CN117821313A - Lactobacillus rhamnosus capable of remarkably improving numbers of enteric neurons and glial cells and application thereof - Google Patents
Lactobacillus rhamnosus capable of remarkably improving numbers of enteric neurons and glial cells and application thereof Download PDFInfo
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Abstract
The invention discloses a lactobacillus rhamnosus capable of remarkably improving the numbers of enteric neurons and glial cells and application thereof, and belongs to the field of microorganisms. The lactobacillus rhamnosus CCFM1360 of the present invention: has better activity, has certain acid and alkali resistance and adhesiveness, can obviously improve the number of intestinal neurons and glial cells, repair intestinal barrier function, improve the content of SCFAs in colon contents, increase the number of mesenteric lymph node Treg cells, reduce the expression level of inflammatory factors in colon tissues, reduce the inflammatory level of intestinal tracts and relieve the movement disorder of the power-reduced intestinal tracts.
Description
Technical Field
The invention relates to a lactobacillus rhamnosus capable of remarkably improving the numbers of enteric neurons and glial cells and application thereof, belonging to the field of microorganisms.
Background
The enteric nervous system (enteric nervous system, ENS) is a vast neural network composed of two nerve plexuses distributed under the mucosa and between the muscles of the intestinal wall, the constituent components include enteric neurons and enteric glial cells, and the enteric nervous system is widely distributed in the digestive system, has the characteristics of uniqueness, complexity of tissue connection, diversity of nerve cell types and the like, and can independently control and regulate the digestive and absorptive functions of the stomach and intestine, and is called the second brain or the enteric brain of the organism. ENS includes primary afferent neurons, interneurons, and motor neurons, and can locally control most intestinal functions by modulating intestinal absorption and secretion, vascular tone, and intestinal motility. ENS has significant uniqueness in neuronal performance and loops relative to the central nervous system (central nervous system, CNS). Normal ENS has a powerful regulatory effect on the homeostasis of the gut and even the systemic environment, which is involved in, but not limited to, regulating gastrointestinal motility and secretion, stabilizing intestinal immune function, maintaining and repairing intestinal epithelial barriers, and possibly forming a feedback loop (brain-gut axis) with the CNS via sympathetic and parasympathetic nerves and peripheral primary sensory nerves, affecting other functions such as mood, appetite and behavior in an individual. Gastrointestinal dysfunction caused by enteric nerve dysfunction not only causes digestive dysfunction, but also causes visceral pain and emotional and behavioral abnormalities, such as irritable bowel syndrome (irritable bowel syndrome, IBS), severely affecting the quality of life of the patient.
Enteric glial cells (enteric glia cells, EGCs) are derived from neural crest cells, one of the major members of ENS, distributed predominantly in the ganglion of the gastrointestinal myoplexus and submucosal plexus. EGCs not only play a role in nutrition and supporting enteric neurons, but also play an important role in regulating the growth and development of neurons, the functions of nerve circuits and apoptosis and maintaining intestinal homeostasis, such as structural and functional integration of enteric nervous systems, maintenance of mucosal epithelial barriers, regulation of gastrointestinal motility, nutrient uptake, secretion and blood flow, participation in immune or inflammatory reactions and the like. Among them, maintaining the integrity of intestinal epithelial barriers is an important feature of EGCs, and the absence and damage of EGCs may result in the absence of the integrity of intestinal mucosal barriers in rats, and increase the permeability of the rats, thereby causing the manifestations of intestinal inflammation, hemorrhage, necrosis, etc. EGCs are involved in the occurrence of digestive tract diseases, and abnormal quantity, receptor and phenotype appear in various intestinal movement dysfunction diseases such as postoperative enteroparalysis, congenital megacolon diseases, inflammatory bowel diseases and the like, and can sense neuron signals, realize interaction with intestinal neurons through intracellular calcium reaction and gap junction protein channels and further influence intestinal motility. In addition, EGCs are also of clinical significance for disorders outside the digestive tract, such as parkinson's disease, etc.
The gastrointestinal microbiota interacts with the central nervous system and the gut through microbiota-enterochromaffin-vagal afferent signaling, and more evidence supports the presence of "microbe-gut-brain axis" demonstrating the important role of microbes in regulating gut motility. Probiotics are active microorganisms and have positive effects on immunity, development and nutrient absorption of human bodies. They are a general term for active microorganisms that can play a positive role by being colonized in the digestive system of the human body in large quantities and improving the host micro-ecological balance. In recent years, more and more research has used prebiotics and probiotics to optimize the intestinal flora. The regulation and control effect on intestinal flora is mainly reflected in recovering flora and maintaining intestinal immune homeostasis, and can strengthen intestinal epithelial integrity, protect intestinal barrier, regulate gastrointestinal mucosa immune system and inhibit pathogenic bacteria growth. Studies have shown that the most commonly used probiotics such as lactobacillus, bifidobacterium, saccharomyces cerevisiae and other coliforms 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. The probiotics have positive regulation effect on brain function, can increase the number of neurons and glial cells, and improve anxiety and depression-like behaviors through intestinal brain axis. Metabolites such as SCFAs or peptides from the fermentation of the gastrointestinal microbiota can stimulate ENS and affect intestinal transport, while CNS and ANS also participate in the control of intestinal motility, both interacting with the gastrointestinal microbiota, and by modulating the intestinal microbiota, probiotics can exert a good modulating effect on ENS or CNS.
Intestinal dyskinesia usually occurs in female populations, and down-regulation of female estrogen receptor expression can lead to severe intestinal nerve damage, which in turn causes intestinal dyskinesia. Tamoxifen citrate is an estrogen receptor inhibitor that reduces the number of enteric neurons and enteric glial cells and causes intestinal dyskinesia by down-regulating estrogen receptor expression. The prior study usually uses loperamide hydrochloride to construct an intestinal dyskinesia model with intestinal nerve damage, the patent uses tamoxifen to make a model, and the two model modes are different in that once the loperamide hydrochloride stops administration, intestinal dyskinesia symptoms disappear, and after the tamoxifen citrate stops administration, intestinal dyskinesia symptoms can be sustained, and meanwhile compared with the loperamide, the tamoxifen model can aggravate intestinal inflammation and intestinal nerve damage, and is a more serious intestinal dyskinesia model. At present, no lactobacillus rhamnosus has been found which can be specifically used for regulating intestinal dyskinesia caused by down-regulation of estrogen receptor expression in females.
In summary, the enteric nervous system is critical for female intestinal health, and the reduced numbers of enteric neurons and enteric glial cells may lead to intestinal dyskinesia, and intestinal dyskinesia caused by down-regulation of female estrogen receptor expression is more refractory and difficult to treat effectively than other intestinal dyskinesias. There is a lack of lactobacillus rhamnosus in the prior art which can be dedicated to improving intestinal dyskinesia caused by abnormal intestinal nervous system in women.
Disclosure of Invention
Technical problem
The invention aims to solve the technical problem of providing a lactobacillus rhamnosus strain capable of remarkably improving the numbers of enteric neurons and glial cells and application of the strain. The tamoxifen citrate is used for establishing a model of intestinal dyskinesia caused by female enteric nerve damage, and screening probiotics capable of remarkably improving the numbers of enteric neurons and glial cells is of great significance for improving intestinal dyskinesia diseases caused by female enteric nervous system abnormality.
Technical proposal
In order to solve the technical problems, the invention provides the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) which can obviously improve the numbers of intestinal neurons and glial cells, repair intestinal barrier functions, effectively improve intestinal peristalsis, shorten intestinal transit time, improve fecal moisture content, increase the numbers of mesenteric lymph node Treg cells, reduce the expression level of inflammatory factors in colon tissues, and relieve colon tissue inflammation, and the strain can obviously repair intestinal nerve injury and relieve power-reduced intestinal dyskinesia caused by the intestinal nerve injury. Provides corresponding probiotic preparation, fermented food and functional food, effectively increases the numbers of intestinal neurons and glial cells, and relieves the movement disorder of intestinal tracts with reduced power.
The invention provides a lactobacillus rhamnosus (Lacticaseibacillus rhamnosus), which is preserved in the microorganism strain collection of Guangdong province at the year of 2023, 11 and 9, and has the preservation address of building 5 of Guangzhou national institute of advanced 100, and the preservation number of GDMCC No:63993.
the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) is from a faecal sample of female in the city of inner Mongolian Hulen, the strain is subjected to sequencing analysis, the sequence obtained by sequencing is subjected to nucleic acid sequence comparison in NCBI Standard Nucleotide BLAST, and the result shows that the similarity with the nucleic acid sequence of the lactobacillus rhamnosus is 100%; the results showed that the strain was lactobacillus rhamnosus, which was designated lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360.
The lactobacillus rhamnosus CCFM1360 has the following biological properties:
(1) Characteristics of the cells: gram-positive, no sporulation, non-motile bacteria.
(2) Colony characteristics: is round, convex, smooth and neat in edge.
(3) Growth characteristics: the culture was carried out in MRS medium at a constant temperature of 37℃for about 18 hours to the end of the log phase.
(4) Has strong tolerance to simulated gastrointestinal fluid.
(5) Remarkably improves the numbers of intestinal neurons and glial cells, repairs the intestinal barrier function, improves the content of SCFAs in colon contents, increases the numbers of mesenteric lymph node Treg cells, reduces the expression level of inflammatory factors in colon tissues, reduces the inflammatory level of intestinal tracts, and relieves the movement disorder of intestinal tracts with reduced power.
The invention also provides a microbial preparation containing the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360.
In one embodiment of the invention, the number of viable bacteria of the Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 in the microbial preparation is not less than 10 8 cfu/g or 10 8 cfu/mL。
In one embodiment of the invention, the microbial preparation is obtained by drying a bacterial liquid containing lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360, and the number of viable bacteria is not less than 10 8 cfu/g or 10 8 cfu/mL of powder.
In one embodiment of the invention, the drying refers to vacuum freeze drying.
The invention also provides a food which contains the microbial preparation of the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360.
In one embodiment of the invention, the food product is a fermented food product produced by fermentation using lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360, including solid, liquid, semi-solid food products.
In one embodiment of the invention, the lactobacillus rhamnosus is added to the food in an amount of at least 10 8 CFU/mL or 10 8 CFU/g。
In one embodiment of the invention, the fermented food product comprises a dairy product, a soy product or a fruit and vegetable product.
In one embodiment of the invention, the dairy product is a fermented dairy product, including fermented milk, fermented milk beverage, cream, cheese or milk powder; the bean product comprises soymilk, soymilk beverage and soymilk powder; the fruit and vegetable product comprises fermented fruit and vegetable drink or food which is fermented by taking Chinese cabbage, white radish, cucumber, beet, yellow peach or waxberry as raw materials.
The invention also provides a medicine which contains the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 or the microbial agent.
In one embodiment of the invention, the pharmaceutical product comprises lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 and a pharmaceutically acceptable carrier.
In one embodiment of the present invention, the carrier comprises one or more of fillers, binders, wetting agents, disintegrants, lubricants, flavoring agents commonly used in medicine.
In one embodiment of the invention, the dosage form of the drug comprises granules, capsules, tablets, pills or oral liquids.
In one embodiment of the invention, the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 is added to the pharmaceutical product in an amount of at least 10 8 CFU/mL or 10 8 CFU/g。
The invention also provides a health care product for relaxing bowel, which contains the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 or the microbial agent.
The invention also provides application of the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 or the microbial agent in preparing a functional product with at least one of the following components:
(a) Increasing the number of enteric glial cells, repairing the damaged enteric nervous system;
(b) Increasing the number of intestinal neurons in the colon, and increasing the expression of PGP9.5 in colon tissue;
(c) Repairing intestinal barrier function, and improving expression of Claudin5, ZO1 and MUC1 genes;
(d) Increasing the SCFAs content of the colon contents;
(e) Reducing the level of inflammation in colon tissue, increasing the number of mesenteric lymph node Treg cells;
(f) And relieving the movement disorder of the intestinal tract with reduced power.
Advantageous effects
(1) The lactobacillus rhamnosus CCFM1360 of the present invention: the activity is better, the number of enteric neurons can be obviously increased to 2 times of a model group, the number of glial cells can be increased to 2.75 times of the model group, the intestinal barrier function can be repaired, the expression quantity of Claudin5, ZO1 and MUC1 can be recovered to the level equivalent to that of a normal control group, the content of SCFAs in colon contents can be increased to the level equivalent to that of the normal control group, the number of mesenteric lymph node Treg cells can be increased, the expression level of inflammatory factors in colon tissues can be reduced, the inflammatory level of intestinal tract can be recovered, the intestinal transit time can be shortened to 1/2 of that of the normal control group, and the dynamic weakening type intestinal dyskinesia can be relieved.
(2) The invention can be regarded as a medicine for obviously improving the numbers of enteric neurons and glial cells, and can be applied to medicines or some fermented foods and functional foods, thereby widely playing the roles and having very valuable application prospect.
Preservation of biological materials
A strain of lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360, taxonomically named: lacticaseibacillus rhamnosus, deposited on month 11 and 9 of 2023 with the collection of microorganisms and cell cultures, cantonese province under the accession number GDMCC No:63993, the preservation address is 5 buildings of Guangzhou Md.A. No. 100 college, no. 59.
Drawings
Fig. 1: the dry prognosis of the Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 strain, the expression of intestinal glial cell marker S100deg.P in colon tissue of female rat after tamoxifen treatment is shown in the schematic (A) S100deg.P immunofluorescence staining; (B) S100deg.P positive expression relative area.
Fig. 2: post-dry prognosis of the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 strain, expression profile of the female rat colon enteric neuronal marker PGP9.5 after tamoxifen treatment schematic (a) PGP9.5 immunohistochemical staining; (B) PGP9.5 positive expression relative area.
Fig. 3: the transcriptional levels of colonic mucin MUC1 (A), claudin5 (B) and ZO1 (C) in colon tissue of female rats after tamoxifen treatment are schematically shown after the dry state of the Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 strain.
Fig. 4: the short chain fatty acid content of the colon contents of female rats after tamoxifen treatment is schematically shown after the dry state of the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 strain.
Fig. 5: a schematic representation of the proportion of mesenteric lymph node Treg cells and a schematic representation of serum inflammatory factor concentration in colon tissue of female rats treated with tamoxifen after the dry state of the lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 strain.
Fig. 6: the related indexes (intestinal transit time and fecal water content) of the dry prognosis of the Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360 strain and the symptom relief of the intestinal dyskinesia of the female rat after tamoxifen treatment are shown in the schematic diagram.
Note that: the symbols above the bar graph represent data significance levels, p <0.05, # represents p <0.01, # represents p <0.001, # represents p <0.0001 (compared to group NC), and # represents p <0.05, # represents p <0.01, # represents p <0.001 (compared to group NC).
Detailed Description
Female intestinal dyskinesia:
female intestinal dyskinesia refers to disorder of intestinal dyskinesia caused by intestinal inflammation and intestinal nervous system damage due to reduced estrogen receptor expression during menstrual cycle, pregnancy, pre-menopause and climacteric period, and is mainly divided into hypomotility (constipation) and hypermotility (diarrhea).
Female enteric nerve damage:
the female enteric nerve damage refers to the symptom that the female has reduced estrogen receptor expression level during menstrual cycle, pregnancy, pre-menopause and climacteric period, and the number of enteric neurons and enteric glial cells is reduced, and the symptom is accompanied by inflammatory reaction and intestinal barrier damage. Damage to the enteric nerve may lead to abnormal digestive function, causing dysfunctional intestinal motility.
Introduction to tamoxifen citrate model:
tamoxifen citrate is an estrogen receptor inhibitor that reduces estrogen receptor expression and initiates intestinal dyskinesia. Compared with loperamide hydrochloride modeling, the two modeling modes are different in that once the loperamide hydrochloride stops administration, intestinal dyskinesia symptoms disappear, and after tamoxifen citrate stops administration, the intestinal dyskinesia symptoms can be sustained, and compared with loperamide, tamoxifen modeling can aggravate intestinal inflammation and intestinal nerve injury and is a more serious intestinal dyskinesia model.
In the experiment, an animal model with the effect of reducing the expression of estrogen receptors to cause intestinal nerve damage is established through tamoxifen, so as to find probiotics capable of remarkably improving the numbers of intestinal neurons and glial cells.
Example 1: acquisition of Lactobacillus rhamnosus CCFM1360
1. Isolation and screening of lactobacillus rhamnosus:
(l) Collecting a fecal sample of a female in the city of inner Mongolian Hulen Bell by using a disposable sterile feces taking device, and culturing for 72 hours at 37 ℃ in an anaerobic environment; observing and recording the morphology of the bacterial colony, picking the bacterial colony, and streaking and purifying; the colonies obtained were gram stained in MRS liquid medium at 37℃for 48 hours, and colony morphology was recorded. Removing gram-negative bacterial strains and gram-positive cocci in the bacterial colonies, and selecting to obtain the gram-positive bacilli.
(2) After the catalase analysis, the catalase positive strain was discarded, and the catalase negative strain was retained.
2. Molecular biological identification of lactobacillus rhamnosus:
(l) Single bacterial genome extraction: culturing the catalase negative strain obtained in the step 1 overnight, taking the bacterial suspension l mL of the cultured overnight, centrifuging the bacterial suspension l in a 1.5mL centrifuge tube at 10000rpm for 2min, and discarding the supernatant to obtain thalli; washing the thalli with l mL of sterile water, centrifuging at 10000rpm for 2min, and discarding the supernatant to obtain thalli; 200 mu L of SDS lysate is added, and water bath is carried out for 30min at 80 ℃; adding 200 mu L of phenol-chloroform solution into the thallus lysate, wherein the phenol-chloroform solution comprises the components and the volume ratio of Tris saturated phenol and chloroform and isoamyl alcohol=25:24:1, mixing the mixture reversely, centrifuging the mixture at 12000rpm for 5-10 min, and taking 200 mu L of supernatant; adding 400 mu L of glacial ethanol or glacial isopropanol into 200uL of supernatant, standing at-20 ℃ for 1h, centrifuging at 12000rpm for 5-10 min, and discarding the supernatant; adding 500 mu L70% (volume percent) of ice-ethanol to re-suspend the sediment, centrifuging at 12000rpm for 1-3 min, and discarding the supernatant; oven drying at 60deg.C, or naturally air drying; 50 mu L ddH 2 O redissolving and precipitating to prepare PCR;
(2)16S rDNA PCR：
A. bacterial 16S rDNA 50. Mu. LPCR reaction System: 10×Taq buffer, 5. Mu.L; dNTP, 5. Mu.L; 27F, 0.5. Mu.L; 1492R, 0.5. Mu.L; taq enzyme, 0.5. Mu.L; template, 0.5 μl; ddH 2 O，38μL。
PCR conditions: 95 ℃ for 5min;95 ℃ for 10s; 30s at 55 ℃; 30s at 72 ℃; step 2-4X; 72 ℃ for 5min; 2min at 12 ℃;
C. preparing 1% agarose gel, mixing the PCR product with 10000×loading buffer, loading 2 μl, running at 120V for 30min, and performing gel imaging;
D. and (3) sending the obtained PCR product to a professional sequencing company, comparing the obtained sequencing result with a BLAST (BLAST-induced amplification factor) in a GeneBank, identifying the PCR product as lactobacillus rhamnosus, and naming the PCR product as lactobacillus rhamnosus CCFM1360, and preserving the sequencing result at-80 ℃ for later use.
Example 2: lactobacillus rhamnosus CCFM1360 increases intestinal glial cell number in colon tissue of a female rat after treatment with tamoxifen
The method comprises the following specific steps:
(1) Preparation of Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) bacterial suspension
The experimental strain is preserved in an ultralow temperature refrigerator at the temperature of minus 80 ℃ under the protection of 30 percent of glycerol. Prior to use, all strains were streaked onto MRS plates and single colonies were selected for sequencing identification to determine the identity of the strain and the correctness of the species. After confirming the strain information, the strain was activated 4 times continuously in MRS liquid medium with an inoculum size of 2% to allow the strain to gradually recover. After culturing at 37℃for 18 hours in a constant temperature and humidity incubator/anaerobic workstation, the bacterial liquid was centrifuged at 6000g for 15min at low temperature and the bacterial cells were collected. The bacterial mud is repeatedly washed for three times by sterile physiological saline to remove the culture medium in the bacterial body. And finally, re-suspending the strain with 30% glycerol and storing the strain in an ultralow temperature refrigerator at the temperature of-80 ℃ for later use. Before use, viable bacteria were counted using a gradient dilution method. When in use, the bacterial liquid is diluted by new sterile physiological saline to make the final bacterial liquid viable bacterial concentration be 1×10 10 CFU/mL。
(2) Healthy female SPF grade Sprague Dawley rats of 6 weeks of age were taken 28, acclimatized for 1 week, and randomly divided into 4 groups: normal, model, intervention (Lactobacillus rhamnosus FJSWX1L3, lactobacillus rhamnosus CCFM 1360), each containing 7 rats, the dosage of the gastric lavage bacterial suspension being 1×10 10 CFU/mL。
The grouping and treatment methods of the experimental animals are shown in table 1:
table 1 experimental animal groups
After the experiment is finished, the mice are sacrificed, and colon tissues of the mice are taken for detection. Immunofluorescent staining was used to characterize the numbers of intestinal glial cells in colon tissue. The specific method comprises the following steps: placing the embedded tissue slice on a glass slide, melting wax in an incubator at 60 ℃ for 1h, soaking and dewaxing in dimethylbenzene, gradient hydrating ethanol (100%, 95%, 85% and 75%) with different concentrations, soaking and cleaning with distilled water, performing antigen retrieval by a boiling method (after boiling sodium citrate-EDTA antigen retrieval liquid, immersing the glass slide in the solution, keeping the temperature for 10min, taking out the glass slide after naturally cooling the solution), and soaking and cleaning again with distilled water and PBST. And (3) dropwise adding a proper amount of enzyme inactivating reagent into each slice, washing the slices after 15 minutes at room temperature in a dark place, dropwise adding a proper amount of sealing liquid into a wet box, and sealing the wet box for 30 minutes. After blocking, the blocking solution was discarded, and the primary antibody was added dropwise and placed in a wet box for incubation overnight at 4 ℃. The next day, rewarming at room temperature for 40min, adding enzyme-labeled secondary antibody after PBST washing, incubating for 45min at room temperature in a dark place, carefully sucking water around tissues by using water absorbing paper after PBST washing, dripping a proper amount of anti-fluorescence quenching agent containing DAPI, and observing red fluorescence under a fluorescence microscope after nail oil sealing.
Glial cells of the mucosa are mainly involved in epithelial barrier function, glial cells within the ganglion are mainly responsible for nerve repair, closely interact with neurons, support differentiation of these cells and glial cells, and are involved in the generation and formation of nerves. The number of glial cells can well reflect the health of the enteric nerve. s100deg.S is a calbindin, which is mainly present in glial cells, and the number of glial cells in the enteric nerve can be characterized by using S100deg.S to label the enteric glial cells (red fluorescence in FIG. 1 (A)). Immunofluorescence results as shown in fig. 1 (B), intestinal glial cell density decreased by 1/3 (P < 0.05) after tamoxifen treatment, indicating that the model rats had enteric nerve damage. The lactobacillus rhamnosus CCFM1360 has remarkable effect on increasing the number of enteric glial cells (P < 0.001), is improved to 2 times of a model group (the model group is 0.63 and the CCFM1360 group is 1.26), and can restore the enteric nerve health of rats with enteric nerve damage.
Example 3: lactobacillus rhamnosus CCFM1360 increases the number of enteric neurons in colon tissue of a female rat after tamoxifen treatment
The experimental animals were grouped, molded and treated in the same manner as in example 2.
After the experiment is finished, the mice are sacrificed, colon tissues of the mice are taken, and the PGP9.5 expression quantity in the colon tissues is quantified by adopting an immunohistochemical method. The specific method comprises the following steps: placing the embedded tissue slice on a glass slide, melting wax in an incubator at 60 ℃ for 1h, soaking and dewaxing in dimethylbenzene, gradient hydrating ethanol (100%, 95%, 85% and 75%) with different concentrations, soaking and cleaning with distilled water, performing antigen retrieval by a boiling method (after boiling sodium citrate-EDTA antigen retrieval liquid, immersing the glass slide in the solution, keeping the temperature for 10min, taking out the glass slide after naturally cooling the solution), and soaking and cleaning again with distilled water and PBST. And (3) dropwise adding a proper amount of enzyme inactivating reagent into each slice, washing the slices after 15 minutes at room temperature in a dark place, dropwise adding a proper amount of sealing liquid into a wet box, and sealing the wet box for 30 minutes. After blocking, the blocking solution was discarded, and the primary antibody was added dropwise and placed in a wet box for incubation overnight at 4 ℃. The next day, rewarming at room temperature for 40min, adding enzyme-labeled secondary antibody after PBST washing, incubating for 30min at room temperature, washing cleanly, developing DAB for about 5min, and stopping developing by distilled water. The hematoxylin somatic cell fast staining solution is subjected to counterstaining, 1% hydrochloric acid-ethanol is used for decoloring and bluing, ethanol with different concentrations is used for gradient dehydration (75%, 85%, 95% and 100%), xylene is soaked for two times, the tissue periphery is wiped by absorbent paper, and then neutral resin sealing sheets are dripped.
The number of PGP9.5 responsive cells in the colon area was significantly reduced in patients with intestinal dyskinesia, and a similar reduction in the number of enteric neurons was detected in submucosal plexuses in patients with refractory intestinal dyskinesia. Remodeling abnormal intestinal microbiota can restore intestinal function and stimulate intestinal neurogenesis to increase the number of neurons, so PGP9.5 is selected as a detection index for quantifying intestinal neurons. As can be seen from fig. 2, the number of colon neurons in the rat after molding was significantly reduced by 1/2 (P < 0.05), and after gavage of the rat with different strains, lactobacillus rhamnosus CCFM1360 could significantly increase the number of neurons to 2.75 times that of the model group (model group: 0.46, CCFM1360 group: 1.25), indicating that lactobacillus rhamnosus CCFM1360 could restore intestinal function by repairing intestinal neurons.
Example 4: lactobacillus rhamnosus CCFM1360 can repair intestinal barrier function of female rat after treatment with tamoxifen
The experimental animals were grouped, molded and treated in the same manner as in example 2.
About 20mg of rat colon preserved at-80 ℃ is taken, and total RNA of the rat colon is extracted by using a TRIzol method: the sheared rat colon tissue is sheared and then placed into an enzyme-free centrifuge tube together with zirconia beads which are subjected to high-temperature enzyme deactivation, 1mL of TRIzol lysate is added, then the mixture is fully crushed by a high-throughput crusher, 200 mu L of chloroform is added, vortex vibration is carried out for 30s, the mixture is fully emulsified, the mixture is kept stand at 4 ℃ for 5min, and then the mixture is centrifuged for 15min at 12000g at 4 ℃. Carefully aspirate 400 μl of supernatant into a new enzyme-free centrifuge tube, add equal volume of pre-chilled isopropyl alcohol, invert several times, mix well, stand for 10min at 4deg.C, centrifuge for 15min again at 12000 g. After the supernatant is discarded, 1mL of pre-cooled DEPC water prepared by 75% ethanol is slowly added along the pipe wall to wash the extracted RNA twice, the pipe bottom is flicked to suspend the RNA sediment, and after standing for 3-5min, the DEPC water is added to dissolve the sediment. 1. Mu.L of RNA sample was taken and assayed for concentration, purity and integrity by an ultra-micro spectrophotometer. According to the instruction provided by the Northenan kit, cDNA is synthesized by taking the extracted total RNA as a template, real-time fluorescence quantitative PCR detection is carried out, a system is prepared according to the instruction of HiScript III RTSuperMix for qPCR, and RT-qPCR program operation is carried out. The reaction system: 2X ChamQ Universal SYBR qPCR Master Mix. Mu.L; forward and reverse primers (10. Mu.M) 1. Mu.L each; 1 μl of cDNA; ddH 2 O2. Mu.L. The reaction procedure: pre-denaturation at 95℃for 10s; amplifying for 40 times at 95 ℃ for 10s and 57 ℃ for 30s; the melting curves were 95℃15s,60℃60s,95℃15s.
The level of transcription of the gene of interest in rat colon tissue was determined by real-time fluorescent quantitative PCR (RT-qPCR). At NCBI website @https://www.ncbi.nlm.nih.gov/) The primer sequences of the related target genes of the rats are searched up and the Shanghai biological engineering technical service company is entrusted to synthesize, and specific primer information is shown in the following table.
TABLE 2 primer sequences for target genes
Claudin5 and ZO1 regulate cell permeability, mediate interactions between proteins, link actin cytoskeleton, have important implications in regulating intestinal mucosal barrier, indicating impaired intestinal mucosa and mechanical barrier in the colon when Claudin5 and ZO1 are down-regulated. MUC1 is a mucin family member that serves as a "lubricant" for the gut to maintain its normal mechanical barrier function.
mRNA levels of Claudin5 and ZO1 in the model group are greatly reduced (P < 0.01), and the expression of Claudin5 and ZO1 in colon is remarkably improved by lactobacillus rhamnosus CCFM1360, so that the expression amounts of Claudin5 and ZO1 are restored to the level equivalent to that of a normal control group, thereby improving the permeability of intestinal tracts, repairing the mechanical barrier of the intestinal tracts and further relieving the intestinal dyskinesia. Tamoxifen significantly reduces the transcriptional expression of MUC1, as shown in figure 3, the results show that the expression of MUC1 in colon can be significantly promoted by lactobacillus rhamnosus FJSWX1L3 and CCFM1360, and the expression quantity of MUC1 is restored to 1.2 times of that of a normal control group by lactobacillus rhamnosus CCFM1360, so that the effect is better than that of lactobacillus rhamnosus FJSWX1L3. From a comprehensive comparison, tamoxifen administration can cause slight pathological damage to the colon, and probiotic intervention can play a certain role in repairing damage to intestinal mucous layer, wherein the repairing effect of lactobacillus rhamnosus CCFM1360 on intestinal barrier is most excellent.
Example 5: the Lactobacillus rhamnosus CCFM1360 can increase the content of SCFAs in the colon contents of a female rat treated with tamoxifen
The experimental animals were grouped, molded and treated in the same manner as in example 2. The method for detecting the SCFAs in the excrement comprises the following steps:
(1) Sample preparation
Collecting feces collected the day before the experiment is finished, vacuum freeze-drying to remove water, accurately weighing a certain amount of feces into a 1.5mL EP tube, and recording the weight of the feces; adding 500 mu L of saturated NaCl solution, soaking for 30min, homogenizing and crushing. After mixing, 40. Mu.L of a 10% aqueous sulfuric acid solution was added to the homogenate. After thoroughly mixing by vortexing for 30s, 1mL of diethyl ether was added to each sample in a fume hood using a 1mL syringe. After thoroughly mixing, the sample was centrifuged at 15000rpm for 15min at high speed. The supernatant was transferred to an EP tube to which 0.3g of anhydrous sodium sulfate had been added. Again, the mixture was centrifuged at 15000rpm for 15min and the supernatant was carefully aspirated into a gas-phase flask for testing.
(2) Detection condition setting
Short chain fatty acids were detected in the samples using GC-MS in combination with Rtx-Wax chromatography (column length 30m, inner diameter 25 μm). The carrier is helium, and the air flow speed is set to be 2mL/min; the sample injection volume was 1. Mu.L and the injection temperature was 240 ℃. Chromatographic column temperature program: raising the temperature to 140 ℃ at 100 ℃,7.5 ℃/min and 5.33min; raising the temperature to 200 ℃ at 140 ℃ and 60 ℃/min for 1min; kept at 200℃for 3min.
(3) Mixed label preparation and standard curve
The 6 short chain fatty acids in the sample were quantitatively calculated by an external standard method. 10. Mu.L of acetic acid, propionic acid, isobutyric acid, butyric acid, valeric acid and isovaleric acid were each taken and mixed well with diethyl ether to a volume of 1000. Mu.L. Taking 100 mu L of mixed solution, using diethyl ether to fix the volume to 1000 mu L, and then respectively taking 200 mu L, 100 mu L, 50 mu L, 25 mu L, 15 mu L and 10 mu L of the mixed solution, using diethyl ether to fix the volume to 1000 mu L, and diluting the mixed solution into standard mixed solutions with different concentrations.
SCFAs are the primary products of colonic epithelial cell uptake and provide energy for colonic bacterial fermentation. It can accelerate colonic motility by stimulating intestinal epithelial cell growth and fluid secretion. In addition, it can protect the functional intestinal barrier by increasing the expression of the claudin and modulating the activity of the intestinal microbiota and immune cells. Targeted metabonomics analysis of the colon contents by GC-MS showed a significant decrease in SCFAs concentration after tamoxifen induced bowel movement disorder as shown in figure 4. Acetic acid can protect the functional barrier of gastrointestinal mucosa, and the interference of two probiotics has an up-regulating effect on acetic acid, but the up-regulating degree is different, the effect of lactobacillus rhamnosus CCFM1360 is more obvious, and the up-regulating effect is (112.45 +/-31.82)%; lactobacillus rhamnosus CCFM1360 restores propionic acid, butyric acid, valeric acid content to levels comparable to normal control groups; lactobacillus rhamnosus CCFM1360 restores isobutyric acid, isovaleric acid content to 1.2 times that of the normal control group. Compared with the lactobacillus rhamnosus FJSWX1L3, the lactobacillus rhamnosus CCFM1360 has larger influence on short chain fatty acid, so the lactobacillus rhamnosus CCFM1360 can improve the microbial abundance of the short chain fatty acid.
Example 6: lactobacillus rhamnosus CCFM1360 may increase the number of mesenteric lymph node Treg cells of female rats after tamoxifen treatment
The experimental animals were grouped, molded and treated in the same manner as in example 2.
The rat mesenteric lymph node Treg cell detection method is as follows: at the end of the experiment, mesenteric lymph nodes (Mesenteric lymph node, MLN) were taken, ground with syringe cores in pre-chilled PBS solution, filtered through a 200 mesh screen, and prepared as a single cell suspension. Centrifuging at 300g for 5min, discarding supernatant, resuspending cell pellet with flow cell buffer, and adjusting cell concentration to 2×10 8 cells/mL. Dyeing: FITC-labeled anti-rate CD4 antibody (1.25 mug/mL) and APC-labeled anti-rate CD25 antibody (0.6 mug/mL) are added to the suspension, and incubated at room temperature for 30min in a dark place to stain the cell surface; then adding Foxp3 fix/Permeabilation Fixation membrane rupture liquid, and incubating for 1h at room temperature in a dark place; finally, 0.5 mug PE-labeled anti-rat/mouse Foxp3 antibody is added, and the cell nucleus is stained by incubation for 1h at room temperature in a dark place, wherein the PE-labeled anti-rat IgG2a antibody is used as a isotype control of Foxp 3. And (5) after the dyeing is finished, re-suspending the flow buffer on the machine for detection.
The detection method of the rat intestinal cytokines comprises the following steps: taking colon tissue preserved at the temperature of minus 80 ℃, removing fat tissue, adding precooled sterile PBS solution according to the weight ratio of 1:9, putting into a 1.5mL centrifuge tube, shearing, adding sterilized zirconium beads, crushing into homogenate by using a high-throughput crusher, centrifuging for 10min at the temperature of 6000r/min, and transferring supernatant into a new centrifuge tube. The IL-6 content of the colon was measured by an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions.
An important energy source for intestinal epithelium is the induction of Treg cell activation by the involvement of immunomodulation by modulating the pro-inflammatory activity of neutrophils and macrophages. The proportion of Treg in mesenteric lymph nodes of female rats with intestinal dyskinesia was evaluated by flow cytometry, and intracellular FACS staining was performed on Foxp3, as shown in fig. 5, the model group decreased the proportion of Treg cells in mesenteric lymph nodes, and increased the expression of pro-inflammatory factor IL-6 in rat serum. The lactobacillus rhamnosus CCFM1360 can obviously increase Treg cells in mesenteric lymph nodes, reduce the expression of IL-6 pro-inflammatory factors in serum, and restore the proportion of the Treg cells and the expression of the IL-6 to the level equivalent to that of a normal control group. It is speculated that CCFM1360 may restore normal signaling to improve intestinal inflammation.
Example 7: the lactobacillus rhamnosus CCFM1360 can obviously relieve the intestinal dyskinesia symptoms of female intestinal dyskinesia rats
The experimental animals were grouped, molded and treated in the same manner as in example 2. The change condition of the related indexes of the intestinal dyskinesia of the rat is monitored during the period of the stomach irrigation of the probiotics, including the small intestine propulsion rate, the intestine transportation time and the fecal water content.
(1) Fecal moisture content
The rats were collected during the trial for termination of faeces the day before sacrifice, which was done after the end of gavage. All rats are placed into a clean cage box individually according to the groups, fresh feces are collected and put into a 5mL centrifuge tube, and after collection, the rats are placed back into the cage box again according to the groups. After weighing, the water content in the feces was removed by freeze drying, and the water content of the rat feces was calculated according to the following formula:
fecal moisture (%) = (fecal wet weight-fecal dry weight)/fecal wet weight x 100%;
(2) Intestinal transit time
Uniformly mixing the gum arabic powder with water according to a ratio of 1:10, heating to be transparent on an electromagnetic oven, continuously stirring during heating, then adding 10% (w/v) active carbon powder, stirring and boiling until the mixture is uniform, cooling the solution, and then placing in a refrigerator at 4 ℃ for storage. The time of the rat intestinal transit is measured in the morning before the end of the experiment, and in order to ensure the accuracy of the result, the rat needs to be fasted without water-stop overnight before the time of the rat intestinal transit is measured. When the measurement is carried out, 1mL of the ink is filled into each rat, the time of filling the stomach is recorded, the defecation state of the rat is concerned at any time, when the first stool containing activated carbon is to be discharged, the time is recorded, and the time difference is the intestinal transit time of the rat.
As shown in fig. 6, after modeling, the total intestinal transit time of the model rats was prolonged by about (0.61±0.24) times compared to the normal group, while the intervention group of lactobacillus rhamnosus FJSWX1L3 and CCFM1360 both significantly shortened the intestinal transit time, and lactobacillus rhamnosus CCFM1360 shortened the intestinal transit time to 1/2 of the normal control group. Fecal moisture content is another important indicator in assessing bowel movement disorders, where the stool of a patient with bowel movement disorder is often dry and hard and lacks moisture, resulting in difficult defecation. After three weeks of intervention, both the two strains of lactobacillus rhamnosus FJSWX1L3 and CCFM1360 have better effects (P < 0.05) on improving the fecal moisture content, indicating that both strains can effectively alleviate intestinal dyskinesia.
Therefore, from the results, it can be seen that the lactobacillus rhamnosus CCFM1360 significantly increases the numbers of intestinal neurons and glial cells, repairs the intestinal barrier function, increases the SCFAs content in the colon content, increases the numbers of mesenteric lymph node Treg cells, reduces the expression level of inflammatory factors in colon tissue, reduces the inflammatory level of the intestinal tract, and relieves the power-reduced intestinal dyskinesia.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A strain of lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) CCFM1360, wherein the lactobacillus rhamnosus CCFM1360 is deposited at the collection of microorganism strains in the cantonese province at about 9/11/2023 at floor 59 of the university of first middle road 100 in the Guangzhou city under the accession number GDMCC No:63993.
2. a microbial inoculant comprising the lactobacillus rhamnosus CCFM1360 of claim 1.
3. A food product comprising the lactobacillus rhamnosus CCFM1360 of claim 1 or the microbial agent of claim 2.
4. A food product according to claim 3, wherein the food product comprises a fermented food product.
5. As claimed inThe food product of claim 3 or 4, wherein the lactobacillus rhamnosus CCFM1360 is added to the food product in an amount of at least 10 8 CFU/mL or 10 8 CFU/g。
6. A pharmaceutical product comprising the lactobacillus rhamnosus CCFM1360 of claim 1 or the microbial agent of claim 2.
7. The pharmaceutical product of claim 6, wherein the pharmaceutical product comprises a dosage form comprising a granule, a capsule, a tablet, a pill, or an oral liquid.
8. The pharmaceutical product according to claim 6 or 7, wherein the lactobacillus rhamnosus CCFM1360 is added to the pharmaceutical product in an amount of at least 10 8 CFU/mL or 10 8 CFU/g。
9. A health product for relaxing bowel, which is characterized in that the health product contains the lactobacillus rhamnosus CCFM1360 of claim 1 or the microbial agent of claim 2.
10. Use of the lactobacillus rhamnosus CCFM1360 of claim 1 or the microbial agent of claim 2 for the preparation of a product having at least one of the following functionalities:
(a) Increasing the number of enteric glial cells, repairing the damaged enteric nervous system;
(b) Increasing the number of intestinal neurons in the colon, and increasing the expression of PGP9.5 in colon tissue;
(c) Repairing intestinal barrier function, and improving expression of Claudin5, ZO1 and MUC1 genes;
(d) Increasing the SCFAs content of the colon contents;
(e) Reducing the level of inflammation in colon tissue, increasing the number of mesenteric lymph node Treg cells;
(f) And relieving the movement disorder of the intestinal tract with reduced power.
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