Toll-like receptor signaling pathway, IL-17 signaling pathway, hepatitis B, etc. Bubble diagrams were drawn according to the degree of enrichment ( Figure 4 B). After integrating the physicochemical properties of drugs, pathophysiological factors of diseases, and network pharmacology visualization, comprehensive analysis revealed important pathways, among which the TLR4 / MyD88 / NF-κB signaling pathway mediated by inflammatory factors was most closely related to the inflammatory response of EGCG to RAU disease ( Figure 4 C).

[0064] 2.4EGCG reduces the expression levels of inflammatory factors TNF-ɑ and IL-6 in RAU mice

[0065] The expression levels of tumor necrosis factor ɑ (TNF-ɑ) and IL-6 in the extracts of each group were determined by ELISA. Compared with the NC group, the levels of TNF-ɑ and IL-6 in the first day of modeling (RAU DAY1) group and the model group (RAU) were significantly increased (p<0.0001); compared with the RAU group, the EGCG-H, EGCG-L and positive groups could significantly reduce the level of IL-6 (p<0.0001); EGCG-H could significantly reduce the level of TNF-ɑ (P<0.0001), and the EGCG-L and positive groups could significantly reduce the level of TNF-ɑ (p<0.01); there was no significant difference in the inflammatory factors in the ulcer site between the positive group and the EGCG group, but the inhibitory effect of the EGCG-H group on the inflammatory factors was more obvious. At the same time, the effect of EGCG on the inflammatory factors was dose-dependent ( Figure 5 AB).

[0066] Immunohistochemistry was used to detect the positive areas of TNF-ɑ and IL-6 in the ulcer sites of each group of mice, and the visual field ratio was calculated and analyzed ( Figure 5C). Compared with the NC group, the positive rates of TNF-ɑ and IL-6 in the RAU DAY1 group and the RAU group were significantly increased (p<0.0001); compared with the RAU group, the positive rates of TNF-ɑ in the EGCG-H, EGCG-L and positive groups were significantly decreased (p<0.01), and the positive rates of IL-6 were significantly decreased (p<0.05); the effect of EGCG on inflammatory factors was dose-dependent.

[0067] 2.5EGCG inhibits TLR4 / MyD88 / NF-κB pathway

[0068] Western blot experiments of mouse ulcer tissues showed that compared with the NC group, the expressions of TLR4, MyD88, P-NF-κBp65 / NF-κB p65 and P-IKB-α proteins in the RAU group were significantly increased (p<0.01), and the expression of IKB-α protein was significantly decreased (p<0.0001). Compared with the RAU group, the protein expressions of MyD88 and P-IKB-α in the positive group were decreased (p<0.05); the protein expressions of TLR4, MyD88, and P-IKB-α in the EGCG-H group were significantly decreased (p<0.01), the protein expressions of P-NF-κB p65 / NF-κB p65 were decreased (p<0.05), and the protein expression of IKB-α was significantly increased (p<0.01); the protein expressions of TLR4 and MyD88 in the EGCG-L group were significantly decreased (p<0.01), the protein expression of P-IKB-α was decreased (p<0.05), and the protein expression of IKB-α was increased (p<0.05) ( Figure 6 AB). The results showed that EGCG could inhibit the expression of TLR4, MyD88, and NF-κB p65 proteins, promote the expression of IKB-α protein, and participate in regulating the TLR4 / MyD88 / NF-κB signaling pathway.

[0069] RT-qPCR was used to verify the effect of EGCG on the expression levels of TLR4, MyD88, NF-κB1, and NF-κBp65mRNA in the ulcer area of ​​mice. The results showed that compared with the NC group, the expression levels of TLR4, MyD88, NF-κB1, and NF-κB p65mRNA in the RAU group were significantly increased (p<0.05) ( Figure 6 C). Compared with the RAU group, the mRNA expression levels of TLR4, MyD88, NF-κB1, and NF-κB p65 in the EGCG-H group were significantly decreased (p<0.001); the mRNA expression levels of TLR4, MyD88, and NF-κB p65 in the EGCG-L group were all decreased (p<0.05); the mRNA expression levels of MyD88, NF-κB1, and NF-κBp65 in the positive group were all decreased (p<0.05) ( Figure 6D). The results showed that EGCG could inhibit TLR4 / MyD88 / NF-κB pathway in a dose-dependent manner.

[0070] 2.6EGCG binds to core targets

[0071] To further clarify the binding ability of EGCG with core targets, AutoDock software was used to dock EGCG with core targets. The 2D structure of EGCG is shown in Figure 7 As shown in A, the smaller the affinity value, the stronger the binding ability. The binding energy of the compound to the core target protein is less than -5 kcal·mol -1 This shows that the two have good integration ability. Figure 7 As shown in Figure C, the binding energy of EGCG to TLR4 is -6.8941 kcal·mol-1. EGCG forms conventional hydrogen bonds with amino acid residues ASPC:99, THR A:232, SER A:207, ASNA:156 and ASPA:181; forms anionic bonds with ASPA:209; and forms π-alkyl with amino acid residue ARGC:106. The binding energy of EGCG to NF-κB p65 is -6.0153 kcal·mol -1 EGCG forms conventional hydrogen bonds with amino acid residues GLN A: 220, ARG A: 158, GLN A: 247 and LYS A: 218; forms carbon-hydrogen bonds with LYS A: 79 and GLN A: 29; and forms π-alkyl bonds with amino acid residues ALA A: 192, PHE A: 184 and PRO A: 182. The binding energy of EGCG and IKB is -7.1239 kcal·mol -1 EGCG forms conventional hydrogen bonds with amino acid residues ASN D: 145, ASN D: 182, ASPA: 294, GLUD: 153 and ASPA: 210, and forms ionic bonds with ARGD: 143 and ARG A: 253. That is, it is predicted that EGCG binds to the relevant core targets with good binding ability.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of epigallocatechin gallate in the preparation of preparations for treating oral ulcers.

2. The use according to claim 1, characterized in that: The oral ulcer is recurrent aphthous ulcer.

3. The use according to claim 2, characterized in that: The epigallocatechin gallate is used to inhibit TLR4, MyD88 and NF-κB signaling pathways to treat oral inflammation.

Images