Clostridium butyricum combined with ranolazine in the preparation of fatty hepatitis drugs

Abstract

This invention relates to the field of biomedical technology, specifically to the application of *Clostridium butyricum* in combination with retemetinol in the preparation of drugs for steatohepatitis. *Clostridium butyricum* not only alleviates the adverse reactions caused by retemetinol in the prevention or treatment of steatohepatitis, but also enhances the ameliorative effects of retemetinol on liver damage, lipid disorders, insulin resistance, related colitis, and intestinal barrier damage caused by steatohepatitis. Therefore, *Clostridium butyricum* combined with retemetinol can be used to prepare drugs for the prevention or treatment of steatohepatitis, with the two drugs exhibiting a synergistic effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of Clostridium butyricum in combination with retinoic acid in the preparation of drugs for steatohepatitis. Background Technology

[0002] Metabolic-associated steatohepatitis (MASH) is a key pathological stage in the progression of metabolic-associated fatty liver disease (MAFLD), marking the transition from simple hepatic steatosis to a state accompanied by significant inflammatory activity and hepatocellular damage, often accompanied by progressive liver fibrosis. MASH is not only a core pathological link driving MAFLD towards cirrhosis, liver decompensation, and hepatocellular carcinoma, but it is also closely associated with a significantly increased risk of systemic metabolic disorders and cardiovascular disease.

[0003] Given its high prevalence, rapid progression, and multiple systemic associations, MASH has become a major disease burden in global public health. This disease severely impairs patients' quality of life, significantly increases the consumption of healthcare resources, and poses a serious challenge to patients' long-term prognosis.

[0004] Currently, there are still significant unmet clinical needs for interventions targeting MASH, and there is an urgent need to explore new treatment strategies to halt disease progression and reduce the risk of end-stage liver disease and cardiovascular events. Summary of the Invention

[0005] Based on this, the present invention provides the application of Clostridium butyricum combined with Remetol in the preparation of drugs for steatohepatitis, which solves at least one problem in the prior art.

[0006] In a first aspect, the present invention provides the application of Clostridium butyricum in combination with remetoidin in the preparation of drugs for steatohepatitis.

[0007] Clostridium butyricum not only alleviates the adverse reactions caused by retemertil in the prevention or treatment of steatohepatitis, but also enhances the ameliorative effects of retemertil on liver damage, lipid disorders, insulin resistance, related colitis, and intestinal barrier damage caused by steatohepatitis. Therefore, Clostridium butyricum combined with retemertil can be used to prepare drugs for the prevention or treatment of steatohepatitis, with the two drugs exhibiting a synergistic effect.

[0008] Secondly, the present invention provides a drug for fatty liver disease, comprising Clostridium butyricum and Remetibillin.

[0009] In some optional embodiments, the butyric acid bacterium is Clostridium butyricum (Clostridium butyricum) Clostridium butyricum HG007. The Clostridium butyricum (… Clostridium butyricum HG007 has been deposited at the China General Microbiological Culture Collection Center and classified as Clostridium butyricum. Clostridium butyricum The date of collection is June 5, 2026. The collection address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The collection number is CGMCC No. 39281.

[0010] In some alternative embodiments, the steatohepatitis is metabolic-associated steatohepatitis (MASH).

[0011] In some optional embodiments, the ratio of Clostridium butyricum to remetrol is 1 × 10⁻⁶. 7 CFU-9×10 8 CFU: 0.01 mg-0.2 mg.

[0012] In some alternative embodiments, the steatohepatitis drug further includes a pharmaceutically acceptable carrier.

[0013] In some alternative embodiments, the pharmaceutically acceptable carrier is water.

[0014] In some optional embodiments, the dosage form of the steatohepatitis drug is a powder or a suspension.

[0015] Due to the adoption of the above technical solutions, the embodiments of the present invention have at least the following beneficial effects: Clostridium butyricum combined with remetrol can play a synergistic role in the preparation of drugs for the prevention or treatment of fatty liver disease. Attached Figure Description

[0016] Figure 1 These are macroscopic morphological photographs of mouse livers in various groups in this embodiment of the invention.

[0017] Figure 2 The figures show the statistical values ​​of body weight, liver weight, liver total cholesterol (TC), and liver total cholesterol (TG) of mice in each group in this embodiment of the invention.

[0018] Figure 3 These are representative HE-stained photomicrographs of mouse liver sections from each group in the embodiments of the present invention, with the scale bar of the upper image being 200 μm and the scale bar of the lower image being 50 μm.

[0019] Figure 4 These are representative photomicrographs of mouse liver sections stained with Oil Red O in each group of embodiments of the present invention, with the scale bar of the upper image being 200 μm and the scale bar of the lower image being 50 μm.

[0020] Figure 5 The above are statistical graphs of serum ALT, AST, TC, and TG in mice in each group in this embodiment of the invention.

[0021] Figure 6 The figures show the statistical values ​​of FBG level, FINS level, HOMA-IR index, and HOMA-IS index of mice in each group in this embodiment of the invention.

[0022] Figure 7 These are representative masson-stained micrographs of mouse liver sections from each group in the embodiments of the present invention, with the scale bar of the upper image being 200 μm and the scale bar of the lower image being 50 μm.

[0023] Figure 8 These are representative SMA-stained micrographs of mouse liver sections from each group in the embodiments of the present invention, with the scale bar of the upper image being 200 μm and the scale bar of the lower image being 50 μm.

[0024] Figure 9 These are representative photomicrographs of HE-stained and AB-PAS-stained colon sections of mice in each group of embodiments of the present invention, with the scale bar of the upper image being 200 μm and the scale bar of the lower image being 50 μm.

[0025] Figure 10 These are representative micrographs (scale bar 200 μm) of mouse liver sections stained with ZO-1 and Occludin in each group of embodiments of the present invention.

[0026] Figure 11 This is a statistical chart showing the kaolin intake of mice in each group in this invention embodiment.

[0027] Figure 12 The figures show statistical graphs of colonic 5-HT levels, colonic SP levels, fold change in brain 5-HT receptor mRNA expression, fold change in brain NK-1 receptor mRNA expression, brain 5-HT levels, and brain SP levels in each group of mice in the embodiments of the present invention.

[0028] Figure 13 The images show the immunofluorescence staining results of FOS neurons in the brains of mice in each group of embodiments of the present invention (scale bar is 100μm).

[0029] Figure 14 This is a growth curve diagram of Clostridium butyricum HG007 in an embodiment of the present invention.

[0030] Figure 15 This is a statistical chart showing the acid resistance test results of Clostridium butyricum HG007 in the embodiments of the present invention.

[0031] Figure 16 This is a statistical chart showing the bile salt tolerance test results of Clostridium butyricum HG007 in the embodiments of the present invention.

[0032] Figure 17 This is a statistical chart showing the antibacterial test results of Clostridium butyricum HG007 in the embodiments of the present invention.

[0033] Figure 18This is a statistical chart of the antibiotic resistance test results of Clostridium butyricum HG007 in the embodiments of the present invention, where PEN represents penicillin; C represents chloramphenicol; E represents erythromycin; AMP represents ampicillin; MY represents lincomycin; CTR represents ceftriaxone; TET represents tetracycline; SXT represents trimethoprim-sulfamethoxazole; GEN represents gentamicin; and CIP represents ciprofloxacin.

[0034] Figure 19 This is a statistical chart showing the evaluation results of the antioxidant properties of Clostridium butyricum HG007 in the embodiments of the present invention. Detailed Implementation

[0035] The following will provide a clear and complete description of the concept and technical effects of the present invention, so as to fully explain the purpose, solution and effects of the present invention.

[0036] To verify the efficacy of Clostridium butyricum combined with retemetine in preventing or treating fatty liver disease, the following animal experiments were conducted according to embodiments of the present invention.

[0037] The experimental animals were 6-8 week old SPF-grade male C57BL / 6J mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.). All mice were housed in an SPF-grade animal room with an ambient temperature of 22±2℃ and a relative humidity of 50%-60%, under a 12-hour light / dark cycle, and with free access to food and water. The experiments began after the mice were acclimatized to a standard diet for one week.

[0038] The butyric acid bacillus used in the experiment was Clostridium butyricum (Clostridium butyricum Clostridium butyricum Clostridium butyricum HG007 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 5, 2026, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 39281. Before the experiment, the frozen strain was inoculated onto reinforced clostridial medium (RCM, purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number LA4360) and cultured anaerobically at 37 °C for 24 h to obtain activated seed culture. This activated seed culture was then transferred to fresh reinforced clostridial medium at a 1% (V / V) inoculation rate and cultured anaerobically at 37 °C for 24 h to obtain a stable bacterial culture.

[0039] The experimental mice were randomly divided into the following 5 groups, with 6 mice in each group, and each group received an intervention treatment: Group C (control group): fed with normal feed for 16 weeks, followed by continuous gavage administration of 200 μL / d sterile PBS solution for 8 weeks; Group M (model group): fed with a high-fat diet for 16 weeks, followed by continuous gavage administration of 200 μL / d of sterile PBS solution for 8 weeks; MR group (Remetitrol group): fed with a high-fat diet for 16 weeks, and then administered 3 mg / kg (based on mouse body weight) of remetitrol by gavage for 8 consecutive weeks; MP group (Clostridium butyricum group): fed with a high-fat diet for 16 weeks, followed by continuous gavage administration of 200 μL / d Clostridium butyricum bacterial solution (1×10⁻⁶) for 8 weeks. 8 (CFU / mL) MPR group (Clostridium butyricum combined with remetrol group): fed with a high-fat diet for 16 weeks, followed by continuous gavage administration of 3 mg / kg (based on mouse body weight) and 200 μL / d of Clostridium butyricum bacterial solution (1×10⁻⁶) for 8 weeks. 8 (CFU / mL).

[0040] The high-fat diet was purchased from Xiaoshuyoutai (Beijing) Biotechnology Co., Ltd., product number D12079B, and was used to establish the MASH liver fibrosis model. After the intervention, the mice were fasted but allowed free access to water for 12 hours, and blood and organ samples were collected for testing.

[0041] like Figures 1-2 As shown, compared with the control group, the model group mice had significantly increased body weight, liver weight, total liver cholesterol (TC), and liver triglyceride (TG) levels, and exhibited a marked steatosis-like liver appearance. Both Clostridium butyricum and retemetirole alone could improve these abnormalities to varying degrees, while the combined treatment with Clostridium butyricum and retemetirole showed the most significant improvement. Figures 3-4 As shown, HE staining and Oil Red O staining histological examination results further revealed that the liver tissue of the model group mice showed significant steatosis, inflammatory cell infiltration, and lipid droplet deposition. Combined treatment with Clostridium butyricum and retemetiride further reduced liver tissue structural damage and lipid deposition. Furthermore, as... Figures 5-6 As shown, the combined treatment of Clostridium butyricum and retemetirole significantly reduced serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), and triglyceride (TG) levels in mice, and improved glucose metabolism indicators such as fasting blood glucose (FBG), fasting insulin (FINS), HOMA-IR index, and HOMA-IS index. These results indicate that Clostridium butyricum can enhance the ameliorative effect of retemetirole on liver injury, lipid disorders, and insulin resistance in MASH mice.

[0042] like Figures 7-8 As shown, Masson staining and SMA immunohistochemical results revealed that collagen deposition and α-SMA positive expression were significantly increased in the livers of mice in the model group. Clostridium butyricum, retemetirol, and combination therapy could all alleviate fibrotic changes, with the combination therapy of Clostridium butyricum and retemetirol showing a more prominent effect.

[0043] like Figure 9As shown, HE staining and AB-PAS staining results of the colon revealed that the colonic mucosal structure of the model group mice was damaged, with a decrease in the number of goblet cells and the mucus barrier; after combined treatment with Clostridium butyricum and retemetiride, the colonic tissue structure and mucus barrier were significantly restored. Meanwhile, as... Figure 10 As shown, immunofluorescence staining results revealed that combined treatment with Clostridium butyricum and retemetiride significantly restored the expression of the tight junction proteins Occludin and ZO-1 in the mouse colon. These results indicate that Clostridium butyricum can enhance the ameliorative effect of retemetiride on MASH-related colitis and intestinal barrier damage.

[0044] To evaluate the effect of Clostridium butyricum on remetrotinol-related gastrointestinal adverse reactions, a kaolin ingestion test was used to assess nausea / vomiting-like behaviors in mice. Specifically, 1% (w / w) gum arabic was mixed with kaolin, and sterile water was added to form pellets of the same size and shape as standard feed. These pellets were then dried at room temperature. Equal amounts of standard feed and kaolin pellets were given to mice in each group for free access, and the kaolin ingestion rate was recorded over 24 hours for three consecutive days. Figure 11 As shown, the kaolin ingestion rate of mice in the remetrotinol group was significantly increased, while this behavior was significantly reduced after the addition of Clostridium butyricum.

[0045] like Figure 12 As shown, further testing revealed that retimetrol could increase the levels of serotonin (5-HT) and substance P (SP) in colon and brain tissue, and upregulate the mRNA expression of 5-HT receptor (5-HT3R) and NK-1 receptor (NK-1R) in brain tissue; these indicators were significantly reduced after combination with Clostridium butyricum. Furthermore, as... Figure 13 As shown, retemetirole induces an increase in FOS-positive neurons in brain tissue, while combined treatment with Clostridium butyricum and retemetirole attenuates FOS fluorescence signals. These results suggest that Clostridium butyricum can alleviate retemetirole-related nausea / vomiting-like reactions, and its effect may be related to the inhibition of 5-HT / SP-mediated abnormal activation of the gut-brain axis.

[0046] Clostridium butyricum used in the experiment ( Clostridium butyricum The separation, purification, and identification process of HG007 is as follows: 1. Separation and purification One g of fresh, healthy fecal matter from a person residing in Nanchang City, Jiangxi Province, was placed in a 10 mL centrifuge tube (containing 5 mL of PBS solution) and centrifuged (800 rpm, 5 min). After centrifugation, 1 mL of the supernatant was transferred to a 1.5 mL centrifuge tube and serially diluted with sterile PBS at 1000 ppm. 1 ~10 9Dilutes 3, 5, 7, and 9 (numbered 1-9) were used for plating (RCM medium). 30 μL of each diluted solution was spread onto a plate and anaerobically cultured for 24 h. Plates with 200-400 colonies were then picked in a sterile laminar flow hood. Rapidly growing, well-defined, round, and slightly raised colonies were inoculated into 5 mL of the corresponding liquid RCM medium and anaerobically activated for 24 h to obtain the purified strain, Clostridium butyricum HG007.

[0047] 2. Morphological identification The purified strain was stained using Gram staining, and its morphology was observed under a 100x oil immersion microscope. The specific steps were as follows: The purified strain was inoculated into RCM solid medium and cultured at 37°C for 24 h in an anaerobic workstation. A small amount of a single colony of Clostridium butyricum HG007 was taken, dipped into 2 µL of physiological saline, and spread clockwise to form a layer of about 1 cm. 2 The slide was prepared as a uniform, thin, round bacterium and placed at room temperature until the bacterial culture dried. The slide was then fixed by passing it through an alcohol lamp flame 1-2 times. Crystal violet staining solution was added to the bacteria and stained for 1 minute. The slide was washed with water, then iodine solution was added and stained for another 1 minute. After washing with water, destaining solution was added, and the slide was shaken for 30 seconds. The slide was then washed with water, the excess water was absorbed, and safranin staining solution was added and stained for 1 minute. After washing with water, excess water was blotted with filter paper, and the slide was examined under a 100x oil immersion microscope. Gram staining results showed that *Clostridium butyricum* HG007 was a Gram-positive bacterium with a rod-shaped morphology.

[0048] 3. Physiological and biochemical identification (1) Growth kinetics: The growth kinetics of Clostridium butyricum strain HG007 were characterized by 24-hour growth curves based on absorbance measurements at 600 nm. Figure 14 As shown, the growth curve indicates that the strain enters a rapid proliferation phase after 4 hours and reaches a stationary phase after 14 hours.

[0049] (2) Acid tolerance test: Clostridium butyricum HG007 cells in logarithmic growth phase were washed with PBS and resuspended in acidic buffer solutions at pH 2.0, 3.0, 5.0, and 7.0, respectively, and incubated at 37°C for 4 hours. Viable cell counts were then performed using the plate method. Figure 15 As shown, this strain is tolerant of acidic environments with a pH range of 2.0-7.0.

[0050] (3) Bile salt tolerance test: Log-phase Clostridium butyricum HG007 cells were washed with PBS and resuspended in bovine bile salt solutions of different concentrations (0.1%, 0.2%, 0.3%). The cells were incubated at 37°C for 4 hours, and viable counts were performed using the plate method. Figure 16 As shown, this strain tolerates 0.1%-0.3% concentrations of bile salts.

[0051] (4) Antibacterial test: Common intestinal pathogens (Pseudomonas aeruginosa) were tested. P. aeruginosa Group A beta-hemolytic streptococci BHS Candida albicans C. albicans Salmonella typhi S. typhi Staphylococcus aureus S. aureus Escherichia coli E. Coli Listeria monocytogenes L. Monocytogenes Shigella flexneri S. flexneri The bacterial suspensions were spread onto agar plates, and Oxford cups were placed on the plates. Clostridium butyricum HG007 culture supernatant was added to the Oxford cups. After incubation at 37°C for 24 hours, the diameter of the inhibition zone was measured to assess antibacterial activity. Figure 17 As shown, this strain has inhibitory effects on a variety of pathogenic bacteria, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Listeria monocytogenes.

[0052] (5) Antibiotic resistance test: 1×10 9 A CFU / mL suspension of Clostridium butyricum HG007 was evenly spread onto an agar plate, and commercial antibiotic susceptibility testing discs containing 10 μg of antibiotic were attached. After 24 hours of incubation, the diameter of the inhibition zone was measured, and the bacterial susceptibility (susceptibility, intermediate resistance, or resistance) was determined according to CLSI standards. Figure 18 As shown, the antibiotic resistance test results indicate that it is sensitive to antibiotics such as erythromycin, penicillin, ciprofloxacin, ampicillin, lincomycin, ceftriaxone, gentamicin, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline.

[0053] (6) The in vitro antioxidant activity of Clostridium butyricum HG007 was measured to inhibit DPPH free radicals, hydroxyl radicals (•OH), and superoxide anions (•O2). - The scavenging ability of ) was assessed by both the iron ion chelation test and the total reducing power test (Prussian blue method, OD measured). 700 Assess its antioxidant activity. For example... Figure 19 As shown, Clostridium butyricum HG007 possesses significant antioxidant capabilities, with a DPPH free radical scavenging rate of 44.87%, a hydroxyl radical (•OH) scavenging rate of 37.10%, and a superoxide anion (•O2) scavenging rate of 37.10%. - The scavenging rate was 40.78%, the iron ion chelating ability was 16.72%, and the total reducing power was 0.96 (OD). 700 ).

[0054] Therefore, Clostridium butyricum HG007 has outstanding probiotic characteristics and is a promising candidate probiotic strain for development.

[0055] 4. Molecular identification Perform molecular identification according to the following steps: (1) The isolated Clostridium butyricum HG007 single bacteria were inoculated into 5 mL of RCM liquid medium; (2) Preserve bacteria with 30% glycerol (one tube is sufficient for multiple samples), centrifuge (8000 rpm, 2 min), and discard the supernatant; (3) Add 600 μL of 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 g of glass beads to the precipitate, shake for 30 s and repeat 3 times until the cells are completely suspended, and centrifuge (8000 rpm, 1 min). (4) Transfer the supernatant to a new 1.5 mL centrifuge tube, add 250 μL of 10 M ammonium acetate, place on ice for 10 min, and centrifuge (8000 rpm, 1 min). (5) Take the supernatant above the organic layer onto the DNA adsorption column and centrifuge (8000 rpm, 1 min). (6) Wash once with 600 μL of 75% ethanol; (7) Repeat step (6) above; (8) After spinning at 8000 rpm for 2 min, transfer the DNA adsorption column to a new EP tube and air dry for 30 min; (9) Add 50 µL of TE buffer (pH=8.0) to the dried centrifuge tube, and send 25 μL to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0056]

[0057] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any embodiment that achieves the technical effects of the present invention by the same or equivalent means should fall within the protection scope of the present invention. Within the protection scope of the present invention, various modifications and variations can be made to the technical solutions and / or implementation methods.

Claims

1. Application of Clostridium butyricum combined with remetoidin in the preparation of drugs for fatty liver disease.

2. The application according to claim 1, characterized in that, The butyric acid bacterium is Clostridium butyricum (Clostridium butyricum) Clostridium butyricum HG007, the Clostridium butyricum ( Clostridium butyricum HG007 has been deposited at the China General Microbiological Culture Collection Center and classified as Clostridium butyricum. Clostridium butyricum The date of collection is June 5, 2026. The collection address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The collection number is CGMCCNo.39281.

3. The application according to claim 1, characterized in that, The fatty liver disease mentioned is metabolic-associated fatty liver disease.

4. A drug for fatty liver disease, characterized in that, Including Clostridium butyricum and Remetidine.

5. The drug for fatty liver disease according to claim 4, characterized in that, The butyric acid bacterium is Clostridium butyricum (Clostridium butyricum) Clostridium butyricum HG007, the Clostridium butyricum ( Clostridium butyricum HG007 has been deposited at the China General Microbiological Culture Collection Center and classified as Clostridium butyricum. Clostridium butyricum The date of collection is June 5, 2026. The collection address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The collection number is CGMCC No. 39281.

6. The drug for fatty liver disease according to claim 4, characterized in that, The fatty liver disease mentioned is metabolic-associated fatty liver disease.

7. The drug for fatty liver disease according to claim 4, characterized in that, The dosage ratio of Clostridium butyricum and remetrol is 1×10⁻⁶. 7 CFU-9×10 8 CFU: 0.01 mg-0.2 mg.

8. The drug for fatty liver disease according to claim 4, characterized in that, The drug for fatty liver disease also includes a pharmaceutically acceptable carrier.

9. The drug for fatty liver disease according to claim 8, characterized in that, The pharmaceutically acceptable carrier is water.

10. The drug for fatty liver disease according to claim 4, characterized in that, The dosage form of the drug for fatty liver disease is powder or suspension.

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