Use of butyric acid in the preparation of a medicament for treating oral ulcers

Abstract

The application discloses application of butyric acid in preparation of a medicine for treating oral ulcer, and belongs to the field of biological medicine. The butyric acid plays a therapeutic effect by increasing expression of tight junction proteins in oral mucosa tissue, reducing inflammatory reaction and promoting healing of oral ulcer. The application uses butyric acid to treat oral ulcer, evaluates the curative effect of butyric acid and explores the corresponding internal mechanism. The results show that the butyric acid has obvious effect in treating oral ulcer caused by chemotherapy, and provide a scientific basis for clinical application of butyric acid.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of butyric acid in the preparation of drugs for treating oral ulcers. Background Technology

[0002] Chemotherapy is currently one of the most effective treatments for cancer in clinical practice. Chemotherapy drugs circulate throughout the body's organs and tissues via the bloodstream, killing tumor cells and improving the cure rate of malignant tumors. However, they also affect normally proliferating cells, such as the epithelial cells of the oral cavity and gastrointestinal mucosa. Therefore, patients often experience adverse reactions such as nausea, vomiting, diarrhea, and ulcers. Among these, oral ulcers are one of the most common side effects of chemotherapy for malignant tumors. Due to high-dose or combination therapy regimens, many patients experience a decline in immunity, leading to damage to the protective layer of the oral mucosa, which in turn causes inflammation and ulcers. Oral ulcers affect patients' eating, causing pain and difficulty swallowing, frequent infections, reduced quality of life, and impact on treatment regimens. Over the past decade, researchers have been studying the molecular basis of this damage to develop new strategies for controlling drug toxicity. According to the European Society for Medical Oncology (ESMO) guidelines, current methods for preventing and treating oral ulcers after chemotherapy include basic oral care, ampicillin mouthwash, recombinant human keratinocyte growth factor-1, cryotherapy, and laser therapy, but the treatment effects remain unsatisfactory.

[0003] Butyric acid (BFA) is a short-chain fatty acid produced by gut microbiota metabolism and is also a major energy source for colonic epithelial cells. Studies have shown that BFA exerts anti-inflammatory, anti-tumor, and intestinal mucosal protective effects through multiple mechanisms. The role of BFA in the human gastrointestinal tract has been a focus of research, and its positive effect on protecting mucosal epithelial cells is widely recognized. Researchers have used butyrate pretreatment to protect the gastric mucosa from ethanol-induced damage by enhancing mucosal defense and antioxidant and anti-inflammatory activities. Recent studies have found that BFA increases the production of mucin in colonic crypts and the proportion of goblet cells that secrete mucin, promoting the repair of ulcerative colitis; BFA also improves intestinal inflammatory responses and intestinal epithelial barrier dysfunction in mice by activating GPR109A and inhibiting the AKT and NF-κB p65 signaling pathways. Therefore, BFA has a positive effect on the protection of gastrointestinal epithelial cells. However, whether BFA has a similar protective effect on the oral mucosa has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of butyric acid in the preparation of drugs for treating oral ulcers, in order to solve the problems existing in the prior art. The present invention has found that butyric acid can slow down the weight loss of a mouse model of chemotherapy-induced ulcers, increase the expression of tight junction proteins in the oral mucosa of mice, reduce the inflammatory response, and promote the healing of oral ulcers. It has a significant therapeutic effect on chemotherapy-induced oral ulcers, providing a scientific basis for the clinical application of butyric acid.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the use of butyric acid in the preparation of drugs for treating oral ulcers.

[0007] Furthermore, the butyric acid exerts its therapeutic effect by increasing the expression of tight junction proteins in oral mucosal tissue, reducing inflammatory response, and promoting the healing of oral ulcers.

[0008] Furthermore, the tight junction proteins include ocludin, claudin-5, and claudin-18.

[0009] Furthermore, the butyric acid reduces the inflammatory response by decreasing the content of the inflammatory factor TNF-α in oral mucosal tissue.

[0010] Furthermore, the oral ulcers include traumatic ulcers caused by chemotherapy drugs.

[0011] The present invention also provides a treatment for oral ulcers, comprising an effective dose of butyric acid.

[0012] Furthermore, the butyric acid is prepared by mixing it with pharmaceutically acceptable excipients in the form of sodium butyrate to obtain the therapeutic drug.

[0013] Furthermore, the effective dose of butyric acid is 0.1-0.5 g / mL.

[0014] The present invention discloses the following technical effects:

[0015] This invention utilizes the classic chemotherapy drug 5-fluorouracil (5-FU) to treat mice, constructs a chemotherapy ulcer model through local mucosal erosion with acetic acid, and treats the ulcers with butyric acid. The efficacy of butyric acid and the underlying mechanisms are evaluated. Experimental results show that butyric acid can slow down the weight loss in the chemotherapy ulcer mouse model, increase the expression of tight junction proteins in the oral mucosa, reduce inflammation, and promote the healing of oral ulcers. It has a significant therapeutic effect on chemotherapy-induced oral ulcers, providing a scientific basis for the clinical application of butyric acid. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Flowchart for 5-FU-induced oral ulceration in mice;

[0018] Figure 2 The effects of butyric acid and 5-FU on mouse body weight and survival rate; A: Flowchart of 5-FU-induced oral ulcer mouse model and butyric acid intervention time; B: Survival rate analysis of mice under different doses of 5-FU intervention; C: Effects of butyric acid and 5-FU on mouse body weight; D: Body weight analysis of mice under different doses of 5-FU intervention;

[0019] Figure 3 The effects of butyric acid on the healing of oral ulcers and the results of HE staining;

[0020] Figure 4 The expression levels of tight junction proteins and inflammatory factors in mice were detected by qRT-PCR, namely Claudin-1, Claudin-5, ZO-1, Occludin, Claudin-18, TNF-α, and IL-6, in that order. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1

[0027] 1. Materials and Methods

[0028] 1.1 Laboratory animals and reagents

[0029] Fifty male ICR mice, weighing approximately 25-30g, were provided by Zhejiang Muke Biotechnology Co., Ltd. The ICR mice were placed in a temperature-controlled room (22±1℃) with a 12-hour light / dark cycle, and were fed standard pelleted feed and water at will.

[0030] Acetic acid (Shanghai Qiangshun Chemical Reagent Co., Ltd.); Chloral hydrate; Sodium butyrate 5-FU 1.2 Establishment of a 5-FU-induced oral ulcer model in mice: Fifty ICR mice were randomly divided into three groups: treatment group (n=20), ulcer group (n=20), and normal group (n=10), and housed separately. For the first 5, 3, and 1 days (-5, -3, -1 days) of model establishment, each mouse in the treatment and ulcer groups was intraperitoneally injected with a 4 mg / mL 5-FU solution prepared with physiological saline, at a dose of 40 mg / kg. Subsequently, on day 0, under 2% chloral hydrate anesthesia, 15 μL of 20% acetic acid was injected into the left cheek of the mouse using a 31-G needle to induce mucosal ulceration. The normal group received no treatment. 1.3 Macroscopic observation: On the third day after model establishment, ulcers were basically formed in the mice. The treatment group was treated daily with 0.1 g / mL sodium butyrate solution applied to the ulcers. Changes in the ulcers, inflammation, congestion, and healing were observed at fixed intervals over the following days, and the healing time was recorded by photographing. 1.4 Histopathological analysis: On days 8 and 12 of oral ulcer formation, four ICR mice were randomly selected from each group. After anesthesia with 2% chloral hydrate, the ulcer tissue was excised, fixed with 4% paraformaldehyde for 24 h, dehydrated with ethanol gradient, embedded in paraffin, and serially sectioned (4 μm) for HE staining. The inflammatory cell infiltration and changes in oral epithelium at the ulcer site were observed under an optical microscope. 1.5 Real-time quantitative PCR: On day 12 of oral ulcer formation, 6 ICR mice were used in each group. The excised oral ulcer tissue was placed in a 1.5 ml enzyme-free centrifuge tube, 1 ml of Trizol was added, and the tissue was thoroughly ground. The mixture was allowed to stand at room temperature for 5 min, then 200 μl of chloroform was added. After mixing, the mixture was centrifuged at 12000 x g at 4℃ for 15 min in a pre-cooled centrifuge. An appropriate amount of the upper aqueous phase (RNA) was aspirated and transferred to a new 1.5 ml enzyme-free centrifuge tube. An equal volume of isopropanol was added, and the mixture was allowed to stand at room temperature for 10 min. The mixture was then centrifuged at 12000 × g at 4℃ for 10 min. The supernatant was discarded, and RNA precipitate was visible at the bottom. 1 ml of 75% ethanol prepared with DEPC water was added to dissolve the RNA precipitate. The supernatant was removed by centrifugation, and the RNA precipitate was allowed to air dry at room temperature. RNA-free water was added to dissolve the RNA, and the concentration was determined. Reverse transcription was then performed. Real-time quantitative PCR amplification was performed using the SYRB Green dye method on a high-throughput quantitative PCR instrument (QuantStudio 6) at 95℃ for 5 min, 95℃ for 10 s, 60℃ for 30 s, and during the melting curve phase. GAPDH was used as an internal control, and 2... -ΔΔCt The mRNA expression levels of Claudin-1, Claudin-5, ZO-1, Occludin, Claudin-18, TNF-α, and IL-6 were analyzed using a method.

[0031] 2 Results

[0032] 2.15-FU-induced oral ulceration in mice

[0033] Fifty male ICR mice were randomly divided into three groups: a treatment group (n=20), an ulcer group (n=20), and a normal group (n=10), and housed separately. For the first 5, 3, and 1 days (-5, -3, -1 days) of modeling, each mouse in the treatment and ulcer groups was intraperitoneally injected with a 4 mg / ml 5-FU solution prepared with physiological saline, at a dose of 40 mg / kg. Subsequently, on day 0, under 2% chloral hydrate anesthesia, 15 μl of 20% acetic acid was injected into the left cheek of the mouse using a 31-G needle to induce mucosal ulceration. The normal group received no treatment. By the third day after modeling, ulcers had largely formed in the mice. The treatment group received daily application of 0.1 g / ml sodium butyrate solution to the ulcers. Changes in the ulcers, inflammation, congestion, and healing were observed at fixed intervals over the following days, and the healing time was recorded by photographing. Figure 1 The flowchart shows the process of 5-FU inducing oral ulcers in mice.

[0034] 2.2 Effects of different doses of 5-FU on the survival rate and body weight of mice

[0035] Figure 2 Figure A shows the 5-FU-induced oral ulcer mouse model and the timeline of butyric acid intervention. To determine the appropriate 5-FU dosage, this invention, based on relevant literature, first selected three dose gradients (40, 60, and 80 mg / kg) of 5-FU for intraperitoneal injection to conduct mouse survival experiments and monitor daily body weight. On day 2 of administration, the survival rate of mice in all three groups was 100%. On day 5 of administration, significant differences in survival rates were observed among the three groups: 100% in the 40 mg / kg group, 40% in the 60 mg / kg group, and 0% in the 80 mg / kg group. Figure 2 (B). After administration, the body weight of mice in all three groups decreased. Compared with the 40 mg / kg group, the body weight of mice in the 60 mg / kg and 80 mg / kg groups was significantly reduced. Figure 2 (D) and there was no plateau phase. Because this experiment required ensuring the survival rate of the ulcer group, 40 mg / kg was chosen as the modeling dose. On day 2 of modeling, compared with the normal group, the body weight of mice in the ulcer group and the treatment group was significantly lower; from day 6 to 12 of modeling, compared with the ulcer group, the body weight of the treatment group was significantly higher (D). Figure 2 (C) This indicates that acetic acid treatment caused pain and discomfort in mice, leading to reduced food intake and weight loss, while administration of sodium butyrate significantly slowed down the weight loss. The weight of mice in the ulcer group showed an inflection point on day 3 of modeling and continued to increase, approaching the weight of mice in the treatment group on day 12, indirectly demonstrating the self-healing nature of oral ulcers.

[0036] 2.3 Butyric acid promotes the healing of oral ulcers in mice

[0037] Oral ulceration was observed in mice of each group on days 8 and 12 after successful modeling. Results are as follows: Figure 3The results showed that the oral mucosa of normal mice was smooth, without edema or congestion. On day 8 of modeling, a thick, light yellow pseudomembrane formed at the modeling site in the oral mucosa of mice in the ulcer group, with a congested red halo around it. In the treatment group, the oral mucosa at the modeling site was edematous and congested, with epithelial damage. On day 12 of modeling, the yellowish-white pseudomembrane sloughed off in the oral mucosa of mice in the ulcer group, and the red halo disappeared. The swelling of the oral mucosa in the treatment group had basically disappeared. The oral ulcers in the treatment group healed faster than those in the ulcer group. Butyric acid had a certain promoting effect on the healing of oral ulcers, and this also verified that oral ulcers have a tendency to heal spontaneously. HE results showed that the oral mucosal epithelium of normal mice was continuous and intact, without pathological manifestations such as vascular congestion and inflammatory cell infiltration. On day 8 of modeling, the integrity of the oral mucosal epithelium, lamina propria, and muscularis mucosae in the ulcer group mice was disrupted, with irregular cell arrangement, inflammatory cell infiltration, and erythrocyte extravasation. On day 12, the degree of damage to the oral mucosa in the ulcer group mice was slightly reduced, and a repair response began to appear, but inflammatory cell infiltration and erythrocyte extravasation were still present. On day 8 of modeling, the oral mucosal epithelium of the treatment group mice remained intact, with more regular cell arrangement, and significantly less inflammatory cell infiltration and erythrocyte extravasation than the ulcer group at the same time point. On day 12, the oral mucosal epithelial tissue structure of the treatment group mice was nearly intact. The results indicate that butyric acid accelerates the healing of oral ulcers and has a certain promoting effect on the healing of oral ulcers.

[0038] 2.4 Butyric acid inhibits inflammation in oral ulcer tissue in mice and promotes the expression of tight junction proteins.

[0039] On day 12 of modeling, oral tissues from mice in each group were collected for qRT-PCR detection. The results showed that ( Figure 4 Compared with the ulcer group, the treatment group showed varying degrees of increased expression of tight junction proteins Claudin-1, Claudin-5, ZO-1, Occludin, and Claudin-18 in the oral mucosa of mice. Compared with the normal group, the ulcer group showed decreased Claudin-1 and Claudin-5, and more pronounced surface damage in the ulcer tissue. Ulcer formation may promote the production of more tight junction proteins in oral tissues for self-repair; compared with the normal group, ZO-1, Occludin, and Claudin-18 were slightly upregulated in the ulcer group. Further detection of inflammatory factors revealed an increase in TNF-α in the oral mucosa of mice in the ulcer group compared to the normal group, while a significant decrease was observed in the treatment group. There was no statistically significant difference in IL-6 levels. These results indicate that butyric acid treatment increased the expression of tight junction proteins in the oral mucosa of mice, alleviated inflammation, and promoted the healing of oral ulcers.

[0040] 3 Results Analysis

[0041] Establishing animal models of oral ulcers is an important method for studying oral mucositis following chemotherapy in humans. The animal model established using chemotherapy in this invention closely resembles clinical manifestations and is representative. The chemical corrosion method increases the probability of oral mucositis formation in animals; the method is simple, easy to perform, and the results are controllable. Therefore, based on relevant literature, a concentration of 8 mg / ml 5-FU solution was used, with a dosage of 40 mg / kg per mouse, administered via intraperitoneal injection every other day. Excessively high concentrations of 5-FU can cause mouse death. Preliminary experiments and other studies have shown that a dosage of 40 mg / kg 5-FU is appropriate, ensuring the efficacy of the chemotherapy drug without inducing a high mortality rate. After three consecutive injections, the mice were anesthetized the following day by intraperitoneal injection of 2% chloral hydrate at 20 ml / kg, and 15 μl of 20% acetic acid solution was injected into the oral mucosa of the left cheek using a 31-G needle. The induction of oral ulcers by a single chemotherapy drug has a long cycle and low specificity; therefore, it is necessary to use an appropriate dose of acetic acid to damage the oral mucosa. Finally, the combined use of 5-FU and acetic acid successfully established an oral mucositis model.

[0042] Chemotherapy-induced oral mucosal damage is generally caused by direct mucosal damage from antitumor drugs, with an average occurrence period of 5-14 days, leading to apoptosis of oral mucosal epithelial cells. Previously, ampicillin mouthwash, tin-based powder, and boric acid powder were commonly used clinically to prevent and treat chemotherapy-related oral mucositis, but the efficacy was unsatisfactory. Butyric acid, a short-chain fatty acid, is a major nutrient for human intestinal epithelial cells. Over 95% of butyric acid in the human body is produced and absorbed in the colon. A certain level of butyric acid can stabilize colonic cells, thereby preventing or inhibiting carcinogenesis, regulating intestinal flora imbalance, and treating irritable bowel syndrome, antibiotic-associated enteritis, acute and chronic diarrhea, and other diseases. Currently, butyric acid has been shown to have multiple effects, including anti-inflammatory, antitumor, and intestinal mucosal protection, but its extraintestinal effects are still unknown.

[0043] Butyric acid (BEA) is a metabolic byproduct of the oral microecology. While oral bacteria produce BEA in the periodontal pockets, current research has found that BEA produced in the oral cavity plays a crucial role in the development of periodontitis. BEA can induce gingival fibroblast death, inhibit connective tissue synthesis, and accelerate the progression of periodontitis. Furthermore, in vitro studies have shown that BEA can disrupt the epithelial barrier by triggering apoptosis and downregulating the expression of intercellular junction proteins in human gingival epithelial cells (HGECs). These results suggest that BEA appears to be a disruptive factor in the oral cavity. Currently, there are no reported studies on whether BEA has a beneficial effect on oral mucosal inflammation.

[0044] This invention utilizes a constructed oral mucositis model for research. On day 3 of modeling, mice with the most severe ulcers and lowest body weight were treated with 0.1 g / ml sodium butyrate solution, applied to the ulcer sites at a fixed time each day. Oral ulcer tissue was then extracted from both the treatment and ulcer groups on day 12 of modeling, and relevant indicators were measured. Gross analysis and HE staining results showed that the oral ulcers in the treatment group recovered significantly better than those in the ulcer group. Therefore, it can be preliminarily determined that 0.1 g / ml sodium butyrate solution accelerates the recovery of oral ulcers in mice and has a positive effect on healing. Furthermore, the protective effect of butyrate was investigated at the cytokine level. Butyrate reduced the relative expression of TNF-α in the oral ulcer tissue of the treatment group mice. Simultaneously, the content of related tight junction proteins in the oral ulcer tissue was measured, revealing that butyrate treatment increased the expression of tight junction proteins in the oral mucosa, reduced inflammation, and promoted the healing of oral ulcers.

[0045] This invention has demonstrated through experiments that butyric acid significantly improves oral ulcers caused by chemotherapy drugs in mice. Further analysis of conventional indicators further proves the therapeutic effect of butyric acid, revealing that butyric acid can be used as an effective treatment for oral ulcers, especially traumatic oral ulcers caused by chemotherapy drugs.

[0046] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of butyric acid as the sole active ingredient in the preparation of drugs for treating oral ulcers, characterized in that, The oral ulcer is a traumatic ulcer caused by chemotherapy drugs; the concentration of butyric acid is 0.1-0.5 g / mL.

2. The application according to claim 1, characterized in that, Butyric acid exerts its therapeutic effect by increasing the expression of tight junction proteins in oral mucosal tissue, reducing inflammatory response, and promoting the healing of oral ulcers.

3. The application according to claim 2, characterized in that, The tight junction proteins include closure protein, tight protein-5, and tight protein-18.

4. The application according to claim 2, characterized in that, Butyric acid reduces inflammatory responses by decreasing the content of the inflammatory factor TNF-α in oral mucosal tissue.

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