Lactobacillus rhamnosus strain for significantly increasing number of enteric neurons and neuroglial cells and application thereof

Abstract

The application discloses a lactobacillus rhamnosus capable of significantly increasing the number of enteric neurons and neuroglial cells and an application thereof, and belongs to the field of microorganisms.The lactobacillus rhamnosus CCFM1360 has good activity, certain acid and alkali resistance and adhesion, can significantly increase the number of enteric neurons and neuroglial cells, repair intestinal barrier function, increase the content of SCFAs in colon contents, increase the number of Treg cells in mesenteric lymph nodes, reduce the expression level of inflammatory factors in colon tissues, reduce the intestinal inflammation level, and relieve the weakened intestinal motility disorder.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain of Lactobacillus rhamnosus which significantly increases the number of enteric neurons and neuroglial cells, and an application thereof, and belongs to the field of microorganisms. BACKGROUND

[0002] The enteric nervous system (ENS) is a huge neural network composed of two nerve plexuses, submucosal and myenteric, distributed in the intestinal wall. The components include enteric neurons and enteric neuroglial cells, which are widely distributed in the digestive system, have unique characteristics, complex organization connections, and diversity of nerve cell types, and can independently control and regulate the digestive and absorptive functions of the gastrointestinal tract, and are known as the second brain or the intestinal brain of the body. The ENS includes primary afferent neurons, interneurons and motor neurons, and can locally control most of the intestinal functions by regulating the absorption and secretion of the intestinal tract, vascular tension and intestinal motility. The ENS has obvious uniqueness in the performance of neurons and loops compared with the central nervous system (CNS). The normal ENS has a strong regulatory effect on the homeostasis of the intestinal tract and even the whole body. Its role is not limited to regulating the movement and secretion of the gastrointestinal tract, stabilizing the immune function of the intestinal tract, maintaining and repairing the intestinal epithelial barrier, and possibly affecting the individual's mood, appetite and behavior through the feedback loop (brain-gut axis) composed of the sympathetic and parasympathetic nervous systems and the peripheral primary sensory nerves with the central nervous system CNS. Gastrointestinal functional disorders caused by abnormal enteric nerve function not only lead to abnormal digestive function, but also cause visceral pain and emotional and behavioral abnormalities, such as irritable bowel syndrome (IBS), which seriously affects the quality of life of patients.

[0003] Enteric glia cells (EGCs) are derived from neural crest cells and are one of the main members of the ENS, mainly distributed in the ganglion of the gastrointestinal myenteric plexus and submucosal plexus. EGCs not only play a role in nutrition and support of enteric neurons, but also play an important role in regulating neuronal growth and development, neural circuit function and apoptosis, maintaining intestinal homeostasis, such as structural and functional integration of the enteric nervous system, maintaining the mucosal epithelial barrier, regulating gastrointestinal motility, nutrient uptake, secretion and regulating blood flow, participating in immune or inflammatory response. Among them, maintaining the integrity of the intestinal epithelial barrier is an important feature of EGCs, and the absence and damage of EGCs can lead to the loss of intestinal mucosal barrier integrity in rats, increasing its permeability, leading to intestinal inflammation, bleeding and necrosis. EGCs are involved in the occurrence of diseases in the digestive tract, and the number, receptor and phenotype of EGCs are abnormal in various intestinal motor dysfunction diseases such as postoperative intestinal paralysis, congenital megacolon and inflammatory bowel disease. It can sense neuronal signals and communicate with enteric neurons through intracellular calcium response and gap junction protein channels to affect intestinal motility. In addition, EGCs also have important clinical significance for diseases outside the digestive tract, such as Parkinson's disease.

[0004] The gut microbiota interacts with the central nervous system and the gut through the microbiota-enterochromaffin cell-vagal afferent signaling, and more and more evidence supports the existence of the'microbiota-gut-brain axis', proving the important role of microorganisms in regulating intestinal motility. Probiotics are a class of active microorganisms that have a positive effect on the immune, development and nutrient absorption of the human body. They colonize the digestive system of the human body in large numbers and improve the host microecological balance, and are a general term for active microorganisms that can play a positive role. In recent years, more and more studies have used prebiotics and probiotics to optimize the intestinal flora. Its regulatory effect on the intestinal flora mainly reflects the restoration of the flora and the maintenance of intestinal immune homeostasis, which can enhance the integrity of the intestinal epithelium, protect the intestinal barrier, regulate the immune system of the gastrointestinal mucosa and inhibit the growth of pathogenic bacteria. Studies have shown that the most commonly used probiotics, such as lactobacilli, bifidobacteria, saccharomyces cerevisiae and other coliform bacteria, have beneficial regulatory effects in the prevention and treatment of central nervous system diseases, obesity, diabetes, cancer, cardiovascular system diseases, malignant tumors, liver diseases and various gastrointestinal diseases. Probiotics have a positive regulatory effect on brain function and can increase the number of neurons and glial cells, and improve anxiety and depression-like behavior through the gut-brain axis. Metabolites from the fermentation of the gut microbiota, such as SCFAs or peptides, can stimulate the ENS and affect intestinal transport, while the CNS and ANS are also involved in the control of intestinal motility, all of which interact with the gut microbiota. By regulating the intestinal microbiota, probiotics can have a good regulatory effect on the ENS or CNS.

[0005] Intestinal motility dysfunction often occurs in the female population, and down-regulation of female estrogen receptor expression can cause severe intestinal nerve damage, which in turn triggers intestinal motility dysfunction. Tamoxifen citrate is an estrogen receptor inhibitor that reduces the number of intestinal neurons and glial cells by down-regulating estrogen receptor expression and triggering intestinal motility dysfunction. Previous studies have generally used loperamide hydrochloride to construct an intestinal motility dysfunction model with intestinal nerve damage. The difference between the two modeling methods is that once loperamide hydrochloride is stopped, the symptoms of intestinal motility dysfunction will disappear, while tamoxifen citrate will cause the symptoms of intestinal motility dysfunction to persist after it is stopped. Compared with loperamide, tamoxifen modeling can exacerbate intestinal inflammation and intestinal nerve damage, and is a more severe intestinal motility dysfunction model. Currently, no lactobacillus rhamnosus has been found that can be specifically used to regulate intestinal motility dysfunction caused by down-regulation of female estrogen receptor expression.

[0006] In summary, the enteric nervous system is crucial for female intestinal health, and a decrease in the number of intestinal neurons and glial cells can lead to intestinal motility dysfunction. Moreover, intestinal motility dysfunction caused by down-regulation of female estrogen receptor expression is more stubborn and difficult to treat effectively than other intestinal motility dysfunctions. There is a lack of lactobacillus rhamnosus in the prior art that can be specifically used to improve intestinal motility dysfunction caused by abnormalities in the female enteric nervous system. SUMMARY

[0007] TECHNICAL PROBLEM

[0008] The technical problem to be solved by the present application is to provide a lactobacillus rhamnosus that can significantly increase the number of intestinal neurons and glial cells, and to provide the application of the strain. The present application uses tamoxifen citrate to establish a model of intestinal motility dysfunction caused by female intestinal nerve damage, and screens for probiotics that can significantly increase the number of intestinal neurons and glial cells, which is of great significance for improving intestinal motility dysfunction caused by abnormalities in the female enteric nervous system.

[0009] TECHNICAL SCHEME

[0010] In order to solve the above technical problems, the present application provides a Lacticaseibacillus rhamnosus which can significantly increase the number of enteric neurons and neuroglial cells, repair the intestinal barrier function, effectively improve the intestinal peristalsis, shorten the intestinal transport time, increase the fecal water content, increase the number of Treg cells in the mesenteric lymph nodes, reduce the expression level of inflammatory factors in the colon tissue, and relieve the inflammation of the colon tissue, indicating that the strain can significantly repair the intestinal nerve damage and relieve the reduced power type intestinal motility disorder caused by the damage of the intestinal nerve. The present application provides a corresponding probiotic preparation, a fermented food and a functional food, which can effectively increase the number of enteric neurons and neuroglial cells and relieve the reduced power type intestinal motility disorder.

[0011] The present application provides a Lacticaseibacillus rhamnosus which was deposited in the Guangdong Microbial Culture Collection Center on November 9, 2023, and the deposit address is No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, and the deposit number is GDMCC No: 63993.

[0012] The Lacticaseibacillus rhamnosus is from a fecal sample of a female in Hulunbuir City, Inner Mongolia. The sequence obtained by sequencing is subjected to nucleic acid sequence alignment in NCBI Standard Nucleotide BLAST, and the result shows that the nucleic acid sequence similarity with the Lacticaseibacillus rhamnosus is 100%; the result shows that the strain is Lacticaseibacillus rhamnosus, which is named Lacticaseibacillus rhamnosus CCFM1360.

[0013] The Lacticaseibacillus rhamnosus CCFM1360 has the following biological characteristics:

[0014] (1) Bacterial characteristics: Gram-positive, non-spore-forming, non-motile bacteria.

[0015] (2) Colony characteristics: round, convex, smooth, and regular edge.

[0016] (3) Growth characteristics: under the condition of constant temperature at 37℃, the logarithmic phase is reached after about 18h in MRS culture medium.

[0017] (4) Strong tolerance to simulated gastrointestinal fluid.

[0018] (5) significantly increase the number of enteric neurons and glial cells, repair intestinal barrier function, increase the content of SCFAs in the colon contents, increase the number of Treg cells in the mesenteric lymph nodes, reduce the expression level of inflammatory factors in the colon tissue, reduce the level of intestinal inflammation, and relieve the weakened intestinal motility disorder.

[0019] The application also provides a microbial preparation containing the above-mentioned Lacticaseibacillus rhamnosus CCFM1360.

[0020] In an embodiment of the application, the number of viable bacteria of Lacticaseibacillus rhamnosus CCFM1360 in the microbial preparation is ≥10 8 cfu / g or 10 8 cfu / mL.

[0021] In an embodiment of the application, the microbial preparation is a powder obtained by drying a bacterial solution containing Lacticaseibacillus rhamnosus CCFM1360, and the number of viable bacteria is ≥10 8 cfu / g or 10 8 cfu / mL.

[0022] In an embodiment of the application, the drying refers to vacuum freeze-drying.

[0023] The application also provides a food containing the above-mentioned microbial preparation of Lacticaseibacillus rhamnosus CCFM1360.

[0024] In an embodiment of the application, the food is a fermented food produced by fermentation using Lacticaseibacillus rhamnosus CCFM1360, and the fermented food includes solid food, liquid food, and semi-solid food.

[0025] In an embodiment of the application, the amount of Lacticaseibacillus rhamnosus added to the food is at least 10 8 CFU / mL or 10 8 CFU / g.

[0026] In an embodiment of the application, the fermented food includes dairy products, bean products, or fruit and vegetable products.

[0027] In an embodiment of the present application, the dairy product is a fermented dairy product, including fermented milk, fermented milk beverage, cream, cheese or milk powder; the soy product includes soy milk, soy milk beverage, soy milk powder; the fruit and vegetable product includes fermented fruit and vegetable beverage or food fermented from Chinese cabbage, white radish, cucumber, beet, yellow peach or waxberry.

[0028] The present application also provides a pharmaceutical product containing the above-mentioned Lacticaseibacillus rhamnosus CCFM1360 or the above-mentioned microbial agent.

[0029] In an embodiment of the present application, the pharmaceutical product contains Lacticaseibacillus rhamnosus CCFM1360 and a pharmaceutically acceptable carrier.

[0030] In an embodiment of the present application, the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

[0031] In an embodiment of the present application, the dosage form of the pharmaceutical product includes granules, capsules, tablets, pills or oral liquids.

[0032] In an embodiment of the present application, the amount of Lacticaseibacillus rhamnosus CCFM1360 added to the pharmaceutical product is at least 10 8 CFU / mL or 10 8 CFU / g.

[0033] The present application also provides a health product for lubricating the intestines and defecating, containing the above-mentioned Lacticaseibacillus rhamnosus CCFM1360 or the above-mentioned microbial agent.

[0034] The present application also provides the use of the above-mentioned Lacticaseibacillus rhamnosus CCFM1360 or the above-mentioned microbial agent in the preparation of a functional product having at least one of the following functions:

[0035] (a) increasing the number of enteric glial cells and repairing damaged enteric nervous system;

[0036] (b) increasing the number of enteric neurons in the colon and increasing the expression of PGP9.5 in the colon tissue;

[0037] (c) repairing the intestinal barrier function and increasing the expression of Claudin5, ZO1 and MUC1 genes;

[0038] (d) increasing the content of SCFAs in the colon contents;

[0039] (e) reducing the level of inflammation in the colon tissue and increasing the number of Treg cells in the mesenteric lymph nodes;

[0040] (f) relieving hypokinetic intestinal motility disorders.

[0041] Beneficial effects

[0042] (1) The Lactobacillus rhamnosus CCFM1360 of the present application has good activity, can significantly increase the number of intestinal neurons to 2 times that of the model group, increase the number of glial cells to 2.75 times that of the model group, repair the intestinal barrier function, restore the expression levels of Claudin5, ZO1 and MUC1 to the level comparable to the normal control group, increase the content of SCFAs in the colon contents to the level comparable to the normal control group, increase the number of Treg cells in the mesenteric lymph nodes, reduce the expression level of inflammatory factors in the colon tissue, restore the level of intestinal inflammation, shorten the intestinal transit time to 1 / 2 of the normal control group, and relieve hypokinetic intestinal motility disorders.

[0043] (2) The present application can be considered as a drug that significantly increases the number of intestinal neurons and glial cells, and can also be applied in pharmaceuticals or some fermented foods and functional foods, thereby widely exerting its effects and having very valuable application prospects.

[0044] Preservation of biological materials

[0045] A strain of Lactobacillus rhamnosus CCFM1360, which is taxonomically named Lactobacillus rhamnosus, has been preserved in the Guangdong Microbial Culture Collection Center on November 9, 2023, with the preservation number GDMCC No: 63993 and the preservation address being No. 59 Building, 5th Floor, 100 Middle Liangma Street, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 : The expression of S100β, a marker of intestinal glial cells in the colon tissue of female rats treated with tamoxifen after intervention of Lactobacillus rhamnosus CCFM1360 strain, is shown in the schematic diagram (A) S100β immunofluorescence staining; (B) relative area of S100β positive expression.

[0047] Figure 2Figure 6: The expression of colon enteric neuron marker PGP9.5 in female rats treated with tamoxifen after intervention of Lacticaseibacillus rhamnosus CCFM1360 strain (A) PGP9.5 immunohistochemical staining; (B) Relative area of PGP9.5 positive expression.

[0048] Figure 3 Figure 7: The transcriptional level of colon mucin MUC1 (A), tight junction protein Claudin5 (B) and ZO1 (C) in the colon tissue of female rats treated with tamoxifen after intervention of Lacticaseibacillus rhamnosus CCFM1360 strain.

[0049] Figure 4 Figure 8: The content of short-chain fatty acids in the colon contents of female rats treated with tamoxifen after intervention of Lacticaseibacillus rhamnosus CCFM1360 strain.

[0050] Figure 5 Figure 9: The proportion of Treg cells in the mesenteric lymph nodes of the colon tissue of female rats treated with tamoxifen after intervention of Lacticaseibacillus rhamnosus CCFM1360 strain and the concentration of serum inflammatory factors.

[0051] Figure 6 Figure 10: The related indicators (intestinal transit time, fecal water content) of the remission of intestinal motility disorder in female rats treated with tamoxifen after intervention of Lacticaseibacillus rhamnosus CCFM1360 strain.

[0052] Note: The symbols above the column chart represent the significance level of the data, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001 (compared with the M group), # represents p < 0.05, ## represents p < 0.01, ### represents p < 0.001, and #### represents p < 0.0001 (compared with the NC group). DETAILED DESCRIPTION

[0053] Female intestinal motility disorder:

[0054] Female intestinal motility disorder refers to a disease caused by reduced expression of estrogen receptors during the menstrual cycle, pregnancy, premenopause, and menopause, leading to intestinal inflammation and damage to the enteric nervous system, and further causing irregular intestinal motility rhythm. It is mainly divided into two types: hypomotility (constipation) and hypermotility (diarrhea).

[0055] Female enteric nervous damage:

[0056] Female enteric nervous damage refers to the symptoms of reduced number of enteric neurons and enteric glial cells caused by reduced expression of estrogen receptors during the menstrual cycle, pregnancy, pre-menopause, and menopause, accompanied by inflammatory response and intestinal barrier damage. Enteric nervous damage can lead to abnormal digestive function and cause intestinal motility dysfunction.

[0057] Brief introduction of tamoxifen citrate model:

[0058] Tamoxifen citrate is an estrogen receptor inhibitor that can reduce estrogen receptor expression and cause intestinal motility dysfunction. Compared with the loperamide hydrochloride model, the difference between the two modeling methods is that once loperamide hydrochloride is stopped, the symptoms of intestinal motility dysfunction will disappear, while tamoxifen citrate will cause the symptoms of intestinal motility dysfunction to persist after stopping administration. Compared with loperamide, tamoxifen modeling will exacerbate intestinal inflammation and enteric nervous damage, and is a more severe model of intestinal motility dysfunction.

[0059] In this experiment, an animal model of estrogen receptor expression down-regulation leading to enteric nervous damage was established by tamoxifen to find probiotics that can significantly increase the number of enteric neurons and glial cells.

[0060] Example 1: Obtaining Lactobacillus rhamnosus CCFM1360

[0061] 1. Isolation and screening of Lactobacillus rhamnosus:

[0062] (l) A fecal sample from a female in Hulunbuir, Inner Mongolia was collected using a disposable sterile fecal sampler and incubated at 37°C for 72 hours in an anaerobic environment. The colony morphology was observed and recorded, and the colonies were streaked and purified. The resulting colonies were subjected to Gram staining after incubation at 37°C for 48 hours in MRS liquid medium. Gram-negative and Gram-positive coccus strains were discarded, and Gram-positive bacilli were selected.

[0063] (2) After catalase analysis, discard the catalase-positive strains and retain the catalase-negative strains.

[0064] 2. Molecular biology identification of Lactobacillus rhamnosus:

[0065] (l) Single bacterial genome extraction: the catalase-negative strain screened in step 1 was cultured overnight, 1 mL of the overnight cultured bacterial suspension was taken in a 1.5 mL centrifuge tube, centrifuged at 10000 rpm for 2 min, and the supernatant was discarded to obtain the bacterial body; the bacterial body was washed with 1 mL of sterile water, then centrifuged at 10000 rpm for 2 min, and the supernatant was discarded to obtain the bacterial body; 200 μL of SDS lysis solution was added, and the bacterial body was incubated at 80°C for 30 min; 200 μL of phenol-chloroform solution was added to the bacterial lysis solution, wherein the composition and volume ratio of the phenol-chloroform solution were Tris-saturated phenol: chloroform: isopropyl alcohol = 25:24:1, and after inversion and mixing, centrifugation was performed at 12000 rpm for 5-10 min, and 200 μL of supernatant was taken; 400 μL of ice ethanol or ice isopropyl alcohol was added to 200 uL of supernatant, and it was placed at -20°C for 1 h, centrifuged at 12000 rpm for 5-10 min, and the supernatant was discarded; 500 μL of 70% (volume percentage) ice ethanol was added to resuspend the precipitate, centrifuged at 12000 rpm for 1-3 min, and the supernatant was discarded; 50 μL of ddH2O was added to redissolve the precipitate for PCR;

[0066] (2) 16S rDNA PCR:

[0067] A. Bacterial 16S rDNA 50 μL PCR reaction system: 10x Taq buffer, 5 μL; dNTP, 5 μL; 27F, 0.5 μL; 1492R, 0.5 μL; Taq enzyme, 0.5 μL; template, 0.5 μL; ddH2O, 38 μL.

[0068] B. PCR conditions: 95°C for 5 min; 95°C for 10 s; 55°C for 30 s; 72°C for 30 s; step 2-4 30x; 72°C for 5 min; 12°C for 2 min;

[0069] C. Prepare 1% agarose gel, then mix the PCR product with 10000x loading buffer, load 2 μL, run at 120V for 30 min, and then perform gel imaging;

[0070] D. The obtained PCR product was sent to a professional sequencing company, and the obtained sequencing results were searched and similarity aligned in GeneBank using BLAST, and identified as Lactobacillus rhamnosus, named Lactobacillus rhamnosus CCFM1360, and preserved at -80°C for standby.

[0071] Example 2: Lactobacillus rhamnosus CCFM1360 increases the number of enteric glial cells in the colon tissue of female rats treated with tamoxifen

[0072] The specific steps are as follows:

[0073] (1) Preparation of Lacticaseibacillus rhamnosus bacterial suspension

[0074] The experimental strains were preserved in a -80°C ultra-low temperature refrigerator under the protection of 30% glycerol. Before use, all strains were streaked on MRS plates and single colonies were selected for sequencing identification to determine the purity of the strains and the correctness of the species. After confirming that the strain information was correct, the strains were activated in MRS liquid medium for 4 times with a 2% inoculation amount, so that the strains gradually recovered the activity. After incubation in a constant temperature and humidity incubator / anaerobic workstation at 37°C for 18 hours, the bacterial liquid was centrifuged at 6000g for 15 min and the bacterial body was collected. The bacterial slurry was washed with sterile physiological saline for three times to remove the culture medium in the bacterial body. Finally, the bacterial body was resuspended with 30% glycerol and stored in a -80°C ultra-low temperature refrigerator for standby. Before use, gradient dilution method was used for viable count. When used, the bacterial liquid was diluted with new sterile physiological saline to make the final concentration of viable bacteria in the used bacterial liquid 1×10 10 CFU / mL.

[0075] (2) 28 healthy 6-week-old female SPF Sprague Dawley rats were adapted to the environment for 1 week and randomly divided into 4 groups: normal group, model group, intervention group (Lacticaseibacillus rhamnosus FJSWX1L3, Lacticaseibacillus rhamnosus CCFM1360), each group containing 7 rats, and the dose of gavage bacterial suspension was 1×10 10 CFU / mL.

[0076] The experimental animal grouping and treatment method are shown in Table 1:

[0077] Table 1 Experimental animal grouping

[0078]

[0079] After the experiment, the mice were sacrificed, and the colon tissues of the mice were taken for detection. Immunofluorescence staining was used to characterize the number of enteric glial cells in the colon tissue. The specific method is as follows: the embedded tissue section was placed on a glass slide, melted in a constant temperature box at 60°C for 1 h, immersed in xylene for deparaffinization, gradient hydrated with different concentrations of ethanol (100%, 95%, 85%, 75%), and then washed with distilled water. The boiled method was used for antigen repair (after boiling the sodium citrate-EDTA antigen repair solution, the glass slide was submerged and placed in it, and the heating was continued to maintain boiling for 10 min. After the solution was naturally cooled, the glass slide was taken out), and then washed with distilled water and PBST. An appropriate amount of inactivated enzyme reagent was added to each section, and after 15 min of incubation at room temperature in the dark, it was washed, and an appropriate amount of blocking solution was added to the wet box and blocked for 30 min. After blocking, the blocking solution was discarded, and the primary antibody was added and incubated in the wet box at 4°C overnight. The next day, it was warmed at room temperature for 40 min, washed with PBST, and then the secondary antibody was added and incubated at room temperature in the dark for 45 min. After washing with PBST, the water around the tissue was carefully absorbed with absorbent paper, an appropriate amount of DAPI-containing anti-fluorescence quencher was added, and the glass slide was sealed with nail polish. The red fluorescence was observed under a fluorescence microscope.

[0080] The glial cells of the mucosa mainly participate in the epithelial barrier function, and the glial cells in the ganglion mainly act on nerve repair, closely interact with neurons, support the differentiation of these cells and glial cells, and participate in the occurrence and formation of nerves. The number of glial cells can well reflect the health status of enteric nerves. S100β is a calcium-binding protein mainly present in neural glial cells. Using S100β to label enteric glial cells can characterize the number of glial cells in enteric nerves Figure 1 (A) The red fluorescence). The immunofluorescence results are shown in Figure 1 (B), which shows that the density of enteric glial cells decreases by 1 / 3 (P<0.05) after tamoxifen treatment, indicating that the model rats have enteric nerve damage. Lactobacillus rhamnosus CCFM1360 has a significant effect on increasing the number of enteric glial cells (P<0.001), which is increased to 2 times that of the model group (model group: 0.63, CCFM1360 group: 1.26), which can restore the enteric nerve health of enteric nerve damaged rats.

[0081] Example 3: Lactobacillus rhamnosus CCFM1360 increases the number of enteric neurons in the colon tissue of female rats treated with tamoxifen

[0082] The experimental animal grouping, modeling and treatment method are the same as in Example 2.

[0083] After the experiment, the mice were killed, and the colon tissues of the mice were taken, and the expression amount of PGP9.5 in the colon tissues was quantified by immunohistochemical method. The specific method is as follows: the embedded tissue section is placed on a glass slide, melted in a constant temperature box at 60℃ for 1 h, immersed in xylene for deparaffinization, gradient hydrated with different concentrations of ethanol (100%, 95%, 85%, 75%), and then washed with distilled water. The boiled method is used for antigen repair (after boiling the sodium citrate-EDTA antigen repair solution, the glass slide is submerged and placed in it, and the heating is continued to maintain boiling for 10 min. After the solution is naturally cooled, the glass slide is taken out), and then washed with distilled water and PBST again. An appropriate amount of inactivated enzyme reagent is added to each section, and after 15 min of incubation at room temperature in the dark, it is washed, and an appropriate amount of blocking solution is added to the wet box and blocked for 30 min. After blocking, the blocking solution is discarded, and the primary antibody is added and incubated in the wet box at 4℃ overnight. The next day, it is warmed at room temperature for 40 min, washed with PBST, and then the secondary antibody is added and incubated at room temperature for 30 min. After washing, DAB color development is performed for about 5 min, and the color development is terminated with distilled water. After staining with hematoxylin, the cells are rapidly stained with 1% hydrochloric acid-ethanol to decolorize and reverse blue, and then gradient dehydrated with different concentrations of ethanol (75%, 85%, 95%, 100%) and xylene twice. After the tissue periphery is wiped dry with a water-absorbing paper, neutral gum is added dropwise to seal the section.

[0084] The number of PGP9.5-reactive cells in the colon region of patients with intestinal motility disorder was significantly reduced, and the number of enteric neurons detected in the submucosal plexus of patients with intractable intestinal motility disorder was also similarly reduced. Abnormal remodeling of intestinal microbiota can restore intestinal function and stimulate intestinal neurogenesis to increase the number of neurons, so PGP9.5 is selected as an index for detecting and quantifying enteric neurons. It can be seen from Figure 2 It can be seen that the number of colon neurons of the rats decreased by 1 / 2 (P<0.05) after modeling. After the rats were given different strains by gavage, the number of neurons was significantly increased by R. rhamnosus CCFM1360, which was 2.75 times that of the model group (model group: 0.46, R. rhamnosus CCFM1360 group: 1.25), indicating that R. rhamnosus CCFM1360 can restore intestinal function by repairing enteric neurons.

[0085] Example 4: Rhamnosus CCFM1360 can repair intestinal barrier function of female rats treated with tamoxifen

[0086] The experimental animals were grouped, modeled, and treated as in Example 2.

[0087] About 20 mg of rat colon stored at -80°C was used to extract total RNA from rat colon using the TRIzol method: After the cut rat colon tissue was cut into small pieces and placed in an enzyme-free centrifuge tube with high-temperature enzyme-killed zirconium oxide beads, 1 mL of TRIzol lysis solution was added and a high-throughput crusher was used for thorough crushing. 200 μL of chloroform was added and vortexed for 30 s to emulsify it thoroughly, and then it was placed at 4°C for 5 min, and then centrifuged at 12000 g for 15 min at 4°C. Carefully pipette 400 μL of supernatant into a new enzyme-free centrifuge tube, add an equal volume of pre-cooled isopropanol, mix well by inverting several times, and place at 4°C for 10 min, then centrifuge at 12000 g for 15 min at 4°C. After discarding the supernatant, 1 mL of pre-cooled DEPC water was added to wash the extracted RNA twice along the tube wall, and the RNA was suspended by tapping the tube bottom. After 3-5 min, DEPC water was added to dissolve the precipitate. Take 1 μL of RNA sample for ultramicro spectrophotometer to detect the concentration, purity and integrity of RNA. According to the instructions provided by the Novozyme kit, total RNA was used as a template to synthesize cDNA, and real-time fluorescent quantitative PCR was performed. According to the instructions of HiScript III RTSuperMix for qPCR, the system was prepared and the RT-qPCR program was run. Reaction system: 2x ChamQ Universal SYBR qPCR Master Mix 5 μL; forward and reverse primers (10 μM) 1 μL each; cDNA 1 μL; ddH2O 2 μL. Reaction program: pre-denaturation 95°C for 10 s; amplification 95°C for 10 s, 57°C for 30 s, 40 cycles; melting curve 95°C for 15 s, 60°C for 60 s, 95°C for 15 s.

[0088] Real-time fluorescent quantitative PCR (RT-qPCR) was used to determine the transcription level of the target gene in rat colon tissue. The primer sequences of the rat target genes were searched on the NCBI website ( https: / / www.ncbi.nlm.nih.gov / ) and synthesized by Shanghai Biosciences Biotechnology Service Co., Ltd. The specific primer information is shown in the table below.

[0089] Table 2 Primer sequences of target genes

[0090]

[0091] Claudin5 and ZO1 regulate cell permeability, mediate protein-protein interactions, and connect the actin cytoskeleton, which is important in regulating the intestinal mucosal barrier. MUC1 is a member of the mucin family and maintains the normal mechanical barrier function of the intestine as a "lubricant" for the intestine.

[0092] The mRNA levels of Claudin5 and ZO1 in the model group were greatly reduced (P<0.01), and the expression of Claudin5 and ZO1 in the colon was significantly improved by Lactobacillus rhamnosus CCFM1360, so that the expression of Claudin5 and ZO1 was restored to a level comparable to that of the normal control group, thereby improving the permeability of the intestinal tract, repairing the mechanical barrier of the intestinal tract, and further relieving intestinal motility disorders. Tamoxifen significantly reduced the transcriptional expression of MUC1, as shown in Figure 3 The results show that Lactobacillus rhamnosus FJSWX1L3 and CCFM1360 can significantly promote the expression of MUC1 in the colon, and Lactobacillus rhamnosus CCFM1360 restores the expression of MUC1 to 1.2 times that of the normal control group, which is better than Lactobacillus rhamnosus FJSWX1L3. In summary, tamoxifen administration causes slight pathological damage to the colon, and probiotic intervention can repair the damage to the intestinal mucus layer to some extent, and Lactobacillus rhamnosus CCFM1360 has the best repair effect on the intestinal barrier.

[0093] Example 5: Lactobacillus rhamnosus CCFM1360 can increase the content of SCFAs in the colon contents of female rats treated with tamoxifen

[0094] The experimental animals were grouped, modeled and treated as in Example 2. The detection method of SCFAs in feces is as follows:

[0095] (1) Sample preparation

[0096] The feces collected the day before the end of the experiment were vacuum freeze-dried to remove water, and a certain amount was accurately weighed in a 1.5 mL EP tube and its weight was recorded; 500 μL of saturated NaCl solution was added, and after soaking for 30 min, homogenate was broken. After mixing, 40 μL of 10% sulfuric acid aqueous solution was added to the homogenate. After vortexing for 30 s, 1 mL of ether was added to each sample in a fume hood using a 1 mL syringe. After mixing thoroughly, the sample was centrifuged at 15000 rpm for 15 min. The supernatant was transferred to an EP tube containing 0.3 g of anhydrous sodium sulfate. Centrifuged again at 15000 rpm for 15 min, and the supernatant was carefully aspirated into a gas-phase sample bottle for detection.

[0097] (2) Detection condition setting

[0098] Short-chain fatty acids in the sample were detected using GC-MS with an Rtx-Wax column (30 m long, 25 μm inner diameter). Helium was used as the carrier gas, and the gas flow rate was set to 2 mL / min. The sample injection volume was 1 μL, and the injection temperature was 240 °C. The column temperature program was as follows: 100 °C to 140 °C, 7.5 °C / min, 5.33 min; 140 °C to 200 °C, 60 °C / min, 1 min; 200 °C held for 3 min.

[0099] (3) Preparation of mixed standards and standard curve

[0100] Six short-chain fatty acids in the sample were quantitatively calculated using the external standard method. 10 μL of each of the following fatty acids were taken and diluted to 1000 μL with diethyl ether, and mixed thoroughly. 100 μL of the mixture was then diluted to 1000 μL with diethyl ether. Then, 200 μL, 100 μL, 50 μL, 25 μL, 15 μL, and 10 μL of this mixture were taken and diluted to 1000 μL with diethyl ether to prepare standard mixtures of different concentrations.

[0101] SCFAs are the main products of absorption by colonic epithelial cells, providing energy for bacterial fermentation in the colon. They can accelerate colonic motility by stimulating intestinal epithelial cell growth and fluid secretion. Furthermore, they can protect the functional intestinal barrier by increasing tight junction protein expression and regulating the activity of the gut microbiota and immune cells. Targeted metabolomics analysis of colonic contents using GC-MS, such as... Figure 4 As shown, tamoxifen-induced intestinal motility disorders significantly reduced SCFA concentrations. Acetic acid can protect the functional barrier of the gastrointestinal mucosa. Both probiotic interventions upregulated acetic acid, but to different degrees. *Lactobacillus rhamnosus* CCFM1360 showed a more significant effect, upregulating acetic acid by (112.45±31.82)%. *Lactobacillus rhamnosus* CCFM1360 restored propionic acid, butyric acid, and valerate levels to levels comparable to the normal control group; *Lactobacillus rhamnosus* CCFM1360 restored isobutyric acid and isovaleric acid levels to 1.2 times that of the normal control group. Compared to *Lactobacillus rhamnosus* FJSWX1L3, *Lactobacillus rhamnosus* CCFM1360 had a greater impact on short-chain fatty acids, therefore, *Lactobacillus rhamnosus* CCFM1360 is more effective in increasing the abundance of microorganisms producing short-chain fatty acids.

[0102] Example 6: Lactobacillus rhamnosus CCFM1360 can increase the number of Treg cells in the mesenteric lymph nodes of female rats treated with tamoxifen.

[0103] The methods for grouping, modeling, and processing experimental animals are the same as in Example 2.

[0104] The rat mesenteric lymph node Treg cell detection method is as follows: after the experiment, take the mesenteric lymph node (MLN), grind it with a syringe core in pre-cooled PBS solution, filter it through a 200 mesh screen, and prepare a single cell suspension. Centrifuge at 300g for 5 min, discard the supernatant, resuspend the cell pellet with flow cytometry buffer, and adjust the cell concentration to 2x10 8 cells / mL. Staining step: add FITC-labeled anti-rat CD4 antibody (1.25 μg / mL) and APC-labeled anti-rat CD25 antibody (0.6 μg / mL) to the suspension, incubate at room temperature for 30 min for cell surface staining; then add Foxp3 Fixation / Permeabilization fixing and permeabilizing solution, incubate at room temperature for 1 h in the dark; finally, add 0.5 μg of PE-labeled anti-rat / mouse Foxp3 antibody, incubate at room temperature for 1 h for nuclear staining, and PE-labeled anti-rat IgG2a antibody as the isotype control of Foxp3. After staining, resuspend in flow cytometry buffer and detect on the machine.

[0105] Rat intestinal cytokine detection method: take the colon tissue preserved at -80°C, remove the adipose tissue, add pre-cooled sterile PBS solution at a weight ratio of 1:9 into a 1.5 mL centrifuge tube, cut it into small pieces, add sterilized zirconium beads, use a high-throughput crusher to crush into a homogenate, then centrifuge at 6000 r / min at 4°C for 10 min, and transfer the supernatant to a new centrifuge tube. According to the manufacturer's instructions, detect the IL-6 content in the colon by enzyme-linked immunosorbent assay (ELISA) kit.

[0106] An important energy source for intestinal epithelium is to participate in immune regulation by regulating the pro-inflammatory activity of neutrophils and macrophages, and to induce Treg cell activation. The proportion of Treg cells in the mesenteric lymph nodes of female rats with intestinal motility disorders was evaluated by flow cytometry, and intracellular FACS staining was performed for Foxp3, as shown in Figure 5 The model group reduced the proportion of Treg cells in the mesenteric lymph nodes and increased the expression of the pro-inflammatory factor IL-6 in the serum of rats. Lactobacillus rhamnosus CCFM1360 can significantly increase the Treg cells in the mesenteric lymph nodes and reduce the expression of the pro-inflammatory factor IL-6 in the serum, so that the proportion of Treg cells and the expression of IL-6 return to a level comparable to that of the normal control group. It is speculated that CCFM1360 can restore normal signaling to improve intestinal inflammation.

[0107] Example 7: Lactobacillus rhamnosus CCFM1360 can significantly alleviate the symptoms of intestinal motility disorders in female rats with intestinal motility disorders

[0108] The grouping, modeling, and treatment methods for experimental animals were the same as in Example 2. Changes in intestinal motility-related indicators in rats were monitored during probiotic gavage, including small intestinal propulsion rate, intestinal transit time, and fecal water content.

[0109] (1) Fecal moisture content

[0110] During the experiment, cessation feces were collected from rats the day before sacrifice, after gavage administration. All rats were placed individually in clean cages according to their groups, and fresh feces were collected and placed in 5 mL centrifuge tubes. After collection, the rats were returned to their respective cages. The feces were weighed and then freeze-dried to remove moisture. The water content of the rat feces was calculated using the following formula:

[0111] Fecal moisture content (%) = (fecal wet weight - fecal dry weight) / fecal wet weight × 100%;

[0112] (2) Intestinal transit time

[0113] Mix gum arabic powder and water at a 1:10 ratio until homogeneous. Heat on an induction cooker until transparent, stirring constantly. Then add 10% (w / v) activated charcoal powder, stir, and boil until homogeneous. After cooling, store the solution at 4°C. The intestinal transit time of rats was measured the morning before the end of the experiment. To ensure accuracy, rats were fasted overnight but allowed free access to water before the measurement. During the measurement, each rat was gavaged with 1 mL of the above-mentioned ink, and the gavage time was recorded. The rats' defecation status was closely monitored. The time difference between the first excreted feces containing activated charcoal and the first recorded time was the rat's intestinal transit time.

[0114] like Figure 6 As shown, after modeling, compared with the normal group, the total intestinal transit time of rats in the model group was prolonged by approximately (0.61±0.24) times. In contrast, both *Lactobacillus rhamnosus* FJSWX1L3 and CCFM1360 in the intervention group significantly shortened the intestinal transit time, with *Lactobacillus rhamnosus* CCFM1360 reducing the intestinal transit time to half that of the normal control group. Fecal water content is another important indicator for assessing intestinal motility disorders. Patients with intestinal motility disorders typically have dry, hard feces lacking moisture, leading to difficulty in defecation. After three weeks of intervention, both *Lactobacillus rhamnosus* strains, FJSWX1L3 and CCFM1360, showed good effects in increasing fecal water content (P<0.05), indicating that both strains can effectively alleviate intestinal motility disorders.

[0115] Therefore, it can be seen from the results that Lactobacillus rhamnosus CCFM1360 significantly increases the number of enteric neurons and glial cells, repairs the intestinal barrier function, increases the content of SCFAs in the colon contents, increases the number of Treg cells in the mesenteric lymph nodes, reduces the expression level of inflammatory factors in the colon tissue, reduces the level of intestinal inflammation, and relieves the reduced motility type intestinal motility disorder.

[0116] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A Lactobacillus rhamnosus (Lactobacillus casei) strain, Lacticaseibacillus rhamnosus ) CCFM 1360, characterized in that, The Lacticaseibacillus rhamnosus CCFM1360 was preserved in Guangdong Microbial Culture Collection Center on November 9, 2023, and the address of the preservation center is No. 100, Martyrs' Avenue, Guangzhou, China, and the preservation number is GDMCC No: 63993.

2. A microbial inoculant containing the Lacticaseibacillus rhamnosus CCFM1360 of claim 1.

3. A food product, characterized by, The food contains the Lacticaseibacillus rhamnosus CCFM1360 of claim 1 or the microbial inoculant of claim 2.

4. The food product as described in claim 3, characterized in that, The food includes fermented food.

5. The food product according to claim 3 or 4, wherein The amount of Lactobacillus rhamnosus CCFM1360 added to the food product is at least 10 8 CFU / mL or 10 8 CFU / g.

6. A medicine, characterized in that, The pharmaceutical product contains the Lacticaseibacillus rhamnosus CCFM1360 of claim 1 or the microbial inoculant of claim 2.

7. The pharmaceutical product according to claim 6, characterized in that The dosage form of the pharmaceutical product includes granules, capsules, tablets, pills, or oral liquids.

8. The pharmaceutical product according to claim 6 or 7, characterized in that The amount of Lactobacillus rhamnosus CCFM1360 added in the pharmaceutical product is at least 10 8 CFU / mL or 10 8 CFU / g.

9. A health care product for promoting intestinal peristalsis and defecation, characterized by comprising the composition according to any one of claims 1 to 8. The health product contains the Lacticaseibacillus rhamnosus CCFM1360 of claim 1 or the microbial inoculant of claim 2.

10. Use of the Lacticaseibacillus rhamnosus CCFM1360 of claim 1 or the microbial inoculant of claim 2 in the preparation of a functional product having at least one of the following functions: (a) increasing the number of enteric glial cells and repairing damaged enteric nervous system; (b) increasing the number of enteric neurons in the colon and increasing the expression of PGP9.5 in colon tissue; (c) repairing intestinal barrier function and increasing the expression of Claudin5, ZO1, and MUC1 genes; (d) increasing the content of SCFAs in the colon contents; (e) reducing inflammation in colon tissue and increasing the number of Treg cells in mesenteric lymph nodes; (f) relieving hypokinetic intestinal motility disorders; The product is a pharmaceutical product for treating intestinal motility disorders. The product is a pharmaceutical product for treating intestinal motility disorders.

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