Application of clostridium butyricum NCU-02 in preparation of medicine for improving hyperlipemia and / or autism

Abstract

The invention discloses application of clostridium butyricum NCU-02 in preparation of a medicine for improving hyperlipidemia and / or autism, and relates to the technical field of biological medicine. The clostridium butyricum NCU-02 is derived from feces of 100-year-old old people, and is preserved in the China General Microbiological Culture Collection Center on August 8, 2022, the address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.25504. The clostridium butyricum NCU-02 is named as clostridium butyricum NCU-02, and the clostridium butyricum NCU-02 is named as clostridium butyricum NCU-02. The clostridium butyricum NCU-02 is named as clostridium butyricum NCU-02. Experiments prove that the clostridium butyricum NCU-02 can significantly improve hyperlipidemia by improving one or more of body inflammation, oxidative stress, lipid metabolism abnormality, liver injury and intestinal microbiota disorder, and the clostridium butyricum NCU-02 and its metabolite butyric acid can improve autism through a GPR109A / AMPK / Nrf2 signal pathway; therefore, the clostridium butyricum NCU-02 provided by the invention can be used for preparing the medicine for improving the hyperlipidemia and / or the autism, and has huge application potential.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to the use of Clostridium butyricum NCU-02 in the preparation of drugs for improving hyperlipidemia and / or autism. Background Art

[0002] Hyperlipidemia is a common lipid metabolism disorder characterized by elevated serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels or decreased high-density lipoprotein cholesterol (HDL-C) levels. It is one of the main risk factors for cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease, myocardial infarction, and stroke. Currently, although statins are widely used to treat hyperlipidemia, their potential adverse reactions such as liver damage and rhabdomyolysis limit their application.

[0003] APOE knockout (APOE- / -) mice completely lack APOE, which exacerbates lipid accumulation in the mouse blood, leading to impaired clearance of chylomicrons (CM), very low-density lipoprotein (VLDL) and low-density lipoprotein in plasma, thereby increasing the cholesterol level in plasma. The APOE- / - mouse model can be used to simulate human hypertriglyceridemia and hypercholesterolemia.

[0004] The prevention and treatment of hyperlipidemia requires comprehensive consideration of multiple factors. However, there is currently a lack of comprehensive intervention strategies for hyperlipidemia.

[0005] Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by persistent deficits in social interaction and communication, as well as restricted and repetitive behavior patterns, that manifest in early childhood. ASD affects more than 1% of children in Western countries, while the prevalence in China is as high as 0.7%, and the rate is increasing due to improvements in identification, screening, clinical assessment, and diagnostic testing, which places a huge economic burden on patients and society.

[0006] At present, the treatment methods for ASD are mainly divided into general treatment and drug treatment. General treatment mainly refers to early education for ASD and intervention training on behavioral pathways, but these early intervention treatments have a long cycle and limited treatment effects. There is currently no specific drug for ASD, and it can only be treated with small doses of atypical psychiatric drugs such as risperidone and aripiprazole based on obvious emotional and behavioral problems. However, these treatments are symptomatic treatments, mainly used to improve emotions or behaviors, and have no obvious effect on core symptoms. In addition, some drug side effects may occur during medication. In this context, it is of great significance to find a new universal and effective treatment method for ASD. Summary of the invention

[0007] The present application provides a Clostridium butyricum NCU-02 and its application in improving hyperlipidemia, which can solve one of the problems existing in the prior art.

[0008] The following technical solutions are specifically adopted: In the first aspect, the present application provides an application of Clostridium butyricum NCU-02 in the preparation of drugs for improving hyperlipidemia and / or autism. Clostridium butyricum NCU-02 is derived from the feces of centenarians and was deposited in the General Microbiology Center of China National Culture Collection Administration on August 8, 2022. The address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.25504.

[0009] Furthermore, the gene sequence of 16S rDNA of Clostridium butyricum NCU-02 is shown in SEQ ID No: 1.

[0010] SEQ ID No: 1:

[0011] Furthermore, Clostridium butyricum NCU-02 improves hyperlipidemia by improving one or more of the body's inflammation, oxidative stress, dyslipidemia metabolism, liver damage and intestinal microbiota disorders.

[0012] Furthermore, the application also includes the use of butyric acid, a metabolite of Clostridium butyricum NCU-02, in the preparation of drugs for improving autism.

[0013] Furthermore, Clostridium butyricum NCU-02 and its metabolite butyrate improve autism through the GPR109A / AMPK / Nrf2 signaling pathway.

[0014] In a second aspect, the present application provides a biological preparation, the active ingredient of which includes Clostridium butyricum NCU-02 and / or butyric acid, a metabolite of Clostridium butyricum NCU-02.

[0015] In a third aspect, the present application provides an auxiliary drug for the treatment of hyperlipidemia and / or autism, wherein the active ingredient of the drug includes Clostridium butyricum NCU-02 and / or butyric acid, a metabolite of Clostridium butyricum NCU-02.

[0016] Furthermore, the drug also includes a drug carrier and / or a pharmaceutical excipient.

[0017] Furthermore, the drug carrier and / or pharmaceutical excipient is at least one of water, lactose, sodium chloride and glucose.

[0018] Furthermore, the dosage form of the drug is powder, granules, capsules or tablets.

[0019] The application of Clostridium butyricum NCU-02 provided in the present application has at least one of the following benefits: 1) Clostridium butyricum NCU-02 can comprehensively improve hyperlipidemia by improving blood lipids, body inflammation, oxidative stress, abnormal cholesterol metabolism and intestinal flora disorders, and can also be used as an auxiliary drug for the treatment of hyperlipidemia. According to the experiments provided in the examples of the present application, the Clostridium butyricum NCU-02 provided in the present application can significantly improve the hyperlipidemia of mice after acting on them. It is found that Clostridium butyricum NCU-02 improves blood lipids, body inflammation and oxidative stress, promotes cholesterol metabolism, improves intestinal flora, and thus significantly improves hyperlipidemia. It can be seen that the Clostridium butyricum NCU-02 provided in the present application can be used to prepare drugs for improving hyperlipidemia, and has great application potential.

[0020] 2) Clostridium butyricum NCU-02 and its metabolite butyric acid have an improving effect on the behavior of autistic mice, and can significantly reduce the oxidative stress and inflammatory response of the hippocampal tissue of autistic mice; and the intervention of Clostridium butyricum NCU-02 reduces the activated microglia in the hippocampus of autistic mice, thereby improving autism. It is also found that Clostridium butyricum NCU-02 and its metabolite butyric acid improve autism through the GPR109A / AMPK / Nrf2 signaling pathway. It can be seen that the Clostridium butyricum NCU-02 provided in this application can be used to prepare drugs for improving autism, and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a graph showing the evaluation results of the probiotic properties of Clostridium butyricum NCU-02 provided in Example 1 of the present application, wherein A is a schematic diagram of the screening of bacteria from centenarians; B is a morphological diagram of Clostridium butyricum NCU-02 under Gram staining microscope; C is a graph showing the growth curve of Clostridium butyricum NCU-02 measured by optical density of bacterial suspension; D is a graph showing the count of live bacteria of C. butyricum NCU-02; E is a graph showing the acid resistance evaluation of C. butyricum NCU-02; F is a graph showing the bile resistance evaluation of C. butyricum NCU-02; G is a graph showing the inhibitory effect of C. butyricum NCU-02 on the growth of intestinal pathogens; H is a graph showing the results of a hemolysis test of C. butyricum NCU-02; (a: C. butyricum NCU-02, b: positive control): Staphylococcus aureus; IJ is a graph showing the drug sensitivity results of C. butyricum NCU-02.

[0022] Figure 2 This is a schematic diagram of the hyperlipidemia model provided in Example 2 of the present application.

[0023] Figure 3 This is a graph of serum TC and TG levels after 2 weeks of high-fat diet modeling provided in Example 2 of the present application.

[0024] Figure 4 This is a graph of serum TC, TG, LDL-c and HDL-c levels after 4 weeks of high-fat diet modeling provided in Example 2 of the present application.

[0025] Figure 5 These are representative images of the appearance and liver of mice after 4 weeks of high-fat diet modeling provided in Example 2 of the present application, wherein: (A) a representative image of the appearance of mice; and (B) a representative image of the liver.

[0026] Figure 6Schematic diagram of Clostridium butyricum NCU-02 improving mouse body weight and blood lipids provided in Example 2 of the present application, wherein: (A) Schematic diagram of the intervention of the entire experiment; (B) Weekly weight changes of mice in each group; (C) Representative diagram of the whole body of mice; (D) Serum TC; (E) Serum TG; (F) Serum LDL-c; (G) Serum HDL-c; C: control group (n = 6); M: model group (n = 6); MA: positive drug group (n = 6); ML: low-dose probiotic group (10 7 CFU / mL, n = 6); MH: high-dose probiotic group (10 9 CFU / mL, n = 6). Each point represents individual biological replicate data. Data are presented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0027] Figure 7 This is a schematic diagram of the amount of water consumed and the amount of water consumed by the hyperlipidemia model mice during the entire experiment provided in Example 2 of the present application.

[0028] Figure 8 Schematic diagram of Clostridium butyricum NCU-02 reducing inflammation and oxidative stress in the body provided in Example 2 of the present application, wherein (AC) ELISA method is used to detect the content of inflammatory factors IL-6, TNF-α, and IL-10 in mouse serum. (DG) Mouse serum antioxidant indexes and GSH-Px, SOD, CAT, and MDA levels. (H) Heat map of inflammatory factors and antioxidant indexes. Red indicates positive correlation, and green indicates negative correlation. C: control group (n = 6); M: model group (n = 6); MA: positive drug group (n = 6); ML: low-dose probiotic group (10 7 CFU / mL, n = 6); MH: high-dose probiotic group (10 9 CFU / mL, n= 6). Each point represents individual biological replicate data. Data are presented as mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0029] Fig. 9Schematic diagram of Clostridium butyricum NCU-02 improving liver lipid deposition through PPARγ-dependent mechanism provided in Example 2 of the present application, wherein: (A) representative image of liver; (B) Oil red O staining of liver sections (200x, 400x); (C) serum AST; (D) serum ALT; (EH) Western blot analysis of PPARγ-LXRα-ABCA1 and PPARγ, LXRα, ABCA1 expression in liver tissue. C- D: C: control group (n = 6); M: model group (n = 6); MA: positive drug group (n = 6); ML: low-dose probiotic group (10 7 CFU / mL, n = 6); MH: high-dose probiotic group (10 9 CFU / mL, n = 6). EH:C: control group (n = 3); M: model group (n = 3); MA: positive drug group (n = 3); ML: low-dose probiotic group (10 7 CFU / mL, n = 3); MH: high-dose probiotic group (10 9 CFU / mL, n = 3). Each point represents individual biological replicate data. Data are presented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0030] Fig.10 This is a schematic diagram of the effect of Clostridium butyricum NCU-02 on the intestinal barrier provided in Example 2 of the present application, wherein: (A) representative image of the colon; (B) colon length; (C) representative image of H&E staining of colon sections; (DF) Western blot analysis of the expression of Occludin and ZO-1 proteins in colon tissue. C: control group (n = 3); M: model group (n = 3); MA: positive drug group (n = 3); ML: low-dose probiotic group (10 7 CFU / mL, n = 3); MH: high-dose probiotic group (10 9 CFU / mL, n = 3). Each point represents individual biological replicate data. Data are presented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0031] Fig.11This is a schematic diagram of Clostridium butyricum NCU-02 improving intestinal flora provided in Example 2 of the present application, wherein, (A) rarefaction curve; (B) Chao1 index, representing α diversity; (C) Observed_species index, representing α diversity; (D) principal coordinate analysis (PCoA) represents the β diversity of intestinal flora; (E) Venn diagram represents the number of unique OTUs between groups; (F) analysis of intestinal flora abundance at the phylum level. (GI) Relative abundance of Firmicutes, Bacteroidetes and Actinobacteria. C: control group (n = 6); M: model group (n = 6); MA: positive drug group (n = 6); ML: low-dose probiotic group (10 7 CFU / mL, n = 6); MH: high-dose probiotic group (10 9 CFU / mL, n = 6). Each point represents individual biological replicate data. For box plots, the center line represents the median, the box boundaries represent the quartiles, and the upper and lower sides represent the maximum to minimum values. Data are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0032] Fig.12 Schematic diagram of the effect of Clostridium butyricum NCU-02 on intestinal flora composition provided in Example 2 of the present application, wherein: (A) Analysis of intestinal flora abundance at the genus level. (BD) Relative abundance of Muribaculaceae, Blautia and Dubosiella. (E) Analysis of intestinal flora abundance at the species level. (F) Relative abundance of Lachnospiraceae. C: control group (n = 6); M: model group (n = 6); MA: positive drug group (n = 6); ML: low-dose probiotic group (10 7 CFU / mL, n = 6); MH: high-dose probiotic group (10 9 CFU / mL, n = 6). Each point represents individual biological replicate data. For box plots, the center line represents the median, the box boundaries represent the quartiles, and the upper and lower sides represent the maximum to minimum values. Data are presented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0033] Fig.13 This is a heat map of the correlation between intestinal flora and hyperlipidemia parameters estimated by Spearman correlation analysis provided in Example 2 of the present application.

[0034] Fig.14This is a graph of the results of an open field test conducted on different groups of mice provided in Example 3 of the present application, wherein: (A) different groups of mice - total moving distance; (B) different groups of mice - center residence time; (C) different groups of mice - center moving distance.

[0035] Fig.15 This is a schematic diagram of the movement trajectories of different groups in the open field experiment provided in Example 3 of the present application.

[0036] Fig.16 This is a graph of the results of the bead embedding experiment for different groups provided in Example 3 of the present application.

[0037] Fig.17 This is a diagram of the results of a three-box social experiment for different groups provided in Example 3 of the present application, wherein (A) is the social behavior test result, and (B) is the social novelty preference test result.

[0038] Fig.18 This is a diagram of the Y-maze test results for different groups provided in Example 3 of the present application.

[0039] Fig.19 This is a schematic diagram of different groups of Clostridium butyricum NCU-02 reducing the level of oxidative stress in the hippocampus provided in Example 3 of the present application, wherein: (A) GSH content; (B) GSH-Px content; (C) SOD content; and (D) MDA content.

[0040] Fig. 20 This is a schematic diagram of the effects of different groups on the inflammatory response of hippocampal tissue provided in Example 3 of the present application, wherein (A) IL-1β content; (B) IL-6 content; and (C) TNF-α content.

[0041] Fig.21 The diagrams of butyrate levels in different groups and the abundance of Clostridium butyricum NCU-02 are provided in Example 3 of the present application, including (A) butyrate level in the brain; (B) butyrate level in feces; (C) butyrate level in serum; and (D) abundance of Clostridium butyricum NCU-02.

[0042] Fig. 22 This is a diagram of the fluorescence staining results of hippocampal tissues in different groups provided in Example 3 of the present application.

[0043] Fig.23 This is a graph of the number of microglia in different groups provided in Example 3 of the present application.

[0044] Fig.24This is a schematic diagram of the effects of different groups of mice on intestinal inflammatory response and barrier provided in Example 3 of the present application, wherein (A, B) are colon lengths; (CE) are Western blot analysis of the expression of Occludin and ZO-1 proteins in colon tissue.

[0045] Fig.25 These are the relevant experimental diagrams for verifying autistic behaviors of different groups of mice provided in Example 4 of the present application, wherein: (AB) three-box social experiment; (CF) open field test; (G) buried bead experiment; and (H) Y maze experiment.

[0046] Fig.26 This is a schematic diagram of Clostridium butyricum NCU-02 and its metabolite butyric acid reducing the oxidative stress level in the hippocampus provided in Example 4 of the present application, wherein: (A) GSH content; (B) GSH-Px content; (C) MDA content; and (D) SOD content.

[0047] Fig. 27 This is a schematic diagram of the effects of different groups on the inflammatory response of hippocampal tissue provided in Example 4 of the present application, wherein (A) IL-1β content; (B) IL-6 content; and (C) TNF-α content.

[0048] Fig.28 The figure (A) shows the results of fluorescence staining of hippocampal tissues in different groups and the figure (B) shows the number of microglia in different groups provided in Example 4 of the present application.

[0049] Fig.29 This is a schematic diagram of the effects of different groups of mice on intestinal inflammation and barrier function provided in Example 4 of the present application, wherein (A, B) are colon lengths; and (CE) are Western blot analysis of the expression of Occludin and ZO-1 proteins in colon tissue.

[0050] Fig.30 This is a diagram of proteins related to the effect of butyric acid, a metabolite of Clostridium butyricum NCU-02, on the GPR109A / AMPK / Nrf2 signaling pathway, as provided in Example 4 of the present application; wherein (A) Western blot analysis of the expression of GPR109A, AMPK, P-AMPK and Nrf2 in the GPR109A / AMPK / Nrf2 signaling pathway; (B) GPR109A content; (C) AMPK content; (D) P-AMPK content; and (E) Nrf2 content. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0052] Example 1 1. Screening of Clostridium butyricum NCU-02 and related experiments 1. Sample Collection and Processing like Figure 1 As shown in A, fresh feces from centenarians were stored in sterile centrifuge tubes containing 30% glycerol. The fecal samples collected from centenarians were immediately diluted and spread on a plate and placed in a 10 mL centrifuge tube (containing 5 mL of PBS solution). Then, the sample was centrifuged (800 rpm, 5 min). After centrifugation, 1 mL of the supernatant was taken and placed in a 1.5 mL centrifuge tube for gradient dilution, with the gradient being 10 0 ~10 9 Times (1-9), select the appropriate concentration gradient for plating (RCM culture medium), this time select 3, 5, 7, 9, apply 30 μL, spread on the plate (repeatedly burn the glass rod, and shake the sample before adding it), spread it for growth in an anaerobic incubator, and culture for 24-48 h.

[0053] 2. Single colony picking and cultivation Take a plate with 200-400 colonies, and inoculate 5-10 single colonies into the corresponding 5 mL liquid RCM culture medium for anaerobic activation culture for 24-28 h in a sterile clean bench according to the shape, size, color, neat edges, bulges and faster growing colonies on the plate.

[0054] 3. DNA Extraction and Sequencing (1) Inoculate the isolated bacteria into 5 ml of the corresponding liquid culture medium.

[0055] (2) Use 30% glycerol to preserve the bacteria (if there are many samples, one tube is sufficient), centrifuge (8000 rpm, 2 min), and discard the supernatant.

[0056] (3) Add 600 μL of lysis buffer (lysis buffer: 500 mM NaCl, 50 mM tris-HCl, pH 8.0, 50 mM EDTA, 4% SDS), 200 μL of Tris-saturated phenol and 0.3-0.4 g of glass beads to the precipitate. Oscillate for 30 s, 3 times, until the bacteria are completely suspended. Centrifuge (8000 rpm, 1 min).

[0057] (4) Transfer the supernatant to a new 1.5 mL centrifuge tube, add 250 μL 10 M ammonium acetate, place on ice for 10 min, and centrifuge (8000 rpm, 1 min).

[0058] (5) Take the supernatant above the organic layer and transfer it to the DNA adsorption column. It is better to take less than more. Do not take the organic layer. Centrifuge (8000 rpm, 1 min).

[0059] (6) Wash once with 600 μL of 75% ethanol.

[0060] (7) Repeat (6).

[0061] (8) After shaking at 8000 rpm for 2 min, transfer the DNA adsorption column to a new EP tube and let it dry for 30 min.

[0062] (9) Add 50 μL of TE (pH=8.0) to the dried centrifuge tube and centrifuge. Take 25 μL and send it to the company for sequencing.

[0063] 4. Identification of strains (1) Morphological identification Observe the morphological characteristics of the strain and record them. Perform Gram staining and observe the results.

[0064] (2) Molecular biological identification According to the sequence of 16S rRNA sequencing results, the strain results were searched and compared on NCBI. After selecting special bacteria for secondary identification, they were deposited in the bacteria library. The secondary sequencing results were submitted to the Gen Bank database of the National Center for Biotechnology Information (NCBI) for basic local alignment search tool (BLAST) homology comparison, and the 16S rDNA gene sequence of the model strain with higher homology was selected. The Neighbor Joining (NJ) method in MEGA software was used to construct a phylogenetic tree to determine the bacterial species and classify the bacteria into genera or species.

[0065] The results are as follows Figure 2 As shown in B, one of the strains was determined to be Clostridium butyricum NCU-02 through colony morphology identification, bacterial morphology identification and molecular biology methods.

[0066] After sequencing, the sequence of Clostridium butyricum NCU-02 is: SEQ ID No: 1:

[0067] 2. In vitro performance evaluation of Clostridium butyricum NCU-02 1. Growth curve of Clostridium butyricum NCU-02 Activate Clostridium butyricum NCU-02 overnight, take 1 mL and inoculate into 100 mL of RCM liquid medium, culture anaerobically at 37 °C, and measure its optical density at a wavelength of 600 nm at 0, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 28h, 32h, and 36h. Draw a growth curve with time as the abscissa and optical density as the ordinate.

[0068] It is worth noting that the present application adopts turbidimetry to determine the growth curve, because the concentration of the bacterial suspension is proportional to the turbidity, and the concentration of the bacterial solution can be inferred by measuring the optical density of the bacterial suspension with a spectrophotometer.

[0069] The experimental results are as follows Figure 1 As shown in C, by analyzing the growth curve, it can be seen that Clostridium butyricum NCU-02 has a strong growth characteristic. The strain enters a rapid growth phase at 8 hours and a steady phase at 22 hours. The optical density at 600 nm (OD 600 ) is 2.19, from Figure 1 D shows that the number of active bacteria reaches 10 8 CFU / mL.

[0070] 2. Acid resistance test Adjust the pH of PBS buffer to 2, 3, 4, 5, and 7 respectively and set aside.

[0071] Prepare RCM solid culture medium, pour the plate after sterilization, seal it with sealing film and put it in a 4℃ refrigerator, take 100μL of Clostridium butyricum NCU-02 liquid, add 5mL of RCM liquid culture medium, and culture it in a 37℃ incubator overnight.

[0072] Take 100ul of Clostridium butyricum NCU-02 solution and dilute it with PBS buffer by 10 steps. 1 , 10 3 , 10 5 times, (take 100 μL and add it to 900 μL PBS buffer, then take 10 μL and add it to 990 μL PBS, then take 10 μL and add it to 990 μL PBS) 10 EP tubes for each gradient. Centrifuge at 2500 rpm for 4 min, discard the supernatant, add PBS buffer with pH values ​​of 2, 3, 4, 5, and 7, place it anaerobically for 4 hours, mix well, take 10 μL and apply it to the plate, culture it in a 37°C incubator overnight, count the viable bacteria, and keep good experimental records.

[0073] The above test results are as follows Figure 1As shown in E, it can be seen that Clostridium butyricum NCU-02 exhibits strong acid resistance and can maintain 10 6 The viable bacterial count of CFU / mL can reach 10 at pH = 5.0 8 CFU / mL and remain stable.

[0074] 3. Bile salt tolerance test (1) RCM liquid culture medium is prepared by adding bile salts into groups, including No. 1 (without bile salts), No. 2 (with 0.1% bile salts), No. 3 (with 0.2% bile salts), and No. 4 (with 0.3% bile salts). The prepared RCM solid culture medium is sterilized.

[0075] (2) After sterilization, pour the plates, seal them with sealing film and place them in a 4℃ refrigerator.

[0076] (3) Take 100 μL of Clostridium butyricum NCU-02 solution and add it into 5 mL of RCM liquid culture medium with different bile salt concentrations, and culture it in a 37°C incubator overnight.

[0077] (4) In the morning, take out the cultured bacterial solution, mix it evenly, take 10 μL and spread it on the solid culture medium, and culture it in a 37°C incubator for 12h-48h. Count the live bacteria and keep experimental records.

[0078] The above test results are as follows Figure 1 As shown in Figure F, it can be seen that Clostridium butyricum NCU-02 exhibits good choline tolerance and can maintain up to 10 8 The viable bacterial count in CFU / mL.

[0079] 4. Antibacterial Experiment Inoculate Clostridium butyricum NCU-02 in RCM medium for 16 h, centrifuge at 8000 rpm for 10 min, and remove the supernatant. Take another 20 μL of 7 pathogenic bacteria (Escherichia coli O157, Staphylococcus aureus, beta-hemolytic Streptococcus, Candida albicans, Salmonella typhimurium, Salmonella enteritidis, Listeria monocytogenes) cultured overnight and spread them on LB solid medium plates, put Oxford cups on them, and draw 250 μL of bacterial supernatant into the Oxford cups. After culturing in a 37 ℃ incubator for 8 h, measure the diameter of the inhibition zone.

[0080] The above test results are as follows Figure 1As shown in G in the figure, it can be seen that Clostridium butyricum NCU-02 has a good antagonistic effect on common pathogens. Among them, the average inhibition zone for Escherichia coli O157 is 18.7 mm, the average inhibition zone for Staphylococcus aureus Cowan1 is 14.5 mm, the average inhibition zone for β-hemolytic streptococci is 15.8 mm, the average inhibition zone for Salmonella typhimurium ATCC13311 is 15.8 mm, the average inhibition zone for Candida albicans SC531 is 14.9 mm, the average inhibition zone for Salmonella enteritidis ATCC 13076 is 18.5 mm, and the average inhibition zone for Listeria monocytogenes ATCC 19111 is 10.4 mm.

[0081] 5. Hemolysis Experiment (1) Prepare 5% sheep blood dish: Prepare RCM solid culture medium and sterilize it at high temperature, then cool it to room temperature (about 45°C), add sheep blood, and shake gently to mix the blood and culture medium. Be careful not to generate bubbles.

[0082] (2) Quickly pour into the culture dish to form a blood agar plate, taking care not to create bubbles.

[0083] (3) After drying, seal the dish with a sealing film and store it in a 4°C refrigerator for later use. (Adding blood at 35°C-45°C is to protect some heat-sensitive nutrients and preserve the integrity of blood cells, so as to facilitate the observation of bacterial hemolysis. At the same time, agar will not coagulate at this temperature.) (4) After the strain is subcultured and activated in RCM medium, 20 μL is streaked onto a sheep blood agar plate. After anaerobic culture at 37°C for 24 h, observe whether a hemolytic ring appears around the colony. Use Staphylococcus aureus as a positive control to determine the hemolytic activity of the test strain.

[0084] The above test results are as follows Figure 1 As shown in H in the figure, it can be seen that compared with Staphylococcus aureus, the hemolytic Clostridium butyricum NCU-02 exhibits γ-hemolysis, that is, it has no hemolytic activity, while β-hemolysis is detected in Staphylococcus aureus, indicating that the Clostridium butyricum NCU-02 strain is non-pathogenic and has a higher safety.

[0085] 6. Antibiotic resistance test The antibiotic susceptibility of probiotics to erythromycin (E), penicillin (PEN), cephalosporin (CTR), ampicillin (AMP), gentamicin (GEN), lincomycin (MY), ciprofloxacin (CIP), tetracycline (TET), chloramphenicol (C), and succinimidyl (SXT) was determined by disk diffusion method.

[0086] The concentration of the activated bacterial solution was adjusted to 10 6 CFU / mL; Use a sterile cotton swab to evenly spread the bacterial solution on the surface of the RCM plate; Place the plate at room temperature for 10 minutes to dry; Use sterile tweezers to pick up the drug-sensitive paper and place it evenly on the surface of the plate; Incubate the plate in a constant temperature incubator at 37°C for 24 hours, and measure and record the diameter of the inhibition zone of each drug-sensitive paper.

[0087] The above test results are as follows Figure 1 I and Figure 1 As shown in J in the figure, it can be seen that Clostridium butyricum NCU-02 is highly sensitive to antibiotics such as erythromycin (E), penicillin (PEN), cephalosporin (CTR), ampicillin (AMP), gentamicin (GEN), lincomycin (MY), ciprofloxacin (CIP), tetracycline (TET) and chloramphenicol (C), but is not sensitive to co-trimoxazole (SXT), indicating that Clostridium butyricum NCU-02 is a probiotic with good probiotic properties.

[0088] Example 2 This example provides the effect of Clostridium butyricum NCU-02 intervening in hyperlipidemia male ApoE knockout mice (hereinafter referred to as ApoE- / - mice), and the scheme is as follows: 1. Experimental Animals 40 SPF male ApoE- / - mice and 10 C57BL / 6 male mice of the same strain, weighing 18-22 g, aged 6-8 weeks, were purchased from Beijing Weitong Lihua Biological Co., Ltd. All mice were housed in an SPF animal laboratory at a temperature of (24±2)℃ and a humidity of (60±10)%, and were allowed to drink water and eat freely.

[0089] 2. Group feeding and establishment of mouse hyperlipidemia model A 12-h light-dark cycle was implemented, and all mice were fed adaptively for one week, and their body weights were weighed and recorded.

[0090] After one week of adaptive feeding, the C57BL / 6 mice were still fed with ordinary feed. After one day of fasting, the ApoE- / - mice were fed with 60% high-fat feed to establish a high-fat model for 4 weeks (the average weight of the mice was 28g). Blood samples were collected to test 4 blood lipids, and the model establishment was judged based on general conditions. After the high-fat model was established, lipid-lowering drugs and Clostridium butyricum NCU-02 were given for intervention for 10 weeks. The grouping is as follows: (1) Blank control group (group C, n = 10): C57BL / 6 mice were gavaged with 100 μL of normal saline as the normal control group; (2) Model group (M group, n = 10): ApoE- / - mice were gavaged with 100 μL of normal saline as the hyperlipidemia model control group; (3) Drug group (MA group, n = 10): ApoE- / - mice were intragastrically administered with 100 μL 10 mg / kg atorvastatin; (4) ApoE- / - mice in the low-dose group (ML group, n=10) of Clostridium butyricum NCU-02 were intragastrically administered with 100 μL of 1×10 7 CFUmL Clostridium butyricum NCU-02; (5) ApoE- / - mice in the high-dose group of Clostridium butyricum NCU-02 (MH group, n=10) were intragastrically administered with 100 μL of 1×10 9 CFUmL Clostridium butyricum NCU-02.

[0091] During the experiment, the drug and bacterial solution were suspended in gelatin saline (0.9% saline containing 0.1% gelatin) before each intragastric administration and placed in a 37°C constant temperature water bath for heating. The weight of the mice was recorded regularly every week and the weight changes were observed. After the experiment, the mice were anesthetized, killed, and the samples were collected and preserved.

[0092] It is worth noting that the indicators of successful modeling are abnormal blood lipid levels; obesity, fatigue; reduced food intake or water intake. If serum triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) increase and the difference is statistically significant, the high-fat model is considered to be established. Figure 2 shown.

[0093] It is worth noting that blood lipid testing mainly collects blood samples from mice after overnight fasting by blood sampling from the tail tip. The mice are fixed in a fixture, and the tails of the mice are gently wiped with warm water to dilate the tail blood vessels; the tail tip of the mouse is quickly cut off 0.5-1cm with sterile scissors, and the tail is gently massaged from top to bottom to promote blood outflow, and the blood is collected into a sterilized 1.5 mL EP tube; gauze is used to press the wound at the tail tip to stop bleeding. After the collected blood samples are placed at room temperature for 30 minutes, they are centrifuged at 3500 rpm and 4℃ for 15 minutes to collect serum for blood lipid testing.

[0094] After one week of adaptive feeding, ApoE − / − mice were fed a high-fat diet, and tail vein blood was collected two weeks later. Figure 3 As shown in Figure 4, there was no significant difference in serum TC and TG compared with C57BL / 6 mice. Figure 4As shown in the figure, ApoE - / - mice had increased serum TC, TG, and LDL-c, and decreased HDL-c, which was significantly different from the blood lipid levels of C57BL / 6 mice. At the same time, a high-fat diet can easily lead to excessive fat accumulation in liver cells, which in turn leads to fatty liver, which is one of the complications of hyperlipidemia. Figure 5 As shown in the figure, liver steatosis was observed in hyperlipidemic ApoE- / - mice compared with C57BL / 6 mice. These results indicate that the hyperlipidemia model was successfully established.

[0095] 3. Sample Collection 1. Preparation of instruments and reagents Surgical instruments (such as scissors, tweezers, hemostatic forceps, needles, etc.) and tissue cryopreservation tubes are soaked in water overnight, dried, disinfected and sterilized for later use.

[0096] 0.9% saline: NaCl 9 g, add dd H2O to make up to 1L, and sterilize with high-pressure steam at 121℃ for 15 min.

[0097] 4% paraformaldehyde: add dd H2O to 40 g paraformaldehyde to make up to 1 L, heat to dissolve, filter with a Buchner funnel to remove impurities, and store at 4 ℃.

[0098] 2. Anesthetize the Mouse Anesthesia method: isoflurane inhalation anesthesia.

[0099] Anesthesia takes effect: When the mouse lies in supine position, the heartbeat and breathing are even, the muscles are relaxed, the limbs are inactive, the whiskers do not respond to touch, and the pedal reflex disappears, which is considered to be a state of complete anesthesia.

[0100] 3. Mouse Tissue Collection (1) Blood: Let it stand for 1 hour, and when the light yellow serum appears on the upper layer, take it out and put it into a centrifuge for 15 minutes (3500r / min). Aspirate the serum and transfer it into an EP tube. Store it in a -80℃ refrigerator and use it as a sample for testing serological indicators.

[0101] (2) Liver: After the mice were killed by cervical dislocation, the liver tissue was obtained. The abdominal cavity was opened and the liver was completely separated. The blood stains on the surface of the liver were rinsed with saline, and the excess connective tissue was removed. The liver was cut into pieces. Small pieces of 0.5 cm × 0.5 cm of liver were taken and soaked in 4% formaldehyde solution. Oil red O staining was performed to observe the degree of fatty degeneration of the liver. Except for the liver used for pathological examination, the remaining liver was quickly placed in a -80℃ low-temperature refrigerator for future use.

[0102] (3) Ileum: Cut the ileum tissue 1 cm away from the cecum, about 2 cm long and weighing about 20 mg, and place it in a 1.5 ml RNase-free centrifuge tube filled with RNA protection solution and store it in a refrigerator at 4°C. After 24 hours, take out the ileum tissue and place it in a centrifuge tube. Store it in a refrigerator at -80°C for detecting the expression of related genes in the ileum.

[0103] (4) Colon: Cut the colon tissue (from the cecum to the anus), wash it briefly with saline, measure the length of the mouse colon with a ruler and record it, then scrape the intestinal contents with forceps and store it in a -80 ℃ refrigerator.

[0104] (5) Feces: On the last two days of the experiment, randomly collect feces from each experimental group. Fix the mice with one hand and aseptically collect 3-4 feces from each mouse. After sampling, place them at 4°C and then transfer them to -80°C after 1 hour. Use 16sRNA to detect changes in the bacterial flora.

[0105] 4. Observation of relevant indicators during the experiment: 1. General observation The mental state, activity, fur condition, water intake and food intake of each group of mice were observed.

[0106] Intervention experiments were performed on mice, such as Figure 6 As shown in A, C57BL / 6 mice served as the control group, and ApoE- / - mice were randomly divided into 4 groups: the model group M was given PBS daily, the positive drug group MA was given 10 mg / kg atorvastatin dissolved in 0.9% saline daily, and the Clostridium butyricum intervention groups ML and MH were given 2.0×10 7 CFU / mL and 2.0×10 9 CFU / mL of Clostridium butyricum NCU-02. Each mouse was intragastrically administered with 0.1 mL each time for 10 weeks.

[0107] The results are as follows Figure 6 A in Figure 6 B in Figure 6 C and Figure 7 As shown in the figure, during the experiment, ApoE− / − mice gained weight faster than C57BL / 6 mice. Compared with the M group, the MA and MH groups significantly reduced the weight gain (p<0.0001), and there was no significant difference in the ML group. According to daily observations, the C group mice had normal food intake and water intake, active activities, and dense and shiny fur. The M group mice had reduced luster, slow movements, depressed state, and obvious obesity. After intragastric administration of atorvastatin and different concentrations of Clostridium butyricum NCU-02, it can be observed that the body shape of the mice in the MA and MH groups has improved.

[0108] 2. Plasma testing (1) Blood lipid measurement The levels of TG, TC, HDL and LDL in mouse serum were determined according to the method in the kit instructions (Nanjing Jiancheng Bioengineering Institute Co., Ltd.).

[0109] It is worth noting that when one or more of the indicators of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) are abnormally elevated, such as increased levels of triglycerides, total cholesterol or low-density lipoprotein cholesterol, while decreased levels of high-density lipoprotein cholesterol, it may indicate that the mice are at risk of hyperlipidemia.

[0110] The results are as follows Figure 6 D in Figure 6 F and Figure 6 As shown in G, the TC, TG, and LDL-c levels in the M group were significantly higher than those in the C group (p<0.0001), and the HDL-c level was significantly lower than that in the C group (p<0.0001). This indicates that atorvastatin and Clostridium butyricum NCU-02 treatment can significantly reverse the negative effects of high-fat diet feeding leading to increased blood lipids. Compared with the M group, Clostridium butyricum NCU-02 significantly reduced the TC, TG, and LDL-c levels in the serum of the ML and MH groups, and increased the HDL-c level. Among them, the improvement in the MH group was significantly better than that in the ML group, as shown in that although the HDL-c level in the ML group was higher than that in the M group, there was no statistical difference ( Figure 6 G). The above results show that Clostridium butyricum NCU-02 has a good lipid-lowering effect, and the high dose has a better effect.

[0111] (2) Determination of inflammatory factors The inflammatory factors TNF-α, IL-6, and IL-10 were determined by enzyme-linked immunosorbent assay (ELISA).

[0112] It is worth noting that inflammatory response accompanies the development of hyperlipidemia, which can accelerate the accumulation of fat in liver cells, and the large accumulation of fat continues to aggravate the inflammatory response, leading to a vicious cycle and increased blood lipids. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are closely related to hyperlipidemia. Hyperlipidemia can induce oxidative stress, which can stimulate inflammatory response. This study further explored the effects of Clostridium butyricum NCU-02 on inflammation and oxidative stress in the body.

[0113] (3) Oxidative stress measurement According to the instructions of the biochemical kit (Nanjing Jiancheng Bioengineering Institute Co., Ltd.), the key molecules of oxidative stress, SOD, CAT, GSH-Px and MDA, were detected in serum.

[0114] It is worth noting that the body's oxidation and antioxidant abilities are in a state of balance. Under certain pathological conditions (such as hyperlipidemia), the body's reactive oxygen species (ROS) content increases, leading to an imbalance between oxidation and antioxidant activity and a significant increase in the oxidative reaction, which is oxidative stress.

[0115] Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and other enzymes are the body's antioxidant defense system, which can protect plasma lipoproteins from oxidative modification. Malondialdehyde (MDA) is one of the markers of cell membrane lipid peroxidation. Under hyperlipidemia, MDA increases significantly, which can reflect the degree of lipid peroxidation and cell damage in the body. Research results show that the liver MDA content of animals in the high-fat diet treatment group increased significantly, and the SOD activity decreased significantly, indicating that the ability of the liver of high-fat animals to scavenge oxygen free radicals and lipid peroxidation products decreased significantly.

[0116] (4) Determination of liver damage The ALT and AST levels in mouse serum were measured according to the method in the kit instructions (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.).

[0117] It is worth mentioning that alanine aminotransferase (ALT) and aspartate aminotransferase (AST) exist in liver cells and are released into the blood when liver cells are damaged. Testing the levels of ALT and AST can reflect whether liver cells are damaged. Hyperlipidemia may indirectly lead to impaired fat metabolism function of the liver.

[0118] 3. Histological and Pathological Examination (1) Liver Oil Red O staining: The mouse liver adipose tissue was stained with Oil Red O staining solution (hereinafter referred to as ORO solution) to observe the accumulation of oil droplets. The liver samples were frozen in liquid nitrogen and sliced ​​at 8 μm thickness. The slices were stained with ORO solution for 10 minutes. After washing with water, the slices were stained in hematoxylin for 1 minute. The slices were observed using an inverted microscope. The effects of fatty degeneration of the liver were observed.

[0119] Normal mouse liver tissue: normal structure, clear hepatic lobule structure, evenly distributed central veins, radially arranged peripheral cell cords, normal hepatic sinusoids, large and round nuclei in the center of cells, uniform cytoplasm, and no fatty hepatocytes.

[0120] (2) Colon Colon length: Colon length is an important indicator for judging colon inflammation. After a brief wash with saline, the length of the mouse colon was measured with a ruler and recorded.

[0121] HE staining: The sections were washed thoroughly in distilled water for 10 min; hematoxylin staining solution for 5 min, differentiation solution for 1 min, blueing solution for 2 min, eosin staining solution for 5 min, gradient alcohol dehydration, xylene for 1 min, 2 times; neutral resin sealing, observation and recording under a microscope.

[0122] 5. Detect cholesterol metabolism and expression of colon tight junction proteins by western blotting (WB) The small intestinal tissue was taken and weighed, and 10 times the volume of tissue protein extraction reagent was added (protease inhibitors were added within a few minutes before use), and the mixture was thoroughly homogenized in an ice bath, centrifuged at 12000 r / min at 4℃ for 5 min, and the supernatant was collected. The supernatant protein was quantified by the BCA method. According to the molecular weight of the protein, 10% separation gel and 5% concentration gel were prepared, and the protein samples of each group were processed and electrophoresis was started. After the electrophoresis, it was transferred to the PVDF membrane, and the transferred PVDF membrane was placed in an appropriate amount of 5% skim milk powder, and the membrane was blocked on a decolorizing shaker for 1 h; the primary antibody was diluted with the primary antibody diluent according to the ratio in the reagent and consumables table, and the blocked PVDF membrane was placed in a hybridization bag and incubated at 4℃ overnight. Wash with TBST 5 times, 5 min each time, and dilute the secondary antibody with 5% skim milk powder according to the ratio in the reagent and consumables table, and incubate at room temperature for 1 h. Wash with TBST 5 times, 5 min each time, and drop the freshly prepared ECL mixed solution onto the protein side of the membrane for luminescence detection. The film was scanned and archived, and the AlphaEaseFC software processing system analyzed the optical density value of the target band.

[0123] (1) Cholesterol metabolism testing The expression of PPARγ, LXRα and ABCA1 was detected by WB experiments.

[0124] It is worth noting that peroxisome proliferator-activated receptor γ (PPARγ) is activated by binding to specific ligands, thereby regulating the expression of downstream genes. It is mainly involved in the differentiation of adipocytes, the regulation of insulin sensitivity and the regulation of lipid metabolism. The activation of PPARγ is generally believed to improve lipid metabolism and reduce triglyceride and cholesterol levels in plasma.

[0125] LXRα (liver X receptor α) is mainly involved in the metabolic regulation of cholesterol, fatty acids and bile acids. After being activated by cholesterol and its derivatives, it can regulate the expression of downstream genes, promote the reverse transport of cholesterol, and reduce the cholesterol level in plasma, thus helping to prevent and treat hyperlipidemia.

[0126] ABCA1 is a membrane protein mainly located on the plasma membrane. It is involved in the efflux of cholesterol and phospholipids from cells and is one of the key steps in reverse cholesterol transport.

[0127] (2) Intestinal barrier testing The expression of ZO-1 and occludin was detected by WB experiment.

[0128] It is worth noting that ZO-1 (Zonula Occludens-1) and occludin are two major tight junction proteins in the intestinal barrier. They are located on the cell membrane of intestinal epithelial cells and form tight junctions through interaction, thereby maintaining the physical integrity and function of the intestinal barrier. When the intestinal barrier is damaged, the expression and distribution of ZO-1 and occludin may change.

[0129] 6. High-throughput sequencing of 16S rRNA of intestinal flora Fecal samples were obtained and stored in liquid nitrogen within 1 hour, where DNA was extracted using the QIAamp DNA stool kit. The bacterial 16S ribosomal RNA (rRNA) gene was amplified by PCR using primers that bind to the V4 region.

[0130] By analyzing the above test results, the following conclusions can be drawn: 1. Clostridium butyricum NCU-02 reduces oxidative stress and inflammation levels in hyperlipidemia mice; The results are as follows Figure 8 As shown, compared with group C, the levels of serum pro-inflammatory factors IL-6 and TNF-α in group M were significantly increased (p<0.0001), and the level of anti-inflammatory factor IL-10 was significantly decreased (p<0.0001) ( Figure 8 AC in the figure). Compared with the M group, the systemic inflammatory response in the MA, ML, and MH groups was improved (p<0.01). Among them, the improvement in the MH group was significantly better than that in the ML group.

[0131] Compared with group C, the activities of GSH-Px, SOD and CAT in group M were significantly decreased (p<0.0001), and the level of MDA was significantly increased (p<0.0001) ( Figure 8DG in the figure). After intervention with atorvastatin and Clostridium butyricum NCU-02, the activities of serum GSH-Px, SOD, and CAT enzymes were significantly upregulated, and the MDA content was significantly decreased compared with the M group. It is worth noting that the MH group had a better effect in reducing GSH-Px, SOD, and CAT compared with the ML group. This indicates that intervention with Clostridium butyricum NCU-02 can reduce systemic oxidative damage in hyperlipidemic mice by increasing the activity of antioxidant enzymes and inhibiting oxidative stress.

[0132] And if Figure 8 As shown in H in the figure, the inflammatory factors and antioxidant indicators were further analyzed by heat map, and the results were consistent with the above content: IL-6, MDA, and TNF-α, which are related to the occurrence and development of hyperlipidemia, were all increased and positively correlated with the M group. The above results show that Clostridium butyricum NCU-02 has the ability to improve the body's oxidative stress damage and inflammation levels, and the high-dose effect is significantly better than the low-dose effect, indicating that the improvement effect of Clostridium butyricum NCU-02 is dose-dependent.

[0133] 2. Clostridium butyricum NCU-02 improves liver damage and lipid deposition in hyperlipidemia mice like Fig. 9 As shown in A, the liver anatomical morphology showed that fat accumulation was improved. ORO staining experiments confirmed the accumulation of lipid droplets histologically.

[0134] like Fig. 9 As shown in B, more lipid droplets, microvesicles, and macrovesicles were observed in most hepatocytes in group M compared with group C. Atorvastatin and Clostridium butyricum NCU-02 intervention reduced steatosis and improved hepatic lipid deposition.

[0135] like Fig. 9 C in Fig. 9 As shown in D, liver lipid accumulation leads to increased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The test results showed that the serum AST and ALT levels in group M were significantly higher than those in group C (p<0.0001), and those in groups MA, ML, and MH were lower than those in group M (p<0.001). Among them, the serum AST and ALT levels in group MH were in a similar range to those in group MA, which means that the improvement effect of Clostridium butyricum NCU-02 is not weaker than that of drugs. The above results show that Clostridium butyricum NCU-02 has the ability to improve liver damage. like Fig. 9 E in Fig. 9 F in Fig. 9 G and Fig. 9As shown in H, the relative protein levels of PPARγ, LXRα and ABCA1 in liver tissue were determined. Compared with group C, the expression of PPARγ (p<0.0001), LXRα (p<0.001) and ABCA1 (p<0.0001) in group M were significantly reduced. The expression of PPARγ and LXRα was upregulated in groups MA, ML and MH, and the expression and activity of ABCA1 were enhanced. The activation of the PPARα-LXRα-ABCA1 pathway increased the cholesterol efflux of cells, reduced serum cholesterol levels, and alleviated lipid accumulation. This indicates that Clostridium butyricum NCU-02 may reduce blood lipids and improve liver damage by mediating liver cholesterol metabolism, thereby alleviating hyperlipidemia.

[0136] 3. Clostridium butyricum NCU-02 improves intestinal inflammation and barrier function in hyperlipidemia mice like Fig.10 As shown in the figure, the protective effect of Clostridium butyricum NCU-02 on intestinal mucosal injury caused by hyperlipidemia was explored. The results of colon dissection showed that the colon length of group M was significantly shortened compared with group C. After treatment with atorvastatin and Clostridium butyricum NCU-02, the shortening of colon length was effectively reduced ( Fig.10 A and Fig.10 In line with this, HE staining results showed that compared with group C, the muscular layer of the colon in group M was significantly thinner, accompanied by obvious inflammatory cell infiltration, a significant decrease in the number of goblet cells, and destruction of colonic crypts. After intervention with atorvastatin and Clostridium butyricum NCU-02, the inflammation of the colonic tissue structure of mice was improved (e.g. Fig.10 C in.

[0137] In addition, studies have shown that the tightness of the intestinal epithelium plays a role in regulating intestinal barrier function. WB was used to detect intestinal epithelial tight junction proteins ZO-1 and Occludin. The test results showed that compared with the M group, the expression of ZO-1 and Occludin in the colon tissue of the MA group, ML group and MH group was significantly upregulated ( Fig.10 DF in ). This indicates that Clostridium butyricum NCU-02 can reduce intestinal inflammation and enhance intestinal mucosal barrier function.

[0138] 4. Clostridium butyricum NCU-02 has the effect of regulating and improving intestinal dysbiosis like Fig.11 and Fig.12 As shown in the figure, intestinal integrity damage caused by hyperlipidemia is related to intestinal flora dysbiosis. Given that supplementation with Clostridium butyricum NCU-02 can improve intestinal barrier function, fecal samples were further sequenced by 16S rRNA high-throughput sequencing to detect the effect of Clostridium butyricum NCU-02 on the intestinal microbiota structure of hyperlipidemia mice. The length and flatness of the rarefaction curve reflect the sequencing depth and diversity of the sample ( Fig.11The alpha diversity index reflects the richness of the intestinal microbiota. Compared with group C, the alpha diversity index Chao1 and observed species in group M were reduced (p < 0.0001). Atorvastatin and Clostridium butyricum NCU-02 improved alpha diversity, but there was no statistical difference ( Fig.11 BC in).

[0139] Principal coordinate analysis (PCoA) visualized the beta diversity of the gut microbiome and revealed significant differences in the gut bacterial community. Atorvastatin and Clostridium butyricum NCU-02 altered the composition of the gut microbiota ( Fig.11 D in.

[0140] The Venn diagram showed the shared OUT richness between the groups by displaying the overlap between the groups. The results showed that the C, M, MA, ML, and MH groups had a total of 123 OUTs, with 1014, 113, 164, 112, and 126 OUTs, respectively, showing differences in the composition of the intestinal flora ( Fig.11 E in.

[0141] Further analysis of the effect of Clostridium butyricum NCU-02 on regulating intestinal flora changes was conducted to explore changes at the phylum, genus, and species levels. At the phylum level, Firmicutes, Bacteroidetes, and Actinobacteria were the main microbiota in the mouse intestine, accounting for 99.08%, 98.66%, 97.73%, 97.59%, and 97.36% of the sequencing results in each group, respectively. Fig.11 Compared with the M group, the relative abundance of Firmicutes increased in the MA group (p<0.001), the ML group (p<0.01), and the MH group (p<0.001), and the relative abundance of Bacteroidetes decreased in the MA group (p<0.0001), the ML group (p<0.01), and the MH group (p<0.0001). Fig.11 No significant effect was observed in the actinomycete group ( Fig.11 At the genus level, Muribaculaceae, Blautia, and Dubosiella were the main microbiota in the mouse intestine. Compared with the M group, the relative abundance of Muribaculaceae decreased in the MA, ML, and MH groups, while the relative abundance of Blautia and Dubosiella increased, but there was no statistical difference ( Fig.12 At the species level, atorvastatin and Clostridium butyricum NCU-02 upregulated the abundance of the main microbial group Lachnospiraceae compared with the M group ( Fig.12 The above results indicate that Clostridium butyricum NCU-02 has the effect of regulating and improving intestinal dysbiosis and reshaping the diversity of intestinal flora.

[0142] 5. Intestinal flora is closely related to hyperlipidemia parameters like Fig.13 As shown, hyperlipidemia indicators include blood lipid levels (TC, TG, LDL-c and HDL-c), liver damage (ALT and AST), oxidative stress (GSH-Px, SOD, CAT and MDA) and inflammatory factors (IL-6, TNF-α and IL-10). ( Fig.13 ) From the Spearman correlation analysis, Muribaculaceae, Odoribacter, and Alistipes were positively correlated with ALT, IL-6, MDA, AST, TNF-α, TC, TG, and LDL-c levels, suggesting that these bacteria may lead to increased blood lipids, liver damage, and inflammation.

[0143] Acetatifactor, Ruminiclostridium_5, Harryflintia, Clostridium_sensu_stricto_1, Blautia, Lachnospiraceae_NK4A136_group, Lachnospiraceae_UCG-006, Lachnospiraceae_FCS020_group, Lachnoclostridium, Tyzzerella, and Ruminococcaceae_UCG-009 were significantly positively correlated with HDL-c, IL-10, GSH-Px, SOD, and CAT levels, and were completely oppositely correlated with ALT, IL-6, MDA, AST, TNF-α, TC, TG, and LDL-c levels. It is speculated that these bacterial species may alleviate hyperlipidemia.

[0144] These results suggest that intestinal flora is closely associated with hyperlipidemia parameters and their composition and abundance play an important role in the occurrence and development of hyperlipidemia.

[0145] Example 3 This example explores the effects of Clostridium butyricum NCU-02 on improving autism, and the specific scheme is as follows: 1. Establishment and treatment of autism mouse model 1. Model building The experiment used C57BL / 6 mice (10 males and 20 females), which were 6-8 weeks old when purchased, and were kept under controlled temperature, lighting conditions (12 hours light: 12 hours dark cycle), noise, ventilation and other conditions under standard laboratory conditions. During the experiment, the mice had free access to water and food. Animals were fed with standard pellet feed every day. Cleaning was done regularly every day. To ensure the accuracy of the experimental results, adaptive feeding was carried out for two weeks. The purchased C57BL / 6 female mice were randomly divided into two groups (i.e., blank control group and model group, with a ratio of 1:3). The female mice were mated regularly (at 17:00 in the afternoon, they were caged together with 1 male and 2 females overnight, and separated at 9:00 the next morning). The vagina of the female mice was checked when the cages were separated. If a vaginal plug was observed, it was considered to be fertilized successfully, which was recorded as 0.5 days of pregnancy (E0.5). VPA 600 mg / kg (model group) or an equal volume of sterile saline (blank control group) was injected intraperitoneally at 12.5 days of pregnancy. Each mother mouse was returned to the cage and undisturbed until the offspring were born. All pups were kept with their mothers until weaning at 21 days after birth (P21), at which time the pups were grouped and housed in separate cages (no more than 5 mice per cage) according to gender, and male mice with obvious autism-like behaviors were selected as ASD model mice for subsequent experiments.

[0146] 2. Treatment process After the offspring male mice were weaned (P21), they were divided into groups for animal experiments and given medication treatments. The interventions were continued every day for 4 weeks. After 4 weeks, subsequent evaluations of autism characterization indicators and sample collection indicators were conducted.

[0147] The animal experiment groups and dosing methods are as follows: (1) Blank control group (Group C): gavage with 100uL normal saline once a day for four weeks; (2) Autism model group (Group M): gavage with 100uL normal saline once a day for four weeks; (3) Autism model + Clostridium butyricum (MC group): intragastrically administered with 100uL of 1×10 9 CFU / mL Clostridium butyricum NCU-02, once a day for four weeks; (4) Autism model + postbiotics (MP group): intragastrically administered with 100uL of 1×10 9 CFU / mL postbiotic solution, once a day for four weeks.

[0148] 2. Evaluation of the phenotypic effects of Clostridium butyricum NCU-02 on ASD mice 1. Exploring the effect of Clostridium butyricum NCU-02 on improving the behavior of autistic mice The order of stress from the least stressful to the most stressful is to reduce the possibility that the previous behavioral test will affect the subsequent behavioral test. (1) Open field test; (2) Buried bead test; (3) Three-box social test test; (4) Y-maze test.

[0149] (1) Open field test This experiment is mainly used to detect the behavioral and mental changes of experimental animals in the new environment, such as exploratory behavior and anxiety.

[0150] Experimental procedures: The mouse open field reaction box (50×50×45 cm) has a bottom divided into 9 small squares, with the middle square designated as the "center area". A digital camera is set up 2 m above the box, and its field of view can cover the entire open field. The experiment is conducted in a quiet environment. The animal is placed in the center of the bottom of the box, and video and timing are performed at the same time. The software tracks the movement trajectory of the mouse. Stop the video after 10 minutes of observation, and record the total distance the animal walks, the time it stays in the central area, and the distance walked in the central area during this period. Wipe the inner wall and bottom of the box with 75% ethanol before changing the animal to prevent the remaining information of the animal from the last time (such as the animal's defecation, urine, and odor) from affecting the next test result.

[0151] (2) Buried bead experiment This experiment is mainly used to detect stereotyped behaviors in mice.

[0152] Experimental steps: The temperature of the test room was stabilized to 25°C in advance, and the light was adjusted to 15 lux. A 42.5cm×26.5cm×19cm rat cage was placed in the test room, and a 5cm thick sawdust bedding was laid and flattened. The mice to be tested were transferred to the room in a standard cage and adapted to the room for 30 minutes without being disturbed. The mice to be tested were placed in the test cage to adapt for 10 minutes (without marbles), returned to the standard cage, and 20 15mm black marbles were evenly placed in the test cage in a 4×5 arrangement. The mice were put back in the cage, and the number of marbles buried by the mice in 10 minutes (assessment standard: 2 / 3 covered by sawdust) was calculated. The urine and feces of the mice were cleaned after each experiment, and wiped with 75% alcohol to remove the odor.

[0153] (3) Three-box social test experiment This experiment is mainly used to detect the social behavior of experimental mice and their preference for new things.

[0154] Experimental steps: Before the experiment, the mice to be tested were placed in the device (60x40x22 cm). After the mice were allowed to freely move in the three connected chambers for 10 minutes, the three boxes were separated by transparent glass resin plates, and the test mice were placed in the middle box for 5 minutes. Social stage: The strange mouse (Stranger 1) was randomly placed in the metal cage in the left or right box. The metal cage in the other box was empty. The glass resin plate separating the boxes was removed, so that the test mice could move freely in the three boxes for 10 minutes. The duration of direct contact between the experimental mice and Stranger 1 or the empty metal cage, the number of times they entered each box, and the duration were recorded. Novel social stage: During the experiment, another strange mouse of the same species (Stranger 2) was placed in the empty metal cage, and then recorded for 10 minutes to observe the time of contact between the experimental mice and Stranger 1 and Stranger 2. The following indicators were recorded: the time the pups sniffed each cage and the time they stayed in each box. After each experiment, the urine and feces of the mice were cleaned and wiped with 75% alcohol to remove the odor.

[0155] It is worth noting that by testing the time the experimental mice spend on the strange mouse and the non-social object on the other side, normal mice should prefer to spend time with the strange mouse, while autism model animals spend less time near the strange mouse. In addition, we can also use the three-box device to test the social novelty preference task, that is, place familiar and unfamiliar animals on both sides of the three boxes. Since rodents are more willing to seek novelty, the time they explore the strange animal can be tested to evaluate their degree of social preference.

[0156] (4) Y-maze test This experiment is mainly used to evaluate the spatial working memory and short-term memory ability of mice.

[0157] The Y-maze consists of three interconnected gray arms with an angle of 120 degrees. Each arm is 59 cm long. The three arms are randomly divided into the start arm, the novel arm, and the other arm. The experiment includes a training period and a test period, with an interval of 1 hour in between. During the first stage of training, the novel arm was blocked by a partition, and the rat was placed in the start arm and moved freely in the start arm and other arms for 10 minutes. After 1 hour, the test period experiment was carried out. The partition of the novel arm was removed, and the rat was placed in the start arm. The three arms were freely explored for 5 minutes. The number of times the rat entered each arm and the length of stay were recorded, and the ratio of the time spent in the novel arm to the number of times entering the novel arm was calculated (Novel / [Novel+Start+other]×100%). After each experiment, the urine and feces of the mice were cleaned up and wiped with 75% alcohol to remove the odor.

[0158] 3. Exploring the effect of Clostridium butyricum NCU-02 on the pathological morphology of brain tissue in ASD mice ①The number of microglia in hippocampal tissue was measured by immunofluorescence using lba-1 antibody.

[0159] ②After measuring the protein concentration using a bovine serum albumin (BCA) protein assay kit, the glutathione peroxidase (GSH-Px) assay kit and the total superoxide dismutase (SOD) assay kit were used to detect the activities of GSH-Px and SOD, respectively. The glutathione (GSH) and malondialdehyde (MDA) assay kits were used to determine the contents of GSH and MDA, respectively. To detect the contents of GSH-Px, SOD, GSH and MDA in mouse hippocampal tissue. (Purpose: to reflect the oxidative stress level of mouse hippocampal microglia).

[0160] ③ Detect the expression levels of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in rat hippocampus by q-PCR. (Purpose: to reflect the level of inflammatory response in mouse hippocampus tissue) TNF-α: 5'-AGAAAGCATGATCCGCGAC-3'5'-TTGTGAGTGTGAGGGTCTGG-3' Il-1β: 5'-TTGGGCCTCAAAGGAAAGAAT-3'5'-TGCTTGTGAGGTGCTGATGTA-3' IL-6: 5'-AATGATGGATGCTACCAAACTG-3'5'-AGGACTCTGGCTTTGTCTTTC-3'IV. Exploring the improving effect of Clostridium butyricum NCU-02 on the intestinal flora of ASD mice 1. Intestinal tissue (1) Colon length measurement: Colon tissue was isolated from each group of mice, and the colon from the anus to the ileocecal region was obtained. The contents of the colon were flushed with normal saline. The colon tissue of 5 mice in each group was selected for length measurement and photographing.

[0161] (2) Intestinal permeability test Tight junction proteins play an important role in maintaining the mechanical barrier of the intestinal mucosal epithelium. Tight junction proteins mainly include occludin, claudins, junctional adhesion molecules (JAMs) and occludens (ZOs). As an important component of cell tight junctions, occludin plays a role in closing cell gaps, forming the body's permeability barrier, and maintaining the difference in substances on both sides of the cell. Tight junction protein 1 (zonulaoccludens-1, ZO-1) is a member of the membrane-associated guanylate protein kinase family. ZO-1 is mainly expressed in endothelial and epithelial cells and is an important component in the formation of the blood-brain barrier. Many studies have confirmed that the decrease in ZO-1 protein expression level and activity will reduce the integrity and stability of the tight junction structure between cells.

[0162] Purpose of detection: To detect the expression levels of Occludin and ZO-1 proteins in the colon using WB experiments.

[0163] 2. Feces Detection of Clostridium butyricum NCU-02 in feces by q-PCR experiment: C. butyricum NCU-02: forward: 5′-CGTGGGGAGCAAACAGGATT-3; Reverse: 5′-CGCGAGGTTGCATCTCATTG-3′.

[0164] Universal: Forward: 5′-AGAGTTTGATCCTGGCTCAG-3′; Reverse: 5′-ACGGCTACCTTGTTACGACTT-3′.

[0165] 5. Detection of butyrate in feces, serum and hippocampus Gas chromatography-mass spectrometry (GC-MS) was used to measure the butyrate content in feces, serum, and hippocampus.

[0166] By analyzing the above test results, the following conclusions can be drawn: 1. Clostridium butyricum NCU-02 and its postbiotics improve the behavior of autistic mice like Fig.14 and Fig.15 As shown, the open field test reflected that compared with group C, the total moving distance, center residence time and center moving distance of mice in group M were significantly decreased, indicating that the mice in group M were in an anxious state, and the anxiety was effectively relieved after intervention with Clostridium butyricum NCU-02 and its postbiotics.

[0167] like Fig.16As shown, the bead burying experiment reflected that compared with the C group, the number of marbles buried by mice in group M increased significantly, reflecting that the mice in group M were in an anxious state and had repetitive stereotyped behaviors, which were effectively recovered after intervention with Clostridium butyricum NCU-02 and its postbiotics.

[0168] like Fig.17 As shown in the figure, the three-box social test experiment reflects that in the social tendency test phase, compared with the mice in the C group, the M group (ASD model group) did not show obvious preference for the strange mouse (S1) and the empty cage (E); and in the social novelty preference test phase, the M group (ASD model group) did not show obvious preference for the strange mouse (S1) and the strange mouse 2 (S2), which indicates that the general social behavior and social novelty preference of the mice in the M group were significantly reduced, showing social disorders. After the intervention of Clostridium butyricum NCU-02 and its postbiotics, the mice showed obvious preference in both the first and second stages, indicating that the social disorders of the mice were effectively restored after the treatment of Clostridium butyricum NCU-02 and postbiotics.

[0169] like Fig.18 As shown, the Y-maze experiment reflected that compared with the C group, the spontaneous alternation rate of mice in the M group was significantly decreased, indicating that their spatial memory ability was damaged, which was effectively restored after intervention with Clostridium butyricum NCU-02 and its postbiotics.

[0170] 2. Clostridium butyricum NCU-02 and its postbiotics can effectively reduce oxidative stress in autistic mice like Fig.19 As shown in the figure, the oxidative stress level of the hippocampus tissue of each group of mice was evaluated through the experiment. The results showed that compared with the C group, the MDA content in the hippocampus tissue of the mice in the M group was increased, the SOD and GSH-Px activities were decreased, and the GSH content was decreased. However, after treatment with Clostridium butyricum NCU-02 and its postbiotics, the increased MDA content decreased, the decreased SOD and GSH-Px activities were restored, and the decreased GSH content increased. This indicates that the oxidative stress level in the hippocampus of ASD mice is increased, and the intervention of Clostridium butyricum NCU-02 can effectively reduce the oxidative stress in the hippocampus of ASD mice.

[0171] 3. Clostridium butyricum NCU-02 and its postbiotics can effectively reduce the inflammatory response in the hippocampus of mice like Fig. 20 As shown, the inflammatory response of the hippocampal tissue of each group of mice was detected by q-PCR. The results showed that the levels of IL-1β, IL-6 and TNF-α in the hippocampus of mice in group M were increased. After intervention with Clostridium butyricum NCU-02 and its postbiotics, this increased inflammation was effectively alleviated.

[0172] 4. Butyrate levels increased significantly, and the abundance of Clostridium butyricum NCU-02 decreased significantly like Fig.21 As shown, the butyrate content in feces, serum, and brain was measured by gas chromatography-mass spectrometry (GC-MS). The results showed that compared with the M group, after intervention with Clostridium butyricum NCU-02 and its postbiotics, the butyrate levels in the mouse brain (A), feces (B), and serum (C) increased significantly; The abundance of Clostridium butyricum NCU-02 in the feces of each group of mice was detected by q-PCR. The results showed that compared with group C, the abundance of Clostridium butyricum NCU-02 in group M was significantly reduced.

[0173] 5. Clostridium butyricum NCU-02 can reduce activated microglia like Fig. 22 and Fig.23 As shown, the results of immunofluorescence staining showed that the number of activated microglia in the hippocampus of mice in group M was significantly higher than that in group C. However, after intervention with Clostridium butyricum NCU-02, the increased number of activated microglia in the hippocampus tissue was reduced. These results indicate that intervention with Clostridium butyricum NCU-02 reduces activated microglia in the hippocampus of autistic mice, thereby improving autism.

[0174] 6. Clostridium butyricum NCU-02 improves intestinal inflammation and barrier function in autistic mice like Fig.24 As shown, compared with the C group, the colon length of the M group was significantly shortened. After intervention with Clostridium butyricum NCU-02 and its postbiotics, the shortening of colon length was effectively reduced (AB). Intestinal epithelial tight junction proteins ZO-1 and Occludin were detected by WB. The test results showed that compared with the M group, the expression levels of ZO-1 and Occludin in the colon tissue of the M-Cb group and the MP group were significantly upregulated (DE). This shows that Clostridium butyricum NCU-02 can reduce intestinal inflammation and enhance the intestinal mucosal barrier function.

[0175] In summary, Clostridium butyricum NCU-02 and its postbiotics have an improving effect on the behavior of autistic mice, can significantly reduce the oxidative stress and inflammatory response in the hippocampal tissue of autistic mice, can also significantly reduce intestinal inflammation, and enhance the intestinal mucosal barrier function. Not only that, the intervention of Clostridium butyricum NCU-02 improves autism by reducing the activated microglia in the hippocampus of autistic mice.

[0176] Example 4 It is worth noting that the results of Example 3 above show that not only Clostridium butyricum has an improvement effect on autism, but its postbiotics also have an improvement effect on autism. In addition, relevant literature has found that butyric acid is the main metabolite of Clostridium butyricum. Therefore, this example aims to explore whether Clostridium butyricum NCU-02 affects the behavior of autism model mice through its metabolite butyric acid, and further clarify its molecular mechanism for improving autism-like behavior in mice. The specific scheme is as follows: ("Like behavior" refers to behavioral manifestations that simulate or resemble human autism.) 1. Animal experiment grouping to explore the mechanism of Clostridium butyricum NCU-02 and its metabolite butyrate in treating autism (1) Blank control group (Group C): gavage with 100uL normal saline once a day for four weeks; (2) Autism model group (Group M): gavage with 100uL normal saline once a day for four weeks; (3) Autism model + Clostridium butyricum NCU-02 (M-CB group): intragastrically administered with 100uL 1×109 CFU / mL Clostridium butyricum NCU-02, once a day for four weeks; (4) Autism model + sodium butyrate (M-SB group): gavage of 100uL 300 mg / kg sodium butyrate solution once a day for four weeks.

[0177] 2. Evaluation of the phenotypic effects of butyrate, a metabolite of Clostridium butyricum NCU-02, on ASD mice It is worth noting that sodium butyrate is generally used for intervention experiments to explore the effects of butyrate on ASD mice.

[0178] 1. Butyrate improves the behavior of autistic mice The following experiments were conducted in the order of least stress to greatest stress in order to reduce the possibility of the previous behavioral test affecting the subsequent behavioral test: (1) three-box social test experiment; (2) open field experiment; (3) buried bead experiment; (4) Y-maze experiment. For the specific experimental process, refer to Example 3.

[0179] 2. Effects of butyrate on brain pathological morphology in ASD mice ①The number of microglia in hippocampal tissue was measured by immunofluorescence using lba-1 antibody.

[0180] ②After measuring the protein concentration using a bovine serum albumin (BCA) protein assay kit, the glutathione peroxidase (GSH-Px) assay kit and the total superoxide dismutase (SOD) assay kit were used to detect the activities of GSH-Px and SOD, respectively. The glutathione (GSH) and malondialdehyde (MDA) assay kits were used to determine the contents of GSH and MDA, respectively. To detect the contents of GSH-Px, SOD, GSH and MDA in mouse hippocampal tissue. (Purpose: to reflect the oxidative stress level of mouse hippocampal microglia).

[0181] ③The expression levels of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in rat hippocampus were detected by q-PCR. (Purpose: to reflect the inflammatory response level of mouse hippocampus tissue).

[0182] TNF-α: 5'-AGAAAGCATGATCCGCGAC-3'5'-TTGTGAGTGTGAGGGTCTGG-3' Il-1β: 5'-TTGGGCCTCAAAGGAAAGAAT-3'5'-TGCTTGTGAGGTGCTGATGTA-3' IL-6: 5'-AATGATGGATGCTACCAAACTG-3'5'-AGGACTCTGGCTTTGTCTTTC-3' III. Effect of butyrate on the improvement of intestinal flora in ASD mice 1. Intestinal tissue (1) Colon length measurement: Colon tissue was isolated from each group of mice, and the colon from the anus to the ileocecal region was taken. The contents of the colon were flushed with normal saline. The colon tissue of 5 mice in each group was selected for length measurement and photographing. (2) Intestinal permeability test Tight junction proteins play an important role in maintaining the mechanical barrier of the intestinal mucosal epithelium. Tight junction proteins mainly include occludin, claudins, junctional adhesion molecules (JAMs) and occludens (ZOs). As an important component of cell tight junctions, occludin plays a role in closing cell gaps, forming the body's permeability barrier, and maintaining the difference in substances on both sides of the cell. Tight junction protein 1 (zonulaoccludens-1, ZO-1) is a member of the membrane-associated guanylate protein kinase family. ZO-1 is mainly expressed in endothelial and epithelial cells and is an important component in the formation of the blood-brain barrier. Many studies have confirmed that the decrease in ZO-1 protein expression level and activity will reduce the integrity and stability of the tight junction structure between cells.

[0183] Purpose of detection: To detect the expression levels of Occludin and ZO-1 proteins in the colon using WB.

[0184] 2. Feces Detection of Clostridium butyricum NCU-02 in feces by q-PCR 4. Molecular Mechanism of Butyrate in Treating Autism-like Behaviors in Mice 1. Use WB to verify the expression of GPR109A / AMOK / Nrf2 signaling pathway Western blot was used to detect the expression levels of GPR109A, AMPK, and Nrf2 proteins in the hippocampal tissues of each group of mice. GPR109A: belongs to the G protein-coupled receptor family and can be activated by niacin, butyrate, and β-hydroxybutyrate AMPK: is an important energy sensor and a major regulator of cellular energy balance and oxidative stress; Nrf2: is a key transcription factor regulating anti-oxidative stress. AMPK phosphorylates Nrf2 and promotes its nuclear accumulation, alleviating oxidative stress and toxicity.

[0185] It is worth mentioning that the hypothesized principle of this experiment is that Clostridium butyricum NCU-02 produces butyric acid through intestinal colonization. Butyric acid enters the blood through the blood-brain barrier and enters the microglia in the hippocampus of the brain, where it binds to the butyrate receptor GPR109A, activates the AMPK / Nrf2 signaling pathway, reduces the level of oxidative stress in microglia, and thus improves autism.

[0186] By analyzing the above test results, the following conclusions can be drawn: By analyzing the above test results, the following conclusions can be drawn: 1. Clostridium butyricum NCU-02 and its metabolite butyric acid improve the behavior of autistic mice like Fig.25 As shown, in the three-box social experiment, the social impairment of mice was effectively restored after intervention with Clostridium butyricum NCU-02 and butyric acid ( Fig.25 AB in).

[0187] In the open field test ( Fig.25 In C), the total distance moved by mice in group M was significantly greater than that of mice in group C ( Fig.25 D in the center area ( Fig.25 E) and the moving distance of the central area ( Fig.25 butyricum NCU-02 and butyrate intervention restored the expression of F in the cultured cells.

[0188] In the bead burying experiment, the number of marbles buried by mice in the M group increased significantly, reflecting that the autistic mice were in an anxious state and had repetitive stereotyped behaviors. After intervention with Clostridium butyricum NCU-02 and butyric acid, the mice were effectively restored ( Fig.25 in G).

[0189] In the Y-maze test, the spontaneous alternation rate of mice in the M group decreased significantly, indicating that their spatial memory ability was impaired, while it was effectively restored after intervention with Clostridium butyricum NCU-02 and butyric acid ( Fig.25 in H).

[0190] 2. Clostridium butyricum NCU-02 and its postbiotics can effectively reduce oxidative stress in autistic mice like Fig.26 As shown in the figure, the oxidative stress level of the hippocampus of each group of mice was evaluated through the experiment. The results showed that compared with the C group, the MDA content in the hippocampus of the mice in the M group increased, the SOD and GSH-Px activities decreased, and the GSH content decreased. However, after treatment with Clostridium butyricum NCU-02 and butyric acid, the MDA content decreased, the reduced SOD and GSH-Px activities were restored, and the GSH content was also increased; it can be inferred that Clostridium butyricum NCU-02 may alleviate the oxidative stress in the hippocampus of ASD mice through its metabolite butyric acid.

[0191] 3. Butyrate, a metabolite of Clostridium butyricum NCU-02, can reduce the level of inflammation in the hippocampus of autistic mice like Fig. 27 As shown, the inflammatory response of the hippocampal tissue of each group of mice was detected by q-PCR. The results showed that the levels of IL-1β, IL-6 and TNF-α in the hippocampus of mice in group M were increased. After intervention with Clostridium butyricum NCU-02 and butyric acid, this increased inflammation was effectively alleviated.

[0192] 4. Clostridium butyricum NCU-02 can reduce activated microglia through its metabolite butyrate like Fig.28 As shown, the results of immunofluorescence staining showed that the number of activated microglia in the hippocampus of mice in group M was significantly higher than that in group C. However, after intervention with Clostridium butyricum NCU-02 and butyrate, the increased number of activated microglia in the hippocampus tissue was reduced. These results indicate that Clostridium butyricum NCU-02 reduces activated microglia in the hippocampus of autistic mice through its metabolite butyrate, thereby improving ASD-like behaviors.

[0193] 5. Butyric acid, a metabolite of Clostridium butyricum NCU-02, improves intestinal inflammation and barrier function in autistic mice like Fig.29As shown in the figure, the results of colon dissection showed that the colon length of group M was significantly shortened compared with group C. After intervention with Clostridium butyricum NCU-02 and butyric acid, the shortening of colon length was effectively reduced (AB). Intestinal epithelial tight junction proteins ZO-1 and Occludin were detected by WB. The test results showed that compared with group M, the expression levels of ZO-1 and Occludin in colon tissue of group M-Cb and group MS were significantly upregulated (DE). This indicates that butyric acid, a metabolite of Clostridium butyricum NCU-02, can reduce intestinal inflammation and enhance intestinal mucosal barrier function.

[0194] 6. Butyric acid, a metabolite of Clostridium butyricum NCU-02, reduces microglial oxidative stress through the GPR109A / AMPK / Nrf2 signaling pathway, thereby improving autism like Fig.30 As shown, WB was used to verify the expression of the GPR109A / AMPK / Nrf2 signaling pathway. The results showed that compared with the M group, the expression levels of GPR109A, P-AMPK and Nrf2 proteins in the hippocampus of mice in the MS group increased significantly, indicating that Clostridium butyricum NCU-02 produced butyric acid through intestinal colonization, butyric acid entered the blood through the blood-brain barrier and entered the hippocampus of the brain, binding to the butyric acid receptor GPR109A, activating the AMPK / Nrf2 signaling pathway to reduce the level of oxidative stress in microglia, thereby improving autism.

[0195] In summary, butyric acid, the metabolite of Clostridium butyricum NCU-02, has an improving effect on the behavior of autistic mice, can significantly reduce the oxidative stress and inflammatory response in the hippocampal tissue of autistic mice, can also significantly reduce intestinal inflammation, and enhance the intestinal mucosal barrier function. Not only that, the intervention of Clostridium butyricum NCU-02 and butyric acid improves autism by reducing the activated microglia in the hippocampus of autistic mice.

[0196] Moreover, through the study of its mechanism, it was found that Clostridium butyricum NCU-02 produces butyric acid through intestinal colonization. Butyric acid enters the blood through the blood-brain barrier and enters the hippocampus of the brain, binds to the butyric acid receptor GPR109A, activates the AMPK / Nrf2 signaling pathway, reduces the level of oxidative stress in microglia, and thus improves autism. Therefore, it can be seen that Clostridium butyricum NCU-02 and its metabolite butyric acid have great potential in improving autism.

[0197] The embodiments of the present application are described above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.

Claims

1. A use of Clostridium butyricum NCU-02 in the preparation of a drug for improving hyperlipidemia and / or autism, characterized in that: The Clostridium butyricum NCU-02 originated from the feces of a centenarian and was deposited at the General Microbiology Center of the China Culture Collection Administration on August 8, 2022. The address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.25504.

2. The use of Clostridium butyricum NCU-02 according to claim 1, characterized in that The gene sequence of 16S rDNA of the Clostridium butyricum NCU-02 is shown in SEQ ID No:

1.

3. The use according to claim 1, characterized in that: The Clostridium butyricum NCU-02 improves hyperlipidemia by improving one or more of inflammation, oxidative stress, dyslipidemia, liver damage and intestinal microbial flora disorder.

4. The use according to claim 1, characterized in that: The application also includes the use of butyric acid, a metabolite of Clostridium butyricum NCU-02, in the preparation of a drug for improving autism.

5. The use according to claim 1 or 4, characterized in that: The Clostridium butyricum NCU-02 and its metabolite butyric acid improve autism through the GPR109A / AMPK / Nrf2 signaling pathway.

6. A biological agent, characterized in that: The active ingredients thereof include the Clostridium butyricum NCU-02 according to claim 1 and / or butyric acid, a metabolite of the Clostridium butyricum NCU-02 according to claim 4.

7. An auxiliary drug for treating hyperlipidemia and / or autism, characterized in that: The active ingredient of the drug includes the Clostridium butyricum NCU-02 according to claim 1 and / or butyric acid, a metabolite of Clostridium butyricum NCU-02 according to claim 4.

8. The auxiliary drug according to claim 7, characterized in that: The medicine also includes a drug carrier and / or a pharmaceutical excipient.

9. The auxiliary drug according to claim 7, characterized in that: The drug carrier and / or pharmaceutical excipient is at least one of water, lactose, sodium chloride and glucose.

10. The auxiliary drug according to any one of claims 7 to 9, characterized in that: The dosage form of the medicine is powder, granule, capsule or tablet.

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