Application of fucoxanthin in preparation of medicine for preventing and treating stomatitis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of fucoxanthin in preparation of a medicine for preventing and treating chemotherapy-induced stomatitis. Experiments show that the fucoxanthin has a protective effect on chemotherapeutic drugs and LPS-induced stomatitis, and specifically, the fucoxanthin has a protective effect on the stomatitis induced by the chemotherapeutic drugs and the LPS-induced stomatitis, and the expression quantity of barrier proteins Occludin and ZO-1 is increased; and the content of epithelial mesenchymal transition (EMT) related protein is reduced. The fucoxanthin is high in safety, wide in source, low in cost and very suitable for being applied to prevention and treatment of stomatitis. The invention not only provides a new drug source for the treatment of stomatitis, but also opens up a new field for the application of the fucoxanthin.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of fucoidan in the preparation of drugs for the prevention and treatment of chemotherapy-induced stomatitis. Background Art

[0002] Stomatitis is a common inflammatory disease, mainly characterized by erythema, ulceration, swelling, pain, and the formation of white pseudomembranes on the oral mucosa. In severe cases, it can affect basic functions such as eating, swallowing, and speaking. Clinically, commonly used chemotherapy drugs (such as the anticancer drug 5-fluorouracil [5-FU], widely used in the treatment of breast cancer, colon cancer, and skin cancer) can induce stomatitis by inhibiting thymidylate synthase, interfering with the integration of nucleic acids into RNA / DNA, and promoting cell death. Studies have shown that genetic factors and nutritional deficiencies may also contribute to stomatitis. As a common human disease, its pathogenesis is not yet fully understood. Summary of the Invention

[0003] Fucoxanthin (FX) is a marine carotenoid widely found in brown algae (accounting for more than 10% of global carotenoid production). Its unique molecular structure includes a propylene bond, a conjugated carbonyl group, a 5,6-monocyclic epoxide ring, and an acetyl group. This substance has attracted considerable attention due to its multifunctional bioactivities, including antioxidant, anticancer, and anti-inflammatory properties, and has become a potential candidate drug for treating oral diseases. In a lipopolysaccharide (LPS)-induced rat inflammation model, FX significantly reduced the expression levels of pro-inflammatory mediators (TNF-α, IL-1β, IL-6, iNOS, and COX-2). Furthermore, FX can alleviate LPS-induced inflammation in RAW 264.7 cells and reduce oxidative stress by scavenging singlet oxygen, reactive oxygen species (ROS), and free radicals. However, the protective effect of FX against stomatitis and its potential mechanisms have not been reported. Therefore, this invention proposes the application of fucoxanthin in the preparation of drugs for the prevention and treatment of stomatitis, aiming to provide a novel prevention and treatment strategy to improve the therapeutic effect of stomatitis.

[0004] The above-mentioned objective of this invention is achieved through the following technical solution: In the embodiments of this application, fucoidan can effectively improve stomatitis in rats.

[0005] Furthermore, the stomatitis is chemotherapy-induced stomatitis or lipopolysaccharide (LPS)-induced stomatitis.

[0006] Furthermore, the chemotherapy-induced stomatitis is induced by chemotherapy drugs.

[0007] Furthermore, the chemotherapy drug is one or more of 5-fluorouracil, methotrexate, cyclophosphamide, and irinotecan.

[0008] Specifically, this invention demonstrates through experiments that fucoidan can significantly improve oral mucosal whitening and swelling in rat stomatitis induced by the chemotherapy drug 5-fluorouracil.

[0009] Meanwhile, the present invention demonstrates through experiments that the fucoidan can increase the expression levels of Occludin and ZO-1 proteins in normal human oral epithelial keratinocytes after LPS-induced treatment.

[0010] Furthermore, the fucoidan can reduce the content of EMT-related proteins in normal human oral epithelial keratinocytes after LPS-induced treatment.

[0011] Furthermore, the fucoidan can reduce LPS-induced mitochondrial dysfunction in normal human oral epithelial keratinocytes.

[0012] Furthermore, the fucoidan inhibits the cGAS-STING inflammatory pathway, reduces the level of oral inflammatory factors, improves oral inflammatory response, and thus reduces the occurrence of stomatitis.

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

[0014] Furthermore, the dosage form of the drug is an oral patch, oral lozenge, mouthwash, spray, ointment, oral preparation, or injection.

[0015] Additionally, the present invention protects the use of a pharmaceutical composition in the preparation of a drug for the prevention and treatment of stomatitis, said pharmaceutical composition comprising fucoidan.

[0016] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0017] The present invention has the following beneficial effects: This invention provides the application of fucoidin in the preparation of drugs for the prevention and treatment of chemotherapy-induced and LPS-induced stomatitis. Animal and cell experiments demonstrate that fucoidin has a protective effect against chemotherapy-induced and LPS-induced stomatitis, specifically by increasing the expression levels of barrier proteins Occludin and ZO-1 and reducing the content of EMT-related proteins. Fucoidin is highly safe, widely available, and inexpensive, making it very suitable for the prevention and treatment of stomatitis. Attached Figure Description

[0018] Figure 1 This is a pathological image of the rat oral mucosa in Example 1 of the present invention; wherein, Figure 1 (a) is a diagram of the oral mucosa of a rat (red circles indicate whitish parts of the mucosa). Figure 1 Image (b) shows a micrograph of sections stained with HE and SR. Figure 1(c) in the figure is a schematic diagram of Masson stained sections and a statistical graph of quantitative analysis data on collagen fiber accumulation.

[0019] Figure 2 This is a graph showing the expression levels of barrier proteins Occludin and ZO-1 in normal oral keratinocytes (HOKs); among them, Figure 2 In the figure, (a) represents the IC50 of phycocyanin against normal human oral epithelial keratinocytes obtained from the CCK-8 assay. Figure 2 Image (b) shows a representative fluorescence imaging micrograph and its statistical data of Occludin protein expression and distribution determined by immunofluorescence assay. Figure 2 (c) shows a representative fluorescence imaging micrograph and its statistical data of the expression and distribution of ZO-1 protein determined by immunofluorescence.

[0020] Figure 3 This image shows the expression levels of EMT protein in normal human oral epithelial keratinocytes; among them... Figure 3 (a) in the figure represents the expression levels of E-cadherin and N-cadherin proteins obtained from Western blotting experiments. Figure 3 (b) in the figure is a statistical graph of E-cadherin and N-cadherin protein level data obtained from Western blotting experiments. Figure 3 (c) A representative fluorescence imaging micrograph of N-cadherin protein expression and distribution determined by immunofluorescence assay. Figure 3 (d) A representative fluorescence imaging micrograph of E-cadherin protein expression and distribution determined by immunofluorescence assay. Figure 3 (e) A representative fluorescence imaging micrograph of the expression and distribution of Vimentin protein determined by immunofluorescence assay. Figure 3 (f) Statistical graph showing the expression and distribution of N-cadherin and Vimentin proteins determined by immunofluorescence assay.

[0021] Figure 4 A diagram illustrating how fucoidan improves lipopolysaccharide-induced mitochondrial functional impairment in human oral epithelial keratinocytes; among which, Figure 4 (a) in the figure is a representative graph of mitochondrial reactive oxygen species (mtSOX) levels and its statistical data. Figure 4 (b) in the figure is a representative diagram of mitochondrial membrane potential (MMP) levels. Figure 4 (c) in the figure represents the level of mitochondrial permeability transition pore (MPTP).

[0022] Figure 5 This image shows the expression of proteins related to the cGAS-STING signaling pathway in human oral epithelial keratinocytes; among them... Figure 5(a) shows a representative graph of the expression levels of STING and cGAS proteins obtained from Western blotting experiments, along with their statistical data. Figure 5 (b) in the figure shows a representative graph of TBK1 protein expression levels obtained from Western blotting experiments and its statistical data. Figure 5 Image (c) shows a representative fluorescence imaging micrograph of cGAS protein expression and distribution determined by immunofluorescence. Figure 5 Image (d) in the image is a representative fluorescence imaging micrograph of STING protein expression and distribution determined by immunofluorescence assay. Figure 5 Image (e) shows a representative fluorescence imaging micrograph of TBK1 protein expression and distribution determined by immunofluorescence assay. Figure 5 (f) in the figure is a statistical graph showing the expression and distribution of cGAS and TBK1 proteins as determined by immunofluorescence.

[0023] Note: Figures 1-5 Compared with the Control group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the 5-FU group or LPS group, #P < 0.05, ##P < 0.01, ###P < 0.001. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0025] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0026] Example 1: Fucoidin can improve stomatitis and pathological conditions in rats. Experimental materials: SD rats (Speford (Beijing) Biotechnology Co., Ltd.; Animal Certificate No.: NO.110324231106182758). Fucoxanthin (Chengdu Purifa Technology Development Co., Ltd.); 5-Fluorouracil (5-FU, Shanghai Aladdin Biochemical Technology Co., Ltd.; CAS: 51-21-8); Triamcinolone Acetone (TA, TargetMol China).

[0027] Experimental Methods: Twenty-six SPF-grade male SD rats weighing 200±20 g were housed in a standard SPF environment and fed with standard diets, with free access to water. After one week of routine feeding, they were randomly divided into a normal control group (n=6), a 5-fluorouracil group (n=6), a fucoidan group (n=7), and a triamcinolone group (n=7). The rat model was induced by intraperitoneal injection of 5-FU (150 mg / kg), and FX (200 mg / kg) was administered by gavage. For 14 days prior to modeling, the 5-FU+FX group was administered fucoidan (200 mg / kg) by gavage, while the Control, 5-FU, and 5-FU+TA groups were administered distilled water by gavage, once daily. After the last gavage, the rats were fasted for 24 hours. After fasting, the 5-FU group, 5-FU+FX group, and 5-FU+TA group were intraperitoneally injected with 5-fluorouracil to establish the model, at a dose of 150 mg / kg. The day of injection of 5-fluorouracil was designated as day 1. After model establishment, each group was fed as follows: Control group (normal group, or Con group): Distilled water was administered by gavage from day 4 to day 6, and normal water and feed were provided daily. 5-FU group (stomatitis model group): On the second day, the buccal mucosa was superficially scratched with the tip of an 18-gauge needle. From the fourth to the sixth day, distilled water was administered by gavage. Normal water and feed were provided daily. 5-FU+FX group (fucoidin group): On the second day, the cheek pouch mucosa was superficially scratched with the tip of an 18-gauge needle. From the fourth to the sixth day, fucoidin was administered by gavage at a dose of 200 mg / kg. At the same time, normal water and feed were provided daily. 5-FU+TA group (triamcinolone acetonide group): On the second day, the cheek pouch mucosa was superficially scratched with the tip of an 18-gauge needle. From the fourth to the sixth day, triamcinolone acetonide was administered by gavage at a dose of 1 mg / kg. At the same time, normal water and feed were provided daily.

[0028] During modeling and the above treatments, the mice were weighed. On day 10 of the treatment, all rats were anesthetized and euthanized, and oral mucosal tissue and rat blood were collected. The collected oral mucosa was fixed in 4% paraformaldehyde for dehydration, embedding, and sectioning to obtain paraffin sections. The sections were stained with hematoxylin-eosin (HE), Masson's stain, and Sirius Red (SR), respectively. In Masson-stained sections, blue represents collagen fibers, and the deposition of collagen fibers in the submucosal matrix was quantitatively analyzed using ImageJ software. Fibrosis percentage = collagen fiber area of ​​Masson-stained section ÷ total mucosal tissue area × 100%. On the last day of the experiment, the oral mucosa of the rats was photographed for observation.

[0029] Experimental results: See Figure 1 (a) shows that no ulcers appeared in the oral mucosa of rats in the Control group (normal group), while the oral mucosa of rats in the 5-FU group (stomatitis model group) showed obvious whitening and swelling after induction with the chemotherapy drug 5-FU; the whitening and swelling of the oral mucosa in the 5-FU+FX group was significantly reduced after FX treatment. The whitening and swelling symptoms were milder in the 5-FU+TA group compared with the model group.

[0030] HE staining results showed that the oral mucosa of rats with 5-FU-induced oral mucositis exhibited characteristics of thinning epithelium, shortening of reticular processes, and thickening of the lamina propria. Figure 1 b). Masson staining and SR staining results showed increased collagen deposition in the lamina propria of the oral mucosa in the model group, indicating collagen metabolism disorder and successful model establishment. Compared with the 5-FU model group, the FX-treated group showed reduced thinning of the oral mucosal epithelium, shortening of the reticular processes, and decreased collagen deposition. Figure 1 (bc). In conclusion, FX can effectively improve 5-FU-induced oral mucositis in rats.

[0031] Figure 1 The right side of (c) in the figure is the content of collagen fibers in the mucosal tissue calculated based on Masson staining. It can be seen from the figure that the collagen fiber content in the 5-FU group is significantly increased compared with the Control group, while gavage with fucoidan can effectively reduce its collagen fiber content.

[0032] Example 2: Fucoidin improves LPS-induced HOK cell barrier dysfunction. Experimental materials: normal human oral epithelial keratinocytes (HOKs) (Shanghai Meiwan Biotechnology Co., Ltd.); oral keratinocyte culture medium (Shanghai Meiwan Biotechnology Co., Ltd.); Cell counting kit-8 (CCK-8) (Shanghai Beyotime Biotechnology Co., Ltd.).

[0033] Experimental methods: CCK-8 cell viability assay: The viability of GMCs was assessed using the Cell Counting Kit-8 (CCK-8) in 96-well plates at a cell concentration of 5 × 10⁶ cells / well. 4Cells were incubated at 37°C in 5% CO2 medium for 24 hours, strictly following established operating procedures. Different concentration groups were set up according to drug concentration gradients, with three replicate wells (including a blank control) for each experimental group. After incubation for 24 hours, cells were treated with different concentrations of FX (0, 1, 5, 10, 20, 40, 80 μM) for 24 and 48 hours, respectively. Using the CCK-8 assay, 10 μL of CCK-8 solution was added to each well, and incubation continued for 1 hour at 37°C in 5% CO2. Finally, absorbance was measured at 450 nm using a microplate reader.

[0034] Normal human oral epithelial keratinocytes (HOKs) were cultured in a specialized medium (37°C, 5% CO2), with the medium changed every two days. After confluent growth, the cells were passaged. After incubation for 24 hours, the confluent cells were divided into four groups: normal group (Con), model group (LPS), drug-treated group (LPS+FX), and positive control group (LPS+TA). The final concentration of LPS in the medium was 5 μg / mL, and the final concentration of FX was 10 μM. Following treatment, subsequent experiments were performed 24 hours later.

[0035] After the above treatment is completed, the cell culture supernatant and cells are collected for further experiments.

[0036] Cellular immunofluorescence assay: HOKs cells were prepared at approximately 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well on glass coverslips of six-well plates and then treated with the drug for 24 hours. After appropriate stimulation, cells were washed three times with PBS buffer, fixed for 15 minutes with PBS solution containing 4% paraformaldehyde, and permeabilized for 15 minutes with 0.5% Triton X-100 (dissolved in PBS). Cells were blocked with 10% normal goat serum for 30 minutes and then incubated overnight with primary antibody in 10% normal goat serum at 4°C. The next day, cells were washed three times with PBS, incubated for 1 hour in a solution containing fluorescently labeled secondary antibody in the dark, then stained with DAPI for 15 minutes to label the nuclei, washed again with PBS, mounted, and finally observed under a laser confocal microscope.

[0037] Experimental results: First, we used the CCK-8 assay to detect FX-induced HOK cell viability, calculated the 50% inhibitory concentration (IC50), and observed cell morphology. The results showed that FX inhibited HOK cell viability in a time- and dose-dependent manner. Figure 2 a) and most cells detached and died 48 hours after FX treatment.

[0038] The oral epithelial barrier is the first line of defense for oral tissues. Occludin protein and ZO-1, as markers of tight junctions in various epithelial tissues, play important roles in maintaining oral homeostasis. Figure 3 As shown in Figure a, immunofluorescence (IF) results showed that Occludin protein in the normal group was localized on the cell membrane; however, after LPS treatment, it could not aggregate normally on the cell membrane, while FX treatment could restore the localization of Occludin protein on the cell membrane. Figure 2 b). The fluorescence intensity of ZO-1 decreased significantly after LPS treatment; after FX treatment, the peripheral membrane protein ZO-1 was distributed in a honeycomb pattern on the cell membrane. Figure 2 c). In summary, FX effectively alleviates LPS-induced HOK cell barrier function damage by restoring the expression and localization of Occludin protein and ZO-1.

[0039] Example 3: Fucoidin can improve LPS-induced EMT in HOKs Experimental materials: Cells collected in Example 2.

[0040] Experimental methods: Proteins were extracted from the cells collected in Example 3. The protein expression levels of cGAS, STING and TBK1 in normal human oral epithelial keratinocytes were detected by Western blotting (WB) and immunofluorescence (IF). The detection methods are routine procedures in the field and will not be described in detail here.

[0041] Experimental results: Studies have reported that excessive reactive oxygen species (ROS) can induce epithelial-mesenchymal transition (EMT), leading to disruption of cell-cell contact, impaired polarity of tight junction-related proteins, and ultimately, the collapse of tight junction structures. Based on this, we investigated whether FX regulates lipopolysaccharide (LPS)-induced EMT in HOKs. We analyzed the expression level and localization of the epithelial marker E-cadherin and the expression changes of mesenchymal proteins N-cadherin and Vimentin using Western blotting and immunofluorescence techniques. The results showed that LPS significantly upregulated N-cadherin expression, while FX treatment significantly attenuated this effect. Figure 3 ab, ef). Furthermore, FX treatment restored the correct localization of E-cadherin protein in LPS-induced HOKs cells and reduced Vimentin protein expression (ab, ef). Figure 3These data collectively indicate that LPS stimulation leads to EMT in HOKs, and FX treatment can effectively reverse the LPS-induced EMT process. Specifically, compared with the LPS group, the FX-treated group showed increased E-cadherin protein expression and decreased N-cadherin and Vimentin protein expression in HOKs cells, suggesting that FX may be a potential therapeutic agent for LPS-induced EMT.

[0042] Example 4: Fucoidin improves LPS-induced mitochondrial dysfunction in HOK cells Experimental materials: Cells collected in Example 2.

[0043] Experimental methods: Proteins were extracted from the cells collected in Example 3 and detected using a kit for detecting mitochondrial superoxide dismutase, mitochondrial membrane potential and mitochondrial permeability transition pore (Shanghai Beyotime Biotechnology Co., Ltd.). The detection methods are standard procedures in the field and will not be described in detail here.

[0044] Experimental results: Mitochondria play a crucial role in cellular energy metabolism. Reactive oxygen species (ROS) are primarily produced by mitochondria; excessive ROS disrupt redox balance, leading to cellular damage. Oxidative stress and mitochondrial dysfunction are major pathogenic factors in stomatitis. Therefore, regulating mitochondrial function may offer new insights for treating oral mucositis. Existing research indicates that FX, as a potential antioxidant with multiple pharmacological effects, can enhance antioxidant enzyme activity, promote mitochondrial biosynthesis, reduce superoxide production, and alleviate oxidative stress. Figure 4 It can be seen that under LPS stimulation, the production of reactive oxygen species (mtROS) in HOK mitochondria increased, while the mitochondrial membrane potential (MMP) decreased significantly. Compared with the model group, the FX intervention group effectively protected mitochondrial structure from damage, cleared excess mtROS, and maintained the balance of mitochondrial MMP levels. Figure 4 ab). This indicates that FX can alleviate LPS-induced increased reactive oxygen species (ROS) production and protect HOKs from oxidative damage. Furthermore, the mitochondrial permeability transition pore (MPTP), a protein complex located between the inner and outer mitochondrial membranes, forms a non-specific channel connecting the inner and outer membranes under stress conditions such as high calcium concentrations. LPS stimulation induces oxidative stress, leading to persistent MPTP opening, which in turn causes increased intracellular Ca2+ concentration, increased ROS, and insufficient membrane potential. The FX intervention group alleviated MPTP opening and maintained mitochondrial respiratory function (…). Figure 4 c). These results indicate that FX improves mitochondrial dysfunction by reducing LPS-stimulated reactive oxygen species generation and MPTP opening, thus protecting against LPS-induced mitochondrial damage.

[0045] Example 5: Fucoidin regulates the mitochondrial inflammatory pathway cGAS-STING Experimental materials: Cells collected in Example 2.

[0046] Experimental methods: Proteins were extracted from the cells collected in Example 3, and the protein expression of cGAS, STING and TBK1 in normal human oral epithelial keratinocytes was detected by Western blotting (WB) and immunofluorescence.

[0047] Experimental results: Related studies have reported that LPS induces mitochondrial dysfunction in the mouse brain and activates the cGAS-STING signaling pathway. Therefore, investigating whether FX affects the activation of the cGAS-STING signaling pathway is of great significance. In the LPS-induced HOKs model, this study has important value for the treatment of stomatitis. This invention uses Western blotting and immunofluorescence techniques to detect changes in the cGAS-STING signaling pathway in HOKs stimulated by LPS. The results showed that LPS upregulated the expression of cGAS and STING proteins and increased the protein level of TBK1, a downstream target protein of STING. After FX administration, the expression levels of cGAS, STING, and TBK1 proteins were all downregulated (…). Figure 5 ab). Immunofluorescence results showed that, compared with the LPS group, FX treatment reduced the fluorescence intensity of cGAS, STING, and TBK1 proteins in HOKs (ab). Figure 5 (cf). The fluorescence intensity of the cGAS-STING-TBK1 signaling axis decreased significantly. These results suggest that the mechanism by which FX improves LPS-induced stomatitis may be related to the regulation of the cGAS-STING signaling pathway, and FX may exert its therapeutic effect by acting on the cGAS-STING-TBK1 axis. Therefore, the cGAS-STING signaling pathway holds promise as a potential target for the treatment of stomatitis.

[0048] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of fucoidan in the preparation of drugs for the prevention and treatment of stomatitis.

2. The application according to claim 1, characterized in that, The stomatitis mentioned is either chemotherapy-induced stomatitis or lipopolysaccharide (LPS)-induced stomatitis.

3. The application according to claim 1, characterized in that, The chemotherapy-induced stomatitis was induced by chemotherapy drugs.

4. The application according to claim 3, characterized in that, The chemotherapy drug is one or more of 5-fluorouracil, methotrexate, cyclophosphamide, and irinotecan.

5. The application according to claim 1, characterized in that, The treatment for stomatitis involves increasing the expression levels of Occludin and ZO-1 proteins.

6. The application according to claim 1, characterized in that, The prevention and treatment of stomatitis involves reducing the content of EMT-related proteins in the epithelial-mesenchymal transition.

7. The application according to any one of claims 1 to 6, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. The application according to any one of claims 1 to 6, characterized in that, The dosage form of the drug is an oral patch, oral lozenge, mouthwash, spray, ointment, oral preparation, or injection.

9. The use of a pharmaceutical composition in the preparation of a drug for the prevention and treatment of stomatitis, characterized in that, The pharmaceutical composition includes fucoidan.

10. The application according to claim 9, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

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