A mellea extract for treating ulcerative colitis, and its preparation method and application

Abstract

The present invention provides a kind of radix schizonepetae extract for treating ulcerative colitis and its preparation method and application, and relates to the field of biomedicine technology. The preparation method of the radix schizonepetae extract of the present invention comprises: soaking radix schizonepetae in water, heating and boiling, condensing and refluxing, filtering, and collecting the filtrate, which is the radix schizonepetae extract. The radix schizonepetae extract of the present invention can reduce collagen deposition and intestinal epithelial cell apoptosis in the colon epithelium of ulcerative colitis, has the effect of downregulating the abnormally high expression of genes and proteins such as N-cadherin, IntegrinA1, Vinculin, TGF-β2, Srcin, etc. in the colon tissue of ulcerative colitis, reduces colon fibrosis and inflammatory damage, protects the barrier function of the colon epithelium, and has the effect of inhibiting the overexpression of Wnt 5A / B and β-Catenin proteins caused by DSS, and alleviates colon fibrosis, thereby achieving the effect of treating ulcerative colitis and alleviating colon fibrosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a mellea fructus extract for treating ulcerative colitis, and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by diarrhea, abdominal pain, and mucus, purulent stools, and its incidence is increasing worldwide. The etiology of ulcerative colitis is complex and includes genetic susceptibility, defects within the epithelial barrier, immune disorders, and environmental factors. The goal of UC treatment is primarily to achieve rapid symptom relief and healing of the mucosal barrier, while restoring the patient's overall quality of life. For mild to moderate disease, 5-aminosalicylic acid (5-ASA) drugs remain the mainstay of treatment. When the therapeutic effect of 5-ASA is poor, steroids and immunosuppressants should be added as the next step. However, 5-ASA has serious side effects, such as renal impairment, which has become an obstacle to long-term use. Inhibiting inflammation and promoting the healing of damaged mucosa remain a challenging issue in the treatment of ulcerative colitis.

[0003] Fibrosis is a pathophysiological mechanism of tissue repair after damage to the body, which leads to the deposition of excess collagen in the extracellular matrix (ECM). Fibrosis is a common complication of chronic inflammation and can occur in all organs and tissues. Intestinal fibrosis is a process driven by chronic intestinal inflammation, leading to an increase in myofibroblasts and collagen deposition. Collagen is one of the main extracellular matrices synthesized during fibrosis and can promote specific signaling pathways by binding to integrins and discoidin domain receptors (DDRs). The transmission of signals within the extracellular matrix is ​​mainly carried out by integrins, a group of cell surface receptors that anchor cells to the matrix and transmit mechanical and chemical signals to promote cell adhesion and migration.

[0004] Fibroblast migration is an important factor in fibrosis. One study showed that this migration is enhanced by TGF-β1, which regulates the migration, proliferation, and differentiation of fibroblasts. Integrin-mediated TGF-β1 activation plays an important role in the healing of the intestinal mucosa and the process of fibrosis. The calcium-dependent cell surface adhesion molecule N-cadherin is strongly induced by TGF-β1 and plays a key role in the interaction between fibroblasts and the ECM and is required for fibroblast migration. Severe intestinal fibrosis can lead to intestinal obstruction, which may require surgical intervention. Although fibrosis is more common in Crohn's disease, there is evidence that fibrosis also occurs in UC and may interfere with treatment. End-stage ulcerative colitis may lead to severe fibrosis, with the phenomenon of shortened, stiff, and contracted colon, known as "lead pipe" colon. Therefore, research on technologies and drugs to hinder intestinal fibrosis is crucial for the current treatment of UC.

[0005] Melastoma dodecandrum Lour., also known as ground taro, is a folk medicine widely distributed in southern China. It has the effects of clearing heat and detoxifying, promoting blood circulation and stopping bleeding. Its extract has anti-inflammatory, antioxidant, hemostatic, and hypoglycemic effects. However, research on the use of water extracts of Melastoma dodecandrum Lour. for the treatment of ulcerative colitis is limited. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a mellea extract for treating ulcerative colitis, and a preparation method and application thereof, wherein the mellea extract has the effects of resisting ulcerative colitis, reducing intestinal epithelial cell apoptosis and alleviating colon fibrosis.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention provides a preparation method of an ophiopogon japonicus extract, comprising: soaking the ophiopogon japonicus in water, heating and boiling, condensing and refluxing, filtering, and collecting a filtrate to obtain the ophiopogon japonicus extract; the condensing and refluxing time is 0.5 to 1.5 hours.

[0009] Preferably, the mass ratio of the ground radish to water is 1:9-11, and the soaking time is 25-35 minutes.

[0010] Preferably, after filtering, the method further comprises mixing the filtered medicinal residue with water, extracting at 100° C. for 40 to 50 minutes, filtering, and combining the filtrates; and the mass ratio of the medicinal residue to water is 1:5 to 9.

[0011] The present invention also provides the Melasma extract obtained by the above preparation method.

[0012] The present invention also provides the use of the above-mentioned Euphorbia pulex extract in preparing a medicine for treating ulcerative colitis.

[0013] The present invention also provides the use of the above-mentioned Euphorbia miliariae extract in preparing anti-intestinal fibrosis medicine.

[0014] Preferably, the extract of Euphorbia miltiorrhiza can reduce colon epithelial collagen deposition and intestinal epithelial cell apoptosis.

[0015] Preferably, the extract of Euphorbia miltiorrhiza has the effect of down-regulating the abnormally high expression of N-cadherin, IntegrinA1, Vinculin, TGF-β2, and Srcin genes and proteins in colon tissue.

[0016] Preferably, the extract of Euphorbia miltiorrhiza has the effects of regulating fibroblast migration, reducing colon fibrosis and inflammatory damage, and protecting goblet cells and colon epithelial barrier function.

[0017] Preferably, the medicine comprises the extract of Euphorbia cerevisiae and pharmaceutically acceptable excipients.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a method for preparing a kelp extract for treating ulcerative colitis, comprising: soaking kelp in water, heating and boiling, condensing and refluxing, filtering, and collecting the filtrate to obtain the kelp extract. The kelp extract can reduce collagen deposition and intestinal epithelial cell apoptosis in the colonic epithelium of patients with ulcerative colitis, downregulate the abnormally high expression of N-cadherin, Integrin A1, Vinculin, TGF-β2, and Srcin genes and proteins in colonic tissue of patients with ulcerative colitis, reduce colonic fibrosis and inflammatory damage, protect colonic epithelial barrier function, and inhibit DSS-induced overexpression of Wnt 5A / B and β-Catenin proteins, thereby alleviating colonic fibrosis, thereby achieving the effect of treating ulcerative colitis and alleviating colonic fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 3. Effect of Radix Dioscoreae on the body weight of mice induced by DSS. (A) Effect of Radix Dioscoreae on the body weight of mice. (B) DAI of different groups. (C) Colon images of different groups (n=3). (D) Colon length of different groups. (E) Representative images of colon tissues of different groups (magnification, 100×) and AB-PAS staining (magnification, 200×). Yellow arrows indicate mucosal damage. (F) Histological scores of colon tissues of different groups (n=6). # indicates the comparison between NC and MC, and * indicates the comparison between MC and SASP, DRH, and DRL. ## , ** p<0.01, ### , *** p<0.001,#### , **** p<0.0001;

[0021] Figure 2 Dioscorea chinensis reduces intestinal fibrosis, inflammatory expression, and cell apoptosis in UC mice; (A) Masson staining of colon tissues from different groups (magnification, 200×); (B) Sirius red staining of colon tissues from different groups (magnification, 200×); (C) Immunohistochemical staining of colonic IL-1β (magnification, 200×); (D) TUNEL fluorescence staining of colon tissues from different groups (magnification, 200×); (EH) Quantitative analysis of the positive expression of the above staining (n=3); # indicates the comparison between NC and MC, * indicates the comparison between MC and SASP, DRH and DRL; # , * p<0.05, ## , ** p<0.01, ### , *** p<0.001, #### , **** p<0.0001;

[0022] Figure 3 Transcriptome sequencing analysis of differentially expressed genes and enriched pathways in mice treated with Diqi (Schisorbus dasyphylla) to alleviate ulcerative colitis; (A) Pearson correlation coefficient; (B) principal component analysis; (C) volcano plot of differentially expressed genes; (D) heat map of differentially expressed genes; (E) bar plot of GO enrichment; (F) scatter plot of GO enrichment; (G) histogram of KEGG enrichment; (H) scatter plot of KEGG enrichment; (I) GSEA (gene set enrichment analysis) of the KEGG focal adhesion pathway; (J) gene expression levels (n=3); (K) qPCR quantification of target genes; # indicates comparison between NC and MC, * indicates comparison between MC and .SASP, DRH, and DRL; # , * p<0.05, ## , ** p<0.01, ### , *** p<0.001;

[0023] Figure 4Figure 3. Dioscorea chinensis inhibits intestinal fibrosis by reducing the expression of genes and proteins involved in focal adhesion and the Wnt / β-Catenin pathway. (AC) Colonic immunohistochemistry for N-cadherin, IntegrinA1, and Vinculin. (D) Fluorescence colocalization of N-cadherin and α-SMA in the colon. (E) Quantitative immunohistochemical analysis (n=3). (F) Fluorescence intensity of N-cadherin and PCCs colocalized between N-cadherin and fibroblasts (yellow arrows). (G) Colonic immunofluorescence staining for Wnt5A / B and β-Catenin. (H) Quantitative analysis of Wnt5A / B and β-Catenin fluorescence expression. # indicates the comparison between NC and MC, * indicates the comparison between MC and SASP, DRH and DRL; # , * p<0.05, ## , ** p<0.01, ### , *** p<0.001. DETAILED DESCRIPTION

[0024] The invention provides a preparation method of an ophiopogon japonicus extract, comprising: soaking the ophiopogon japonicus in water, heating and boiling, condensing and refluxing, filtering, and collecting a filtrate to obtain the ophiopogon japonicus extract; the condensing and refluxing time is 0.5 to 1.5 hours.

[0025] The mass ratio of the radish to water of the present invention is preferably 1:9-11, more preferably 1:10, and the soaking time is preferably 25-35 minutes, more preferably 30 minutes.

[0026] The filtration of the present invention further preferably includes mixing the filtered medicinal residue with water, extracting at 100° C. for 40 to 50 minutes, filtering, combining the filtrates, recovering under reduced pressure at 50 to 55° C. and -0.08 to -0.1 MPa, and concentrating to contain 1 g of the original drug per milliliter; the mass ratio of the medicinal residue to water is preferably 1:5 to 9, more preferably 1:8.

[0027] The present invention also provides the Melasma extract obtained by the above preparation method.

[0028] The present invention also provides the use of the above-mentioned Euphorbia pulex extract in preparing a medicine for treating ulcerative colitis.

[0029] The present invention also provides the use of the above-mentioned Euphorbia miliariae extract in preparing anti-intestinal fibrosis medicine.

[0030] The mellea fructus extract of the present invention can reduce colon epithelial collagen deposition and intestinal epithelial cell apoptosis.

[0031] The mellea fructus extract of the present invention has the effect of down-regulating the abnormally high expression of N-cadherin, IntegrinA1, Vinculin, TGF-β2 and Srcin genes and proteins in colon tissue.

[0032] The mellea fructus extract of the present invention has the functions of regulating fibroblast migration, reducing colon fibrosis and inflammatory damage, and protecting goblet cells and colon epithelial barrier function.

[0033] The medicament of the present invention comprises an extract of Euphorbia miliariae and pharmaceutically acceptable excipients. The active ingredient in the medicament of the present invention may be the Euphorbia miliariae extract as the sole active ingredient, or the Euphorbia miliariae extract may be used in combination with other active ingredients for treating ulcerative colitis. The medicament of the present invention includes, but is not limited to, an injectable formulation, an emulsion, an ointment, a granule, a powder, and an oral solution. The excipients contained in the medicament are not particularly limited in the present invention; commonly used excipients in pharmaceuticals of this field may be used.

[0034] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] In the following examples, unless otherwise specified, all methods are conventional.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] The experimental results data involved in the following examples are presented as mean ± standard deviation (SD). Data analysis was performed using GraphPad 8 software. Multiple group comparisons were performed using one-way analysis of variance (ANOVA), and comparisons between two groups were performed using t-test. When p < 0.05, the difference was considered statistically significant.

[0038] Example 1

[0039] Preparation of Melasma extract

[0040] 100 g of dried radix schizonepetae was soaked in 1 kg of pure water for 30 minutes, then heated to boiling, condensed and refluxed for 1 hour, filtered, and the filtrate was collected. 800 g of water was then added to the residue, and extraction was carried out at 100°C for 45 minutes. The two collected filtrates were combined, recovered under reduced pressure at 50-55°C and -0.08-0.1 MPa, and concentrated to a concentration of 1 g of the original drug per milliliter, thereby obtaining the radix schizonepetae extract.

[0041] Example 2

[0042] Animal experiments

[0043] 2.1 Experimental animals

[0044] Male C57BL / 6 mice (specific pathogen-free, SPF, weighing 20–22 g) were purchased from Shanghai Bikeway Biotechnology Co., Ltd. (license number SCXK(Shanghai)2018-0006). All mice were housed at the Experimental Animal Research Center of Zhejiang Chinese Medical University under a controlled temperature (23 ± 1°C) with a 12-h light-dark cycle. Experiments were conducted in accordance with ethical guidelines (ethics number IACUC 202309-18).

[0045] 2.2 Modeling and grouping

[0046] After one week of adaptive feeding, 30 mice were randomly divided into a normal control group (NC), a model control group (MC), a sulfasalazine group (SASP), a high-dose Radix Dioscoreae group (DRH), and a low-dose Radix Dioscoreae group (DRL). The NC group served as a normal control and was given distilled water for 8 days. The MC group served as a model control and was given a 2.5% dextran sulfate sodium (DSS, MP Biomedicals, UK) aqueous solution for 8 days. Except for the NC group, all other groups received the same 2.5% DSS as the MC group. The SASP group served as a positive control and was given a 0.5 g / kg sulfasalazine suspension (SASP, H31020557, Sinepharm, China) by gavage for 8 days. The DRH and DRL groups were each given 9.2 g and 2.3 g of the Radix Dioscoreae aqueous extract prepared in Example 1 by gavage for 8 days. The mice were observed daily for body weight, stool characteristics, and blood in stool. The disease activity index (DAI) was evaluated according to the recognized standards. Eight days after modeling and administration, the mice were euthanized and colon tissues were collected.

[0047] 2.3 Body weight, DAI, HE and AB-PAS staining

[0048] The weight of mice was observed and recorded daily and DAI scores were calculated. HE and AB-PAS staining were performed on the colon tissue of mice to observe the effect of Dioscorea oleracea on the colon tissue damage of UC mice induced by DSS. Figure 1 shown.

[0049] The AB-PAS staining method involves immersing a colon section (approximately 1 cm long) in 4% neutral formaldehyde and fixing it at room temperature for 48 hours. Four-μm thick paraffin sections were stained using a hematoxylin-eosin staining kit (HE, D006-1-4, Jiancheng, Nanjing, China) and an AB-PAS staining kit (D033-1-1, Jiancheng, Nanjing, China). Histopathological examination was performed according to established methods, and colonic inflammatory cell infiltration and crypt damage were observed under a light microscope. Crypt goblet cells appear blue in AB-PAS staining.

[0050] Figure 1 The results showed that the body weight of mice treated with DSS was significantly reduced, the DAI score was significantly increased, and the colon length was significantly reduced ( Figure 1 AD, P<0.0001). HE staining and AB-PAS staining showed that DSS-treated mice exhibited typical epithelial defects, inflammatory cell infiltration, crypt damage, and goblet cell depletion ( Figure 1 E, as indicated by yellow arrows). The histopathological score of the MC group increased ( Figure 1 F). After treatment with Dioscorea scoparia, mice experienced slower weight loss, decreased DAI scores (P<0.01), and colon length gradually approached that of the NC group, with significant improvement in pathological findings. Compared with mice treated with DSS, Dioscorea scoparia significantly reduced colon histopathological scores (P<0.0001), maintained intestinal epithelial and crypt integrity, and increased goblet cell numbers, maintaining mucosal integrity. Overall, these results suggest that Dioscorea scoparia can alleviate symptoms and accelerate mucosal healing in UC mice.

[0051] 2.4 Masson staining, Sirius red staining, IL-1β immunohistochemistry staining, and TUNEL fluorescence staining for apoptosis detection

[0052] Colon sections were stained with Masson staining and Sirius red staining to observe colonic collagen deposition and fibrosis. Collagen appears blue in Masson staining and red in Sirius red staining.

[0053] IL-1β immunohistochemical staining and TUNEL fluorescence staining were performed to detect cell apoptosis. The specific results are as follows Figure 2 shown.

[0054] Masson and Sirius red staining methods involve dewaxing and rehydrating colon paraffin sections. The distribution of collagen fibers is then observed using a Masson trichrome staining kit (G1340, Solarbio, China) and a Sirius red staining kit (G1472, Solarbio, China). Collagen fibers appear blue with Masson trichrome staining and red with Sirius red staining.

[0055] TUNEL fluorescence staining was performed as follows: colon sections were dewaxed and rehydrated, then fixed in a microwave oven and washed with PBS. Sections were then fluorescently stained according to the instructions of the TdT-mediated dUTP nick-end labeling (TUNEL) kit (MA0224, meilunbio, China) and mounted with an anti-fluorescence sealant (including DAPI) (MA0222, meilunbio, China) in the dark. Under a fluorescence microscope, red fluorescence was detected from the fragmented DNA of apoptotic cells.

[0056] IL-1β immunohistochemical staining was performed as follows: colon sections were dewaxed and rehydrated, then washed three times with PBS. Subsequently, sections were microwave-fixed with sodium citrate solution (pH 6.0, P0081, Beyotime, China). Endogenous peroxides were blocked with 3% hydrogen peroxide. Immunostaining blocking solution (P0260, Beyotime, China) was then applied and blocked for 15 minutes at room temperature. Antibody IL1-β (ET1701-39, Huabio, China; dilution 1:200) was added dropwise to the sections and incubated overnight at 4°C. The corresponding HRP secondary antibody (PV-6001, ZSbio, China) was incubated with the sections for 30 minutes at 37°C, followed by washing with PBS. The sections were developed with DAB (ZLI-9019, ZSbio, China), stained with hematoxylin, and rapidly dehydrated in anhydrous ethanol. Finally, the sections were mounted with neutral resin. Images were captured using a light microscope and analyzed using Image-Pro Plus 6.0 software.

[0057] Figure 2 The results showed that there was a lot of collagen deposition in the mucosal layer and submucosa of the MC group ( Figure 2 A, B and E, F). The expression of IL-1β in the model group was elevated, and the expression was significantly reduced after treatment with Melastoma serrata, which proved that Melastoma serrata has the effect of improving DSS-induced colon inflammation in mice ( Figure 2 C, G, p<0.0001). At the same time, TUNEL staining can make broken DNA emit red fluorescence, and the results showed that a large number of apoptotic cells were present in the intestinal epithelium and crypt base of the MC group ( Figure 2 D, H). Treatment with Melastoma rutaecarpa significantly reduced collagen deposition, intestinal inflammatory cytokine IL-1β expression, and cell apoptosis.

[0058] 2.5 Transcriptome sequencing analysis of differentially expressed genes and enriched pathways

[0059] The colon was subjected to RNA-seq transcriptome sequencing and RT-qPCR quantitative analysis of target genes. The specific results are as follows Figure 3 shown.

[0060] RNA-seq transcriptome sequencing was performed as follows: RNA from the entire sample was isolated and purified using TRIzol reagent (thermofisher, 15596018). The quantity and purity of RNA were measured using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and the integrity of RNA was assessed using a Bioanalyzer 2100 (Agilent, CA, USA). The concentration was >50 ng / μL, the RIN value was >7.0, and the total RNA was >1 μg. Illumina Novaseq was used by Lianchuan Biotechnology Co., Ltd. (Hangzhou, China). TM RNA libraries were prepared and sequenced using the 6000 platform. Data were then analyzed and visualized using the R language (https: / / www.r-project.org / ) and the OmicStudio platform from Lianchuan Bio (https: / / www.omicstudio.cn / tool). The high-quality sequencing data were then aligned to the reference genome of the project species, and related projects such as gene expression quantification, gene set enrichment analysis (GSEA), gene differential analysis, and enrichment analysis were performed.

[0061] The real-time fluorescence quantitative PCR (RT-qPCR) method was as follows: total RNA was extracted from the colon using SteadyPure Quick RNA Extraction Kit (AG21023, Accurate Biotechnology, Hunan, China) and stored at -80°C for subsequent reverse transcription. TM cDNA was synthesized by reverse transcription using R TIII super Mix with dsDNase reverse transcription kit (MR05201M, Mon ad, Suzhou, China). TM SYBR Gr een qPCR Mix (High ROX) (MQ10301S, Monad, Suzhou, China) in StepOne TM RT-qPCR amplification was performed in a RealTime PCR instrument. The target gene primer sequences are shown in Table 1. The program was pre-denaturation: 95℃ for 3 min; denaturation: 95℃ for 10 s; annealing extension: 60℃ for 30 s, 40 cycles. -△△CT The relative expression values ​​of gene mRNA were calculated by the method.

[0062] Table 1 Target gene primer sequences

[0063]

[0064]

[0065] Figure 3 The Pearson correlation coefficient graph showed that there was a good correlation between the samples of the Dioscorea treatment group and the DSS administration model group ( Figure 3 A). PCA (Principal Component Analysis) results showed that there were significant differences in the sample distribution between the radix schizonepetae group and the model group ( Figure 3 B). Figure 3 C and D show the overall distribution and clustering of differentially expressed genes. Compared with the model group, most genes in the radix scutellariae group were down-regulated. GO enrichment and KEGG enrichment ( Figure 3 EH) showed that compared with the model group, the gene changes in the DIREN group were mainly concentrated in "synapses", "cell connections", "neuroactive ligand receptor interactions" and "circadian rhythm entrainment". GSEA (Gene Set Enrichment Analysis) based on the KEGG database showed that the genes in the DIREN group were enriched in the focal adhesion pathway. Compared with the model group, the gene expression in the DIREN group was significantly decreased ( Figure 3 I, p.adjust=0.0039). There were significant differences in the expression of CDH2, ITGA1 and TGF-β2 genes between the two groups ( Figure 3 J). RT-qPCR quantitative analysis of target genes also confirmed the previous results ( Figure 3 Based on the above results, it can be concluded that the extract of the present invention may regulate cell migration through the focal adhesion pathway mediated by N-cadherin (encoded by the CDH2 gene), improve the intestinal barrier, and reduce fibrosis and UC damage.

[0066] 2.6 Immunohistochemistry and fluorescence staining

[0067] N-cadherin is an adherens junction that is important for cell attachment, migration, and the formation of fibrous connective tissue. To observe whether there is a correlation between N-cadherin and fibroblast migration in the colon, intestinal tissue sections were subjected to immunohistochemistry and fluorescence staining. Fibroblasts were labeled with α-SMA, a classic marker. PCC was used to analyze the colocalization of N-cadherin and intestinal fibroblasts in the intestinal mucosa to explain the correlation between their co-expression. Figure 4 shown.

[0068] Immunohistochemical staining was performed as follows: colon sections were dewaxed and rehydrated, then washed three times with PBS. Sections were then microwave-fixed with sodium citrate solution (pH 6.0, P0081, Beyotime, China). Endogenous peroxides were blocked with 3% hydrogen peroxide. Immunostaining blocking solution (P0260, Beyotime, China) was then applied and blocked for 15 minutes at room temperature. Antibodies against N-cadherin (ET1701-39, Huabio, China; dilution 1:200), IntegrinA1 (22146-1-AP, Proteintech, China; dilution 1:200), and Vinculin (ET1705-94, Huabio, China; dilution 1:200) were incubated with sections overnight at 4°C. Sections were then incubated with the corresponding HRP secondary antibody (PV-6001, ZSbio, China) for 30 minutes at 37°C and washed with PBS. DAB (ZLI-9019, ZSbio, China) was used for color development and hematoxylin staining followed by rapid dehydration in anhydrous ethanol. Finally, the slides were mounted with neutral resin. Images were captured using an optical microscope and analyzed using Image-ProPlus 6.0 software.

[0069] Immunofluorescence staining was performed as follows: colon sections were dewaxed and rehydrated, then fixed in a microwave oven with 200 ml of sodium citrate fixative. Endogenous peroxides were then blocked with 3% hydrogen peroxide. Sections were then washed three times with PBS for 5 min. Sections were then incubated with recombinant rabbit monoclonal antibodies against N-cadherin (SY02-46, Huabio, China) (1:200), α-SMA (ET1607-53, Huabio, China) (1:200), WNT5A / B polyclonal antibodies (55184-1-AP, Proteintech, China) (1:200), and β-catenin (M24002, Abmart, China) (1:200) at 4°C overnight. After each incubation, sections were stained according to the instructions of the TSA fluorescent double staining kit (RK05902, ABclonal, China). Finally, sections were sealed with an anti-fluorescence sealant (including DAPI). Application of TSA fluorescent dye and all subsequent procedures were performed in a light-protected environment. Images were acquired using a fluorescence microscope equipped with Zen software (AXIO SCOPE.A1, Carl Zeiss, Germany). Colocalization analysis of fluorescence images was performed using ImagePro Plus 6.0, and the results were expressed as Pearson's correlation coefficient (PCC).

[0070] Immunohistochemistry results showed that N-cadherin expression was significantly increased in the MC group ( Figure 4 A, E, p<0.001). The expressions of IntegrinA1 and Vinculin increased in the MC group, but decreased after treatment with Melastoma sibiricum ( Figure 4 B, C). The RT-PCR results above also showed that the expression of TGF-β2, integrin, CDH2 (N-cadherin), and Srcin1 genes increased in the model group, while their expression decreased after treatment with Melastoma serrata ( Figure 3 K).

[0071] Immunofluorescence results showed that the colocalization of the MC group was significantly lower than that of the NC group or other groups. At the same time, the fluorescence expression of N-cadherin in the MC group also increased ( Figure 4 D, F). This indicates that N-cadherin leads to fibroblast migration in the MC group. In contrast, Melastoma sibiricum improved the co-expression of both and decreased the expression of N-cadherin. These results suggest that Melastoma sibiricum reduces fibroblast migration by inhibiting the overexpression of N-cadherin and focal adhesion pathway proteins induced by DSS ( Figure 4 G, H). Activation of the Wnt / β-Catenin pathway is associated with fibrosis in multiple pathological organs. Based on the expression of Wnt 5A / B and β-Catenin proteins in the colon, it was found that Euphorbia miliariae could inhibit DSS-induced overexpression of Wnt 5A / B and β-Catenin proteins, thereby alleviating intestinal fibrosis. Therefore, the Euphorbia miliariae extract of the present invention can treat intestinal fibrosis caused by ulcerative colitis by inhibiting activation of the Wnt / β-Catenin pathway.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of an extract of Melasma scoparia in preparing a drug for treating ulcerative colitis, characterized in that: The preparation method of the radix schizonepetae extract comprises: soaking the radix schizonepetae in water, heating and boiling, condensing and refluxing, filtering, and collecting the filtrate to obtain the radix schizonepetae extract; the condensing and refluxing time is 0.5 to 1.5 hours; The mass ratio of the radish to water is 1:9-11, and the soaking time is 25-35 minutes; After the filtration, the method further comprises mixing the filtered medicinal residue with water, extracting at 100° C. for 40 to 50 minutes, filtering, and combining the filtrates; the mass ratio of the medicinal residue to water is 1:5 to 9.

2. Use of the Herba Lysimachiae extract according to claim 1 in the preparation of anti-intestinal fibrosis drugs.

3. The use according to claim 1 or 2, characterized in that The extract of Eucommia barbata can reduce colon epithelial collagen deposition and intestinal epithelial cell apoptosis.

4. The use according to claim 1 or 2, characterized in that The mellea fructus extract has the effect of down-regulating the abnormally high expression of N-cadherin, IntegrinA1, Vinculin, TGF-β2 and Srcin genes and proteins in colon tissue.

5. The use according to claim 1 or 2, characterized in that: The extract of Euphorbia miltiorrhiza has the functions of regulating fibroblast migration, reducing colon fibrosis and inflammatory damage, and protecting goblet cells and colon epithelial barrier function.

6. The use according to claim 1 or 2, characterized in that The medicine comprises an extract of Euphorbia miliariae and pharmaceutically acceptable excipients.

Images