Application of rosa rugosa in preparation of medicine for treating IBD (infectious bursal disease)

Abstract

The invention relates to application of rosa rugosa in preparation of a medicine for treating IBD (infectious bursal disease). According to the invention, firstly, it is found that the branchlet rose has a repairing effect on damage caused by IBD; researches find that the aqueous extract of rugosa branchlet can play a role by repairing intestinal barriers and inhibiting TLR4, TLR9 and proinflammatory cytokines, so that the aqueous extract of rugosa branchlet is beneficial to treatment of IBD. The invention provides an application prospect of the rosa rugosa in preparation of the medicine for treating IBD.

Description

Technical Field

[0001] The invention belongs to the technical field of application of Rosa serrata medicines, and particularly relates to application of Rosa serrata in preparing medicines for treating IBD. Background Art

[0002] Inflammatory bowel disease (IBD) is a complex, chronic inflammatory disease caused by a defective gastrointestinal immune system. It includes ulcerative colitis (UC) and Crohn's disease (CD), and is characterized by abdominal pain, diarrhea, and mucus-like blood. The pathogenesis of IBD remains unclear, and current treatments are prone to immune tolerance and other side effects, with no cure.

[0003] The safety of traditional Chinese medicine and its active ingredients demonstrates advantages in the treatment of IBD and has been recommended as a treatment for IBD due to its significant efficacy. Based on this, the present invention focuses on Miniature rose (MR), a Xinjiang specialty medicinal plant belonging to the Rosaceae family. Traditional Chinese medicine believes that MR has intestinal motility-promoting and anti-inflammatory effects. LC-TOF-MS / MS analysis of MR components yielded 34 compounds, including flavonoids such as luteolin, hyperoside, and quercetin dihydrate; phenolic acids such as gallic acid, ellagic acid, and flavonolgallic acid; organic acids such as citric acid and linoleic acid; and triterpenes such as hederagenin, chenodeoxycholic acid, and other polyphenols such as di-O-galloylhexahydroxydiphenoyl-glucoside. Studies have shown that luteolin exerts anti-inflammatory properties by inhibiting TLR2 and NF-κB signaling pathways, while gallic acid directly reduces the harmful metabolite ammonia by forming aminated metabolites, thereby improving intestinal dysbiosis and alleviating colitis. Therefore, the present invention proposes a new use of Rosa microcarpa, that is, the use of Rosa microcarpa in preparing a medicine for treating IBD. Summary of the Invention

[0004] The purpose of the present invention is to provide a new use of Rosa microcarpa. Its application in the preparation of drugs for treating IBD can promote barrier integrity and anti-inflammatory effects, and provide a reference for the in-depth development of Rosa microcarpa and the application of Rosa microcarpa as a functional ingredient.

[0005] In order to achieve the above objectives, the technical solutions adopted are:

[0006] Application of rose twigs in preparing medicines for treating IBD.

[0007] Furthermore, the rose twig is used in the preparation of medicines for repairing the intestinal barrier.

[0008] Furthermore, the rose twig is used in the preparation of drugs that inhibit TLR4 and TLR9 receptors.

[0009] Furthermore, the application of the Rosa microcarpa in the preparation of drugs for upregulating the expression of barrier proteins OCC and ZO-1.

[0010] Furthermore, the rose twig is used in the preparation of a medicine for alleviating the inflammatory response of IBD.

[0011] Furthermore, the rose of small branches is used in the preparation of drugs for inhibiting the expression of pro-inflammatory cytokines TNF-α, IL-6, CXCL-1 and MPO.

[0012] Furthermore, the water extract of the rose is used in the preparation of drugs for treating IBD.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] In order to clarify the potential mechanism of Rosa microcarpa in treating IBD, the present invention studies the correlation between intestinal changes, TLRs, tight junction proteins and other indicators of IBD mice after MR intervention, explores the occurrence and development of IBD and the mechanism of MR treatment, and provides data support for the clinical treatment of IBD and the determination of new therapeutic targets. The present invention establishes a DSS-induced IBD mouse model, administers different doses (25 mg / kg, 50 mg / kg, 100 mg / kg) of MR intervention, evaluates the effect of the water extract of Rosa microcarpa, a characteristic medicinal plant in Xinjiang, on intestinal health, and finds that MR can promote barrier integrity and anti-inflammatory effects. Therefore, the present invention believes that Rosa microcarpa may regulate the composition of intestinal microbiota and improve host metabolite disorders through the synergistic effect of multiple active ingredients, thereby exerting an anti-inflammatory effect. Thus, the application of Rosa microcarpa in the preparation of drugs for treating IBD is proposed, which provides a reference for the in-depth development of Rosa microcarpa and the application of Rosa microcarpa as a functional ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 MR alleviates the inflammatory response in IBD mice (n=10). Figures show (A) Body weight changes of mice, (B) Disease Activity Index (DAI) score, (C) Intestinal permeability test, colon weight changes, and colon length changes, (D) Hematoxylin-eosin staining of colon tissue (n=6), and (E) ELISA assays for TNF-α, IL-6, CXCL-1, and MPO concentrations; ***P<0.001, **P<0.01, *P<0.05. Data were expressed as mean±SEM.

[0016] Figure 2MR improves intestinal barrier function in IBD mice by regulating tight junction proteins (n=6). Figures show (A) TLR4 protein immunohistochemical staining results and quantitative analysis of TLR4 protein expression levels in colon tissue, (B) TLR9 protein immunohistochemical staining results and quantitative analysis of TLR9 protein expression levels in colon tissue, (C) OCC protein immunohistochemical staining results and quantitative analysis of OCC protein expression levels in colon tissue, (D) ZO-1 protein immunohistochemical staining results and quantitative analysis of ZO-1 protein expression levels in colon tissue; ***P<0.001, **P<0.01, *P<0.05. Data were expressed as mean±SEM. DETAILED DESCRIPTION

[0017] To further illustrate the use of Rosa rugosa in preparing a drug for treating IBD and achieve the intended purpose of the invention, the following describes, in conjunction with preferred embodiments, the use of Rosa rugosa in preparing a drug for treating IBD, including its specific implementation, structure, features, and efficacy. In the following description, different references to "one embodiment" or "embodiment" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics of one or more embodiments may be combined in any suitable manner.

[0018] The application of the present invention in preparing a drug for treating IBD will be further described in detail below with reference to specific examples:

[0019] The present invention uses a DSS-induced IBD mouse model to evaluate the effect of an aqueous extract of Miniaturerose, a Xinjiang specialty medicinal plant, on intestinal health, and systematically studies the mechanism of action of MR on IBD mice.

[0020] In the IBD model, different doses (25 mg / kg, 50 mg / kg, 100 mg / kg) of MR were administered, and pro-inflammatory factors were detected by HE staining, immunohistochemistry analysis, and ELISA.

[0021] The present invention found that the expression of tight junction proteins occludin (OCC) and ZO-1 was upregulated, while the expression of TLR4 and TLR9 was downregulated, indicating that MR has both barrier-promoting and anti-inflammatory effects. Therefore, the present invention proposes that MR is a beneficial drug for the treatment of IBD, exerting its effects by repairing the intestinal barrier and inhibiting TLR4 and TLR9 and pro-inflammatory cytokine pathways.

[0022] See the following examples for details:

[0023] Example 1.

[0024] A. Materials and Methods

[0025] 1. Experimental drug

[0026] Preparation of Rosa brachyota water extract: Take 10 g of Rosa brachyota powder, soak in 10 times water for 30 min, extract at 80℃ for 1 h, extract for 3 times, mix the 3 times extract, concentrate to 10 mL, prepare into dry extract containing 1 g / mL of crude drug mass concentration (i.e. 1.0 g of original medicinal material per milliliter of extract), take 0.2 mL of dry extract, add 100 mL of water, prepare into a concentration of 2 mg / mL. The main components of Rosa brachyota water extract are gallic acid and hyperoside.

[0027] Crude drug refers to the general term of natural, unprocessed or only simple processed plant, animal and mineral medicinal materials.

[0028] 2. Experimental animals

[0029] 60 male C57BL / 6J mice aged 6-8 weeks (20-24 g) were purchased from the Animal Experimental Center of Xinjiang Medical University and kept in a clean room at a temperature of 21±2℃, humidity of 40-45%, and a 12-hour light / dark cycle. After one week of adaptive feeding, the experiment was approved by the Ethics Committee of the Experimental Animal Center of Xinjiang Medical University, with permit number IACUC-20211118-02.

[0030] 3. Establishment of IBD mouse model and drug intervention

[0031] The mice were randomly divided into 6 groups, including: Control group, DSS group, DEX (0.4 mg / kg) group, MR-L (25 mg / kg) group, MR-M (50 mg / kg) group and MR-H (100 mg / kg) group. Except for the Control group, the rest of the mice were established as IBD models. The IBD mice were allowed to freely drink 3% DSS (dextran sulfate sodium) water for 10 consecutive days, and the body weight and fecal characteristics of the mice were observed and recorded daily to evaluate whether the model was established.

[0032] The Control group and DSS group were given oral gavage with equal volume of sterile water 0.2 ml daily, the DEX group was given intraperitoneal injection of dexamethasone injection 0.2 ml, and the MR-L, MR-M and MR-H groups were given gavage with MR dry extract solution 0.2 ml / 10 g of mouse body weight, respectively.

[0033] On the 11th day, the mice were anesthetized and sacrificed by intraperitoneal injection of pentobarbital sodium (100-150 mg / kg), and the length and weight of the colon tissue were recorded. The removed colon tissue was fixed in paraformaldehyde or stored at -80℃, and fresh feces were collected and stored at -80℃. The disease activity index (DAI) score was calculated based on the changes in body weight, fecal characteristics and bleeding to evaluate the severity of colitis in each group.

[0034] 4. Colon permeability detection

[0035] After the last administration, the mice were fasted for 4 h and then orally administered with fluorescein isothiocyanate-dextran (FITC-dextran, 1 mg / mL). After 3 h, the blood was collected from the orbital vein into a heparinized tube. The blood was centrifuged at 3500 rpm for 15 min, and 100 μL of the sample was taken and used to detect the FITC-D content in the plasma at 490 nm by spectrophotometry to calculate the intestinal mucosal permeability of the mice.

[0036] 5. Hematoxylin and eosin staining

[0037] The colon tissues fixed in a 4% formaldehyde solution were embedded in paraffin and prepared into sections (about 5 um thick), which were then stained with a hematoxylin-eosin solution, and the pathological morphological changes of the colon tissues in each group were observed and recorded under an optical microscope.

[0038] 6. ELISA analysis

[0039] The levels of TNF-a, IL-6, CXCL1, and MPO in the serum were detected using an ELISA kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.

[0040] 7. Immunohistochemistry

[0041] The colon tissue sections were sequentially deparaffinated, gradiently dehydrated with ethanol, antigen-repaired, and washed with phosphate buffer solution (PBS). The sections were blocked with 10% goat serum, sequentially incubated with a primary antibody (4°C, overnight) and a secondary antibody (37°C, 30 min), and then developed with DAB, restained with hematoxylin, and mounted with neutral resin. The expression of TLR4, TLR9, OCC, and ZO-1 proteins in the colon tissues was observed and recorded under a microscope, and the positive rate of the images was analyzed using ImagePro Plus 6.0 software.

[0042] 8. Statistical analysis

[0043] Statistical analysis was performed using GraphPad Prism version 9. Continuous variables were expressed as mean ± standard deviation, while categorical variables were expressed as percentages. The results of two groups of experiments were analyzed using an unpaired t test, and the results of more than two groups of experiments were analyzed using one-way analysis of variance (ANOVA), with a test standard of a = 0.05.

[0044] B. Results

[0045] 1. Anti-inflammatory effect of MR

[0046] Compared with the control group, mice in the DSS group showed typical IBD pathological features: body weight decreased significantly (P<0.01), disease activity index (DAI), FITC-DEX level, colon weight increased significantly (P<0.001), and colon length decreased significantly (P<0.01). After treatment with different doses of MR, the MR-H group significantly reversed the above pathological indicators (P<0.01), and the therapeutic effect was comparable to that of the DEX group ( Figure 1 AC).

[0047] HE staining results showed that compared with the Control group, the DSS group showed crypt structure destruction, mucosal loss and a large number of inflammatory cells infiltration. After MR treatment, the crypt structure was restored, the mucosa was neatly arranged and the inflammatory cell infiltration was reduced ( Figure 1 D).

[0048] Proinflammatory cytokine detection showed that the levels of proinflammatory cytokines TNF-α, IL-6, MPO, and CXCL-1 in the DSS group were significantly increased compared with the control group (P<0.001), and the levels of proinflammatory cytokines were significantly decreased after MR treatment (P<0.05, Figure 1 E). The results showed that MR could effectively alleviate the inflammatory response and improve the pathological manifestations of IBD in IBD mice.

[0049] 2. MR inhibits the expression of proinflammatory pathway proteins in IBD mice and protects the intestinal mucosal barrier

[0050] TLR4 and TLR9 are mainly located in the cell membrane and cytoplasm of the colon tissue mucosa, and their expression levels reflect the inflammatory state of the colon tissue. Occludin (OCC) and ZO-1 are mainly located on the cell membrane of the colon tissue. They are key structural proteins that maintain intercellular barrier function and cell polarity, and appear as diffuse yellow granules. Protein quantitative analysis showed that the expression of TLR4 and TLR9 in the DSS group was significantly higher than that in the Control group (P<0.01), and the expression of OCC and ZO-1 was significantly lower than that in the Control group (P<0.001). After MR treatment, the expression of TLR4 and TLR9 was significantly decreased (P<0.05), and the expression of OCC and ZO-1 was significantly increased (P<0.01). The results showed that MR can effectively inhibit the expression of proinflammatory pathway proteins TLR4 and TLR9 in IBD mice, increase the expression levels of intestinal barrier proteins OCC and ZO-1, and has the effect of maintaining the barrier function of intestinal mucosa ( Figure 2 AD).

[0051] These experiments demonstrate that MR alleviates IBD progression by upregulating the expression of barrier proteins OCC and ZO-1, enhancing mucosal integrity, inhibiting TLR4 and TLR9 receptors, and reducing the expression of proinflammatory cytokines. Compared to single-target biological inhibitors, MR offers promising comprehensive intervention potential and shares the low toxicity of traditional Chinese medicine, offering a new avenue for the safe, long-term treatment of IBD.

[0052] The above is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the embodiments of the present invention. Any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the embodiments of the present invention are still within the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Rosa microcarpa in the preparation of drugs for the treatment of IBD.

2. The use according to claim 1, characterized in that The application of the small twig rose in preparing medicine for repairing intestinal barrier.

3. The use according to claim 1, characterized in that The application of the small-branched rose in the preparation of drugs for inhibiting TLR4 and TLR9 receptors.

4. The use according to claim 2, characterized in that The application of the Rosa microcarpa in preparing a medicine for upregulating the expression of barrier proteins OCC and ZO-1.

5. The use according to claim 1, characterized in that The application of the small-twig rose in preparing a medicine for alleviating IBD inflammatory response.

6. The use according to claim 5, characterized in that The application of the small-branched rose in the preparation of a medicine for inhibiting the expression of pro-inflammatory cytokines TNF-α, IL-6, CXCL-1 and MPO.

7. The use according to claim 1, characterized in that The application of the water extract of Rosea dasyphylla in preparing medicine for treating IBD.

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