A Clostridium butyricum NCU-27 and Its Application

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to a Clostridium butyricum NCU-27 and its application. This Clostridium butyricum ( Clostridium butyricum ) NCU-27 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on July 22, 2024. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO. 31386. According to the data of the embodiments of the present invention, the Clostridium butyricum NCU-27 provided by the present invention improves the postoperative IR of the gastrectomized SD rat model by regulating the intestinal microbiota and short-chain fatty acid metabolism, especially increasing the production of butyric acid. This improvement may be achieved through the gut-liver axis and the mTORC1 signaling pathway, providing a potential strategy for the future clinical use of probiotics and butyric acid derivatives to treat postoperative IR.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a Clostridium butyricum NCU-27 and its application. Background Art

[0002] At present, the comprehensive treatment mainly based on radical surgery is the main treatment mode for advanced gastric cancer. However, on the one hand, removing the tumor lesion can improve the survival rate of patients; on the other hand, surgical trauma, as a stressor, due to local tissue inflammation and surgical reactive stress, a series of neuroendocrine reactions mainly characterized by sympathetic nerve excitement and increased pituitary-adrenal cortex secretion occur after surgery, causing metabolic disorders in gastric cancer patients after surgery, and insulin resistance is one of them. Its specific manifestation is that the blood sugar of patients increases after surgery, and at the same time, the fasting serum insulin level also increases. This phenomenon is the insulin resistance phenomenon.

[0003] Research shows that insulin resistance almost exists in all surgeries, can occur within a few minutes at the beginning of the surgery, and can last for several weeks. At the same time, the research points out that the incidence of insulin resistance in patients after elective general surgery is relatively low, and the incidence of insulin resistance varies depending on the surgical site. For example, the incidence of insulin resistance after gastric cancer surgery is the highest, followed by colorectal cancer surgery. Insulin resistance reduces the effect of hormones synthesized in the body, enhances the catabolism of the body, and at the same time, the hypermetabolic state during surgical stress increases the energy demand of the body, but the glucose oxidative energy supply of tissue cells is insufficient, and the body's energy supply then turns to mainly fat, increasing fat mobilization and glycolysis, and accelerating protein breakdown at the same time. To sum up, postoperative insulin resistance not only leads to increased catabolism of the body, negative nitrogen balance, poor wound healing and increased infection rate, especially for patients after gastrectomy, the postoperative protein level decreases, and the gastroenteric anastomosis is edematous and poorly healed, resulting in anastomotic fistula. In addition, it also affects the stability of the body's internal environment, and thus seriously affects the postoperative recovery and prognosis of patients.

[0004] At present, it is considered that the mechanism of postoperative insulin resistance mainly involves the direct or indirect interference of neuroendocrine and inflammatory factors with the insulin action pathway. For example, studies have found that under surgical stress, the body will produce some counter-regulatory hormones (such as glucagon, catecholamines, cortisol, etc.), which inhibit the normal effect of anti-insulin, and the inflammatory factors produced by surgery (such as TNF-α) can indirectly stimulate the secretion of insulin-antagonistic hormones or directly affect the insulin signal transduction pathway and the membrane translocation of glucose transporter (GLUT)-4, affecting blood glucose, and thus leading to insulin resistance. However, most domestic and foreign studies on insulin resistance focus on diabetes, endocrine diseases and their pathogenesis, and less attention is paid to insulin resistance after radical gastrectomy. The treatment mainly relies on symptomatic treatments such as preoperative administration of carbohydrates and insulin hypoglycemia. However, by injecting insulin to lower blood sugar, on the one hand, invasive operations are not readily accepted by patients, and the dose is difficult to regulate, and it is easy to cause hypoglycemia in patients, increasing the perioperative risk. Therefore, it is of great significance to explore an effective method for preventing and treating insulin resistance after gastrectomy and which is safe and effective. Summary of the Invention

[0005] The object of the present invention is to solve the deficiencies of the prior art and provide a Clostridium butyricum NCU-27 and its application, and the following technical solutions are specifically adopted:

[0006] In the first aspect of the present invention, a Clostridium butyricum NCU-27 is provided. This Clostridium butyricum ( Clostridium butyricum ) NCU-27 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on July 22, 2024. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO. 31386. And the gene sequence of the 16S rDNA of this Clostridium butyricum NCU-27 is as shown in SEQ ID No: 1.

[0007] SEQ ID No: 1:

[0008]

[0009] In the second aspect of the present invention, there is provided the use of the above-mentioned Clostridium butyricum NCU-27 in the preparation of a medicament for preventing, improving or treating insulin resistance after gastrectomy.

[0010] In the third aspect of the present invention, there is provided a biological preparation, the active ingredient of which comprises Clostridium butyricum NCU-27.

[0011] In the fourth aspect of the present invention, there is provided the use of the above-mentioned biological preparation in the preparation of a medicament for preventing, improving or treating insulin resistance after gastrectomy.

[0012] In the fifth aspect of the present invention, there is further provided a medicament for preventing, improving or treating insulin resistance after gastrectomy, which comprises Clostridium butyricum NCU-27.

[0013] As a further preferred embodiment, the above-mentioned medicament further comprises a pharmaceutical excipient; more preferably, the above-mentioned pharmaceutical excipient is at least one of water, lactose, sodium chloride, and glucose.

[0014] As a further preferred embodiment, the dosage form of the above-mentioned medicament is powder, granule, capsule or tablet; more preferably, the medicament is for oral administration.

[0015] The beneficial effects of the present invention are as follows: The present invention provides a strain of Clostridium butyricum NCU-27, which can be used in the preparation of a medicament for preventing, improving or treating insulin resistance after gastrectomy. According to the data of the examples of the present invention, Clostridium butyricum NCU-27 improves the postoperative IR of the gastrectomized SD rat model by regulating the intestinal microbiota and short-chain fatty acid metabolism, especially by increasing the production of butyric acid. This improvement may be achieved through the enterohepatic axis and the mTORC1 signaling pathway, providing a potential strategy for the future clinical use of probiotics and butyric acid derivatives in the treatment of postoperative IR. Description of the Drawings

[0016] Figure 1 Shown is the determination result graph of the in vitro probiotic properties of Clostridium butyricum ( Clostridium butyricum ) NCU-27; wherein, A is the growth curve graph, B is the acid tolerance evaluation graph, C is the bile salt tolerance evaluation graph, D is the hemolysis experiment result graph, E-F is the drug sensitivity experiment result graph, G is the antioxidant property evaluation graph; H is the bacterial Gram staining graph.

[0017] Figure 2 Shown is Clostridium butyricum ( Clostridium butyricum)NCU-27's evaluation diagram of IR level in SD rats after gastrectomy; among them, A is the fasting blood glucose level of rats in each group on the first day after surgery, B is the fasting insulin level of rats in each group on the first day after surgery, C is the insulin resistance index of rats in each group on the first day after surgery, D is the area under the curve of the intraperitoneal glucose tolerance test of rats in each group on the second day after surgery, E is the fasting blood glucose level of rats in each group on the third day after surgery, F is the fasting insulin level of rats in each group on the third day after surgery, G is the insulin resistance index of rats in each group on the third day after surgery, and H is the hepatic glycogen content of rats in each group on the third day after surgery.

[0018] Figure 3 The figure shows the results of Clostridium butyricum ( Clostridium butyricum )NCU-27 on intestinal flora imbalance in SD rats after gastrectomy; among them, A is the Shannon index, B is the Venn diagram, C is the principal coordinate analysis, D is the change in the abundance of Firmicutes, Verrucomicrobia, and Bacteroidetes at the phylum level, E is the change in the abundance of Streptococcus, Akkermansia, Turicibacter, and Roseburia at the genus level, and F is the random forest analysis.

[0019] Figure 4 The figure shows the results of Clostridium butyricum ( Clostridium butyricum )NCU-27 on the fecal butyric acid level in SD rats after gastrectomy; among them, A is the ZO-1 immunofluorescence staining result of the colon of rats in each group, and B-H are the levels of acetic acid, butyric acid, caproic acid, isobutyric acid, isovaleric acid, propionic acid, and valeric acid in the feces of rats in each group.

[0020] Figure 5 The figure shows the effect of butyric acid on the IR level in SD rats after gastrectomy; A is the fasting blood glucose level of rats in each group on the first day after surgery, B is the fasting insulin level of rats in each group on the first day after surgery, C is the insulin resistance index of rats in each group on the first day after surgery, D is the area under the curve of the intraperitoneal glucose tolerance test of rats in each group on the second day after surgery, E is the fasting blood glucose level of rats in each group on the third day after surgery, F is the fasting insulin level of rats in each group on the third day after surgery, G is the insulin resistance index of rats in each group on the third day after surgery, and H is the hepatic glycogen content of rats in each group on the third day after surgery.

[0021] Figure 6 The figure shows the results of Clostridium butyricum ( Clostridium butyricum )NCU-27 and butyric acid on the mechanism of IR level in SD rats after gastrectomy; A is the mRNA expression levels of AMPK, JNK, ERK, Raptor, IRS1, and AKT detected by qPCR in the liver of rats in each group after surgery; B is the representative image of the protein expression of Raptor, P Ser -IRS1, IRS1, P-AKT, AKT, and β-actin in the liver of rats in each group after surgery; C is the ratio of Raptor to β-actin in the liver of rats in each group after surgery; D is PSer -Ratio of IRS1 to IRS1; E is the ratio of P-AKT to AKT in the liver of rats in each group after surgery; F is the ratio of GLUT4 to β-actin in the liver of rats in each group after surgery; G is a representative image of the protein expression of GLUT4, G6pase, Pepck, GYS2, and β-actin in the liver of rats in each group after surgery; H is the ratio of G6pase to β-actin in the liver of rats in each group after surgery; I is the ratio of Pepck to β-actin in the liver of rats in each group after surgery; J is the ratio of GYS2 to β-actin in the liver of rats in each group after surgery.

[0022] Figure 7 The figure shows the effects of mTORC1 agonist and inhibitor on the improvement of IR level in SD rats after gastrectomy by Clostridium butyricum NCU-27 and butyric acid; among them, A is the fasting blood glucose level of rats in each group on the first day after surgery, B is the fasting insulin level of rats in each group on the first day after surgery, C is the insulin resistance index of rats in each group on the first day after surgery, D is the area under the curve of the intraperitoneal glucose tolerance test of rats in each group on the second day after surgery, E is the fasting blood glucose level of rats in each group on the third day after surgery, F is the fasting insulin level of rats in each group on the third day after surgery, G is the insulin resistance index of rats in each group on the third day after surgery, and H is the liver glycogen content of rats in each group on the third day after surgery.

[0023] Figure 8 The figure shows the mechanism of action of mTORC1 agonist and inhibitor on the improvement of IR level in SD rats after gastrectomy by Clostridium butyricum NCU-27 and butyric acid; A is a representative image of the protein expression of Raptor, P Ser -IRS1, IRS1, P-AKT, AKT, and β-actin in the liver of rats in each group after surgery; B is the ratio of Raptor to β-actin in the liver of rats in each group after surgery; C is P Ser -Ratio of IRS1 to IRS1; D is the ratio of P-AKT to AKT in the liver of rats in each group after surgery; E is a representative image of the protein expression of GLUT4, G6pase, Pepck, GYS2, and β-actin in the liver of rats in each group after surgery; F is the ratio of GLUT4 to β-actin in the liver of rats in each group after surgery; G is the ratio of G6pase to β-actin in the liver of rats in each group after surgery; H is the ratio of Pepck to β-actin in the liver of rats in each group after surgery; I is the ratio of GYS2 to β-actin in the liver of rats in each group after surgery. Detailed implementation mode

[0024] The concept, specific structure, and technical effects of the present invention will be clearly and completely described below in conjunction with embodiments and the accompanying drawings to fully understand the purpose, solution, and effects of the present invention. It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other.

[0025] Example 1

[0026] Clostridium butyricum ( Clostridium butyricum ) Screening and related experiments of NCU-27

[0027] 1. Material collection: Fresh feces were collected from normal and healthy men and stored in a sterile centrifuge tube containing 50% glycerol. The collected fecal samples from normal and healthy men were immediately diluted and spread on plates.

[0028] 2. Isolation, identification, and evaluation of Clostridium butyricum: For fresh feces, TSB, LB, MRS, RCM, and BSM media were used for microbial isolation. Then, the isolated strains were cultured in liquid respectively. Then, the genes of the strains were extracted, and the 16S rDNA fragments were amplified using the designed primers. The amplified 16S rDNA fragments were sequenced, and the sequences were as shown in SEQ ID No: 1. The sequencing results were compared with the data in the NCBI database to determine the species classification of the strains.

[0029] One of the strains was determined to be Clostridium butyricum NCU-27 through colony morphology identification, cell morphology identification, and molecular biology methods.

[0030] Example 2

[0031] Clostridium butyricum ( Clostridium butyricum ) In vitro probiotic properties determination of NCU-27

[0032] (1) Growth curve

[0033] The probiotic Clostridium butyricum NCU-27 was incubated in an anaerobic culture reinforced Clostridium medium (RCM, LA4360, Solarbio, China) at 37°C for 24 hours. The optical density (OD600 nm) of the bacterial suspension at each time point was measured by spectrophotometry, and the bacterial concentration was measured by plate counting method. The bacterial growth curve (as shown in A in Figure 1 ) was drawn. The results showed that Clostridium butyricum NCU-27 had strong growth characteristics under anaerobic conditions at 37°C, reached the logarithmic growth phase at 4 hours, and reached the plateau phase at 14 hours, with an OD600 nm of 2.06.

[0034] (2) Acid tolerance experiment

[0035] Take 10 mL of Clostridium butyricum cultured for 14 h, centrifuge at 3000 rpm for 10 min, discard the supernatant, wash twice with PBS, resuspend the cells with RCM medium, and adjust the pH to 2.0, 3.0, 5.0, and 7.0 with hydrochloric acid. Incubate in an anaerobic incubator at 37 °C for 2 h. Then take 100 μL each, serially dilute with PBS, and spread on RCM agar plates for viable cell counting. The results are as shown in Figure 1 B in Figure 1 , and as can be seen from B in Figure 1 , Clostridium butyricum NCU-27 showed high adaptability at different pH values (2.0, 3.0, 5.0, and 7.0). Especially at pH = 7, the number of surviving bacteria was the highest, reaching 1.6×10 8 CFU / mL.

[0036] (3)Bile salt tolerance experiment

[0037] Take 10 mL of Clostridium butyricum cultured for 14 h, centrifuge at 3000 rpm for 10 min, discard the supernatant, wash twice with PBS, resuspend the cells with RCM medium and add 0.3%, 0.2%, and 0.1% bile salts. Incubate in an anaerobic incubator at 37 °C for 2 h. Then take 100 μL each, serially dilute with PBS, and spread on RCM agar plates for viable cell counting. The results are as shown in Figure 1 C in Figure 1 , and as can be seen from C in Figure 1 , Clostridium butyricum NCU-27 had strong tolerance to bile salts. Even at a bile salt concentration of 0.3%, the number of surviving bacteria was still as high as 7.0×10 7 CFU / mL.

[0038] (4)Hemolysis experiment

[0039] Take 10 mL of Clostridium butyricum cultured for 14 h, inoculate 20 μL on a blood agar plate containing 5% sheep blood, and incubate in an anaerobic incubator at 37 °C for 24 h. Use Staphylococcus aureus (ATCC25923) as a positive control to perform a hemolysis test on Clostridium butyricum NCU-27, and observe and measure the diameter of the clear hemolysis zone. The results are as shown in Figure 1 D in Figure 1 , and as can be seen from D in Figure 1 , Clostridium butyricum NCU-27 did not have a hemolysis ring, while the positive control group (Staphylococcus aureus) had an obvious hemolysis ring.

[0040] (5)Antibiotic susceptibility test

[0041] After taking 30 μL of the activated Clostridium butyricum NCU-27 bacterial solution twice and spreading it on an RCM agar plate, stick the antibiotic susceptibility test discs on the surface of the agar plate. After incubating in an anaerobic incubator at 37 °C for 16 h - 18 h, measure and record the diameter of the inhibition zone. The results are as shown in Figure 1 E in Figure 1as shown by F in, and by Figure 1 E- in Figure 1 As can be seen from F in, the average inhibition zone diameters of Clostridium butyricum NCU-27 against erythromycin (EM), penicillin (PEN), ciprofloxacin (CIP), ampicillin (AMP), lincomycin (MY), ceftriaxone (CTR), gentamicin (GEN), chloramphenicol (CAP) and tetracycline (TET) in the antimicrobial susceptibility test were 1.77 cm, 3.23 cm, 1.20 cm, 4.37 cm, 0.60 cm, 2.80 cm, 0.70 cm, 3.33 cm and 4.37 cm, respectively, while it showed resistance to sulfamethoxazole-trimethoprim (SXT). These results confirmed that Clostridium butyricum NCU-27 is a probiotic strain with excellent properties.

[0042] (6) Antioxidant experiment

[0043] Take the Clostridium butyricum NCU-27 bacterial solution activated twice respectively, filter it through a 0.22 μm sterile filter membrane, and retain the culture supernatant for subsequent determination.

[0044] a. DPPH radical scavenging ability

[0045] Add the bacterial culture supernatant to the DPPH methanol solution (0.2 mmol / L), react at room temperature in the dark for 30 min, and measure OD517. Calculate through the formula: Scavenging rate = [1 - A517 (sample) - A517 (control) / A517 (blank)] × 100%.

[0046] b. Hydroxyl radical (·OH) scavenging ability

[0047] Mix the FeSO 4 solution (2 mmol / L), H 2 O 2 (6 mmol / L), salicylic acid (6 mmol / L) and the bacterial culture supernatant, let it stand at room temperature for 30 min, and then measure OD510. Calculate through the formula: Scavenging rate = [1 - A510 (sample) / A510 (control)] × 100%.

[0048] c. Superoxide radical (O 2- ) scavenging ability

[0049] Mix the Tris-HCL solution (150 mmol / L, pH = 8), pyrogallol solution (1.2 mmol / L) and the bacterial culture supernatant, react at room temperature for 30 min, and measure OD330. Calculate through the formula: Scavenging rate = [1 - (A11 - A10) / (A01 - A00)] × 100%.

[0050] Among them: A00: without sample and pyrogallol; A01: without sample, with pyrogallol

[0051] A10: with sample, without pyrogallol; A11: with sample and pyrogallol

[0052] d. Chelation ability of Fe 2+

[0053] Invert and mix 0.4% ferrous sulfate solution and bacterial culture supernatant, then add 1% VC solution and NaOH solution (0.2 mol / L), react at room temperature for 20 min, then add 10% trichloroacetic acid, centrifuge at 4°C and 6000 rpm / min for 10 min to remove proteins, and take the supernatant. Mix the supernatant with 0.1% o-phenanthroline, react at room temperature for 10 min, and measure OD546. Calculate through the formula: Chelation ability = [A546 (blank) - A546 (sample) / A546 (blank)] × 100%.

[0054] e. Total reducing power

[0055] Mix PBS (pH = 6.6), 1% K 3 [Fe(CN) 6 solution and bacterial culture supernatant, invert and mix well, react at 50°C for 2 min. After adding 10% trichloroacetic acid solution, add deionized water and 0.1% FeCl 3 solution to the mixture, let it stand for 10 min, and measure OD700.

[0056] The above experimental results are as shown in G in Figure 1 , and it can be seen from G in Figure 1 that the scavenging abilities of Clostridium butyricum NCU-27 against DPPH, •OH and •O 2- are 46.02%, 37.37% and 37.21% respectively. In addition, its chelation ability for Fe 2+ is 10.35%, and the OD 700 nm of the total reducing ability is 1.09.

[0057] (7) Gram staining

[0058] Under aseptic conditions, use an inoculation loop to pick the bacterial solution and spread it on a glass slide. After making the slide, perform Gram staining and observe the cell morphology under a microscope. It is straight rod-shaped or slightly curved, single or paired, short-chain, with spores (as shown in H in Figure 1 ).

[0059] Example 3

[0060] Clostridium butyricum (( Clostridium butyricum ​)Investigation on the Experiment of NCU-27 in Improving the Postoperative Insulin Resistance Level of Gastrectomized SD Rats

[0061] 1. Establishment, Grouping and Treatment of Gastrectomized SD Rat Animal Model

[0062] Modeling:

[0063] The rats were fasted for 12 hours before surgery without water deprivation. Ensure sufficient anesthesia before surgery, and shave and disinfect the surgical area. Make a midline abdominal incision about 3 cm, and expand the incision with a retractor. Lift the left lobe of the liver, clamp the gastric body and pull it out of the abdominal cavity, and protect it with a wet gauze. Cut the ligaments, free the lesser curvature, greater curvature and lower esophagus of the stomach, and ligate the left gastroepiploic vessels. Cut the duodenum 0.5 cm proximal to the pylorus and the esophagus 0.5 cm above the cardia, and disinfect the incision. Anastomose the lower end of the esophagus with the duodenum, stop bleeding and check the suture. Wash the abdominal cavity, suture the incision and disinfect it. Inject 20 mL of normal saline after surgery, keep warm and resuscitate, and put the rats back into the cage after waking up.

[0064] Grouping: ① Model (M) group: Gastric gavage with 1 mL of normal saline daily for 4 weeks before surgery; ② Clostridium butyricum (CB) group: Gastric gavage with 1 mL of Clostridium butyricum bacterial solution at a concentration of 1×10 8 CFU / mL daily for 4 weeks before surgery.

[0065] 2. Comparison of Fasting Blood Glucose and Fasting Insulin Levels in Rats of Each Group

[0066] Fasting blood glucose measurement (FBG) and fasting insulin measurement (FINS) were performed on the 1st and 3rd days after surgery respectively.

[0067] Biochemical analysis: Commercial enzyme-linked immunosorbent assay (ELISA) kits (enzyme immunoassay, China) were used to detect serum insulin and liver glycogen levels. Fasting blood glucose (FBG) was detected using a blood glucose meter (Sinocare, China).

[0068] The serological parameter results of the SD rats after surgery are as shown in Figure 2 A- Figure 2 B in Figure 2 and E- Figure 2 F in Figure 2 A- Figure 2 B in Figure 2 and E- Figure 2 F in

[0069] 3. Comparison of Insulin Resistance in Rats of Each Group

[0070] Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) = fasting insulin level (uIU / mL) × fasting blood glucose level (mmol / L) / 22.5

[0071] The results of its insulin resistance index are as Figure 2 shown by C in Figure 2 and G in Figure 2 Moreover, C in Figure 2 and G in

[0072] Intraperitoneal Glucose Tolerance Test (IPGTT, on the second day after surgery):

[0073] The rats were fasted for 12 h and then intraperitoneally injected with 40% glucose solution (5 mL / kg). Blood glucose levels were measured fasting and at 0, 15 min, 30 min, 60 min, and 120 min after glucose load. An IPGTT curve was plotted based on the results, and the area under the curve (AUC) was calculated as (0 min glucose + 2 × 30 min glucose + 3 × 60 min glucose + 2 × 120 min glucose) / 4.

[0074] The results are as Figure 2 shown by D in Figure 2 Moreover, it can be seen from D in

[0075] 4. Comparison of liver tissues of rats in each group

[0076] Quantitative detection of liver glycogen: Detected using an Elisa kit

[0077] The results are as Figure 2 shown by H in Figure 2 Moreover, it can be seen from H in

[0078] 5. Comparison of intestinal flora differences between M group and CB group rats - 16S rDNA sequencing

[0079] The potential mechanism of Clostridium butyricum NCU-27 in improving IR after gastrectomy in SD rats was further explored. First, 16S rDNA gene sequencing was used to compare the changes in the intestinal microbiota of the two groups of SD rats after surgery. The results are as Figure 3 shown by A- Figure 3As shown by C in [reference], and the results indicate that there is no significant difference in the α-diversity index (including the Shannon index) between the two groups of rats. It is worth noting that the principal coordinate analysis shows a significant difference in the β-diversity of the gut microbiota between the two groups (P < 0.001). In addition, the Venn diagram also shows differences in the gut microbiota between the two groups.

[0080] Subsequently, the composition of the gut microbiota of the two groups was further analyzed at the phylum level and genus level. The results are as Figure 3 shown in D- Figure 3 shown in E in [reference], and as Figure 3 shown in D- Figure 3 shown in E in [reference], at the phylum level, there is no significant difference in Firmicutes between the CB group and the M group, but the Verrucomicrobia and Bacteroidetes in the CB group are significantly increased (P < 0.05). At the genus level, compared with the M group, the genera Akkermansia, Turicibacter, and Roseburia in the CB group are significantly increased, while the genus Streptococcus is significantly decreased (P < 0.05).

[0081] In addition, the results of the random forest analysis show ( Figure 3 shown in F in [reference]) that at the genus level, the CB group is enriched in the genera Akkermansia, Bifidobacterium, Prevotella, and Roseburia, while the M group is enriched in the genus Streptococcus. The above results indicate that Clostridium butyricum NCU-27 can regulate the gut microbiota after gastrectomy in SD rats and increase the abundance of related butyrate-producing bacteria.

[0082] 6. Comparison of the intestinal mucosal barrier permeability of rats in each group

[0083] Collect the colon tissues of mice in each group, and detect the intestinal (colon) permeability protein ZO-1 (tight junction protein) by immunological experiments.

[0084] Immunofluorescence: Incubate the paraffin-embedded sections with the primary antibody against ZO-1 overnight at 4°C, then incubate with the appropriate secondary antibody, and counterstain with DAPI. Fluorescence can be seen under a fluorescence microscope.

[0085] Immunofluorescence staining of intestinal tight junction protein-1 (ZO-1) in two groups of rats was performed to evaluate the intestinal barrier function. The results are shown by Figure 4 A in [reference], and the intervention of Clostridium butyricum NCU-27 increased the expression level of intestinal ZO-1 in gastrectomized SD rats after surgery.

[0086] 7. Targeted metabolomics detection - Detection of SCFA content (GC-MS)

[0087] The metabolites in fecal samples were analyzed by gas chromatography - mass spectrometry (GC - MS). Feces (20 mg) were mixed with phosphoric acid solution and steel beads, ground, vortexed, sonicated, and then centrifuged. 0.1 mL of the supernatant was taken, 0.5 mL of methyl tert - butyl ether (MTBE) was added, vortexed and sonicated again, and then centrifuged, and the supernatant was collected. Analysis was performed using an Agilent 7890B GC - MS and a DB - 5MS chromatographic column.

[0088] In addition to the gut microbiota, targeted metabolomics analysis was performed on the fecal samples of rats in each group to clarify the metabolic changes of short - chain fatty acids (SCFAs) within the gut microbiota. The results are as shown in Figure 4 B - Figure 4 in H, and as can be seen from Figure 4 B - Figure 4 in H, compared with the M group, the levels of acetic acid, caproic acid, isobutyric acid, isovaleric acid, propionic acid, and valeric acid in the feces of the CB group did not change significantly; on the contrary, the level of butyric acid in the CB group increased significantly (P < 0.05).

[0089] Example 4

[0090] Experiment on exploring the regulatory effect of Clostridium butyricum on insulin resistance in SD rats after gastrectomy through butyric acid

[0091] 1. Model selection: SD rat gastrectomy model, the construction process is the same as that in Example 3.

[0092] 2. Grouping: ① Blank control (C) group: gavaged with 1 mL of normal saline daily; ② Model (M) group: gavaged with 1 mL of normal saline daily 4 weeks before surgery; ③ Clostridium butyricum (CB) group: gavaged with 1 mL of Clostridium butyricum suspension at a concentration of 1×10 8 CFU / mL daily for 4 weeks before surgery; ④ Sodium butyrate preparation (BA) group: gavaged with 1 mL of sodium butyrate, 400 mg / kg / d, for 4 consecutive weeks.

[0093] 3. Compare the fasting blood glucose and fasting insulin levels of rats in each group

[0094] Fasting blood glucose measurement (FBG) + fasting insulin measurement (FINS) were performed on the first and third days after surgery respectively

[0095] To explore the effect of the metabolite butyric acid on the postoperative IR level in gastrectomized SD rats, in this study, a gastrectomy model was constructed after gavaging SD rats with sodium butyrate for 4 weeks. As shown in Figure 5 A - Figure 5 in B and Figure 5 E - Figure 5As can be seen from F in [reference], the fasting blood glucose levels and fasting insulin levels on the 1st and 3rd days after surgery were significantly increased in group M compared with group C (P<0.05). However, the intervention of Clostridium butyricum NCU-27 and butyric acid significantly improved these parameters (P<0.05).

[0096] 4. Compare the insulin resistance of rats in each group

[0097] Insulin resistance index (HOMA-IR) = fasting insulin level (uIU / mL) × fasting blood glucose level (mmol / L) / 22.5

[0098] The results of its insulin resistance index are as Figure 5 C in [reference] and Figure 5 G in [reference] show that, as can be seen from C in [reference] and Figure 5 C in [reference] and Figure 5 G in [reference], the insulin resistance index on the 1st and 3rd days after surgery was significantly increased in group M compared with group C (P<0.05). However, the intervention of Clostridium butyricum NCU-27 and butyric acid significantly improved these parameters (P<0.05).

[0099] Intraperitoneal glucose tolerance test IPGTT (the 2nd day after surgery):

[0100] The rats were fasted for 12 h and then intraperitoneally injected with 40% glucose solution (5 mL / kg). The blood glucose levels at fasting and 0, 15 min, 30 min, 60 min, 120 min after glucose load were measured. According to the results, an IPGTT curve was plotted, and the area under the curve (AUC) = (0 min glucose + 2×30 min glucose + 3×60 min glucose + 2×120 min glucose) / 4 was calculated.

[0101] The results of the intraperitoneal glucose tolerance test on the 2nd day after surgery are as Figure 5 D in [reference] shows that compared with group C, the glucose tolerance of group M was significantly decreased, while it was significantly improved under the intervention of Clostridium butyricum NCU-27 and butyric acid (P<0.05).

[0102] 5. Compare the liver tissues of rats in each group

[0103] Quantitative detection of liver glycogen: It was detected using an Elisa kit

[0104] The results are as Figure 5 H in [reference] shows that, as can be seen from H in [reference] Figure 5 the liver glycogen content in group M on the 3rd day after surgery was significantly lower than that in group C, but it was significantly improved after the intervention of Clostridium butyricum NCU-27 and butyric acid (P<0.05). These findings indicate that butyric acid can improve the IR level in SD rats after gastrectomy.

[0105] 6. Detection of changes in key proteins in the insulin signaling pathway in the liver tissues of rats in each group by q-PCR (AMPK, JNK, ERK, Raptor, IRS1, AKT)

[0106] Total RNA was extracted from rat liver tissues using Trizol lysis reagent. Reverse transcription was performed using a reverse transcription kit. On the ViiA 7 real-time PCR system, real-time quantitative PCR analysis of mRNA expression levels was carried out using the TB Green® Premix Ex TaqTM kit. The 2 -△△Ct -method was used for data processing, with β-actin as the internal reference gene.

[0107] To deeply explore the specific mechanism by which Clostridium butyricum NCU-27 and butyric acid improve postoperative IR in the gastrectomized SD rat model, PCR quantification was used to evaluate the changes in the related insulin signaling pathway in liver tissues. As Figure 6 shown in A of, there were no significant changes in the expression levels of AMPK, JNK, and ERK among the groups. However, compared with other groups, the expression level of Raptor in the M group was significantly increased, while the expression levels of IRS1 and AKT were significantly decreased (P<0.05).

[0108] 7. Detection of changes in key proteins in mTORC1 in the liver tissues of rats in each group by Western blot (Raptor, P Ser -IRS1, IRS1, P-AKT, AKT, GLUT4, G6pase, Pepck, GYS2, β-actin)

[0109] Tissue samples were treated with RIPA lysis buffer containing protease inhibitors, homogenized on ice, and centrifuged at 4°C to obtain the supernatant. The protein concentration was determined using a BCA protein quantification kit. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. After blocking with a skim milk solution, they were incubated with primary and secondary antibodies, and finally protein visualization was performed using an enhanced chemiluminescence solution.

[0110] The results are as Figure 6 shown in B- Figure 6As shown by J in [reference], and the results indicate that compared with group C, the expression level of Raptor in group M was significantly higher, the phosphorylation level of serine residues of IRS1 was significantly higher, and the phosphorylation level of AKT was significantly lower (P<0.05). In addition, the expression levels of GLUT4 and GYS2 in group M were significantly decreased, while the expression levels of G6pase and Pepck were significantly increased. These indicators were significantly improved after intervention with Clostridium butyricum NCU-27 and butyric acid (P<0.05). Based on the above results, it was found that Clostridium butyricum NCU-27 and butyric acid could improve the postoperative IR level in the gastrectomized SD rat model. In addition, the mTORC1 signaling pathway may be involved in this process.

[0111] Example 5

[0112] Explore the regulatory effect of Clostridium butyricum ( Clostridium butyricum ) on insulin resistance after gastrectomy in SD rats through butyric acid-mediated mTORC1 pathway

[0113] 1. Model selection: SD rat gastrectomy model, the construction process was the same as that in Example 3.

[0114] 2. Grouping: ① Blank control (C) group: gavaged with 1 mL of normal saline daily; ② Model (M) group: gavaged with 1 mL of normal saline daily for 4 weeks before surgery; ③ Clostridium butyricum (CB) group: gavaged with 1 mL of Clostridium butyricum bacterial solution at a concentration of 1×10 8 CFU / mL daily for 4 weeks before surgery; ④ Sodium butyrate preparation (BA) group: gavaged with 1 mL of sodium butyrate, 400 mg / kg / d, for 4 consecutive weeks; ⑤ mTORC1 inhibitor treatment (RA) group: intraperitoneally injected with rapamycin solution at 1 mg / (kg·d); ⑥ Clostridium butyricum preparation + mTORC1 agonist treatment (CBL) group: gavaged with 1 mL of Clostridium butyricum bacterial solution at a concentration of 1×10 8 CFU / mL daily for 4 weeks before surgery + given 1.5% L-leucine solution for free drinking; ⑦ Sodium butyrate preparation + mTORC1 agonist group treatment (BAL) group: gavaged with 1 mL of sodium butyrate + given 1.5% L-leucine solution for free drinking, 400 mg / kg / d, for 4 consecutive weeks.

[0115] 3. Compare the fasting blood glucose and fasting insulin levels, insulin resistance, and liver glycogen quantitative detection of rats in each group

[0116] (1) Fasting blood glucose measurement (FBG) + fasting insulin measurement (FINS) (the 1st day + the 3rd day after surgery)

[0117] (2) Insulin resistance index (HOMA-IR)

[0118] (3) Intraperitoneal Glucose Tolerance Test IPGTT (on the 2nd day after surgery)

[0119] (4) Hepatic glycogen quantification - Elisa

[0120] The specific procedures of the above (1)-(4) are the same as those in Example 3.

[0121] The above detection results are as Figure 7 shown in A- Figure 7 shown in H of [reference], and from the results, it can be seen that on the 1st and 3rd days after surgery, the fasting blood glucose level, fasting insulin level, and insulin resistance index show that the mTORC1 agonist can counteract the beneficial effects of Clostridium butyricum NCU-27 and butyric acid on the above indicators. In addition, the effect of using the mTORC1 inhibitor alone on improving IR after surgery in SD rats is similar to that of the Clostridium butyricum NCU-27 and butyric acid groups (P<0.05). The results of the intraperitoneal glucose tolerance test on the 2nd day after surgery and the hepatic glycogen content on the 3rd day after surgery also show similar findings (P<0.05).

[0122] 4. Western blot was used to detect the changes of key proteins in mTORC1 in the liver tissues of rats in each group (Raptor, P Ser -IRS1, IRS1, P-AKT, AKT, GLUT4, G6pase, Pepck, GYS2, β-actin)

[0123] The detection steps are the same as those in Step 7 of Example 4, and the results are as Figure 8 shown in A- Figure 8 shown in I of [reference], and from the results, it can be seen that the mTORC1 agonist counteracts the inhibitory effect of Clostridium butyricum NCU-27 or butyric acid on Raptor inhibition, the inhibitory effect on the phosphorylation level of serine residues of IRS1, and the stimulatory effect on the phosphorylation level of AKT, while the effect of using the mTORC1 inhibitor alone is similar to that of the CB and BA groups (P<0.05). In addition, compared with the M group, the expression levels of GLUT4 and GYS2 in the C, CB, BA, and RA groups are significantly increased, while the expression levels of G6pase and Pepck are significantly decreased. These changes can be counteracted by the mTORC1 agonist (P<0.05). In summary, these data indicate that Clostridium butyricum NCU-27 and butyric acid play an important role in improving postoperative IR in the gastrectomized SD rat model through the gut-liver axis and the mTORC1 signaling pathway.

[0124] As can be seen from the above results, Clostridium butyricum NCU-27 provided by the present invention improved the postoperative IR in the gastrectomized SD rat model by regulating the gut microbiota and short-chain fatty acid metabolism, especially by increasing the production of butyric acid. This improvement may be achieved through the enterohepatic axis and the mTORC1 signaling pathway, providing a potential strategy for the clinical use of probiotics and butyric acid derivatives to treat postoperative IR in the future.

[0125] Although the description of the present invention has been quite detailed and several of the described embodiments have been described in particular, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but rather should be regarded as providing a broad interpretation of these claims in light of the prior art by reference to the appended claims, so as to effectively cover the intended scope of the present invention. In addition, the present invention has been described above in terms of embodiments foreseeable by the inventors for the purpose of providing a useful description, and non-substantive modifications to the present invention that are not currently foreseeable may still represent equivalent modifications of the present invention.

Claims

1. Use of Clostridium butyricum NCU-27 in the preparation of a drug for preventing, improving or treating insulin resistance after gastrectomy, characterized in that: The gene sequence of 16S rDNA of Clostridium butyricum NCU-27 is shown in SEQ ID No: 1; The Clostridium butyricum ( Clostridium butyricum )NCU-27 was deposited on July 22, 2024 at the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.31386.

2. A biological agent, characterized in that: Its active ingredients include Clostridium butyricum NCU-27; the Clostridium butyricum NCU-27 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 22, 2024, with the address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO.31386.

3. Use of the biological preparation according to claim 2 in the preparation of a drug for preventing, improving or treating insulin resistance after gastrectomy.

4. A drug for preventing, improving or treating insulin resistance after gastrectomy, characterized in that: The drug includes Clostridium butyricum NCU-27; the Clostridium butyricum NCU-27 has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 22, 2024, with the address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.31386.

5. The drug according to claim 4, characterized in that The medicine also includes pharmaceutical excipients.

6. The drug according to claim 5, characterized in that The pharmaceutical excipient is at least one of water, lactose, sodium chloride and glucose.

7. The drug according to any one of claims 4 to 6, characterized in that The dosage form of the medicine is powder, granule, capsule or tablet.

8. The drug according to claim 7, characterized in that The medicine is an oral medicine.

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