Lactobacillus paracasei for repairing enteric nerve and application thereof

Abstract

The application discloses a lactobacillus paracasei for repairing intestinal nerves and an application thereof, and belongs to the field of microorganisms. The lactobacillus paracasei CCFM1321 provided by the application can significantly increase the number of intestinal nerve glial cells, improve the level of glial cell-derived neurotrophic factor GDNF and the expression of its receptor GFR alpha 1 / RET, and repair the damaged intestinal nervous system of mice. The lactobacillus paracasei can down-regulate the over-activation of Toll-like receptor 2 (TLR2), reduce the expression of inhibitory neurotransmitters, increase the expression of serotonin 5-HT and its receptor 5-HT 2B , and has the effect of protecting the intestinal nervous system. Moreover, the lactobacillus paracasei can reduce the inflammation level in the colon tissue caused by the damage of intestinal nerves, improve the pathological damage of the colon tissue, and relieve the intestinal motility disorder. Therefore, a more personalized treatment scheme can be adopted for gastrointestinal diseases caused by the abnormal intestinal nervous system, and the application prospect is considerable.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus paracasei that repairs intestinal nerves and its applications, belonging to the field of microbiology. Background Technology

[0002] The gastrointestinal tract plays a vital role in maintaining the host's normal physiological activities, such as the transport of food and waste, the digestion and absorption of nutrients, the secretion of signaling molecules and antimicrobial substances, maintaining the integrity of the intestinal barrier, maintaining the balance of gut microbiota, and preventing the invasion of pathogens. The regulation of these key processes mainly depends on the enteric nervous system (ENS), which is the core of the regulation of gastrointestinal digestive and defense functions.

[0003] The enteric nervous system (ENS) is composed of enteric neurons and numerous enteric glial cells (EGCs). It can autonomously regulate intestinal homeostasis independently of the central nervous system, hence its nickname "the brain of the gastrointestinal tract" or "the second brain of the body." EGCs are an important component of the ENS, playing a role in supporting, nourishing, and protecting enteric neurons, regulating intestinal motility, and maintaining intestinal homeostasis. The active signaling mechanisms between EGCs and enteric neurons regulate gastrointestinal reflexes: Under physiological conditions, EGCs can secrete various neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), nitrosoglutathione, and transforming growth factor-β, which have protective effects on enteric nerves and intestinal epithelial cells. Among these, the GDNF-RET signaling pathway is crucial for normal ENS development. RET is a transmembrane tyrosine kinase that is activated when GDNF binds to its co-receptor, the GDNF family receptor (GFRα1). GDNF-GFRα1-RET signaling is essential for the survival, proliferation, and migration of ENS progenitor cells. There is bidirectional communication between EGCs and immune cells. EGCs can respond to immune regulatory signals, such as cytokines, bacteria, and neurotransmitters in the extracellular environment. Glial cell line-derived neurotrophic factor (GDNF) secreted by EGCs through autocrine or paracrine regulation can inhibit apoptosis, reduce the release of pro-inflammatory cytokines, exert immunosuppressive and anti-inflammatory effects, and participate in the function of the immune barrier. Therefore, the neuro-immunomodulatory role of EGCs plays an important role in gastrointestinal function, and impairment of any one or more of these functions can lead to widespread and significant enteric neuropathy. For example, intractable constipation caused by long-term use of stimulant laxatives (sennosides, emodin, etc.) – laxative colon – shows severe enteric nerve damage histopathologically, mainly manifested as loss of enteric glial cells and functional impairment, accompanied by degenerative changes in enteric neurons. Some studies have also observed colonic mucosal lesions.

[0004] Degenerative changes in the enteric nervous system (ENS) can lead to gastrointestinal dysfunction and consequently, intestinal motility disorders. Intestinal motility disorders are classified into hypomotility and hypermotility types, clinically manifesting as constipation and diarrhea. However, current research on the etiology and mechanisms of diagnosis and treatment of ENS abnormalities is incomplete. Potential therapeutic targets for ENS degeneration include: increasing protective substances, such as increasing certain neurotrophic factors (GDNF) or 5-HT4 receptor agonists (procapride) to protect the ENS; and regulating the gut microbiota, as gut microbiota have been shown to have a profound impact on the development and maintenance of the ENS. The ENS in mice lacking gut microbiota is immature. Studies have shown that in germ-free mice, colonization-induced ENS maturation depends on 5-HT4 receptor (5-HT4R) signaling; inhibiting endogenous 5-HT4 or blocking 5-HT4R can prevent this pairing process. Furthermore, treatment of germ-free mice with 5-HT4R agonists promotes the differentiation and maturation of enteric nerve cells. Gut microbes may also regulate the development and function of the ENS through microbial pattern recognition receptors (such as TLR2 and TLR4). EGCs can respond differentially to stimulation by probiotics (Lactobacillus parasiticus F19) and harmful bacteria (Escherichia coli), which also suggests that EGCs may be the main target cells of the gut microbiota.

[0005] Therefore, in-depth research into the interactions and roles between microorganisms and EGCs, and revealing the connections between symbiotic microorganisms and the host's nervous and immune systems, will help elucidate their pathogenesis and pathogenicity, and provide new treatment strategies for gastrointestinal diseases. Summary of the Invention

[0006] Technical issues

[0007] The technical problem to be solved by the present invention is to provide a strain of Lactobacillus paracasei that repairs the enteric nerve by regulating the GDNF / RET signaling pathway, and to provide the application of this strain.

[0008] Technical solution

[0009] To address the aforementioned technical problems, this invention provides a strain of Lactobacillus paracasei (… Lacticaseibacillus paracasei This strain can effectively repair the enteric nervous system. Compared to strain CCFM1164, this strain also shows higher levels of enteric glial cell markers (…). GFAP and S100β The expression of glial cell-derived neurotrophic factor (GDNF) was upregulated, and the content of GDNF increased by 38.25%. The level of inflammation and colonic pathological damage were also significantly improved, indicating that this strain can repair the enteric nervous system and alleviate various adverse effects caused by enteric nerve damage, thus helping patients with enteric nervous system abnormalities.

[0010] This invention provides a strain of Lactobacillus paracasei (Lacticaseibacillus paracasei The Lactobacillus paracasei was deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 63717.

[0011] The Lactobacillus paracasei ( Lacticaseibacillus paracasei The strain was found in a fecal sample from a girl in Jinhua City, Zhejiang Province. Sequencing analysis of the sequence and alignment of the sequenced data with NCBI Standard Nucleotide BLAST showed a 100% similarity to the nucleic acid sequence of *Lactobacillus paracasei*. Therefore, the strain was identified as *Lactobacillus paracasei* and named *Lactobacillus paracasei*. Lacticaseibacillus paracasei (CCFM1321)

[0012] The Lactobacillus paracasei CCFM1321 has the following biological characteristics:

[0013] (1) Bacterial characteristics: Gram-positive, non-spore-forming, non-motile bacteria.

[0014] (2) Colony characteristics: round, raised, smooth, with neat edges.

[0015] (3) Growth characteristics: Under constant temperature of 37℃, it reaches the end of the logarithmic phase after about 18 h of culture in MRS medium.

[0016] (4) It has a strong tolerance to simulated gastrointestinal fluid.

[0017] (5) Significantly increases the number of enteric glial cells in colonic tissue, increases the content or expression of glial cell-derived neurotrophic factors and their receptors in colonic tissue, downregulates excessive activation of TLR2, reduces the expression of inhibitory neurotransmitters, and increases serotonin and its receptor 5-HT. 2B It can express and improve pathological damage to colonic tissue, reduce intestinal inflammation levels, and alleviate hypomotility-related intestinal motility disorders.

[0018] The present invention also provides a solution containing the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1321 microbial preparation.

[0019] The present invention also provides a medicine containing the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1321 or the above-mentioned microbial agents.

[0020] In one embodiment of the invention, the pharmaceutical product comprises Lactobacillus paracasei CCFM1321 and a pharmaceutically permissible carrier.

[0021] In one embodiment of the present invention, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0022] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquid.

[0023] In one embodiment of the present invention, the *Lactobacillus paracasei* ( Lacticaseibacillus paracasei The minimum addition level of CCFM1321 to the drug is 10. 8 CFU / mL or 10 8 CFU / g.

[0024] The present invention also provides the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei The use of CCFM1321 or the above-mentioned microbial agents in the preparation of medicaments for relieving intestinal nerve damage, characterized in that the relief of intestinal damage includes at least one of the following:

[0025] (a) Increase the number of enteric glial cells and repair the damaged enteric nervous system;

[0026] (b) Increase the level of glial cell-derived neurotrophic factor (GDNF) and the expression of its receptor GFRα1 / RET;

[0027] (c) Downregulation of Toll-like receptor 2 (TLR2) overactivation and reduction of neuronal nitric oxide synthase (nNOS) expression;

[0028] (d) Increase serotonin (5-HT) and its receptor 5-HT 2B The expression;

[0029] (e) Reduces inflammation levels in colon tissue and improves pathological damage to colon tissue;

[0030] (f) Relieve intestinal motility disorders caused by weakened motility.

[0031] The present invention also provides Lactobacillus paracasei (… Lacticaseibacillus paracasei CCFM1321 bacterial agent.

[0032] In one embodiment of the present invention, the bacterial agent contains Lactobacillus paracasei ( Lacticaseibacillus paracasei The number of live bacteria in CCFM1321 is ≥10. 8 cfu / g or 10 8 cfu / mL.

[0033] In one embodiment of the present invention, the bacterial agent is a mixture containing Lactobacillus paracasei (… Lacticaseibacillus paracaseiThe number of viable bacteria obtained by drying the bacterial solution of CCFM1321 is ≥10. 8 cfu / g or 10 8 Powder with cfu / mL.

[0034] In one embodiment of the present invention, the drying refers to vacuum freeze drying.

[0035] Beneficial effects

[0036] (1) The Lactobacillus paracasei GDMCC No: 63717 of the present invention has better activity. Compared with the model group, the CCFM1321 intervention group can enhance the gene expression of enteric glial cell marker protein in colon tissue. S100 β The expression level was significantly increased by 49.98% (P<0.05), the content of glial cell-derived neurotrophic factor in colon tissue increased by 185.66% (P<0.01) and the expression level of its RET receptor increased by 48.68% (P<0.01), the overactivation of Toll-like receptor 2 (TLR2) was downregulated by 43.15% (p<0.01), the expression of neuronal nitric oxide synthase (nNOS) decreased by 70.21% (p<0.01), and the expression level of serotonin (5-HT) increased by 151.35% (p<0.05). This reduced the level of inflammation in colon tissue, improved the pathological damage of colon tissue, and could alleviate intestinal motility disorders caused by weakened motility. Importantly, *Lactobacillus paracasei* CCFM1321 showed significantly better results than prucalopride in terms of EGC count, GDNF content, and RET receptor expression. Compared to CCFM1164, it significantly increased GDNF content by 38.25%, with a more significant increase in EGC count. Therefore, this strain exhibits a more pronounced effect on the repair of enteric nerves.

[0037] (2) This invention can be regarded as a drug for relieving or treating intestinal motility disorders with weakened motility. It can also be applied in pharmaceuticals, thereby exerting its effects widely and having very valuable application prospects.

[0038] Preservation of biological materials

[0039] A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1321, its taxonomic name is: Lacticaseibacillus paracasei It was deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63717. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology. Attached Figure Description

[0040] Figure 1 Lactobacillus paracasei ( LacticaseibacillusEffects of paracasei) CCFM1321 on the enteric nervous system, (a) relative expression level of S100β; (b) relative expression level of GFAP.

[0041] Figure 2 Lactobacillus paracasei ( Lacticaseibacillus paracasei The effects of CCFM1321 on the GDNF / GFRα1 / RET signaling pathway: (a) GDNF concentration; (c) relative expression level of GFRα1; (d) relative expression level of RET.

[0042] Figure 3 Lactobacillus paracasei ( Lacticaseibacillus paracasei The effects of CCFM1321 on TLR2 and nNOS: (a) relative expression level of TLR2; (b) relative expression level of nNOS.

[0043] Figure 4 Lactobacillus paracasei ( Lacticaseibacillus paracasei The effects of CCFM1321 on 5-HT and 5-HT2B, (a) relative expression level of 5-HT; (b) 5-HT 2B Relative expression level.

[0044] Figure 5 Lactobacillus paracasei ( Lacticaseibacillus paracasei Effects of CCFM1321 on inflammatory factors and intestinal mechanical barrier: (a) relative expression of IL-1β; (b) relative expression of IL-6; (c) H&E staining of colon tissue.

[0045] Figure 6 Lactobacillus paracasei ( Lacticaseibacillus paracasei The effects of CCFM1321 on intestinal motility: (a) time to first black stool; (b) number of stool particles in 5 hours; (c) small intestinal propulsion rate; (d) stool water content. Detailed Implementation

[0046] The male C57BL / 6J mice used in the following examples were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0047] The strain information involved in the following examples is as follows:

[0048] Lactobacillus paracasei ( Lacticaseibacillus paracasei (CCFM1321, categorized as follows) Lacticaseibacillus paracasei It was deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 63717. The deposit address is Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0049] Lactobacillus paracasei ( Lacticaseibacillus paracaseiCCFM1164, its taxonomic name is: Lacticaseibacillus paracasei It has been published in patent CN112877260A, with accession number GDMCC No: 61479.

[0050] The culture media involved in the following examples are as follows:

[0051] MRS liquid culture medium: 10g beef extract; 10g tryptone; 5g yeast extract; 20g glucose; 5g anhydrous sodium acetate; 0.1g MgSO4·7H2O; 0.05g MnSO4·H2O; 2g diammonium hydrogen citrate; 2.6g K2HPO4·3H2O; 1mL Tween 80; adjust pH to 6.8±0.2; bring volume to 1L. Autoclave at 115℃ for 20min.

[0052] MRS solid medium: 2% agar powder is added to the MRS liquid medium.

[0053] Preparation of Lactobacillus paracasei suspension in the following examples

[0054] Lactobacillus paracasei CCFM1321 and Lactobacillus paracasei CCFM1164 were inoculated into MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. The obtained single colonies were then inoculated into MRS liquid medium and cultured at 37°C for 24 h to activate them.

[0055] After activation for three generations, the bacterial culture was inoculated into 1 L of MRS liquid medium at a 2% (v / v) inoculation rate. After shaking to mix, the culture was incubated at 37 °C for 18 h. The culture was then centrifuged at 8000 g / min at 4 °C for 10 min. After discarding the supernatant, the cells were washed twice with sterile physiological saline, and centrifuged again under the same conditions. After discarding the supernatant, the cells were resuspended in 10% sterile defatted emulsion to prepare a final concentration of 5 × 10⁻⁶ cells / mL. 9 CFU / mL Lactobacillus paracasei CCFM1321 and CCFM1164 bacterial suspensions were obtained to prepare the bacterial suspensions for gavage. These suspensions were then frozen at -80°C for one week.

[0056] Before conducting animal experiments, the frozen bacterial culture was removed from the freezer, and the number of viable bacteria was determined using the plate spread method at the initial stage and after one week of freezing. The order of magnitude of the initial and one-week viable bacterial counts did not change, indicating that freezing the bacterial culture did not affect the experiment and it could be used for animal experiments.

[0057] The following embodiments involve S100β / GFAP Gene, GFRα1 / RET Gene, TLR2 / nNOS Genes and IL-1β / IL-6 Methods for detecting gene expression levels

[0058] Real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine... S100β / GFAP Gene, GFRα1 / RET Gene, TLR2 / nNOS Genes and IL-1β / IL-6 To determine gene expression levels, RNA must first be extracted from fresh tissue. The specific method is as follows:

[0059] 0.2g of fresh colon tissue obtained from mouse dissection was repeatedly ground in a mortar (180℃, 4 h high-temperature enzyme inactivation) with liquid nitrogen. Then, 1 mL of TrizoL reagent was added to the mortar, and grinding continued until the liquid was basically clear. The mixture was then collected into a 1.5 mL enzyme-free centrifuge tube and allowed to stand at room temperature for 15 min. 200 μL of chloroform solution was added to the centrifuge tube, and the mixture was gently shaken for 15 s. The mixture was allowed to stand at room temperature for 10 min, and then centrifuged at 4℃ and 12000 r / min for 15 min. 600 μL of the colorless upper aqueous phase was transferred to another enzyme-free centrifuge tube, and 500 μL of isopropanol was added. Invert the tube to mix thoroughly, let stand at room temperature for 10 min, then centrifuge at 12000 r / min for 10 min at 4℃. Discard the supernatant, leaving the white precipitate formed at the bottom of the centrifuge tube containing RNA. Add 1 mL of 75% ethanol solution prepared with DEPC water, vortex to resuspend, and centrifuge at 7500 r / min for 5 min at 4℃. Discard the supernatant and allow to evaporate and dry at room temperature. Add 30 μL of RNase-free water to the dried RNA. After the RNA dissolves, determine the RNA concentration and purity using Nanodrop, and assess the RNA quality by agarose gel electrophoresis. Using the extracted total RNA as a template, reverse transcribe cDNA according to the instructions of the Comvita HiFiScript gDNA RemovaL RT MasterMix reverse transcription kit and store at -20℃.

[0060] mice S100β / GFAP Gene, GFRα1 / RET Gene, TLR2 / nNOS Gene, IL-1β / IL-6 Genes and internal reference genes GAPDH Gene primers are shown in Table 1.

[0061] Table 1 Primer Sequences

[0062]

[0063] RT-qPCR reaction system and conditions:

[0064] PCR amplification was performed using the Bio-Rad® CFX96™ Real-Time PCR instrument, and the fluorescence signal was read.

[0065] The RT-qPCR reaction system is as follows:

[0066] Table 2 RT-qPCR reaction system

[0067]

[0068] The RT-qPCR reaction conditions are as follows:

[0069] The temperature was set at 95℃ for 30 seconds, followed by cycles of 95℃ for 10 seconds and 60℃ for 30 seconds, for a total of 40 cycles. The GAPDH gene was used as an internal reference, and the results were analyzed using CFX96Manager software.

[0070] The following examples illustrate the methods for detecting GDNF content in colon tissue.

[0071] The expression level of GDNF in colon tissue was quantified using ELISA. The specific method was as follows: Colon tissue was rinsed with pre-cooled PBS to remove residual blood and surrounding adipose tissue. After weighing, the tissue was minced. The minced tissue was then mixed with PBS solution at a weight-to-volume ratio of 1:9 using a high-throughput tissue homogenizer. Finally, the homogenate was centrifuged at 5000×g for 5-10 minutes, and the supernatant was collected for analysis. The experiment was conducted according to the instructions of the corresponding kit, and the GDNF level in the tissue was calculated based on the standard curve.

[0072] Example 1: Lactobacillus paracasei ( Lacticaseibacillus paracasei Obtaining CCFM1321

[0073] 1. Isolation and screening of Lactobacillus paracasei:

[0074] (l) A fecal sample was collected from a girl in Jinhua City, Zhejiang Province using a disposable sterile fecal collection device and incubated at 37°C for 72 hours under anaerobic conditions. The colony morphology was observed and recorded, and colonies were picked and streaked for purification. The colonies were then incubated in MRS liquid medium at 37°C for 48 hours. The obtained colonies were Gram-stained, and the colony morphology was recorded. Gram-negative bacterial strains and Gram-positive cocci were discarded from the colonies, and Gram-positive bacilli were selected.

[0075] (2) After catalase analysis, discard catalase-positive strains and retain catalase-negative strains.

[0076] 2. Molecular biological identification of Lactobacillus paracasei:

[0077] (l) Single-cell genome extraction: The catalase-negative strains screened in step 1 were cultured overnight. 1 mL of the overnight bacterial suspension was placed in a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm for 2 min. The supernatant was discarded to obtain bacterial cells. The bacterial cells were washed with 1 mL of sterile water and centrifuged at 10,000 rpm for 2 min. The supernatant was discarded to obtain bacterial cells. 200 μL of SDS lysis buffer was added, and the mixture was incubated at 80℃ for 30 min. 200 μL of phenol-chloroform solution was added to the bacterial lysis buffer. The composition and volume ratio of the phenol-chloroform solution were Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1. After inverting and mixing, the mixture was centrifuged at 12,000 rpm for 5-10 min. 200 μL of the supernatant was collected. 400 μL of ice-cold ethanol or ice-cold isopropanol was added to 200 μL of the supernatant. The mixture was incubated at -20℃ for 1 h and centrifuged at 12,000 rpm for 5-10 min. The supernatant was discarded. 500 μL of SDS lysis buffer was added to the supernatant. Resuspend the precipitate in 70% (v / v) ice-cold ethanol, centrifuge at 12000 rpm for 1-3 min, discard the supernatant; dry in a 60℃ oven or air dry; redissolve the precipitate in 50 μL ddH2O for PCR preparation.

[0078] (2) 16S rDNA PCR:

[0079] A. Bacterial 16S rDNA 50μL PCR reaction system: 10×Taq buffer, 5μL; dNTP, 5μL; 27F, 0.5μL; 1492R, 0.5μL; Taq enzyme, 0.5μL; template, 0.5μL; ddH2O, 38μL.

[0080] B. PCR conditions: 95℃ for 5 min; 95℃ for 10 s; 55℃ for 30 s; 72℃ for 30 s; step 2-4 30×; 72℃ for 5 min; 12℃ for 2 min.

[0081] C. Prepare a 1% agarose gel, then mix the PCR product with 10000× loading buffer, load 2 μL, run at 120V for 30 min, and then perform gel imaging.

[0082] D. The obtained PCR product was sent to a professional sequencing company. The sequencing results were compared with those obtained by searching and similarity analysis in GeneBank using BLAST. It was identified as Lactobacillus paracasei and named Lactobacillus paracasei. Lacticaseibacillus paracasei CCFM1321, stored at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 63717, stored at -80℃ for future use.

[0083] Example 2: Lactobacillus paracasei CCFM1321 increases the number of enteric glial cells in the colon tissue of mice with enteric nerve damage.

[0084] The specific steps are as follows:

[0085] (1) Lactobacillus paracasei ( Lacticaseibacillus paracasei Preparation of bacterial suspension

[0086] After the Lactobacillus paracasei CCFM1321 strain was taken out of the -80℃ freezer, it was streaked on MRS solid medium and cultured at 37℃ for 48h. Single colonies were picked and cultured on MRS liquid medium at 37℃ for 24h to prepare seed culture.

[0087] The prepared seed culture was inoculated into a new MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37°C for 18 h. The culture was repeated for another generation in the same manner to prepare the fermentation broth of Lactobacillus paracasei CCFM1321.

[0088] The prepared Lactobacillus paracasei CCFM1321 fermentation broth was then centrifuged at 8000 r / min and 4℃ for 10 min, and then resuspended in a 10% (w / w) skim milk aqueous solution to obtain a bacterial suspension for animal experiments.

[0089] (2) Thirty healthy male C57BL / 6J mice aged 8 weeks were taken and acclimatized to the environment for 1 week. They were then randomly divided into 5 groups: control group, model group, prucalopride group (positive drug group), Lactobacillus paracasei CCFM1164 group (positive bacteria group) (disclosed in patent CN112877260A), and Lactobacillus paracasei CCFM1321 group. Each group contained 6 mice. The dose of bacterial suspension administered by gavage was 5 × 10⁻⁶. 9 CFU / mL, administered via gavage starting at 9 AM every morning, 0.2 mL each time.

[0090] The grouping and treatment methods for experimental animals are shown in Table 5:

[0091] Table 3 Grouping of experimental animals

[0092]

[0093] After the experiment, characteristic proteins of mouse midgut glial cells were used. S100 β and GFAP Gene expression levels are used to characterize the number of glial cells in the enteric nervous system.

[0094] Glial cells in the mucosa primarily participate in epithelial barrier function, while glial cells within ganglia mainly function in nerve repair, interacting closely with neurons and supporting the differentiation of these cells and glial cells, participating in neurogenesis and formation. The number of glial cells is a good indicator of the health of the enteric nervous system. S100β and GFAP are characteristic proteins of glial cells, and their levels can reflect the number of glial cells. Figure 1 It can be seen that the model group mice S100 β and GFAP The expression levels decreased by 49.96% and 43.79% respectively compared to the control group (p<0.01), indicating enteric nerve damage in the model mice, and the enteric nerve damage model was successfully established. After gavage administration of CCFM1321, the expression levels of marker protein genes... S100 β and GFAP The expression levels increased by 49.98% and 60.67% respectively compared to the model group (p<0.05). GFAP The expression level was increased by 25.95% compared with the CCFM1164 group, indicating that the Lactobacillus paracasei group CCFM1321 participated in enteric nerve reconstruction and could restore the enteric nerve health of mice with damaged enteric nerves.

[0095] Example 3: Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1321 can increase the content of glial cell-derived neurotrophic factor (GDNF) in the colonic tissue of mice with enteric nerve damage.

[0096] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The expression level of GDNF in colon tissue was quantified using ELISA. The specific method is as follows:

[0097] The colon tissue was rinsed with pre-cooled PBS to remove residual blood and surrounding adipose tissue. After weighing, the tissue was minced. The minced tissue was then mixed with PBS solution at a weight-to-volume ratio of 1:9 using a high-throughput tissue homogenizer. The homogenate was then centrifuged at 5000×g for 5-10 minutes. The supernatant was collected for analysis. Experiments were performed according to the instructions of the corresponding kit, and GDNF in the colon tissue was calculated based on the standard curve. Results are as follows: Figure 2 As shown in a.

[0098] GDNF is a polypeptide neurotrophic factor that has nutritional and protective effects on damaged nerve cells. Its expression is reduced in degenerated neurons, indicating neuronal damage. Figure 2The results showed that the GDNF level in the model group mice with enteric nerve damage (20.16 pg / mg) was 54.28% lower than that in the control group (44.09 pg / mg) (p<0.01), indicating enteric nerve damage. The GDNF level in the prucalopride group (27.53 pg / mg) was 36.56% higher than that in the model group, and the GDNF level in the CCFM1164 group (41.65 pg / mg) was 106.56% higher than that in the model group (p<0.05). After gavage administration of CCFM1321 (57.59 pg / mg), the GDNF level was significantly higher than that in the model group by 185.66% (p<0.001). The GDNF level in the CCFM1321 group was 38.25% higher than that in the CCFM1164 group and 109.19% higher than that in the prucalopride group.

[0099] Lactobacillus paracasei CCFM1321 nourishes the enteric nerves and restores normal enteric nerve function by increasing GDNF expression.

[0100] Example 4: Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1321 can increase the expression level of GDNF downstream receptor GFRα1 / RET in the colon tissue of mice with enteric nerve damage.

[0101] The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 2. Specific methods are as follows:

[0102] Real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine GFRα1 and RET Gene expression levels. Glial cell line-derived neurotrophic factor (GDNF) is a ligand that activates several signaling pathways via the co-receptor GDNF family receptor α-1 (GFRα1) and the receptor tyrosine kinase “RET,” pathways crucial for the development and maintenance of multiple neuronal populations. Figure 2 b / c indicates that mice with enteric nerve damage... GFRα1 Gene expression levels were significantly reduced by 45.92% compared to the control group (p<0.01). RET Gene expression levels were significantly reduced by 58.93% compared to the control group (p<0.001), indicating abnormalities in the GDNF / GFRα1 / RET signaling pathway and impaired enteric nervous system function in mice with enteric nerve damage. Following oral administration of CCFM1321, GFRα 1 Genes and RET Gene expression levels increased by 49.36% and 48.68% respectively compared to the model group (p<0.01), approaching the blank control group, and were superior to the CCFM1164 group and the positive drug group. Among them, the CCFM1321 group was significantly better than the CCFM1164 group.RET Gene expression levels increased by 23.42%.

[0103] It has been reported that the signaling emitted by the GDNF / GFRα1 / RET ternary complex plays a crucial role in the self-renewal of the enteric nervous system. Therefore, it is inferred that the CCFM1321 strain's repair of the enteric nervous system may depend on the GDNF / GFRα1 / RET signaling pathway.

[0104] Example 5: Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1321 modulates the overactivation of TLR2 in the colon tissue of mice with enteric nerve damage and reduces the expression of neuronal nitric oxide synthase (nNOS).

[0105] The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 2. Specific methods are as follows:

[0106] Real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine TLR2 and nNOS Gene expression levels. Studies have shown that TLR2 activation can promote the proliferation of nitric neurons and has a regulatory effect on intestinal motility. Figure 3 It can be seen that mice with damaged enteric nerves TLR2 Gene expression levels increased significantly by 44.59% compared to the control group (p<0.05). nNOS Gene expression levels increased by 131.42% compared to the control group (p<0.01), indicating that the model mice exhibited TLR2 overactivation, leading to excessive release of inhibitory neurotransmitters and toxic effects on the enteric nervous system. Following oral administration of CCFM1321, [the following text appears to be incomplete and requires further context: "will..."] TLR2 The overexpression of the gene was downregulated by 43.15% (p<0.01), and the expression of neuronal nitric oxide synthase was reduced by 70.21% (p<0.001).

[0107] It is speculated that the CCFM1321 strain's repair of enteric nerves may be related to the TLR2-nNOS signaling pathway.

[0108] Example 6: Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1321 enhances the colonic tissue of mice with enteric nerve damage. 5-HT and its receptors 5-HT 2B expression

[0109] The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 2. Specific methods are as follows:

[0110] Real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine 5-HT and 5-HT 2BGene expression levels.

[0111] Serotonin (5-HT) is primarily released by enterochromaffin cells and exerts its effects by acting on specific receptors. Specifically, 5-HT can exert its effects by acting on 5-HT receptors. 2B This activates acetylcholine or calcitonin gene-related peptide, thereby increasing proximal smooth muscle contraction, 5-HT 2B It can also regulate the number of Cajal-interstitial cells, thereby regulating intestinal peristalsis. Results are as follows... Figure 4 As shown, mice with enteric nerve damage 5-HT Gene expression levels were significantly reduced by 65.39% compared to the control group (p<0.05). 5-HT 2B Gene expression levels were significantly reduced by 30.11% compared to the control group (p<0.01), indicating a decrease in excitatory neurotransmitters in the colonic tissue of mice with enteric nerve damage. Following oral administration of CCFM1321, 5-HT Genes and 5-HT 2B Gene expression levels increased by 151.35% (p<0.05) and 48.49% compared to the model group, and by 44.53% and 47.01% compared to the CCFM1164 group, respectively, approaching the blank control group and superior to the positive drug group.

[0112] Therefore, the results show that *Lactobacillus paracasei* CCFM1321 can promote the growth of bacteria in colonic tissue. 5-HT and 5-HT 2B Gene expression is used to repair the enteric nervous system.

[0113] Example 7: Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1321 reduced inflammation levels and colonic tissue pathological damage caused by enteric nerve injury in mice.

[0114] The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 2. Specific methods are as follows:

[0115] Real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine IL-1β and IL-6Gene expression levels were characterized using H&E-stained mouse colon tissue sections to assess pathological damage in mouse colon tissue. Fresh tissue was collected after mouse dissection and soaked in paraformaldehyde fixative, then rinsed overnight. Samples were dehydrated sequentially with 70%, 80%, and 90% ethanol solutions for 30 minutes each, followed by two 20-minute immersions in 95% and 100% ethanol solutions. The samples were then immersed in a mixture of equal parts pure ethanol and xylene for 15 minutes, followed by 15 minutes of pre-xylene washing and 15 minutes of post-xylene washing until clear. Finally, the samples were immersed in a 1:1 mixture of xylene and paraffin for 15 minutes, and then in paraffin I and paraffin II for 50-60 minutes each to remove the clearing agent. The treated samples were then embedded, sectioned, spread, baked, H&E-stained, and mounted.

[0116] IL-1β and IL-6 possess strong pro-inflammatory activity, inducing various pro-inflammatory mediators and ultimately leading to widespread inflammatory events. Results are as follows... Figure 5 As shown in a / b, both pro-inflammatory factors were significantly elevated in the model group compared to the control group (p<0.01), indicating intestinal inflammation in mice with enteric nerve damage. After gavage administration of CCFM1321, IL-1β and IL-6 Gene expression levels were significantly lower in the mouse colon tissue compared to the model group (p<0.05). Observation of histopathological sections of mouse colon tissue, such as... Figure 5 As shown in Figure c, compared with the control group, the colonic tissue of mice in the model group showed a thinner mucus layer, shorter crypt structures, fewer goblet cells, and inflammatory infiltration. However, after gavage administration of Lactobacillus paracasei CCFM1321, the mucus layer in the colonic tissue of mice thickened, the boundaries of goblet cells became clearer, and the smooth muscle structure was more intact. After gavage administration of prucalopride and Lactobacillus paracasei CCFM1164, the mucus layer thickness of mice increased, but the increase was not as significant as with CCFM1321; the goblet cells and crypt structures became clearer, and inflammatory infiltration was reduced.

[0117] Example 8: Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1321 alleviates symptoms of intestinal motility disorders in mice caused by enteric nerve damage.

[0118] The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 2. Specific methods are as follows:

[0119] After gavage in week 16, mice were placed individually in cages lined with absorbent paper, and their feces were collected and weighed to obtain the wet weight. After freeze-drying, the wet weight was obtained. The water content of the feces was calculated using the following formula.

[0120] Fecal moisture content = (fecal wet weight - fecal dry weight) / fecal wet weight × 100%

[0121] Each mouse was given 0.2 mL of ink by gavage. The time of the first black stool and the number of black stools excreted within 5 hours were recorded from the start of gavage.

[0122] Before sacrifice, each mouse was given 0.2 mL of ink by gavage. After 30 minutes, the mice were sacrificed and dissected. The upper part of the small intestine from the lower end of the pylorus to the cecum was cut off. The total length of the small intestine was measured as the "total length of the small intestine", and the length from the pylorus to the front of the ink was measured as the "ink propulsion length". The propulsion rate of the small intestine was calculated according to the following formula.

[0123] Small intestine propulsion rate = (Ink propulsion length (cm)) / (Total small intestine length (cm)) × 100%

[0124] Results for fecal water content, time to first black stool, number of fecal pellets in 5 hours, and small intestinal propulsion rate are as follows: Figure 6 As shown in the figure, compared with the control group, the fecal water content of mice after modeling decreased by 10.98% (p<0.01), the time to first black stool was prolonged to 1.38 times that of the blank control group (p<0.01), and the number of fecal particles within 5 hours decreased by 30.26% (p<0.01), while the small intestinal propulsion rate did not change significantly, indicating that the model mice have colonic transit disorder caused by enteric nerve damage. Compared with the model group, gavage treatment with Lactobacillus paracasei CCFM1321 shortened the time to first black stool in mice with enteric nerve damage to 70.38% of the model group (p<0.05), and the number of fecal particles within 5 hours increased by 66.67% compared with the model group (p<0.01), enhancing the colonic peristalsis of mice with enteric nerve damage, and the effect was better than that of the drug prucalopride group and the positive strain control group. This indicates that gavage treatment with Lactobacillus paracasei CCFM1321 can alleviate the symptoms of intestinal motility disorder in mice caused by enteric nerve damage.

[0125] Therefore, the results show that Lactobacillus paracasei CCFM1321 can effectively repair the damaged enteric nervous system, improve intestinal inflammation and colonic pathological damage caused by enteric nerve damage, and alleviate intestinal motility disorders caused by enteric nerve damage.

[0126] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1321, characterized in that, The Lactobacillus paracasei CCFM1321 was deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 63717.

2. A microbial inoculum containing Lactobacillus paracasei as described in claim 1.

3. A pharmaceutical product containing Lactobacillus paracasei as described in claim 1 or the microbial agent as described in claim 2.

4. The medicine as described in claim 3, characterized in that, The dosage forms of the medicine include granules, capsules, tablets, pills, or oral liquids.

5. The medicine as described in claim 3 or 4, characterized in that, The amount of *Lactobacillus paracasei* added to the drug is at least 10. 8 CFU / mL or 10 8 CFU / g.

6. The use of the *Lactobacillus paracasei* of claim 1 or the microbial agent of claim 2 in the preparation of a medicament for relieving intestinal nerve damage, characterized in that, The relief of intestinal damage includes at least one of the following: (a) Increase the number of enteric glial cells and repair the damaged enteric nervous system; (b) Increase the level of glial cell-derived neurotrophic factor GDNF and the expression of its receptor GFRα1 / RET; (c) Downregulates the overactivation of Toll-like receptor 2 and reduces the expression of neuronal nitric oxide synthase nNOS; (d) Increase serotonin 5-HT and its receptor 5-HT 2B Express; (e) Reduces inflammation levels in colon tissue and improves pathological damage to colon tissue; (f) Relieve intestinal motility disorders caused by weakened motility.

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