Plant lactobacillus GXFF202402 and application thereof in preparation of product for preventing and / or treating haemorrhoids

Abstract

The invention provides a plant lactobacillus GXFF202402 strain and application of the plant lactobacillus GXFF202402 strain in preparation of a product for preventing and / or treating haemorrhoids, and belongs to the technical field of microorganisms. The preservation number of the plant lactobacillus GXFF202402 provided by the invention is CGMCC NO.32257, and the plant lactobacillus GXFF202402 is preserved in China General Microbiological Culture Collection Center on October 18, 2024, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The plant lactobacillus GXFF202402 provided by the invention relieves haemorrhoids by reducing inflammation, recovering tissue integrity, regulating expression of inflammatory cytokines and the like, and can be used as a potential therapeutic agent for treating haemorrhoids. In addition, IBS symptoms can be effectively relieved by relieving inflammation, regulating immune response, recovering intestinal barrier function and the like.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus plantarum GXFF202402 and application thereof in preparing products for preventing and / or treating hemorrhoids. Background Art

[0002] Currently, the term “hemorrhoids” is mainly used to describe the pathological and symptomatic downward displacement of normal anal cushions. The true pathophysiology of hemorrhoids may be the result of multifactorial effects, including anal cushion sliding, hyperperfusion, vascular abnormalities, tissue inflammation, and internal rectal prolapse. Different etiological theories have led to a variety of treatments. Hemorrhoids are also closely related to other anorectal diseases such as anal pruritus, anal fissures, and anal spasms. Although the exact pathophysiology of these diseases is not fully understood, researchers have proposed that they may be related to dysfunction of the smooth muscle of the internal anal sphincter. According to current hypotheses, anorectal diseases are often regarded as a direct consequence of constipation. Through an extensive literature review, a new etiological theory has been proposed, which believes that there is an important association between altered anal canal flora, local inflammation, and smooth muscle damage.

[0003] Irritable bowel syndrome (IBS) is a common functional gastrointestinal disorder that affects 9% to 23% of the world's population and has become an important public health issue. IBS is characterized by recurrent abdominal pain or bloating, often accompanied by abnormal bowel movements. Based on the symptoms, IBS can be divided into four main types: constipation type (IBS-C), diarrhea type (IBS-D), mixed type (IBS-M), and unclassified type (IBS-U). Meta-analysis shows that the risk of IBS increases 3 to 11 times after an episode of gastroenteritis. Increasing evidence shows that changes in the gastrointestinal microbiota are closely related to the occurrence of IBS. Recent studies have further revealed the key roles of food sensitivity, post-infection response, brain-gut interaction, changes in the gastrointestinal microbiota, and activation of the gastrointestinal mucosal immune system in the pathogenesis of IBS.

[0004] Probiotics are a type of active microorganisms that can improve host health. They play an important role in maintaining the balance of intestinal flora, regulating immune function, and improving intestinal function. Studies have shown that oral probiotics can not only effectively relieve gastrointestinal symptoms, but also promote the recovery of intestinal flora. Therefore, it is of great significance to develop probiotics that can relieve or treat hemorrhoids and IBS. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a strain of Lactobacillus plantarum GXFF202402 and its use in preparing products for preventing and / or treating hemorrhoids. The Lactobacillus plantarum GXFF202402 provided by the present invention relieves hemorrhoids by reducing inflammation, restoring tissue integrity and regulating the expression of inflammatory cytokines, and effectively relieves IBS symptoms by reducing inflammation, regulating immune response and restoring intestinal barrier function.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum ) GXFF202402, the deposit number of the plant lactobacillus GXFF202402 is CGMCC NO.32257, and it was deposited in the General Microbiology Center of China Culture Collection Administration on October 18, 2024, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] In the present invention, the Lactobacillus plantarum GXFF202402 is isolated from naturally fermented sour fish in Yanjiao Village, Liuzhou City, Guangxi Zhuang Autonomous Region.

[0008] The present invention also provides the use of Lactobacillus plantarum GXFF202402 described in the above technical solution in preparing products for preventing and / or treating hemorrhoids.

[0009] In the present invention, the Lactobacillus plantarum GXFF202402 can reduce inflammation, restore tissue integrity and regulate the expression of inflammatory cytokines.

[0010] In the present invention, the concentration of Lactobacillus plantarum GXFF202402 in the product is preferably 1×10 9 CFU / mL.

[0011] The present invention also provides the use of Lactobacillus plantarum GXFF202402 described in the above technical solution in preparing a product for relieving or treating irritable bowel syndrome.

[0012] In the present invention, the Lactobacillus plantarum GXFF202402 relieves IBS symptoms by reducing inflammation, regulating immune response and restoring intestinal barrier function.

[0013] In the present invention, the concentration of Lactobacillus plantarum GXFF202402 in the product is preferably 1×10 9 CFU / mL.

[0014] In the present invention, the products preferably include but are not limited to food, medicine, and health care products.

[0015] The present invention also provides a hemorrhoids therapeutic agent, which comprises the live bacterial cells of Lactobacillus plantarum GXFF202402 and / or fermentation metabolites described in the above technical solution.

[0016] Beneficial technical effects: The present invention provides a strain of Lactobacillus plantarum GXFF202402 and its application in the preparation of products for preventing and / or treating hemorrhoids, and the Lactobacillus plantarum GXFF202402 has a deposit number of CGMCCNO.32257, which was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on October 18, 2024, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The Lactobacillus plantarum GXFF202402 provided by the present invention relieves hemorrhoids by reducing inflammation, restoring tissue integrity, and regulating the expression of inflammatory cytokines, and can be used as a potential therapeutic agent for treating hemorrhoids. In addition, IBS symptoms can also be effectively relieved by reducing inflammation, regulating immune response, and restoring intestinal barrier function. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the colony characteristics of Lactobacillus plantarum GXFF202402; Figure 2 This is the Gram staining microscopic examination image of Lactobacillus plantarum GXFF202402; Figure 3 is the weight change of mice in Example 2; Figure 4 The results of H&E staining of rectal tissue of 15-day model mice in Example 2; Figure 5 The results of H&E staining of rectal tissue of 30-day model mice in Example 2; Figure 6 The results of rectal cytokine detection in each group of mice on day 15 in Example 2; different letters indicate significant differences. p <0.05, the same below; Figure 7 The results of rectal cytokine detection in each group of mice at 30 days in Example 2; Figure 8 The results of serum inflammatory factors detection in each group of mice on day 15 in Example 2; Fig. 9 The results of serum inflammatory factors detection in each group of mice at 30 days in Example 2; Fig.10 The detection results of inflammatory factor mRNA in rectal cells of mice in each group on day 15 in Example 2; Fig.11 The detection results of mRNA of inflammatory factors in rectal cells of mice in each group at 30 days in Example 2; Fig.12 is the weight change of mice in Example 3; Fig.13 is the change in food intake of mice in Example 3; Fig.14 is the change in the number of feces particles of mice in Example 3; Fig.15 The change in water content of mouse feces in Example 3; Fig.16 The H&E stained pathological sections of the colon tissue of male mice in Example 3 are observed; Fig.17 The H&E stained pathological sections of the colon tissue of female mice in Example 3 are observed; Fig.18 is the corticosterone content in the serum of mice in Example 3; Fig.19 is the IL-1β content in the colon tissue of mice in Example 3; Fig. 20 is the IL-6 content in the colon tissue of mice in Example 3; Fig.21 is the TNF-α content in the colon tissue of mice in Example 3; Fig. 22 is the IL-10 content in the colon tissue of mice in Example 3; Fig.23 is the MCT content in the colon tissue of mice in Example 3; Fig.24 is the PAR-2 ​​content in the mouse colon tissue in Example 3; Fig.25 is the occludin content in the colon tissue of mice in Example 3; Fig.26 is the expression level of Occludin gene in the mouse colon tissue in Example 3; Fig. 27 This is the expression level of PAR-2 ​​gene in the mouse colon tissue in Example 3. DETAILED DESCRIPTION

[0018] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0019] Example 1 Strain Isolation and Identification 1. Experimental Materials The fish was collected from the naturally fermented sour fish made by the villagers of Yanjiao Village, Liuzhou City, Guangxi Zhuang Autonomous Region. The fish meat was cut and placed in a 50mL sterilized centrifuge tube, which was then put into a low-temperature food sampling box and brought back to the laboratory for freezing and storage in an ultra-low temperature refrigerator at -80℃ for later use.

[0020] 2. Isolation and identification of microorganisms 2.1 Isolation and purification of microorganisms Take 1g of fermented acid fish sample and crush it, add 10 times sterile physiological saline and stir it, take the upper stirring liquid and dilute it to 10 -6 , then take 10 -4 , 10 -5 , 10 -6 100 μL of 3 gradient bacterial solutions were spread on the plate and cultured at 37℃ for 24-48h. The colony morphology was observed and recorded. Colonies with different morphologies on the plate were picked for streaking separation. After culturing at 37℃ for 48h, single colonies with different morphologies on the plate were picked for streaking separation again. This process was repeated several times until pure single colonies with consistent morphology were obtained.

[0021] 2.2 Preliminary identification of microorganisms Pick the pure colonies on the plate and inoculate them into 5 mL of MRS liquid medium and culture them at 37°C for 24 hours. The colonies are mostly white or milky white in color, round in shape, with neat edges and a moist and smooth surface ( Figure 1 ).

[0022] Take 1 mL of the above-mentioned culture medium into a sterile centrifuge tube, centrifuge at 4000 r / min for 10 min, discard the upper culture medium, resuspend the bacterial pellet in sterile saline and perform Gram staining microscopy. Under a 100x oil microscope, the cell morphology of the lactic acid bacteria strain is long rod, short rod, and spherical, and there is no budding reproduction ( Figure 2 ). It was preliminarily identified as lactic acid bacteria.

[0023] 2.3 Molecular identification Inoculate the purified suspected target strain into MRS broth, culture at 37℃ for 18h-24h, and then use a bacterial genomic DNA extraction kit to extract DNA. Number the extracted DNA and store it in a -20℃ freezer for later use.

[0024] The extracted DNA was subjected to PCR amplification, including 1 μL of upstream primer 27F (5'-AGA GTT TGA TCC TGGCTC AG-3', Seq_1), 1 μL of downstream primer 1495R (5'-CTA CGG CTA CCTTGT TAC GA-3', Seq_2), 12.5 μL of 2× Taqplus Buffer, 1 μL of template DNA, and the system was supplemented to 25 μL with sterile ddH2O. Sterile ultrapure water was used to replace template DNA as a negative control. The amplification conditions were: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, for a total of 29 cycles, and finally extended at 72°C for 5 min.

[0025] Then 5 μL of the amplified product was taken for agarose gel electrophoresis detection, the agarose concentration was 1.2%, and the electrophoresis conditions were 110V, 45min. The successfully detected PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the sequencing results are shown in Seq_3. The sequence successfully sequenced was compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI. After comparison, the strain was Lactobacillus plantarum, named Lactobacillus plantarum GXFF202402, and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on October 18, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.32257.

[0026] Seq_3:

[0027] Example 2 Effect of Lactobacillus plantarum GXFF202402 on hemorrhoids 1. Materials and reagents: as shown in Table 1.

[0028] Table 1 Experimental materials and reagents 2. Instruments and equipment: as shown in Table 2.

[0029] Table 2 Instruments and equipment 3. Experimental Methods 3.1 Animal Model Sixty 5-week-old ICR female mice (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.) were randomly divided into two experimental groups after one week of adaptive feeding: 15-day experimental group and 30-day experimental group. Each experimental group was further divided into three groups, each with 10 mice: normal group, model group, and Lactobacillus plantarum GXFF202402 group. In the 15-day experimental group, the experimental period lasted for 15 days, during which the GXFF202402 group was gavaged with Lactobacillus plantarum (1×10 9 CFU / mL). On the 15th day, except for the normal group, 0.05 mL of croton oil mixture (mixed with distilled water, pyridine, ether and 6% croton oil in a ratio of 1:4:5:10) was injected into the mouse anus at 0.5 cm and withdrawn after 10 seconds to establish an anal swelling model. After 6 hours, the swelling, redness, bleeding, ulcer formation, ulcer size, color change, etc. of the mouse anus were observed. After confirming that the model was successful, subsequent experiments were carried out. The experimental period of the 30-day experimental group was 30 days, and the GXFF202402 group was injected with Lactobacillus plantarum (1×10 9 CFU / mL) by gavage. On the 15th day, the same croton oil mixture was injected as in the 15th day experimental group to establish the anal swelling model. The animals maintained the same feeding regimen until the 30th day, when subsequent experiments were performed. Body weight was recorded every two days throughout the experiment.

[0030] 3.2 Determination of anorectal coefficient (ARC) in mice To calculate the anorectal coefficient (ARC), the animals and their isolated anorectal tissue (15 mm in length) were weighed. The ARC was calculated using the formula: Anorectal coefficient (ARC) = anorectal tissue weight (mg) / animal weight (g).

[0031] 3.3 Determination of serum cytokine levels in mice with rectal swelling The sample was refrigerated at 4°C for about 2 hours to promote coagulation and serum separation. Subsequently, centrifuged at 4°C and 3000rpm for 10 minutes. After centrifugation, the upper light yellow serum was carefully removed and transferred to a sterile tube. To ensure the stability of the serum components, it was quickly frozen and stored in an ultra-low temperature refrigerator at -80°C for subsequent biochemical analysis. Blood was collected from each group of mice to prepare serum samples, and the contents of PGE2, VEGF, EGF and NO in mouse serum were detected. The detection method refers to the instructions of the kit.

[0032] 3.4 Determination of cellular inflammatory factor levels in mice with rectal swelling After collecting serum in the same manner as above, the levels of IL-2, IL-4, IL-6, IL-1β and TNF-α in mouse serum were detected respectively. The detection method refers to the instructions of the kit.

[0033] 3.5 Eosin-hematoxylin (H&E) staining of mouse tissues with rectal swelling The rectum was fixed in 10% formalin solution for 24 hours, and H&E staining was used to observe the tissue damage of the mice with rectal swelling. The specific steps are as follows: (1) Fixation: Cut a 0.5 cm piece of mouse rectum without feces and fix it in a fixative (10% formalin) for 24 hours; (2) Embedding: After the rectum is fixed and dehydrated, it is embedded in paraffin; (3) Sectioning: Use a paraffin slicer to cut the sections into 0.4 μm slices; (4) Slide preparation: Place a 0.4 μm thick slice on a prepared slide, then place it on a slide oven and allow it to dry slowly. (5) Dewaxing: xylene (I) 5 min -- xylene (II) 5 min -- anhydrous ethanol 5 min -- 95% ethanol 2 min -- 80% ethanol 2 min -- 70% ethanol 2 min; (6) Place the slices in hematoxylin stain for 2-8 minutes, rinse off the excess stain with tap water, and then place them in differentiation solution (1% hydrochloric acid alcohol) for 30 minutes. Finally, soak the slices in tap water for 15 minutes. (7) Soak the sections in eosin stain for 1-2 minutes, rinse off the excess stain with tap water, and then soak the sections in tap water for 5 minutes; (8) 95% ethanol solution (I) 1 min--95% ethanol solution (II) 1 min--anhydrous ethanol (I) 1 min--anhydrous ethanol (II) 1 min--xylene carbolic acid (3:1) 1 min--xylene (I) 1 min--xylene (II) 1 min, seal the slices with neutral gum, and finally observe the slices under a microscope.

[0034] 3.6 Determination of mRNA in mouse anal margin tissue In this experiment, fluorescence quantitative PCR (qPCR) was used to detect the mRNA levels of related genes in the anal margin tissues of each group of mice.

[0035] (1) Extraction of total RNA from tissues After the anal margin tissue was homogenized, chloroform was added, mixed thoroughly, and centrifuged at 4°C for 15 min. The supernatant was added with an equal volume of isopropanol, mixed, and centrifuged at 4°C for 20 min. After discarding the supernatant, it was washed with 75% ethanol, centrifuged at 4°C for 15 min, the upper aqueous phase was discarded, and it was allowed to stand for 3-5 min. 20 μL of RNase-Free water was added for later use.

[0036] (2) Determination of RNA concentration and purity Dilute the RNA stock solution and RNase-Free water at a ratio of 1:49 and mix well. Use a micro-spectrophotometer to measure the concentration and purity of RNA. An A260 / A280 ratio between 1.8 and 2.0 indicates good purity.

[0037] (3) Reverse transcription of RNA into cDNA According to the instructions of the Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigester plus) synthesis kit, RNA was reverse transcribed into cDNA, and the concentration of cDNA was measured using a micro-spectrophotometer. The cDNA concentration needed to be diluted to 1000 ng / ml before use in qPCR amplification.

[0038] (4) PCR amplification 1) Amplification system: Master mix 10μL; Forward primer 0.4μL; Reverse primer 0.4μL; Template (cNDA, 1000 ng / mL) 1μL; Sterile ultrapure water 8.2μL. After the amplification system is evenly mixed, centrifuge at low speed and amplify on a Real-Time PCR instrument.

[0039] The primer sequences are shown in Table 3.

[0040] Table 3 Primer sequences 2) Amplification conditions For all assays, after initial denaturation at 95°C for 5 min, 40 cycles were performed at 95°C for 10 s and 60°C for 20 s, 72°C for 20 s.

[0041] 3) Calculation of experimental results Taking β-actin as the internal reference gene, according to formula 2 -ΔΔCT Calculate the relative mRNA expression of each target gene.

[0042] 3.7 Data Processing Statistical analysis software SPSS for Windows, version 27.0, was used for data analysis. To determine the significance of the difference between two means, one-way ANOVA and Duncan's test were used. p <0.05) were considered statistically significant. Different letters indicate that one group is significantly different from the other groups ( p <0.05).

[0043] 4. Results and Analysis 4.1 Results of mouse weight changes Croton oil mixture can induce hemorrhoids in mice. The weight of mice decreased significantly during the modeling process. Therefore, the effect of Lactobacillus plantarum GXFF202402 on croton oil-induced hemorrhoids during the experiment can be evaluated by the changes in mouse weight. Figure 3 ).

[0044] In the 15-day mouse experimental model, the weight of mice in the croton oil group showed a significant downward trend compared with the normal group, while the weight of mice in the Lactobacillus plantarum GXFF202402 group decreased more slowly, but still lower than the level of the normal group. These results show that hemorrhoids induced by croton oil can cause weight loss in mice, and oral administration of Lactobacillus plantarum GXFF202402 can partially alleviate the weight loss.

[0045] In the 30-day mouse experimental model, on the 15th day after modeling, the weight of the mice in the model group first decreased and then gradually increased, while the weight of the mice in the Lactobacillus plantarum GXFF202402 group slowly decreased and then continued to increase, and finally returned to the level of the normal group. These research results show that hemorrhoids induced by croton oil can cause a certain degree of weight loss in mice, and continuous 30-day intragastric administration of Lactobacillus plantarum GXFF202402 can effectively alleviate this weight loss.

[0046] 4.2 Pathological observation of mouse rectal tissue like Figure 4As shown in the figure, in the 15-day experimental model, the anal mucosal epithelial tissue of the mice in the normal group was intact and had normal structure. The rectal mucosal epithelial tissue of the mice in the model group showed local necrosis, inflammatory cell infiltration, submucosal edema, etc. The rectal mucosal epithelial tissue of the mice in the Lactobacillus plantarum GXFF202402 group was significantly improved, the glands were arranged more orderly, the mucosal bleeding was reduced, the boundaries were clearer, and the inflammatory changes were alleviated, indicating that Lactobacillus plantarum GXFF202402 has a protective effect on the rectal tissue of mice with hemorrhoids induced by croton oil.

[0047] like Figure 5 As shown in the figure, in the 30-day experimental model, the anal mucosal epithelial tissue of the normal group mice was intact and normal in structure. The model group had local necrosis of the rectal mucosal epithelial tissue, inflammatory cell infiltration, severe edema of the submucosal layer, inflammatory cell infiltration, and vascular dilation and congestion. The situation in the plant lactobacillus GXFF202402 group was significantly improved, with an intact mucosal layer structure, reduced mucosal bleeding, clear boundaries, and significantly reduced inflammatory cells. This shows that plant lactobacillus GXFF202402 can effectively reduce the swelling and inflammatory response of the croton oil-induced hemorrhoid mouse model, promote tissue repair, and provide an important basis for the application of probiotics in the treatment of hemorrhoids and related diseases.

[0048] 4.3 Swelling coefficient of mouse rectal tissue The rectal tissue swelling coefficient of mice in the 15-day group is shown in Table 4. As can be seen from Table 4, compared with the normal group, the anorectal swelling rate of mice in the model group was significantly increased ( p <0.05, indicating that the model was successfully established. The plant lactobacillus group was significantly lower than the model group ( p <0.05), which proved that Lactobacillus plantarum GXFF202402 had a relieving effect on mice with hemorrhoids induced by croton oil mixture.

[0049] Table 4 Swelling coefficient of rectal tissue of mice in the 15-day group The rectal tissue swelling coefficient of mice in the 30-day group is shown in Table 5. As can be seen from Table 5, compared with the normal group, the anorectal swelling rate of mice in the model group was significantly increased ( p <0.05, indicating that the model was successfully established. The plant lactobacillus group was significantly lower than the model group ( p <0.05), proving that Lactobacillus plantarum GXFF202402 has a protective effect on mice with hemorrhoids induced by croton oil mixture, and continuous oral administration for 30 days has a significant alleviating effect on mice with hemorrhoids.

[0050] Table 5 Swelling coefficient of rectal tissue of mice in the 30-day group 4.4 Determination of the levels of rectal cytokines in mice with rectal swelling The results of rectal cytokine levels in rectal tissue of 15-day model mice are shown in Figure 6 .Depend on Figure 6 It can be seen that after croton oil induction, PGE2 and NO increased, leading to aggravated local inflammatory response, increased vascular permeability and dilatation, and the formation of edema, pain and congestion. VEGF, with the assistance of NO, promotes angiogenesis, which may lead to venous plexus dilation and worsening of lesions. EGF and VEGF work synergistically to promote the repair and regeneration of damaged tissues and help restore the function and structure of the diseased area. The results showed that according to the results of PGE2 and NO cytokine level detection, Lactobacillus plantarum GXFF202402 can reduce inflammation in hemorrhoidal mice. Compared with the model group, there was a significant difference in the Lactobacillus plantarum GXFF202402 group ( p <0.05).

[0051] The results of rectal cytokine levels in rectal tissue of 30-day model mice are shown in Figure 7 .Depend on Figure 7 It can be seen that in the 30-day croton oil-induced mouse hemorrhoid model, rectal PGE2 and NO levels were significantly increased ( p <0.05, while the levels of rectal PGE2 and NO were significantly decreased after intervention with Lactobacillus plantarum GXFF202402 ( p <0.05). The results showed that Lactobacillus plantarum GXFF202402 intervention could regulate the levels of PGE2 and NO in the rectum of hemorrhoid model mice. Lactobacillus plantarum GXFF202402 alleviated the inflammatory response and tissue damage caused by hemorrhoids through anti-inflammatory, antioxidant and immunomodulatory mechanisms, thereby reducing the levels of PGE2 and NO.

[0052] 4.5 Determination of serum inflammatory factors in mice with rectal swelling The croton oil-induced hemorrhoid model in mice resulted in local tissue inflammation, vascular dilation, leukocyte infiltration, immune activation and imbalance in the rectum. To evaluate the preventive effect of Lactobacillus plantarum GXFF202402 on hemorrhoids, the serum levels of proinflammatory cytokines IL-1β, IL-6, TNF-α, IL-2 and IL-4 were analyzed.

[0053] The results of serum inflammatory factors in each group of mice on day 15 were as follows Figure 8 As shown. Figure 8 It can be seen that in the 15-day croton oil-induced mouse hemorrhoid model, the levels of serum IL-1β, IL-6, TNF-α, IL-2, and IL-4 were significantly increased compared with normal levels ( p <0.05). After intervention with Lactobacillus plantarum GXFF202402, the levels of inflammatory factors decreased significantly ( p<0.05), and returned to the level of the normal group. These findings indicate that Lactobacillus plantarum GXFF202402 intervention alleviated the inflammatory response induced by the croton oil mixture and regulated the immune response.

[0054] The detection of serum inflammatory factors in each group of mice on day 30 is as follows Fig. 9 As shown. Fig. 9 It can be seen that in the 30-day croton oil-induced mouse hemorrhoid model, the levels of IL-1β, IL-6, TNF-α, IL-2 and IL-4 in the serum were significantly higher than those in the normal group ( p <0.05, and after intervention with Lactobacillus plantarum GXFF202402, the above levels were significantly reduced ( p <0.05), and returned to the level of the normal group. This shows that Lactobacillus plantarum GXFF202402 reduces cytokine levels through anti-inflammatory and immunomodulatory effects, alleviates inflammatory responses, restores immune balance, and promotes tissue healing.

[0055] 4.6 mRNA expression levels of inflammatory factors in mouse anal margin tissue To explore the regulatory mechanism of hemorrhoidal inflammation, the mRNA levels of TNF-α and IL-6 in the anal margin tissues of hemorrhoidal mice were measured.

[0056] In the 15-day croton oil-induced mouse hemorrhoid model, the levels of IL-6 and TNF-α in the anal margin tissue were significantly increased compared with the normal group ( p <0.05) ( Fig.10 After intervention with Lactobacillus plantarum GXFF202402, the above levels were significantly reduced ( p <0.05), and returned to the level of the normal group. These results indicate that Lactobacillus plantarum GXFF202402 can effectively prevent hemorrhoids induced by croton oil in mice.

[0057] Depend on Fig.11 It can be seen that in the 30-day croton oil-induced mouse hemorrhoid model, the levels of IL-6 and TNF-α in the anal margin tissue were significantly increased compared with the normal group ( p <0.05). After intervention with Lactobacillus plantarum GXFF202402, the level of TNF-α was significantly reduced ( p <0.05), suggesting that Lactobacillus plantarum GXFF202402 exerts anti-inflammatory and tissue repair effects by regulating immune function.

[0058] In summary, this experiment induced a mouse hemorrhoid model by using a mixture of croton oil, causing tissue redness, swelling, and ulcers in the anorectum of mice, accompanied by a large amount of inflammatory fluid exudation. After intervention with Lactobacillus plantarum GXFF202402, the rectal damage of mice was significantly improved, the glands were arranged more neatly, the mucosal tissue bleeding was reduced, the boundaries were clearer, and the inflammatory changes were obvious. At the same time, the bacteria also had a certain alleviating effect on the systemic inflammatory response produced by mice with rectal swelling, and could reduce the expression of proinflammatory factors such as IL-1β, IL-6, IL-4, IL-2, TNF-α, PGE2, and NO, thereby alleviating the swelling of the rectum of mice.

[0059] Example 3 Effect of Lactobacillus plantarum GXFF202402 on IBS 1. Materials and reagents: as shown in Table 6.

[0060] Table 6 Experimental materials and reagents for the effect of Lactobacillus plantarum GXFF202402 on IBS 2. Instruments and equipment: as shown in Table 2.

[0061] 3. Experimental Methods 3.1 Animal Model The experiment used 40 5-week-old C57BL / 6 mice (half male and half female, purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.). After acclimatization, the mice were randomly divided into 4 groups, with 5 mice in each group: normal group, model group, positive drug group and Lactobacillus plantarum GXFF202402 group. The experiment lasted for 4 weeks. Before modeling, the mice were fasted for 12 hours. Except for the normal group, the IBS model was established by enema trinitrobenzene sulfonic acid (TNBS) in the other groups. The specific method is to use a suitable medical polyurethane catheter to inject 0.1mL TNBS into the mouse about 3-4cm proximal to the anal verge, retain it for 1min, and then slowly pull out the enema tube. The mouse was then inverted for 30s to prevent liquid reflux and ensure uniform distribution of the reagent in the intestine. Mice in the Lactobacillus plantarum group were orally gavaged with Lactobacillus plantarum solution (1×10 9 CFU / mL). Mice in the positive drug group were orally gavaged with linaclotide (100 μg / kg) from the 2nd to the 4th week. During the experiment, the body weight of the mice was measured every two days and the changes in food intake were recorded. The number of fecal particles of the mice within 6 hours was recorded every three days, and fecal samples were collected to determine their water content.

[0062] 3.2 Mouse serum cytokine determination The content of corticosterone (CORT) in serum was determined by enzyme-linked immunosorbent assay (ELISA). After centrifugation at 3000 rpm for 10 min, the serum samples were stored in an ultra-low temperature refrigerator at -80°C for future use. The experiment was carried out strictly in accordance with the operating instructions of the ELISA kit.

[0063] 3.3 Determination of inflammatory factor levels in mouse colon tissue ELISA kits were used to measure the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) in mouse colon tissue. Colon tissue samples (50-100 mg) were collected, minced, and transferred to homogenization tubes. The samples were homogenized in normal saline at a weight to volume ratio of 1:9 using a tissue homogenizer. The resulting homogenate was centrifuged at 4000 rpm for 10-15 min at 4°C, and the supernatant was carefully collected for subsequent analysis.

[0064] 3.4 Determination of intestinal permeability factor levels in mouse colon tissue ELISA kits were used to measure the intestinal permeability-associated protein Occludin (OCLN), protease-activated receptor-2 (PAR-2) and mast cell tryptase (MCT) in mouse colon tissue.

[0065] 3.5 Determination of mRNA in mouse colon tissue In this experiment, qPCR was used to detect the mRNA levels of related genes in the colon of each group of mice.

[0066] The extraction of total RNA from tissues, determination of RNA concentration and purity, reverse transcription of RNA into cDNA, and PCR amplification were all the same as 3.6 in Example 2, and the primer sequences are shown in Table 7.

[0067] Table 7 Example 3 Primer sequences 3.6 Data processing: same as 3.7 in Example 2.

[0068] 4 Results and analysis 4.1 Results of mouse weight changes TNBS has been shown to cause adverse reactions such as abdominal pain in mice. In the first week of modeling, except for the normal group, all other groups of male and female mice showed significant weight loss. Fig.12The results showed that Lactobacillus plantarum GXFF202402 had an intervention effect on TNBS-induced weight loss in mice. The experimental results showed that after the intervention of Lactobacillus plantarum and linaclotide, the weight of male and female mice gradually recovered, and the weight of mice in both the drug group and the Lactobacillus plantarum group showed an upward trend, eventually approaching the level of the normal group.

[0069] 4.2 Changes in food intake of mice like Fig.13 As shown in the figure, in the first week of modeling, compared with the normal group, the food intake of mice in the other groups decreased after TNBS modeling, and showed large fluctuations. However, after oral gavage intervention with drugs and Lactobacillus plantarum GXFF202402, the food intake of mice in both male and female groups gradually recovered, especially after the second week of intervention, gradually approaching the level of the normal group. It is worth noting that in all experimental groups, the food intake of mice in the model group was always higher than that of other groups, which may be related to their IBS symptoms. These results indicate that Lactobacillus plantarum GXFF202402 can alleviate the feeding behavior of modeling mice.

[0070] 4.3 Changes in the number of fecal particles in mice like Fig.14 As shown in the figure, according to the analysis of the changes in the number of fecal particles in male mice, after modeling, the number of fecal particles in the model group mice increased rapidly in the early stage (about the 5th to 10th day) and reached a peak, and then gradually decreased. The peak number of fecal particles was significantly higher than that of the normal group, suggesting that the model group may have intestinal dysfunction or inflammatory response after modeling. After intervention (after the 10th day), the number of fecal particles in the drug group and the GXFF202402 group of mice showed a downward trend and approached the level of the normal group around the 20th day. Compared with the model group, the number of fecal particles in the GXFF202402 group of mice decreased more steadily. The trend of the number of fecal particles in female mice was similar to that in male mice, indicating that Lactobacillus plantarum GXFF202402 may have a potential role in regulating intestinal flora.

[0071] 4.4 Changes in water content in mouse feces like Fig.15 As shown in the figure, after TNBS modeling, the fecal water content of mice in the model group decreased significantly, followed by a large fluctuation, which may be related to intestinal inflammation, dysbacteriosis or water regulation dysfunction. The fecal water content of both male and female mice showed an obvious fluctuation trend. The drug group and Lactobacillus plantarum GXFF202402 intragastric intervention significantly improved the fecal water content of mice, gradually approaching the level of the normal group. It is worth noting that the fecal water content of mice in the Lactobacillus plantarum GXFF202402 group recovered more smoothly, with smaller fluctuations in the later period, which may indicate that Lactobacillus plantarum GXFF202402 is more effective in regulating the balance of intestinal flora.

[0072] 4.5 Histopathological observation of mouse colon like Figure 16-17 As shown in the figure, after TNBS induction, in order to observe the pathological lesions of the colon tissue of mice and evaluate the effect of Lactobacillus plantarum GXFF202402 on colon inflammation, H&E staining was used to perform histological examination on the colon of mice after modeling. As the experimental results show, the colon tissue structure of male and female mice in the normal group was intact, with no signs of inflammation or damage. After TNBS modeling, the colon tissue of mice showed obvious pathological changes, including epithelial cell damage, abnormal crypt structure, inflammatory cell infiltration and submucosal edema, indicating that the intestine was in an inflammatory and dysfunctional state. The degree of colon inflammation and damage in mice in the drug group was significantly reduced, and the tissue structure gradually recovered. The intestinal mucosal barrier function of mice in the Lactobacillus plantarum GXFF202402 group was significantly improved, and the inflammatory response was reduced, which indicates that Lactobacillus plantarum GXFF202402 has the potential to relieve IBS symptoms and repair intestinal mucosa. In summary, Lactobacillus plantarum GXFF202402 has a significant protective effect on the colon tissue of TNBS-induced IBS mice.

[0073] 4.6 Corticosterone (CORT) content in mouse serum CORT plays a key role in stress response, regulating intestinal motility and sensitivity and influencing the activity of the immune system. The regulatory effect of probiotics on the gut-brain axis is a known mechanism by which they reduce visceral hypersensitivity. Chronic stress caused by TNBS activates the gut-brain axis, leading to increased release of corticosterone from the adrenal cortex. Prolonged exposure of the amygdala to elevated corticosterone levels induces persistent pain responses. The IBS model significantly increases serum corticosterone levels. Lactobacillus plantarum restored corticosterone levels ( Fig.18 , p <0.05), and the recovery effect was better than that of the drug group. These findings suggest that probiotics may alleviate visceral hypersensitivity symptoms through their ability to regulate corticosterone levels.

[0074] 4.7 Expression levels of inflammatory factors in mouse colon tissue When the intestine is attacked by inflammatory factors, intestinal epithelial cells and macrophages will significantly upregulate the expression of proinflammatory cytokines. Conversely, the expression of proinflammatory cytokines also promotes colon inflammation. On the contrary, as an anti-inflammatory cytokine IL-10 plays a vital role in controlling and preventing intestinal inflammation. IL-10 is believed to block the metabolism of macrophages and promote damaged mitochondrial autophagy, thereby alleviating inflammation. In order to evaluate the anti-inflammatory effect of Lactobacillus plantarum on IBS, the study analyzed the expression of proinflammatory cytokines (IL-1β, IL-6, TNF-α) and anti-inflammatory cytokine IL-10 in the colon of mice ( Figure 19-22). The results showed that TNBS disrupted the intestinal immune homeostasis of mice, significantly increased the expression of pro-inflammatory cytokines in the colon and reduced anti-inflammatory cytokines ( p <0.05). In male mice, Lactobacillus plantarum GXFF202402 significantly reduced the expression of IL-1β and TNF-α, restoring them to levels comparable to those of the normal group ( p <0.05). In contrast, the anti-inflammatory cytokine IL-10 plays a key role in alleviating and preventing intestinal inflammation. Lactobacillus plantarum GXFF202402 significantly increased IL-10 levels ( p <0.05, restoring it to the level of the control group. In female mice, Lactobacillus plantarum GXFF202402 significantly reduced the expression of IL-1β and IL-6 ( p <0.05) and moderately reduced TNF-α levels ( p >0.05, and significantly increased the expression level of IL-10 ( p <0.05). These findings suggest that Lactobacillus plantarum GXFF202402 can effectively alleviate TNBS-induced intestinal inflammation.

[0075] 4.8 Expression level of colonic permeability factor in mice In the gastrointestinal tract, mast cells are activated and release tryptase, which in turn activates PAR-2 ​​on the surface of intestinal nerve cells, ultimately causing a state of sustained neural excitement that feeds back to the gastrointestinal tract, resulting in gastrointestinal motility disorders. Occludin is a membrane protein and a representative protein of tight junction structures. Its function in the intestine is usually to maintain the stability of intestinal tight junctions and ensure normal intestinal barrier function. IBS-related studies have shown that low-grade intestinal inflammation in IBS is characterized by intestinal barrier function damage and decreased occludin expression. Lactobacillus plantarum is often reported to have the function of restoring the intestinal barrier. In this study, after TNBS induction ( Figure 23-25 ) significantly increased the expression of MCT and PAR-2 ​​in the colon tissue of mice ( p <0.05, while reducing the expression of OCLN ( p <0.05). In male mice, Lactobacillus plantarum was able to reduce the expression of MCT and significantly decreased the expression of PAR-2 ​​( p <0.05), increased the expression of OCLN ( p <0.05). In female mice, Lactobacillus plantarum was able to reduce the expression of MCT and significantly decreased the expression of PAR-2 ​​( p <0.05, and increased the expression of OCLN ( p >0.05). These findings suggest that Lactobacillus plantarum GXFF202402 can alleviate visceral hypersensitivity and restore intestinal barrier integrity that is damaged by intestinal inflammation.

[0076] 4.9 Gene expression level of mouse colon permeability factor like Fig.26 As shown, there is no significant difference in the expression level of Occludin gene in colon tissues of male and female mice ( p >0.05).

[0077] like Fig. 27 As shown, there was no significant difference in the expression level of PAR-2 ​​gene in colon tissues of male and female mice ( p >0.05).

[0078] In this study, the intervention of Lactobacillus plantarum GXFF202402 significantly alleviated the inflammatory response of the intestinal mucosa of mice, restored the integrity of the tissue structure, and reduced the infiltration of inflammatory cells, which was significantly different from the model group. In addition, the expression levels of IL-1β, IL-6, and TNF-α in male and female mice were significantly increased after TNBS induction, but the intervention of Lactobacillus plantarum GXFF202402 could effectively inhibit the expression of proinflammatory factors and promote the production of anti-inflammatory factor IL-10. Although female mice are more sensitive to intestinal inflammation than male mice and have poorer tolerance, the intervention of Lactobacillus plantarum showed significant anti-inflammatory effects on both sexes.

[0079] In summary, Lactobacillus plantarum GXFF202402 shows the potential to relieve IBS symptoms through multiple mechanisms such as regulating intestinal flora, enhancing intestinal mucosal barrier function and inhibiting intestinal inflammation. Existing studies have shown that Lactobacillus plantarum has a significant effect in improving the intestinal microecological environment and alleviating IBS symptoms. With the further development of probiotic research, Lactobacillus plantarum is expected to become an effective method in the adjuvant treatment of IBS, providing patients with better prognosis and quality of life.

[0080] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum ) GXFF202402, characterized in that, The deposit number of the plant lactobacillus GXFF202402 is CGMCC NO.32257. It was deposited in the General Microbiology Center of the China Culture Collection Administration on October 18, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Lactobacillus plantarum GXFF202402 according to claim 1 in preparing products for preventing and / or treating hemorrhoids.

3. The use according to claim 2, characterized in that: The concentration of Lactobacillus plantarum GXFF202402 in the product is 1×10 9 CFU / mL.

4. Use of the Lactobacillus plantarum GXFF202402 according to claim 1 in preparing a product for alleviating or treating irritable bowel syndrome.

5. The use according to claim 4, characterized in that: The concentration of Lactobacillus plantarum GXFF202402 in the product is 1×10 9 CFU / mL.

6. A hemorrhoids therapeutic agent, characterized in that: The hemorrhoid treatment agent comprises the live bacterial cells of Lactobacillus plantarum GXFF202402 according to claim 1, and / or fermentation metabolites.

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