A strain of Lactiplantibacillus plantarum GXFF202402 and its application in the preparation of products for preventing and / or treating hemorrhoids

Abstract

The present invention provides a Lactiplantibacillus plantarum GXFF202402 and its application in the preparation of products for preventing and / or treating hemorrhoids, belonging to the field of microbial technology. The preservation number of the Lactiplantibacillus plantarum GXFF202402 provided by the present invention is CGMCC NO. 32257, and it was deposited on October 18, 2024 at the General Microbiological Center of the China National Center for Culture Collection of Microorganisms, with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Lactiplantibacillus plantarum GXFF202402 provided by the present invention can relieve 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, it can also effectively relieve IBS symptoms by reducing inflammation, regulating the immune response, and restoring intestinal barrier function.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a Lactiplantibacillus plantarum GXFF202402 and its application in the preparation of 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 the normal anal cushion. The true pathophysiology of hemorrhoids may be the result of multiple factors, including anal cushion slippage, hyperperfusion, vascular abnormalities, tissue inflammation, and rectal intussusception. Different etiological theories have given rise to a variety of treatment methods. Hemorrhoids are also closely related to other anorectal diseases such as anal pruritus, anal fissure, and anal spasm. Although the exact pathophysiology of these diseases has not been fully elucidated, researchers have proposed that they may be related to the dysfunction of the smooth muscle of the internal anal sphincter. According to the current hypothesis, anorectal diseases are generally regarded as a direct consequence of constipation. Through an extensive literature review, a new etiological theory has been proposed, suggesting an important association between changes in the 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 global population and has become an important public health problem. IBS is characterized by recurrent abdominal pain or bloating, often accompanied by abnormal bowel movements. According to the symptom manifestations, IBS can be divided into four main types: constipation-predominant (IBS-C), diarrhea-predominant (IBS-D), mixed (IBS-M), and unclassified (IBS-U). Meta-analyses have shown that the risk of developing IBS increases 3 to 11 times after a gastroenteritis attack. Increasing evidence suggests 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 responses, brain-gut interactions, changes in the gastrointestinal microbiota, and activation of the gastrointestinal mucosal immune system in the pathogenesis of IBS.

[0004] Probiotics are a class of active microorganisms that can improve host health and play an important role in maintaining the balance of the intestinal flora, regulating immune function, and improving intestinal function. Studies have shown that oral administration of probiotics can not only effectively relieve gastrointestinal symptoms but also promote the recovery of the 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 object of the present invention is to provide a Lactiplantibacillus plantarum GXFF202402 and its application in the preparation of products for preventing and / or treating hemorrhoids. The Lactiplantibacillus plantarum GXFF202402 provided by the present invention alleviates hemorrhoids by reducing inflammation, restoring tissue integrity, and regulating the expression of inflammatory cytokines, and effectively alleviates IBS symptoms by reducing inflammation, regulating the immune response, and restoring intestinal barrier function.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) GXFF202402, the preservation number of the Lactiplantibacillus plantarum GXFF202402 is CGMCC NO.32257, and it was preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on October 18, 2024, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] In the present invention, the Lactiplantibacillus plantarum GXFF202402 is isolated from naturally fermented sour fish in Yanjiao Village, Liuzhou City, Guangxi Zhuang Autonomous Region.

[0009] The present invention also provides the application of the Lactiplantibacillus plantarum GXFF202402 described in the above technical solution in the preparation of products for preventing and / or treating hemorrhoids.

[0010] In the present invention, the Lactiplantibacillus plantarum GXFF202402 can reduce inflammation, restore tissue integrity, and regulate the expression of inflammatory cytokines.

[0011] In the present invention, the concentration of Lactiplantibacillus plantarum GXFF202402 in the product is preferably 1×10 9 CFU / mL.

[0012] The present invention also provides the application of the Lactiplantibacillus plantarum GXFF202402 described in the above technical solution in the preparation of products for alleviating or treating irritable bowel syndrome.

[0013] In the present invention, the Lactiplantibacillus plantarum GXFF202402 alleviates IBS symptoms by reducing inflammation, regulating the immune response, and restoring intestinal barrier function.

[0014] In the present invention, the concentration of Lactiplantibacillus plantarum GXFF202402 in the product is preferably 1×10 9 CFU / mL.

[0015] In the present invention, the products are all preferably drugs.

[0016] The present invention also provides a hemorrhoid treatment agent, which comprises viable bacterial cells of Lactiplantibacillus plantarum GXFF202402 described in the above technical solution, and / or fermentation metabolites.

[0017] Advantageous technical effects: The present invention provides a strain of Lactiplantibacillus plantarum GXFF202402 and its application in the preparation of products for preventing and / or treating hemorrhoids. The preservation number of Lactiplantibacillus plantarum GXFF202402 is CGMCC NO. 32257, and it was deposited at the General Microbiology Center of the China Microbial Culture Collection Center 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. Lactiplantibacillus plantarum GXFF202402 provided by the present invention can relieve hemorrhoids by reducing inflammation, restoring tissue integrity, and regulating the expression of inflammatory cytokines, etc., and can be used as a potential therapeutic agent for treating hemorrhoids. In addition, it can also effectively relieve IBS symptoms by reducing inflammation, regulating the immune response, and restoring intestinal barrier function, etc. Description of the Drawings

[0018] Figure 1 shows the colony characteristics of Lactiplantibacillus plantarum GXFF202402;

[0019] Figure 2 shows the Gram staining microscopic examination diagram of Lactiplantibacillus plantarum GXFF202402;

[0020] Figure 3 shows the body weight change of mice in Example 2;

[0021] Figure 4 shows the H&E staining results of the rectal tissues of 15-day model mice in Example 2;

[0022] Figure 5 shows the H&E staining results of the rectal tissues of 30-day model mice in Example 2;

[0023] Figure 6 shows the detection of rectal cytokines in mice of each group on the 15th day in Example 2; among them, different letters indicate significant differences, p <0.05, the same below;

[0024] Figure 7 shows the detection of rectal cytokines in mice of each group on the 30th day in Example 2;

[0025] Figure 8 shows the detection of serum cell inflammatory factors in mice of each group on the 15th day in Example 2;

[0026] Figure 9 shows the detection of serum cell inflammatory factors in mice of each group on the 30th day in Example 2;

[0027] Figure 10 Detection of inflammatory cytokine mRNA in rectal cells of mice in each group on the 15th day in Example 2;

[0028] Figure 11 Detection of inflammatory cytokine mRNA in rectal cells of mice in each group on the 30th day in Example 2;

[0029] Figure 12 Body weight change of mice in Example 3;

[0030] Figure 13 Food intake change of mice in Example 3;

[0031] Figure 14 Change in the number of fecal pellets of mice in Example 3;

[0032] Figure 15 Change in fecal water content of mice in Example 3;

[0033] Figure 16 Observation of H&E-stained pathological sections of colon tissues of male mice in Example 3;

[0034] Figure 17 Observation of H&E-stained pathological sections of colon tissues of female mice in Example 3;

[0035] Figure 18 Corticosterone content in the serum of mice in Example 3;

[0036] Figure 19 IL-1β content in the colon tissues of mice in Example 3;

[0037] Figure 20 IL-6 content in the colon tissues of mice in Example 3;

[0038] Figure 21 TNF-α content in the colon tissues of mice in Example 3;

[0039] Figure 22 IL-10 content in the colon tissues of mice in Example 3;

[0040] Figure 23 MCT content in the colon tissues of mice in Example 3;

[0041] Figure 24 PAR-2 content in the colon tissues of mice in Example 3;

[0042] Figure 25 Occludin content in the colon tissues of mice in Example 3;

[0043] Figure 26The expression level of Occludin gene in the mouse colon tissue in Example 3;

[0044] Figure 27 The expression level of PAR-2 gene in the mouse colon tissue in Example 3. Detailed implementation manners

[0045] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with examples. However, 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 can be obtained from commercial channels without special instructions; the methods used in the examples and test examples of the present invention are all conventional methods without special instructions.

[0046] Example 1 Isolation and identification of strains

[0047] 1. Experimental materials

[0048] Self-made naturally fermented sour fish from villagers in Yanjiao Village, Liuzhou City, Guangxi Zhuang Autonomous Region was collected. The fish meat was cut and placed in a 50 mL sterilized centrifuge tube, then put into a low-temperature food sampling box and taken back to the laboratory for freezing and storage in an ultra-low temperature refrigerator at -80 °C for later use.

[0049] 2. Isolation and identification of microorganisms

[0050] 2.1 Isolation and purification of microorganisms

[0051] Take 1 g of the fermented sour fish sample and crush it, add 10 times sterile physiological saline and stir. Take the upper layer of the stirred solution and dilute it stepwise to 10 -6 , then take 10 -4 , 10 -5 , 10 -6 3 gradients of 100 μL of the bacterial liquid for plate coating, culture at 37 °C for 24 - 48 h, observe and record the colony morphology. Pick different-shaped colonies on the plate for streak separation. After culturing at 37 °C for 48 h, pick different-shaped single colonies on the plate for streak separation again. Repeat this process many times until pure single colonies with consistent morphology are obtained.

[0052] 2.2 Preliminary identification of microorganisms

[0053] Pick the pure colonies on the plate and inoculate them into 5 mL of MRS liquid medium, culture at 37 °C for 24 h. The colony color is mostly white or milky white, the shape is round, the edge is neat, and the surface is moist and smooth ( Figure 1 ).

[0054] Take 1 mL of the above-mentioned bacteria-containing culture medium in a sterile centrifuge tube, centrifuge at 4000 r / min for 10 min, then discard the upper-layer culture medium. Resuspend the bacterial cell precipitate in sterile normal saline and perform Gram staining and microscopic examination. Under a 100× oil immersion microscope, the cell morphology of the lactic acid bacteria strains includes long rods, short rods, and spheres, and there is no budding reproduction ( Figure 2 ). It was preliminarily identified as lactic acid bacteria.

[0055] 2.3 Molecular identification

[0056] Inoculate the purified suspected target strain into MRS broth, culture at 37 °C for 18 h - 24 h, and then extract DNA using a bacterial genomic DNA extraction kit. Number the extracted DNA and store it at -20 °C in a freezer for later use.

[0057] Perform PCR amplification on the extracted DNA. Among them, 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 use sterile ddH 2 O to make up the system to 25 μL. And use sterile ultrapure water to replace the template DNA as a negative control. The amplification conditions are: 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 extend at 72 °C for 5 min.

[0058] Then take 5 μL of the amplification product for agarose gel electrophoresis detection. The agarose concentration is 1.2%, and the electrophoresis conditions are 110 V for 45 min. Send the successfully detected PCR product to Beijing Tsingke Biotechnology Co., Ltd. for sequencing. The sequencing results are shown as Seq_3. The successfully sequenced sequence is analyzed by alignment using the BLAST (Basic Local Alignment Search Tool) program in NCBI. After alignment, this strain is Lactiplantibacillus plantarum, named Lactiplantibacillus plantarum GXFF202402, and was deposited in the China General Microbiological Culture Collection Center on October 18, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 32257.

[0059] Seq_3:

[0060]

[0061] Example 2. Effect of Lactiplantibacillus plantarum GXFF202402 on hemorrhoids

[0062] 1. Materials and reagents: As shown in Table 1.

[0063] Table 1 Experimental materials and reagents

[0064]

[0065] 2. Instruments and equipment: As shown in Table 2.

[0066] Table 2 Instruments and equipment

[0067]

[0068] 3. Experimental methods

[0069] 3.1 Animal model

[0070] After 60 5-week-old ICR female mice (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.) were adaptively fed for one week, the mice were randomly divided into two experimental groups: the 15-day experimental group and the 30-day experimental group. Each experimental group was further divided into three groups, with 10 mice in each group: the normal group, the model group, and the Lactiplantibacillus plantarum GXFF202402 group. In the 15-day experimental group, the experimental period lasted for 15 days. During this period, the GXFF202402 group was intragastrically administered Lactiplantibacillus plantarum (1×10 9 CFU / mL) every day. On the 15th day, except for the normal group, 0.05 mL of croton oil mixture (prepared by mixing distilled water, pyridine, ether, and 6% croton oil in a ratio of 1:4:5:10) was injected 0.5 cm into the anus of the mice and withdrawn after 10 s to establish an anal swelling model. Six hours later, the anal swelling, redness, bleeding, ulcer formation, ulcer size, color change, etc. of the mice were observed. After confirming the successful establishment of the model, subsequent experiments were carried out. In the 30-day experimental group, the experimental period was 30 days, and the GXFF202402 group was intragastrically administered Lactiplantibacillus plantarum (1×10 9 CFU / mL) every day throughout the experiment. On the 15th day, the same croton oil mixture was injected according to the method of the 15-day experimental group to establish an anal swelling model. The animals were maintained on the same feeding regimen until the 30th day for subsequent experiments. The body weight was recorded every two days throughout the experiment.

[0071] 3.2 Determination of the anorectal coefficient (ARC) of mice

[0072] To calculate the anorectal coefficient (ARC), the animals and their isolated anorectal tissues (15 mm in length) were weighed. The calculation formula for ARC is:

[0073] Anorectal coefficient (ARC) = Anorectal tissue weight (mg) / Animal weight (g).

[0074] 3.3 Determination of the levels of serum cytokines in mice with rectal swelling

[0075] Place the sample in a refrigerator at 4°C for about 2 h to promote coagulation and serum separation. Subsequently, centrifuge at 4°C and 3000 rpm for 10 min using a centrifuge. After centrifugation, carefully remove the upper light yellow serum and transfer it to a sterile tube. To ensure the stability of the serum components, quickly freeze it and store it in an ultra-low temperature freezer at -80°C for subsequent biochemical analysis. Collect blood from mice in each group to prepare serum samples, and detect the contents of PGE2, VEGF, EGF, and NO in the serum of mice. The detection method refers to the instruction manual of the kit.

[0076] 3.4 Determination of the levels of cellular inflammatory factors in mice with rectal swelling

[0077] After collecting serum by the same method as above, detect the contents of IL-2, IL-4, IL-6, IL-1β, and TNF-α in the serum of mice. The detection method refers to the instruction manual of the kit.

[0078] 3.5 Hematoxylin and eosin (H&E) staining of tissues in mice with rectal swelling

[0079] Place the rectum in a 10% formalin solution for fixation for 24 h, and use H&E staining to observe the tissue damage in mice with rectal swelling. The specific steps are as follows:

[0080] (1) Fixation: Cut about 0.5 cm of the mouse rectum without feces and place it in the fixative (10% formalin) for fixation for 24 h;

[0081] (2) Embedding: After the rectum is fixed and dehydrated, embed it with paraffin;

[0082] (3) Sectioning: Cut the sections into thin slices of 0.4 μm using a paraffin slicer;

[0083] (4) Slide preparation: Place the 0.4-μm-thick sections on the pre-prepared glass slides, and then place them on a slide warmer to dry them slowly;

[0084] (5) Dewaxing: Xylene (I) 5 min - Xylene (II) 5 min - Absolute ethanol 5 min - 95% ethanol 2 min - 80% ethanol 2 min - 70% ethanol 2 min;

[0085] (6) Place the sections in hematoxylin staining solution and stain for 2 - 8 min. Rinse off the excess staining solution with tap water, then place them in the differentiation solution (1% hydrochloric acid alcohol) for 30 min, and finally soak the sections in tap water for 15 min;

[0086] (7) Soak the sections in eosin staining solution for 1 - 2 min. Rinse off the excess staining solution with tap water, and then soak the sections in tap water for 5 min;

[0087] (8) 95% ethanol solution (I) for 1 min - 95% ethanol solution (II) for 1 min - absolute ethanol (I) for 1 min - absolute ethanol (II) for 1 min - xylene carbolic acid (3:1) for 1 min - xylene (I) for 1 min - xylene (II) for 1 min. Mount the sections with neutral gum, and finally observe the sections under a microscope.

[0088] 3.6 Determination of mRNA in the anal margin tissue of mice

[0089] In this experiment, fluorescence quantitative PCR (qPCR) was used to detect the mRNA levels of related genes in the anal margin tissues of mice in each group.

[0090] (1) Extraction of total RNA from tissues

[0091] After homogenizing the anal margin tissue, add chloroform, mix well, and centrifuge at 4°C for 15 min. Take the upper supernatant and add an equal volume of isopropanol, mix well, and centrifuge at 4°C for 20 min. After discarding the supernatant, wash with 75% ethanol, centrifuge at 4°C for 15 min, discard the upper aqueous phase, let it stand for 3 - 5 min, and add 20 μL of RNase-Free water for standby.

[0092] (2) Determination of RNA concentration and purity

[0093] Dilute the RNA stock solution and RNase-Free water at a ratio of 1:49 and mix well. Measure the concentration and purity of RNA with a micro-spectrophotometer. A ratio of A260 / A280 between 1.8 - 2.0 indicates good purity.

[0094] (3) Reverse transcription of RNA into cDNA

[0095] Reverse transcribe RNA into cDNA according to the instructions of the Yeasen Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNAdigester plus) synthesis kit. Take the cDNA and measure its concentration with a micro-spectrophotometer. Dilute the cDNA concentration to 1000 ng / ml before use in qPCR amplification.

[0096] (4)PCR Amplification

[0097] 1) Amplification system: Master mix 10 μL; Forward primer 0.4 μL; Reverse primer 0.4 μL; Template (cNDA, 1000 ng / mL) 1 μL; Sterile ultrapure water 8.2 μL. After mixing the amplification system evenly, centrifuge at low speed and then amplify on a Real-Time PCR instrument.

[0098] The primer sequences are shown in Table 3.

[0099] Table 3 Primer Sequences

[0100]

[0101] 2) Amplification conditions

[0102] For all assays, after initial denaturation at 95 °C for 5 min, 40 cycles are performed at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s.

[0103] 3) Calculation of experimental results

[0104] Using β-actin as the internal reference gene, the relative mRNA expression levels of each target gene are calculated by formula 2 -ΔΔCT

[0105] 3.7 Data processing

[0106] The statistical analysis software SPSS for Windows, version 27.0, is used for data analysis. To determine the significance of differences between two means, one-way ANOVA and Duncan's test are used. ( p Values of (<0.05) are considered statistically significant. Different letters indicate significant differences between one group and other groups ( p <0.05).

[0107] 4. Results and analysis

[0108] 4.1 Results of mouse body weight changes

[0109] The croton oil mixture can induce hemorrhoids in mice, and the body weight of mice decreased significantly during the modeling process. Therefore, the alleviating effect of Lactiplantibacillus plantarum GXFF202402 on croton oil-induced hemorrhoids during the experiment can be evaluated by the changes in mouse body weight ( Figure 3 ).

[0110] ​In a 15-day mouse experimental model, the body weight of mice in the croton oil group showed an obvious downward trend compared with that of the normal group. The body weight of mice in the Lactiplantibacillus plantarum GXFF202402 group decreased more slowly, but was still lower than the normal group level. These results indicate that croton oil-induced hemorrhoids can cause weight loss in mice, and intragastric administration of Lactiplantibacillus plantarum GXFF202402 can partially alleviate the weight loss.

[0111] In a 30-day mouse experimental model, on the 15th day after modeling, the body weight of mice in the model group first decreased and then gradually increased, while the body weight of mice in the Lactiplantibacillus plantarum GXFF202402 group showed a slow decrease followed by a continuous increase and finally returned to the normal group level. These research results indicate that croton oil-induced hemorrhoids can cause a certain degree of weight loss in mice, and intragastric administration of Lactiplantibacillus plantarum GXFF202402 for 30 consecutive days can effectively alleviate this weight loss.

[0112] 4.2 Histopathological observation of mouse rectal tissue

[0113] As Figure 4 shown, in the 15-day experimental model, the anal mucosal epithelial tissue of mice in the normal group was intact and the structure was normal. In the model group, local necrosis, infiltration of inflammatory cells, submucosal edema and other phenomena were observed in the rectal mucosal epithelial tissue. In the Lactiplantibacillus plantarum GXFF202402 group, the rectal mucosal epithelial tissue was significantly improved, the glandular arrangement was more orderly, mucosal bleeding was reduced, the boundary was clearer, and the inflammatory changes were alleviated, suggesting that Lactiplantibacillus plantarum GXFF202402 has a protective effect on the rectal tissue of mice with croton oil-induced hemorrhoids.

[0114] As Figure 5 shown, in the 30-day experimental model, the anal mucosal epithelial tissue of mice in the normal group was intact and the structure was normal. In the model group, local necrosis of the rectal mucosal epithelial tissue, infiltration of inflammatory cells, severe submucosal edema, infiltration of inflammatory cells, and dilation and congestion of blood vessels were observed. In the Lactiplantibacillus plantarum GXFF202402 group, the situation was significantly improved, the mucosal layer structure was intact, mucosal bleeding was reduced, the boundary was clear, and the inflammatory cells were significantly reduced. It shows that Lactiplantibacillus plantarum GXFF202402 can effectively reduce the swelling and inflammatory reaction of the hemorrhoid mouse model induced by croton oil, promote tissue repair, and provide an important basis for the treatment application of probiotics in hemorrhoids and related diseases.

[0115] 4.3 Swelling coefficient of mouse rectal tissue

[0116] The swelling coefficients of the rectal tissues of mice in the 15-day group are shown in Table 4. It can be seen from Table 4 that compared with the normal group, the anal and rectal swelling rate of mice in the model group was significantly increased ( p <0.05), indicating that the modeling was successful. The Lactiplantibacillus group was significantly lower than the model group ( p(< 0.05), indicating that Lactobacillus plantarum GXFF202402 has a relieving effect on mice with mixed croton oil-induced hemorrhoids.

[0117] Table 4 Swelling coefficient of rectal tissues of mice in the 15-day group

[0118]

[0119] The swelling coefficient of rectal tissues 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 anal and rectal swelling rate of mice in the model group was significantly increased ( p (< 0.05), indicating that the modeling was successful. The Lactobacillus plantarum groups were all significantly lower than the model group ( p (< 0.05), proving that Lactobacillus plantarum GXFF202402 has a protective effect on mice with mixed croton oil-induced hemorrhoids, and continuous intragastric administration for 30 days has an obvious relieving effect on hemorrhoid mice.

[0120] Table 5 Swelling coefficient of rectal tissues of mice in the 30-day group

[0121]

[0122] 4.4 Determination of the levels of rectal cytokines in rectal swelling mice

[0123] The results of the levels of rectal cytokines in the rectal tissues of 15-day model mice are shown in Figure 6 . From Figure 6 it can be seen that after croton oil induction, PGE2 and NO increased, leading to an exacerbation of local inflammatory reactions, an increase in vascular permeability and dilatation, and the formation of edema, pain and congestion. With the assistance of NO, VEGF promotes angiogenesis, which may lead to the dilation of venous plexuses and the deterioration of lesions. EGF and VEGF act synergistically to promote the repair and regeneration of damaged tissues and help restore the function and structure of the diseased area. The results show that according to the detection results of the levels of PGE2 and NO cytokines, Lactobacillus plantarum GXFF202402 can reduce the inflammation in the bodies of hemorrhoid mice, and there are significant differences between the Lactobacillus plantarum GXFF202402 group and the model group ( p (< 0.05).

[0124] The results of the levels of rectal cytokines in the rectal tissues of 30-day model mice are shown in Figure 7 . From Figure 7 it can be seen that in the 30-day croton oil-induced mouse hemorrhoid model, the levels of rectal PGE2 and NO were significantly increased ( p (< 0.05), while the levels of rectal PGE2 and NO were significantly reduced after intervention with Lactobacillus plantarum GXFF202402 ( p(<0.05). The results showed that Lactiplantibacillus plantarum GXFF202402 intervention could regulate the levels of PGE2 and NO in the rectum of mice with hemorrhoid models. Lactiplantibacillus plantarum GXFF202402 reduced the levels of PGE2 and NO by alleviating the inflammatory response and tissue damage caused by hemorrhoids through anti-inflammatory, antioxidant, and immunomodulatory mechanisms.

[0125] 4.5 Determination of the levels of serum cytokines in mice with rectal swelling

[0126] The hemorrhoid model induced by croton oil in mice led to local tissue inflammation, blood vessel dilation, leukocyte infiltration, immune activation, and imbalance in the rectum. To evaluate the preventive effect of Lactiplantibacillus plantarum GXFF202402 on hemorrhoids, the serum levels of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, IL-2, and IL-4 were analyzed.

[0127] The detection of serum cytokines in mice of each group on the 15th day was as Figure 8 shown. As Figure 8 could be seen, in the croton oil-induced hemorrhoid model of mice on the 15th day, the levels of serum IL-1β, IL-6, TNF-α, IL-2, and IL-4 were significantly increased compared with the normal group ( p <0.05). After intervention with Lactiplantibacillus plantarum GXFF202402, the levels of inflammatory factors decreased significantly ( p <0.05), returning to the normal group level. These findings indicated that Lactiplantibacillus plantarum GXFF202402 intervention alleviated the inflammatory response caused by the croton oil mixture and regulated the immune response.

[0128] The detection of serum cytokines in mice of each group on the 30th day was as Figure 9 shown. As Figure 9 could be seen, in the croton oil-induced hemorrhoid model of mice on the 30th day, the levels of IL-1β, IL-6, TNF-α, IL-2, and IL-4 in the serum were significantly increased compared with the normal group ( p <0.05), and after intervention with Lactiplantibacillus plantarum GXFF202402, the above levels decreased significantly ( p <0.05), returning to the normal group level. This indicated that Lactiplantibacillus plantarum GXFF202402 reduced the cytokine levels through anti-inflammatory and immunomodulatory effects, alleviated the inflammatory response, restored immune balance, and promoted tissue healing.

[0129] 4.6 mRNA expression levels of inflammatory factors in the anal margin tissues of mice

[0130] To explore the regulatory mechanism of hemorrhoid inflammation, the mRNA levels of TNF-α and IL-6 in the anal margin tissues of hemorrhoid mice were measured.

[0131] In a 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 those in the normal group ( p <0.05) ( Figure 10 ). After intervention with Lactiplantibacillus plantarum GXFF202402, the above levels were significantly decreased ( p <0.05), returning to the normal group level. These results indicate that Lactiplantibacillus plantarum GXFF202402 can effectively prevent croton oil-induced hemorrhoids in mice.

[0132] As Figure 11 can be seen, in a 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 those in the normal group ( p <0.05). After intervention with Lactiplantibacillus plantarum GXFF202402, the TNF-α level was significantly decreased ( p <0.05), suggesting that Lactiplantibacillus plantarum GXFF202402 exerts anti-inflammatory and tissue repair effects by regulating immune function.

[0133] In summary, in this experiment, a mouse hemorrhoid model was induced by a croton oil mixture, causing tissue swelling, ulceration in the anorectum of mice, and accompanied by a large amount of inflammatory fluid exudation. After intervention with Lactiplantibacillus plantarum GXFF202402, the rectal injury in mice was significantly improved, the glandular arrangement was relatively neat, the bleeding of the mucosal tissue was reduced, the boundary was clearer, and the inflammatory changes were obvious. At the same time, this bacterium also has a certain alleviating effect on the systemic inflammatory response in mice with rectal swelling, and can reduce the expression of inflammatory factors such as pro-inflammatory factors IL-1β, IL-6, IL-4, IL-2, TNF-α, PGE2, NO, etc., thereby alleviating the rectal swelling in mice.

[0134] Example 3 Effect of Lactiplantibacillus plantarum GXFF202402 on IBS

[0135] 1. Materials and reagents: As shown in Table 6.

[0136] Table 6 Experimental materials and reagents for the effect of Lactiplantibacillus plantarum GXFF202402 on IBS

[0137]

[0138] 2. Instruments and equipment: As shown in Table 2.

[0139] 3. Experimental method

[0140] 3.1 Animal model

[0141] Forty 5-week-old C57BL / 6 mice (half male and half female, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.) were selected for the experiment. After environmental adaptation, the mice were randomly divided into 4 groups of 5 each: normal group, model group, positive drug group, and Lactiplantibacillus plantarum GXFF202402 group. The experiment lasted for 4 weeks. Before modeling, the mice were fasted for 12 h. Except for the normal group, IBS models were established in the other groups by enema with trinitrobenzenesulfonic acid (TNBS). The specific method was as follows: Using a suitable medical polyurethane catheter, 0.1 mL of TNBS was injected about 3 - 4 cm proximal to the anal verge of the mice, and the enema tube was slowly withdrawn after 1 min of retention. Subsequently, the mice were inverted for 30 s to prevent liquid reflux and ensure uniform distribution of the reagent in the intestine. The mice in the Lactiplantibacillus plantarum group were orally gavaged with Lactiplantibacillus plantarum solution (1×10 9 CFU / mL) continuously for 4 weeks from the start to the end of the experiment. The 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 change in food intake was recorded. The number of fecal pellets of the mice within 6 h was recorded every three days, and at the same time, fecal samples were collected to measure their moisture content.

[0142] 3.2 Determination of cytokines in mouse serum

[0143] The content of corticosterone (CORT) in serum was determined by enzyme-linked immunosorbent assay (ELISA). After centrifuging the serum at 3000 rpm for 10 min to separate it, the serum samples were stored in a -80 °C ultra-low temperature refrigerator for later use. The experiment was carried out strictly according to the operation instructions of the ELISA kit.

[0144] 3.3 Determination of the levels of inflammatory factors in mouse colon tissue

[0145] The contents of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) in mouse colon tissue were determined using an ELISA kit. Colon tissue samples (50 - 100 mg) were collected, chopped and transferred to a homogenization tube. The samples were homogenized in physiological 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.

[0146] 3.4 Determination of the levels of intestinal permeability factors in mouse colon tissue

[0147] The intestinal permeability-related proteins Occludin (OCLN), protease-activated receptor-2 (PAR-2), and mast cell tryptase (MCT) in mouse colon tissue were determined using an ELISA kit.

[0148] 3.5 Determination of mRNA in Mouse Colon Tissue

[0149] In this experiment, qPCR was used to detect the mRNA levels of related genes in the colon of mice in each group.

[0150] The extraction of total RNA in the tissue, the determination of RNA concentration and purity, the reverse transcription of RNA into cDNA, and PCR amplification were all the same as in 3.6 of Example 2. The primer sequences are shown in Table 7.

[0151] Table 7 Primer Sequences of Example 3

[0152]

[0153] 3.6 Data Processing: The same as 3.7 in Example 2.

[0154] 4 Results and Analysis

[0155] 4.1 Results of Mouse Body Weight Changes

[0156] TNBS has been proven to cause adverse reactions such as abdominal pain in mice. In the first week of modeling, except for the normal group, male and female mice in the other groups showed significant weight loss. Figure 12 Shows the intervention effect of Lactiplantibacillus plantarum GXFF202402 on TNBS-induced weight loss in mice. The experimental results show that after the intervention of Lactiplantibacillus plantarum and linaclotide, the body weights of male and female mice gradually recovered, and the body weights of mice in the drug group and the Lactiplantibacillus plantarum group showed an upward trend and finally approached the normal group level.

[0157] 4.2 Changes in Mouse Food Intake

[0158] As Figure 13 shown, in the first week of modeling, compared with the normal group, after TNBS modeling, the food intake of mice in the other groups decreased and showed large fluctuations. However, after intragastric intervention with the drug and Lactiplantibacillus plantarum GXFF202402, the food intake of male and female mice in each group gradually recovered, especially after the second week of intervention, it gradually approached the normal group level. It should be noted 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 its IBS symptoms. These results indicate that Lactiplantibacillus plantarum GXFF202402 can relieve the feeding behavior of modeled mice.

[0159] 4.3 Changes in the Number of Mouse Fecal Particles

[0160] As Figure 14As shown by the analysis of the number of fecal pellets in male mice, after modeling, the number of fecal pellets in the model group of mice increased rapidly and reached a peak in the early stage (about day 5 to day 10), and then gradually decreased. The peak value of the number of fecal pellets was significantly higher than that of the normal group, suggesting that intestinal dysfunction or inflammatory responses may occur in the model group after modeling. After intervention (after day 10), the number of fecal pellets in the mice of the drug group and the Lactiplantibacillus plantarum GXFF202402 group showed a downward trend and approached the level of the normal group around day 20. Compared with the model group, the decrease in the number of fecal pellets in the mice of the GXFF202402 group was more stable. The changing trend of the number of fecal pellets in female mice was similar to that in male mice, indicating that Lactiplantibacillus plantarum GXFF202402 may have the potential to regulate the intestinal flora.

[0161] 4.4 Changes in fecal water content of mice

[0162] As Figure 15 shown, after TNBS modeling, the fecal water content of the mice in the model group decreased significantly and then fluctuated greatly, 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 fluctuating trend. Intragastric intervention with the drug group and Lactiplantibacillus plantarum GXFF202402 significantly improved the fecal water content of the mice and gradually approached the level of the normal group. It is worth noting that the recovery of fecal water content in the mice of the Lactiplantibacillus plantarum GXFF202402 group was more stable and the later fluctuations were smaller, which may indicate that Lactiplantibacillus plantarum GXFF202402 has a more effective regulatory effect on the balance of the intestinal flora.

[0163] 4.5 Histopathological observation of mouse colon tissues

[0164] As Figures 16 - 17 shown, after TNBS induction, to observe the pathological lesions of mouse colon tissues and evaluate the effect of Lactiplantibacillus plantarum GXFF202402 on colon inflammation, histological examination of the colon of the modeled mice was performed using H&E staining. As shown by the experimental results, the colon tissue structures of male and female mice in the normal group were intact without signs of inflammation or injury. After TNBS modeling, obvious pathological changes occurred in the mouse colon tissues, including epithelial cell damage, abnormal crypt structure, inflammatory cell infiltration and submucosal edema, indicating that the intestine was in a state of inflammation and dysfunction. The degree of colon inflammation and injury in the mice of the drug group was significantly reduced and the tissue structure gradually recovered. The intestinal mucosal barrier function of the mice in the Lactiplantibacillus plantarum GXFF202402 group was significantly improved and the inflammatory response was reduced, indicating that Lactiplantibacillus plantarum GXFF202402 has the potential to relieve IBS symptoms and repair the intestinal mucosa. In summary, Lactiplantibacillus plantarum GXFF202402 has a significant protective effect on the colon tissues of TNBS-induced IBS mice.

[0165] 4.6 Cortisol (CORT) content in mouse serum

[0166] CORT plays a key role in the stress response, regulating intestinal motility and sensitivity and affecting the activity of the immune system. The regulatory effect of probiotics on the gut-brain axis is a known mechanism for reducing visceral hypersensitivity. Chronic stress induced 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 a persistent pain response. The IBS model significantly increases serum corticosterone levels. Lactiplantibacillus plantarum restored the corticosterone level ( Figure 18 , p <0.05), with a better restoration effect than the drug group. These findings suggest that the ability of probiotics to regulate corticosterone levels may alleviate visceral hypersensitivity symptoms.

[0167] 4.7 Expression levels of inflammatory factors in mouse colon tissue

[0168] When the intestine is attacked by inflammatory factors, intestinal epithelial cells and macrophages significantly upregulate the expression of pro-inflammatory cytokines. Conversely, the expression of pro-inflammatory cytokines also promotes colon inflammation. In contrast, the anti-inflammatory cytokine IL-10 plays a crucial role in controlling and preventing intestinal inflammation. IL-10 is thought to block macrophage metabolism and promote damaged mitophagy, thereby reducing inflammation. To evaluate the anti-inflammatory effect of Lactiplantibacillus plantarum on IBS, the study analyzed the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and the anti-inflammatory cytokine IL-10 in the colon of mice ( Figures 19 - 22 ). The results showed that TNBS disrupted the intestinal immune homeostasis of mice, significantly increasing the expression of pro-inflammatory cytokines in the colon and decreasing the anti-inflammatory cytokines ( p <0.05). In male mice, Lactiplantibacillus plantarum GXFF202402 significantly reduced the expression of IL-1β and TNF-α, restoring it to a level comparable to that 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. Lactiplantibacillus plantarum GXFF202402 significantly increased the level of IL-10 ( p <0.05), restoring it to the control group level. In female mice, Lactiplantibacillus plantarum GXFF202402 significantly reduced the expression of IL-1β and IL-6 ( p <0.05) and moderately reduced the level of TNF-α ( p >0.05), while significantly increasing the expression level of IL-10 ( p <0.05). These findings suggest that Lactiplantibacillus plantarum GXFF202402 can effectively alleviate TNBS-induced intestinal inflammation.

[0169] 4.8 Expression levels of mouse colonic permeability factors

[0170] In the gastrointestinal tract, after mast cells are activated, tryptase is released, which in turn activates PAR-2 on the surface of intestinal nerve cells, ultimately causing a continuous state of nerve excitation to feedback 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. Related studies on IBS have shown that IBS with low-grade intestinal inflammation is characterized by damage to the intestinal barrier function and decreased expression of occludin. Lactiplantibacillus plantarum has often been reported to have the function of restoring the intestinal barrier. In this study, after induction with TNBS ( Figures 23 - 25 ), the expression of MCT and PAR-2 in mouse colonic tissues was significantly increased ( p <0.05), while the expression of OCLN was decreased ( p <0.05). In male mice, Lactiplantibacillus plantarum could reduce the expression of MCT and significantly decrease the expression of PAR-2 ( p <0.05), and increase the expression of OCLN ( p <0.05). In female mice, Lactiplantibacillus plantarum could reduce the expression of MCT and significantly decrease the expression of PAR-2 ( p <0.05), and increase the expression of OCLN ( p >0.05). These findings indicate that Lactiplantibacillus plantarum GXFF202402 can reduce visceral hypersensitivity and restore the integrity of the intestinal barrier damaged by intestinal inflammation.

[0171] 4.9 Gene expression levels of mouse colonic permeability factors

[0172] As Figure 26 shown, there was no significant difference in the expression level of the Occludin gene in the colonic tissues of male and female mice ( p >0.05).

[0173] As Figure 27 shown, there was no significant difference in the expression level of the PAR-2 gene in the colonic tissues of male and female mice ( p >0.05).

[0174] In this study, through the intervention of Lactiplantibacillus plantarum GXFF202402, the intestinal mucosal inflammatory response of mice was significantly alleviated, the tissue structure was restored to integrity, and the infiltration of inflammatory cells was reduced, showing significant differences compared with 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 Lactiplantibacillus plantarum GXFF202402 could effectively inhibit the expression of pro-inflammatory factors and promote the production of the anti-inflammatory factor IL-10. Although female mice are more sensitive to intestinal inflammation and have poorer tolerance than male mice, the intervention of Lactiplantibacillus plantarum showed significant anti-inflammatory effects on both genders.

[0175] In summary, Lactiplantibacillus plantarum GXFF202402 shows the potential to relieve IBS symptoms through multiple mechanisms such as regulating the intestinal flora, enhancing the intestinal mucosal barrier function, and inhibiting intestinal inflammation. Existing studies have shown that Lactiplantibacillus plantarum plays a significant role in improving the intestinal microecological environment and alleviating IBS symptoms. With the further in-depth study of probiotics, Lactiplantibacillus plantarum is expected to become an effective method in the adjuvant treatment of IBS, providing better prognosis and quality of life for patients.

[0176] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope 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 plant lactobacillus GXFF202402 according to claim 1 in the preparation of medicines for preventing hemorrhoids.

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

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