Lactobacillus plantarum SCS7 and application of acellular extract of lactobacillus plantarum SCS7 in preparation of medicine for relieving aflatoxin B1-induced liver injury

Abstract

The invention relates to the technical field of biology, in particular to application of lactobacillus plantarum SCS7 and an acellular extract of the lactobacillus plantarum SCS7 in preparation of a medicine for relieving aflatoxin B1 induced liver injury. The cell-free extract of the lactobacillus plantarum SCS7 is applied to preparation of a medicine for relieving aflatoxin B1 induced liver injury. Aflatoxin B1 causes liver injury and disorder of intestinal flora. Meanwhile, the lactobacillus plantarum SCS7 cell-free extract recovers the mitochondrial function damaged by aflatoxin B1 by inhibiting over-activated mitochondrial autophagy, so that the mitochondrial homeostasis is balanced, meanwhile, the composition of intestinal flora and the abundance of dominant flora are adjusted, and the content of short-chain fatty acid is increased to maintain the intestinal flora homeostasis. The research possibly enriches the protective effect and mechanism of lactobacillus plantarum on liver injury caused by various factors, and the lactobacillus plantarum has wide application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a strain of Lactobacillus plantarum (Lactobacillus plantarum, L.plantarum) SCS7 and a cell-free extract thereof for use in the preparation of a medicament for alleviating liver injury induced by aflatoxin B1. Lactobacillus plantarum The present application relates to the field of biotechnology, in particular to a strain of Lactobacillus plantarum (Lactobacillus plantarum, L.plantarum) SCS7 and a cell-free extract thereof for use in the preparation of a medicament for alleviating liver injury induced by aflatoxin B1. BACKGROUND

[0002] Aflatoxins (AFs) are secondary metabolites mainly produced by Aspergillus flavus and Aspergillus parasiticus, which are widely present in nature, especially in warm and humid environments, and easily contaminate crops and food. AFs have strong toxicity, mutagenicity, carcinogenicity and immunosuppressive effect 1 . Aflatoxin B1 (AFB1) is the most toxic of all aflatoxins, often found in nut foods and animal feed based on corn and peanuts, causing public health incidents, so the contamination of AFB1 has attracted widespread public attention 2 . Long-term exposure to low levels of aflatoxins through contaminated food and feed can cause serious health consequences, especially DNA damage in embryos, immune suppression, growth disorders in children and various other systemic diseases 3 . High-dose exposure can cause acute aflatoxin poisoning, leading to acute liver injury, bleeding and potentially fatal consequences 4 . Liver is the target organ of AFB1, and AFB1 has a great impact on liver injury and hepatocellular carcinoma 5 . Studies have shown that AFB1 is metabolically activated in the liver by cytochrome P450 enzyme system to generate AFB1-8,9-epoxide with high reactivity. This metabolite can covalently bind to guanine residues in DNA molecules to form AFB1-N7-guanine adducts, leading to DNA damage and gene mutation, and thus inducing hepatocellular carcinoma 6 . In addition, AFB1 can also promote cell proliferation and inhibit apoptosis by activating the PI3-Akt signaling pathway, thereby accelerating the development of liver cancer 7 . In recent years, more and more studies have shown that the liver injury of AFB1 is not only reflected in its genetic toxicity, but also closely related to its damage to mitochondria. AFB1 can destroy the structure, function and homeostasis of mitochondria, leading to oxidative stress, apoptosis and inflammation, thereby causing liver injury in the body, and even further developing into hepatocellular carcinoma 8-10 . Given the significant threat of AFB1 to public health and food safety, it is of great significance to conduct in-depth research on its toxicity mechanism and develop effective prevention and intervention strategies.

[0003] Mitophagy is an important cellular self-cleaning mechanism, primarily responsible for clearing damaged or dysfunctional mitochondria to maintain cellular homeostasis. This process is mainly regulated by the PINK1 / Parkin signaling pathway: when mitochondria are damaged, PINK1 protein accumulates on the mitochondrial outer membrane and recruits Parkin protein. Subsequently, Parkin ubiquitinates mitochondrial outer membrane proteins, which then degrade the damaged mitochondria via the autophagosome-lysosome pathway. 11 Studies have shown that AFB1 can lead to mitochondrial damage or dysfunction, thereby activating mitophagy to clear damaged mitochondria and maintain cellular homeostasis. Research in HepaRG cells has shown that AFB1 exposure significantly increases the expression of PINK1 and Parkin, and induces mitophagy, a process closely related to the progression of liver fibrosis. 12 Under the influence of 30 μg / kg AFB1, mitochondrial reactive oxygen species (mtROS) in duck liver significantly increased, mitochondrial morphology changed, and mitophagy was activated. 13 Meanwhile, studies have found that AFB1 exposure activates PINK1 / Parkin-mediated mitophagy, thereby preventing immunosuppression in the mouse spleen. 14 Therefore, the classical mitophagy pathway may become a new target for lactic acid bacteria to prevent or alleviate AFB1 toxicity.

[0004] Research on the prevention and control of aflatoxin B1 hazards currently mainly employs physical and chemical detoxification techniques, including irradiation, ozone treatment, cold plasma, and the application of chemical adsorbents. 15 However, these methods may induce other environmental, ecological, and food safety problems in practical applications. Therefore, exploring novel biological prevention pathways or sustainable detoxification mechanisms has become an important research direction in this field. 16,17 Lactic acid bacteria, as a widely used probiotic, can bring various benefits to the host. 18-20 In addressing the challenge of counteracting aflatoxin toxicity, lactic acid bacteria, as an emerging biological approach, offer advantages such as safety, effectiveness, and environmental friendliness. 21 Studies have shown that lactic acid bacteria can alleviate the damage caused by aflatoxin. Parvaneh Afshar et al. 22 Research using micronucleus experiments found that 10 9 Live *Lactobacillus plantarum* (CFU / mL) exhibits high protective efficacy against AFB1 genotoxicity. Investigating the mechanisms by which lactic acid bacteria alleviate AFB1 toxicity is crucial for better application of biological methods to prevent AFB1-induced damage. *Lactobacillus plantarum* C88 can alleviate AFB1-induced liver injury in mice by inhibiting NF-κB-mediated inflammatory responses and excessive apoptosis.23 Lactobacillus casei contents exert a protective effect by improving the body's antioxidant status in response to AFB1-induced oxidative stress damage. 24 Its mechanism of action may involve activating the activity of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase, and reducing the accumulation of lipid peroxidation products such as malondialdehyde. 25 Although some progress has been made in the study of lactic acid bacteria in the prevention and control of AFB1, the research on the protective effects and mechanisms of cell-free extracts from lactic acid bacteria on the body is still insufficient. Summary of the Invention

[0005] To address the above problems, this invention provides a strain of *Lactobacillus plantarum* (…). Lactobacillus plantarum The application of SCS7 and its cell-free extract in the preparation of drugs to alleviate aflatoxin B1-induced liver injury: This invention uses normal mouse hepatocytes (AML12) as the research object, divided into 5 groups: blank control group (NG), solvent control group (DG, 0.1% DMSO), model group (AG, treated with 10 μmol / L AFB1 for 24 h), pretreatment group (LG group, 0.1 mg / mL AFB1), and pretreatment group (LG group). L. plantarum SCS7 cell-free extract pretreatment for 3 h), pretreatment + model group (A+LG group, 0.1 mg / mL) L. plantarum SCS7 cells were pretreated with cell-free extract for 3 h, followed by treatment with 10 μmol / LAFB1 for 24 h. Cell viability, ROS, mitochondrial damage markers, and PINK1 / Parkin signaling pathway expression were measured using cell viability and toxicity assays, DCFH-DA fluorescent probe, transmission electron microscopy, mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) assays, quantitative real-time fluorescence PCR (qPCR), and Western blotting (WB). Simultaneously, 60 five-week-old male Kunming (KM) mice were used as subjects. After one week of acclimatization, the experiment lasted 28 days and was randomly divided into 6 groups: PBS control group (NP), DMSO control group (ND, 0.1% DMSO), model group [MA, 300 μg / kg (bw) AFB1], low-dose + model group [LLP+A, 1 mg / mL AFB1], and [LLP+A, 1 mg / mL AFB1]. L. plantarumSCS7 cell-free extract + 300 μg / kg (bw) AFB1], high dose + model group [HLP+A, 3 mg / mL] L. plantarum SCS7 cell-free extract + 300 μg / kg (bw) AFB1], cell-free extract treatment group (HLP, 3 mg / mL) L. plantarum SCS7 cell-free extract). After gavage, liver tissue morphology analysis, serum liver function tests, detection of mitochondrial autophagy-related proteins (P62, PINK1, Parkin, PGAM5, LC3), gut microbiota assessment, and short-chain fatty acid determination were performed. The aim was to explore the combined effects of in vitro and in vivo experiments. L. plantarum The protective effect and mechanism of SCS7 cell-free extract against AFB1-induced liver injury.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a strain of Lactobacillus plantarum ( Lactobacillus plantarum SCS7, the Lactobacillus plantarum SCS7, was deposited on January 13, 2025 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 33379.

[0007] The present invention also provides the application of the cell-free extract of Lactobacillus plantarum SCS7 described in the above technical solution in the preparation of a drug to alleviate aflatoxin B1-induced liver injury.

[0008] Preferably, the cell-free extract of Lactobacillus plantarum SCS7 alleviates aflatoxin B1-induced liver injury through PINK1 / Parkin signaling pathway-mediated mitophagy.

[0009] Preferably, the cell-free extract of Lactobacillus plantarum SCS7 alleviates aflatoxin B1-induced liver damage by regulating the intestinal flora.

[0010] Preferably, aflatoxin B1-induced liver damage is alleviated by increasing the abundance of beneficial bacteria and decreasing the abundance of pathogenic bacteria; The beneficial bacteria include *Vibrio spp.* Oscillospira Akkermania ( ) Akkermansia Phylum Verrucomicrobiota, etc. The pathogenic bacteria include *Thorax* spp. ( Thomasclavella ).

[0011] Preferably, the cell-free extract of Lactobacillus plantarum SCS7 alleviates aflatoxin B1-induced liver damage by increasing the content of short-chain fatty acids.

[0012] Preferably, the short-chain fatty acid includes one or more of acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and isobutyric acid.

[0013] Preferably, the method for preparing the cell-free extract of Lactobacillus plantarum SCS7 includes the following steps: 1) Mix the Lactobacillus plantarum SCS7 with PBS buffer and sonicate for 30 min under ice bath conditions to obtain the sonicated fragment; The conditions for ultrasonic disruption include: 4℃, 400W, 5s ultrasound, and 10s interval. 2) The ultrasonically broken material obtained in step 1) is centrifuged at 4°C and 10,000 r / min for 10 min. The resulting supernatant is passed through a 0.22 µm sterile filter membrane. The filtrate is a cell-free extract of Lactobacillus plantarum SCS7.

[0014] Preferably, the concentration of protein in the cell-free extract of Lactobacillus plantarum SCS7 is 2–3.5 mg / mL.

[0015] The beneficial effects of this invention are: In this invention, the search is conducted through both in vitro and in vivo experiments. L. plantarum Mechanism of action of SCS7 cell-free extract in preventing AFB1-induced liver injury. Evaluation in vitro using AML12 cells. L. plantarum The protective effect and mechanism of cell-free extract of SCS7 against AFB1-induced liver injury. The results showed that... L. plantarum SCS7 cell-free extract can inhibit AFB1-induced excessive mitophagy by regulating the PINK1 / Parkin signaling pathway, thereby maintaining the integrity of mitochondrial structure and function. In vivo, male KM mice were divided into 6 groups, and liver tissue morphology, serum liver function indicators, gut microbiota, and short-chain fatty acids were measured after administration to explore [the following parameters]. L. plantarum The protective effect of cell-free extract of SCS7 against AFB1-induced liver injury. The results indicate that... L. plantarum Pretreatment with SCS7 cell-free extract alleviated liver function damage and liver pathological changes induced by AFB1 exposure. Simultaneously, it can also promote the growth of beneficial bacteria (such as *Oscillatoria* spp.) by regulating gut microbiota composition. Oscillospira Akkermania ( ) Akkermansia Enrichment of bacteria such as *Verrucomicrobiota*, while inhibiting the growth of pathogenic bacteria such as *Thomas*. Thomasclavella The proliferation of [etc.]. Additionally, compared to the model group, L. plantarum The content of short-chain fatty acids in the SCS7 cell-free extract pretreatment group showed an increasing trend. These results suggest that... L. plantarumCell-free extracts of SCS7 may play a positive role in alleviating AFB1-induced liver injury by maintaining gut-hepatic axis homeostasis. This study may enrich our understanding of the protective effects and mechanisms of *Lactobacillus plantarum* against liver injury induced by various factors, and its application prospects are broad. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 For AFB1 and L. plantarum Changes in AML12 cell viability after treatment with SCS7 cell-free extract; Note: a shows the effect of different concentrations of AFB1 (0–40 μmol / L) on AML12 cell viability; b shows the effect of different concentrations of AFB1 on AML12 cell viability. L. plantarum Effect of SCS7 cell-free extract (0–0.8 mg / mL) on AML12 cell viability; results are expressed as mean ± standard deviation, n≥3; * indicates that the difference between each group and group 0 is statistically significant. P <0.05); Figure 2 The changes in ROS levels in AML12 cells from different treatment groups are shown. Note: a is a ROS fluorescence image captured under laser confocal microscopy; b is a change in ROS fluorescence intensity. Results are expressed as mean ± standard deviation, n≥3; * indicates that the difference between the data and the NG group is statistically significant. P <0.05); # indicates that the difference between the data and the AG group is statistically significant. P <0.05); NG: blank control group, DG: 0.1% DMSO treatment group, AG: 10 μmol / L AFB1 treatment group, LG: 0.1 mg / mL L. plantarum SCS7 cell-free extract pretreatment group, A+LG: 0.1 mg / mL L. plantarum After pretreatment with SCS7 cell-free extract, the group was then treated with 10 μmol / L AFB1. Figure 3 Mitochondrial morphology and autophagy in AML12 cells from different treatment groups; Note: Image magnification: 3000×, mild mitochondrial swelling (↑), mitophagy (↑), autophagy (↑) are shown in the experimental groups. Figure 2 ; Figure 4 Changes in mitochondrial function in AML12 cells under different treatment groups; Note: a is a representative laser confocal image of MMP; b is quantification of MMP; c is... L. plantarumEffects of SCS7 cell-free extract and AFB1 on ATP; results are expressed as mean ± standard deviation, n≥3; * indicates statistically significant differences compared with the NG group. P <0.05); # indicates that the difference between the data and the AG group is statistically significant. P <0.05); experimental groups were the same. Figure 2 ; Figure 5 The changes in the expression of mitophagy-related genes in AML12 cells under different treatment groups; Note: ae represents the relative expression of mitophagy-related genes; Results are expressed as mean ± standard deviation, n≥3; * indicates that the difference between the data and the NG group is statistically significant. P <0.05); # indicates that the difference between the data and the AG group is statistically significant. P <0.05); experimental groups were the same. Figure 2 ; Figure 6 The changes in the expression of mitochondrial autophagy-related proteins in AML12 cells under different treatment groups are shown. Note: ab represents representative bands of mitochondrial autophagy-related proteins; cg represents the relative protein expression level quantified by density analysis using β-actin as an internal control; results are expressed as mean ± standard deviation, n≥3; * indicates that the difference between the data and the NG group is statistically significant. P <0.05); # indicates that the difference between the data and the AG group is statistically significant. P <0.05); experimental groups were the same. Figure 2 ; Figure 7 The changes in liver organ coefficients in mice under different treatment groups; Note: Results are expressed as mean ± standard deviation, n≥3; * indicates that the differences between the data and the NP group are statistically significant. P <0.05); # indicates that the difference between the data groups is statistically significant compared with the MA group. P <0.05); NP: PBS solvent control group, ND: 0.1% DMSO treatment group, MA: 300 μg / kg (bw) AFB1 treatment group, LLP+A: 1 mg / mL L. plantarum After pretreatment with SCS7 cell-free extract, the group was treated with 300 μg / kg (bw) AFB1 and HLP+A: 3 mg / mL. L. plantarum Pretreatment with SCS7 cell-free extract followed by treatment with 300 μg / kg (bw) AFB1 and HLP: 3 mg / mL L. plantarum SCS7 cell-free extract pretreatment group; Figure 8HE pathological sections of mouse livers from different treatment groups; Note: Magnification: 400×, necrotic cell debris (↑), neutrophils (↑); Experimental groups were the same. Figure 7 ; Figure 9 The changes in serum biochemical parameters in mice under different treatment groups are shown below. Note: a represents serum ALT enzyme activity; b represents serum AST enzyme activity; c represents serum ALP enzyme activity. Results are expressed as mean ± standard deviation, n≥3; * indicates that the difference between the data and the NP group is statistically significant. （P <0.05); # indicates that the difference between the data groups is statistically significant compared to the MA group. P <0.05); experimental groups were the same. Figure 7 ; Figure 10 Changes in ROS levels in the liver of mice in different treatment groups; Note: Results are expressed as mean ± standard deviation, n≥3; * indicates statistically significant differences compared with the NP group. P <0.05); # indicates that the difference between the data groups is statistically significant compared with the MA group. P <0.05); experimental groups were the same. Figure 7 ; Figure 11 The changes in the expression of mitochondrial autophagy-related proteins in the liver of mice under different treatment groups are shown. Note: ab is a representative band diagram of mitochondrial autophagy-related proteins in mouse liver; cg is the relative protein expression level quantified by density analysis using β-actin as an internal control; results are expressed as mean ± standard deviation, n≥3; * indicates that the difference between the data and the NP group is statistically significant. P <0.05); # indicates that the difference between the data groups is statistically significant compared with the MA group. P <0.05); experimental groups were the same. Figure 7 ; Figure 12 The abundance of gut microbiota is shown at the genus and phylum levels; Note: a is a bar chart of bacterial abundance at the genus level; b is a bar chart of bacterial abundance at the phylum level; c is the ratio of Firmicutes to Bacteroidetes; d is the abundance value of Verrucous Microbes; Results are expressed as mean ± standard deviation, n≥3; * indicates that the difference between the data and the ND group is statistically significant. P <0.05); # indicates that the difference between the data groups is statistically significant compared with the MA group. P <0.05); ND: 0.1% DMSO treatment group, MA: 300 μg / kg (bw) AFB1 treatment group, LLP+A: 1 mg / mL L. plantarumAfter pretreatment with SCS7 cell-free extract, the group was then treated with 300 μg / kg (bw) AFB1 and HLP+A: 3 mg / mL. L. plantarum After pretreatment with SCS7 cell-free extract, the group was then treated with 300 μg / kg bw AFB1. Figure 13 for L. plantarum Effects of SCS7 cell-free extract on AFB1-induced gut microbiota composition; a) Changes in Chao1 values ​​in different treatment groups; b) Changes in Shannon values ​​in different treatment groups; c) PCoA analysis of MA and ND groups; d) PCoA analysis of HLP+A, LLP+A, and MA groups; Percentage values ​​in parentheses on the coordinates represent the contribution of principal components to the differences in the constituent samples, different points represent different samples, and points of the same color represent the same group. Results are expressed as mean ± standard deviation, n=3; experimental groups are the same. Figure 12 ; Figure 14 The bacterial taxa showed significant differences between the groups; Note: a represents the ND group vs. the MA group; b represents the MA group vs. the LLP + A group vs. the HLP + A group; n=3; experimental groups were the same. Figure 12 ; Biological Preservation Certificate Lactobacillus plantarum SCS7, Latin name Lactobacillus plantarum It was deposited on January 13, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33379, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, 100101, China. Detailed Implementation

[0018] This invention provides a strain of Lactobacillus plantarum ( Lactobacillus plantarum SCS7, L. plantarum Lactobacillus plantarum SCS7 was deposited at the China General Microbiological Culture Collection Center on January 13, 2025, with accession number CGMCC No. 33379.

[0019] This invention also provides the application of the cell-free extract of *Lactobacillus plantarum* SCS7 described in the above-mentioned technical solution in the preparation of a drug to alleviate aflatoxin B1-induced liver injury. This invention does not impose any special limitations on the dosage form or preparation method of the drug; those skilled in the art can follow conventional methods. In this invention, the cell-free extract of *Lactobacillus plantarum* SCS7 preferably alleviates aflatoxin B1-induced liver injury by regulating mitophagy mediated by the PINK1 / Parkin signaling pathway. In this invention, the cell-free extract of *Lactobacillus plantarum* SCS7 preferably alleviates aflatoxin B1-induced liver injury by regulating the intestinal flora. This invention preferably alleviates aflatoxin B1-induced liver injury by increasing the levels of beneficial bacteria (such as *Oscillatoria* spp.). Oscillospira Akkermania ( ) Akkermansia The abundance of phyla such as Verrucomicrobiota and the reduction of pathogenic bacteria [such as Thomas's spp.] Thomasclavella The abundance of [e.g., Lactobacillus plantarum SCS7] alleviates aflatoxin B1-induced liver damage. In this invention, the cell-free extract of Lactobacillus plantarum SCS7 preferably alleviates aflatoxin B1-induced liver damage by increasing the content of short-chain fatty acids. In this invention, the short-chain fatty acids preferably include one or more of acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and isobutyric acid.

[0020] In this invention, the method for preparing the cell-free extract of Lactobacillus plantarum SCS7 preferably includes the following steps: 1) Mix the Lactobacillus plantarum SCS7 with PBS buffer and sonicate for 30 min under ice bath conditions to obtain the sonicated fragment; The conditions for ultrasonic disruption include: 4℃, 400W, 5s ultrasound, and 10s interval. 2) The ultrasonically fragmented material obtained in step 1) is subjected to an ultrasonic treatment at 4°C and a rotation speed of 10000 r / min. n Centrifuge for 10 min, and pass the supernatant through a 0.22 µm sterile filter membrane. The filtrate is a cell-free extract of Lactobacillus plantarum SCS7.

[0021] In this invention, the protein concentration in the cell-free extract of Lactobacillus plantarum SCS7 is preferably 2 to 3.5 mg / mL.

[0022] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1 1. Experimental Methods 1.1 L. plantarum Preparation of SCS7 cell-free extract L. plantarumSCS7 was isolated and purified from Sichuan fermented sausage by our research group. Under aseptic conditions, 50 μL of the original bacterial culture was inoculated into 100 mL of MRS liquid medium (adjusted to pH approximately 7.4). After inoculation, the culture was placed in a 37℃ incubator for approximately 18 h. The cells were collected by centrifugation (temperature: 4℃, speed: 10000 r / min) for 10 min, resuspended in PBS buffer, and centrifuged again. This process was repeated three times to collect the cells. L. plantarum SCS7 microbial sludge, stored in an ultra-low temperature freezer at -80 ℃ for later use. Take one portion of the dispensed product. L. plantarum After expanding the culture of SCS7 using the above method, the bacterial cells were collected and resuspended in PBS buffer. The cells were then sonicated under ice bath conditions (sonication conditions: 4℃, 400 W, 5 s sonication, 10 s interval) for 30 min, followed by centrifugation for 10 min (temperature: 4℃, speed: 10000 r / min). The supernatant was collected and filtered through a 0.22 μm sterile filter membrane for sterilization. The protein concentration of the cell-free extract was determined using a BCA kit. Finally, the extract was stored at -20℃ for later use.

[0024] 1.2 Cell culture and cell viability assay AML12 cells, normal mouse hepatocytes, were purchased from Wuhan Pronosai Biotechnology Co., Ltd. Cells were grown in DMEM / F12 medium supplemented with 10% fetal bovine serum (FBS), 1% 100 U / mL penicillin and streptomycin, 1% insulin-transferrin-selenium medium supplement (ITS-X), and 40 ng / mL dexamethasone under conditions of 5% carbon dioxide and >75% humidity. Cells were acclimatized for 24 h before any treatment. Additionally, AML12 cell viability was assessed using the Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions. AML12 cells were cultured at 5 × 10⁶ cells / mL. 4 Cells were seeded at a density of [number] cells / mL in 96-well plates and cultured for 24 h. The original culture medium was then removed, and AFB1 (1.25–40 μmol / L) was added for treatment for another 24 h. A similar batch of cells was cultured and treated with AFB1 at a concentration of 0–0.8 mg / mL. L. plantarum Cell viability was observed after 24 h of treatment with SCS7 cell-free extract. Additionally, 0–0.4 mg / mL... L. plantarumCell-free SCS7 extract was used as a pretreatment agent for 3 h, 6 h, 12 h, and 24 h, then aspirated. Cells were then treated with 10 μmol / L AFB1 for 24 h, and changes in cell viability were observed. A blank control group and a control group were set up; the blank control group contained no cells, and the control group was treated with 0.1% DMSO. After the treatment time of the test substance, the culture medium was discarded, and the cells were washed with PBS buffer and discarded. Finally, 100 μL of CCK8 reagent was added to each well, and the cells were incubated in a cell culture incubator for 2 h. The absorbance was then measured at 450 nm using a multi-mode microplate reader.

[0025] 1.3 Cell Experiment Grouping The experiment consisted of five groups: a blank control group (NG); a 0.1% DMSO treatment group (DG); 10 μmol / L AFB1 treatment group (AG); 0.1 mg / mL L. plantarum SCS7 cell-free extract pretreatment group (LG); 0.1 mg / mL L. plantarum After pretreatment with SCS7 cell-free extract, the group was then treated with 10 μmol / L AFB1 (A+LG).

[0026] 1.4 Detection of Reactive Oxygen Species Levels in Cells 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe was used to detect cellular ROS. AML12 cells were grown at a density of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 1 / mL in 15mm glass-bottomed culture dishes. After culture, the original culture medium was discarded, and the cells were washed with PBS buffer. Then, 10 μmol / L DCFH-DA probe was added, and the cells were incubated in a 37℃ cell culture incubator for 20 min. After washing the cells three times with DMEM / F12 medium, the cells were quickly observed and photographed using a laser confocal microscope.

[0027] 1.5 Observation of mitochondrial autophagy under transmission electron microscopy AML12 cells were divided into groups of 2.6 × 10⁻⁶. 6Cells were seeded per well in 60 mm × 12 mm dishes. After culture, cells were digested with 1 mL of trypsin for 3 min, centrifuged at 1000 r / min for 2 min to collect cells, discarded the supernatant, and resuspended in 0.5% glutaraldehyde fixative by slowly adding it along the tube wall. The cells were incubated at 4℃ for 5 min, centrifuged again at 12000 r / min for 10 min, discarded the supernatant, and resuspended in 3% glutaraldehyde fixative (without dispersing the cells). The samples were then refixed with 1% osmium tetroxide. Acetone was used for stepwise dehydration, with the concentration of the dehydrating agent gradually increasing. A gradient mixture of dehydrating agent and epoxy resin embedding medium (Epon-812) was used for infiltration at volume ratios of 3:1, 1:1, and 1:3. After tissue infiltration, the samples were embedded and solidified using a pure resin system. Thin sections with a thickness of 60–90 nm were prepared using ultrathin sectioning techniques and transferred to a copper mesh support. First, uranium acetate was used for staining for 10-15 minutes, followed by lead citrate for counterstaining for about 90 seconds. The entire staining process was carried out at room temperature. Images were acquired using a JEOL JEM-1400 FLASH transmission electron microscope (JEOL Ltd.). The entire sample was first scanned at low magnification, and after selecting the target area, the working magnification was switched to high resolution for image recording, with a focus on observing pathological ultrastructural changes.

[0028] 1.6 Detection of mitochondrial membrane potential and ATP levels using the JC-1 probe AML12 cells were stored at a density of 1.5 × 10⁻⁶. 5 Inoculate at 0.1 mg / mL in 15 mm glass-bottomed culture dishes, using 0.1 mg / mL of [a specific ingredient / component]. L. plantarum After pretreatment with SCS7 cell-free extract for 3 h, cells were treated with 10 μmol / L AFB1 for 24 h. Cells were then incubated with JC-1 at 37°C in the dark for 30 min. Cells were then washed with sterile PBS and immediately observed and imaged under a laser confocal microscope. Excitation / emission wavelengths were set to 490 / 530 nm for JC-1 monomer detection and 525 / 590 nm for JC-1 polymer detection. Results were shown as the red-green fluorescence intensity ratio. ATP changes were analyzed using an adenosine triphosphate (ATP) assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). Cells were incubated at 2.5 × 10⁻⁶ cells / year. 5Cells were evenly seeded per well in a 6-well plate. After treatment, the culture medium was discarded and the cell surface was washed with PBS. Then, 200 μL of lysis buffer was added to each well. After complete lysis, the cells were collected using a cell scraper into 1.5 mL centrifuge tubes and centrifuged at 4°C (12000 r / min, 5 min). The supernatant was then collected for subsequent assays. The specific assay steps are as follows: (1) Preparation for standard curve determination: Thaw the reagents to be used on ice, and prepare a standard series with concentration gradients of 0.01 μmol / L, 0.03 μmol / L, 0.1 μmol / L, 0.3 μmol / L, 1 μmol / L, 3 μmol / L and 10 μmol / L using ATP dilution buffer.

[0029] (2) Preparation of ATP detection working solution: Mix the ATP detection reagent and ATP detection reagent diluent according to... Dilute the solution at a ratio of 1:4 and store at 4°C. Add 100 μL of ATP detection working solution to each well of a black 96-well plate, and let it stand at room temperature for 3–5 minutes before adding the sample.

[0030] (3) ATP concentration detection: Add 20 μL of sample or standard to each well and mix quickly with a pipette tip. After at least 2 seconds, the RUL value was measured using a chemiluminescence analyzer (Luminometer). (4) Protein concentration determination: The protein concentration in the sample was determined by the BCA method, and the ATP concentration was converted into nmol / mg protein to eliminate the influence of protein quantity.

[0031] 1.7 Real-time quantitative PCR detection of mitochondrial autophagy protein gene expression levels AML12 cells were loaded at 2.5 × 10⁻⁶. 5 Individual cells / well were evenly seeded in a 6-well plate using 0.1 mg / mL of [a specific antibiotic / method]. L. plantarumAfter pretreatment with SCS7 cell-free extract for 3 h, cells were then treated with 10 μmol / L AFB1. Following treatment, cells were washed once with PBS buffer and collected according to the manufacturer's instructions. Total RNA was extracted using a cell RNA isolation kit. RNA was reverse transcribed into cDNA using a first-strand complementary DNA (cDNA) synthesis kit. Using cDNA as a template, a reaction system was constructed by mixing specific amplification primers, deionized water, and 2×SYBR Green PCR Master Mix reagent in optimized proportions. qPCR was used to amplify the target fragment. The 2-ΔΔCT relative quantification algorithm was employed, with the internal reference gene (β-actin) used as an endogenous reference to standardize and correct Ct values, analyzing the relative changes in target gene expression. Primer sequences are shown in Table 1.

[0032] Table 1 Primer Sequences 1.8 Protein Immunoblotting Detection of Mitochondrial Autophagy Protein Expression in Cells AML12 cells were divided into 2.6 × 10⁻⁶ cells. 6 Cells were seeded per well in 60 mm × 12 mm dishes and cultured for 24 h. After treatment with the test substance, 1 mL of trypsin was added for digestion for 3 min. Cells were collected and centrifuged at 1200 r / min for 8 min. The supernatant was discarded, and the cell pellet was collected. The cells were resuspended in 2 mL of PBS and centrifuged again to collect the cell pellet. A mixture of RIPA lysis buffer containing 1% PMSF and a cocktail was added, and the mixture was lysed on ice for 30 min, vortexing every 10 min. After lysis, the supernatant was collected after centrifugation at 18000 r / min for 20 min.

[0033] Protein concentration was determined using the BCA method. The prepared BCA working solution (BCA:Cu = 50:1) was thoroughly mixed. Both the standard and sample were appropriately diluted with PBS buffer. The standard solution and sample were added to each well of a 96-well plate, followed by 200 μL of BCA working solution. The plate was then incubated in the dark at a constant temperature for 15–30 min. The absorbance of each well was measured at 562 nm using a multi-mode microplate reader. The extracted total cellular protein was diluted with 5×SDS-PAGE loading buffer at a volume ratio of buffer:protein = 1:4. After vortexing, the sample was heated at 100℃ for 5 min, cooled, and stored at -20℃ for later use.

[0034] After preparing separating gels with concentrations of 10% and 12% according to the proportions in the instructions, vortex and mix well, then slowly and evenly add them to the assembled glass plates until they reach two-thirds of the height. Next, add the previously prepared stacking gel to the top of the glass plates, immediately insert the gel comb, and wait for 20 - 30 min until the gel is completely solidified, thus completing the gel preparation. Correctly insert the gel plate into the electrophoresis tank and fill the inner tank with electrophoresis buffer. Subsequently, carefully remove the gel comb and start adding samples and markers into the wells. After loading the samples, first perform electrophoresis at a constant voltage of 80 V for 30 - 60 min until the standard protein bands are clearly separated, and then adjust the voltage to 120 V until the samples approach the bottom of the gel to end the electrophoresis. Perform membrane transfer in the pre-cooled electrotransfer buffer. Activate the cut PVDF membrane in methanol for 1 min, assemble the sandwich device in the order of sponge pad, filter paper, gel, PVDF membrane, filter paper, and sponge pad, and expel the air bubbles in the structure. Then, correctly place the sandwich into the electrotransfer tank, place ice bricks around it, and replenish the electrotransfer buffer to perform membrane transfer. The electrotransfer conditions are 200 mA and 60 min. After the electrotransfer is completed, immediately transfer the PVDF membrane to the TBST buffer containing 5% skim milk and immerse the PVDF membrane completely in the liquid. Under room temperature conditions, place it on a shaker and slowly shake for 2 h for blocking treatment. After blocking, wash the PVDF membrane three times with 1×TBST buffer. Dilute the antibody to an appropriate ratio according to the instructions, and incubate the bands in the primary antibody at 4℃ overnight. The next day, wash the bands 3 times with TBST buffer, and then incubate the bands in the secondary antibody at room temperature for 1 h. After incubation, wash the bands 3 times again with TBST buffer. Mix the A and B solutions of the ECL developing solution in a volume ratio of 1:1, and evenly dip the bands in the developing solution, then perform exposure and development under darkroom conditions. Read the obtained image through Image J software and analyze the gray values of the protein bands, and perform normalization with the internal reference protein and the control group to calculate the relative expression of each group of proteins.

[0035] 1.9 Animal experiment design The experimental subjects were SPF-grade male KM mice, weighing 20 - 25 g and 5 weeks old. They were purchased from Beijing Spey Foster Biotechnology Co., Ltd., and the license number was SCXK-(Beijing)2019-0010. The maintenance feed and bedding for the experimental animals were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. All animal experiment protocols have been approved by the Experimental Animal Welfare and Ethics Committee of Chengdu University of Traditional Chinese Medicine (Ethical number: 2024162).

[0036] Feeding conditions: The mice were housed in the experimental animal room of the School of Pharmacy, Chengdu University of Traditional Chinese Medicine, and the license number was SYXK-(Chuan) 2020-124, at room temperature of 20-25 °C, relative humidity of 50-60%, maintaining a 12 h light and 12 h dark cycle, with free access to food and water. Adaptively fed for 1 week before the experiment.

[0037] After adaptively feeding 60 male KM mice at 5 weeks old with a body weight of 20-25 g for 1 week, they were randomly divided into 6 groups according to body weight, with 10 mice in each group. NP group: 0.2 mL / mouse of sterile PBS buffer, ND group: the same dose of 0.1% DMSO, MA group: 300 μg AFB1 / kg (bw), LLP+A group: 1 mg / mL L. plantarum SCS7 cell-free extract + 300 μg AFB1 / kg (bw), HLP+A group: 3 mg / mL L. plantarum SCS7 cell-free extract + 300 μg AFB1 / kg (bw), HLP group: 3 mg / mL L. plantarum SCS7 cell-free extract. The experiment lasted for 28 days. For the first 14 days, intragastric administration of L. plantarum SCS7 cell-free extract was used as pretreatment, and for the next 14 days, intragastric administration of AFB1 was used for modeling. When no modeling was performed, normal saline was used for intragastric administration. At the same time, the basic signs of the mice such as body weight, food intake, and water intake were observed and recorded daily.

[0038] 1.10 Detection of liver coefficient After the experiment ended, the liver tissue samples were quickly dissected and separated. After rinsing with pre-cooled PBS and blotting the surface moisture with filter paper, they were weighed on a precision electronic balance and the data was recorded. The calculation formula is: liver coefficient = liver weight (g) / mouse body weight (g) × 100%.

[0039] 1.11 Histopathological examination of liver tissue Fresh liver tissue specimens were immediately immersed in 4% paraformaldehyde fixative and continuously fixed at room temperature for 24- 48 h. Through the gradient ethanol dehydration procedure, after being made transparent through xylene medium, the tissues were embedded in paraffin and after trimming, the tissue blocks were sectioned by a microtome. The sections were stained with hematoxylin-eosin dye, dehydrated with gradient ethanol and then sealed. The pathological and tissue structure changes were observed under an optical microscope, and images were collected and the pathological changes were described.

[0040] 1.12 Detection of serum biochemical indexes and ROS Mice were fasted for 12 hours before blood collection, but water was allowed. Blood from the mouse eyeballs was collected in 1.5 mL centrifuge tubes, placed in a 4°C refrigerator for 2 hours, and then centrifuged (2500 r / min, 4°C) for 10 min. The supernatant was collected in 200 μL centrifuge tubes and stored at -80°C for later use. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) activities were measured using the microplate method, strictly following the kit instructions. An appropriate amount of liver tissue was taken, washed with physiological saline to remove surface blood, and then sonicated. The supernatant was used to detect the ROS content in the tissue, following the kit instructions.

[0041] 1.13 Detection of mitochondrial autophagy protein expression in liver tissue using protein immunoblotting Mouse liver tissue was removed from a -80°C freezer. 20 mg of tissue was weighed and placed in a 1.5 mL centrifuge tube, then minced. The tissue was washed three times with physiological saline, centrifuged at 2000 rpm for 5 min, and the supernatant was discarded to remove blood. A mixture of RIPA lysis buffer containing 1% PMSF and cocktail was added, and the mixture was lysed on ice for 30 min, vortexing every 10 min. The tissue was then ground completely in a pre-chilled grinder. Finally, the tissue was centrifuged at 18000 rpm for 20 min at 4°C, and the supernatant was collected into a 200 μL centrifuge tube for later use. Subsequent procedures were the same as described in section 1.8.

[0042] 1.14 Sequencing of 16SRNA and Detection of Short-Chain Fatty Acids in Mouse Cecal Contents Cecal contents were collected, aseptically transferred to cryovials, and flash-frozen in liquid nitrogen, then stored at -80°C for later use. Total fecal DNA extraction, 16S rRNA gene sequencing analysis, and short-chain fatty acid detection services were provided by Suzhou Panomick Biomedical Technology Co., Ltd. (1) DNA preparation: Fecal samples pre-cooled by liquid nitrogen were used to prepare OMEGA Soil DNA (M5635- After extracting whole-genome DNA using the Omega Bio-Tek (USA) kit, nucleic acid purity was measured using a full-wavelength spectrophotometer (NanoDrop 2000, Thermo Scientific), and DNA integrity was assessed by agarose gel electrophoresis. (2) PCR amplification of the target fragment: In this experiment, specific primer pairs were designed for the hypervariable region of the V3-V4 region of the prokaryotic 16S rRNA gene: forward primer 338F (SEQ ID No. 13: 5'-barcode+ACTCCTACGGGAGGCAGCA-3') and reverse primer 806R (SEQ ID No. 14: 5'-GGACTACHVGGGTWTCTAAT-3'). PCR amplification was then performed.

[0043] (3) Amplicon purification and recovery: Take 25 μL of the amplification system and mix it with 0.8 times the volume of magnetic beads (Vazyme VAHTS™ DNAClean Beads) by vortexing. After incubating on a magnetic rack for 5 min, discard the supernatant. Wash with 0.8 times the magnetic bead washing buffer and 80% ethanol to remove impurities. After standing at room temperature for 5 min, add 25 μL of Elution Buffer to elute. After 5 min, aspirate the supernatant into a 1.5 mL centrifuge tube for later use. (4) Illumina sequencing library construction: Library preparation was performed using the TruSeq Nano DNA LT Library Prep Kit (Illumina). First, end-repair reactions were performed on the PCR amplification products: using End... The Repair Mix 2 enzyme mixture modifies DNA ends, repairing 5' protrusions while simultaneously completing 3' end augmentation and introducing phosphate groups. Adding an A base to the 3' end of the DNA strand effectively prevents self-ligation. A sequencing aptamer containing a specific index sequence is introduced at the 5' end via enzymatic ligation, a design that allows the DNA fragment to be effectively anchored to the flow cell. During the experiment, a BECKMAN AMPure XP magnetic bead purification system was used to screen fragments and remove self-ligation products, ensuring the purity of the effective ligation products. After PCR amplification to enhance library concentration, magnetic bead screening was used again to remove amplification byproducts. Finally, 2% agarose gel electrophoresis was used to precisely screen the library for fragment ranges, obtaining standardized libraries that meet the requirements of the sequencing platform.

[0044] (5) High-throughput sequencing: Paired-end sequencing of 2×250 bp was performed on an Illumina NovaSeq machine using the NovaSeq 6000SP Reagent Kit (500 cycles). The libraries to be sequenced were serially diluted to 2 nmol / L, and the mixing was performed according to the required data volume ratio. Finally, the mixed libraries were denatured into single strands using 0.1 mol / L NaOH before sequencing.

[0045] Meanwhile, take an appropriate amount of fecal sample into a 1.5 mL centrifuge tube, add 500 μL of ultrapure water and 100 mg of sterile glass beads, homogenize for 1 min, centrifuge at 12000 r / min at 4℃ for 10 min, take 200 μL of supernatant, add 100 μL of 15% phosphoric acid, then add 20 μL of 4-methylvaleric acid solution and 280 μL of diethyl ether, homogenize again for 1 min, centrifuge at 12000 r / min at 4℃ for 10 min, and take the supernatant for instrument testing.

[0046] 1.15 Statistical Analysis Results are expressed as mean ± standard deviation ( ). Data are expressed in the form of ±s, and data processing and statistical analysis are performed using IBM SPSS Statistics 25.0. Normality and homogeneity of variance tests are conducted, and analysis of variance (ANOVA) is used to evaluate the significance of each parameter. Tukey's method is used for multiple comparisons. P A value <0.05 indicates statistical significance. After statistical analysis, the data were plotted using GraphPad Prism 8.0 software.

[0047] 2. Experimental Results 2.1 Effects of Lactobacillus plantarum SCS7 cell-free extract and AFB1 on AML12 cell viability The results are as follows Figure 1 As shown in (a), AFB1 exhibits significant and dose-dependent cytotoxicity. When the AFB1 concentration was 10 μmol / L, the viability of AML12 cells decreased to 68.4 ± 2.66%. To ensure the accuracy of subsequent experimental results, a treatment concentration of 10 μmol / L AFB1 was selected. Meanwhile, as... Figure 1 As shown in (b), 0–0.4 mg / mL L. plantarum SCS7 cell-free extract showed no significant toxicity to AML12 cells, while 0.8 mg / mL L. plantarum SCS7 cell-free extract significantly inhibited AML12 cell viability ( P <0.05). Therefore, a dose of 0–0.4 mg / mL will be selected subsequently. L. plantarum SCS7 cell-free extract was further screened for pretreatment time and concentration. Additionally, as shown in Table 2, AML12 cell viability increased with... L. plantarum The concentration of SCS7 cell-free extract pretreatment showed a trend of first increasing and then decreasing. At pretreatment times of 3 h and 6 h, compared with the control group treated with 10 μmol / L AFB1 alone, the concentration of pretreatment at these concentrations was significantly lower. L. plantarum SCS7 cell viability significantly increased when the concentration of cell-free extract was 0.1 mg / mL.P <0.05), which were 87.67±6.57% and 86.84±4.06%, respectively. Therefore, subsequent experiments used 0.1 mg / mL as the concentration. L. plantarum The concentration of SCS7 cell-free extract pretreatment and the pretreatment time were 3 h.

[0048] Table 2 L. plantarum Effects of SCS7 cell-free extract pretreatment time and dosage on AML12 cell viability Note: Results are expressed as mean ± standard deviation, n≥3. * indicates that the difference between each group and group 0 is statistically significant. P <0.05); # indicates that the difference between each group and the 10 μmol / L AFB1 control group is statistically significant. P <0.05) 2.2 Effects of cell-free extract of Lactobacillus plantarum SCS7 and AFB1 on ROS levels in AML12 cells like Figure 2 As shown in (a), compared with the control group, ROS fluorescence in AML12 cells was enhanced under AFB1 induction; compared with the AFB1-only treatment group, L. plantarum ROS fluorescence was attenuated in AML12 cells pretreated with SCS7 cell-free extract. Simultaneously, the fluorescence intensity of ROS was quantified using ImageJ, and the results are as follows: Figure 2 As shown in (b), compared with the control group, 10 μmol / L AFB1 induced a significant increase in ROS levels in AML12 cells. P <0.05), fluorescence intensity was 163.96 ± 4.88; while at 0.1 mg / mL L. plantarum Pretreatment with SCS7 cell-free extract for 3 h significantly reduced cell ROS levels. P <0.05), fluorescence intensity was 98.75 ± 6.32, indicating L. plantarum SCS7 cell-free extract can reduce the level of ROS produced by cells under AFB1 stimulation.

[0049] 2.3 Effects of cell-free extracts of Lactobacillus plantarum SCS7 and AFB1 on mitochondrial function in AML12 cells The results are as follows Figure 3 As shown, compared with the control group, AML12 cells in the AFB1-treated group exhibited significant mitochondrial swelling, cristae breakage and dissolution, and a greater number of lipid droplets, autophagy, and mitophagy were observed in the cytoplasm. Compared with the AFB1-treated group, it can be found that... L. plantarumIn the SCS7 cell-free extract pretreatment group, mitochondrial morphology was relatively intact, and autophagy in AML12 cells and mitophagy were alleviated. Additionally, as... Figure 4 As shown in Figures (ab), green fluorescence in AML12 cells was significantly enhanced under AFB1 stimulation; however, after pretreatment with cell-free extracts, green fluorescence decreased while red fluorescence relatively increased. After quantifying and normalizing the fluorescence intensity, compared with the control group, the AFB1-treated group and... L. plantarum The mitochondrial membrane potential (MMP) value of the SCS7 cell-free extract pretreatment group decreased significantly. P The value <0.05 indicates that AML12 cells experienced mitochondrial damage under AFB1 stimulation, leading to increased mitochondrial membrane permeability, altered potential difference across the mitochondria, and consequently, a decrease in mitochondrial membrane potential. Compared to the AFB1-treated group, L. plantarum The ΔΨm value of the SCS7 cell-free extract pretreatment group increased significantly. L. plantarum Pretreatment with cell-free SCS7 extract can reduce mitochondrial membrane permeability increased by AFB1. Meanwhile, as... Figure 4 As shown in (c), the changes in ATP follow a trend consistent with the changes in mitochondrial membrane potential, indicating that at 0.1 mg / mL... L. plantarum Pretreatment with SCS7 cell-free extract for 3 h alleviated AFB1-induced mitochondrial damage in AML12 cells to some extent.

[0050] 2.4 Effects of cell-free extracts of Lactobacillus plantarum SCS7 and AFB1 on the expression of mitochondrial autophagy-related genes in AML12 cells like Figure 5 As shown in (ae), the expression level of the p62 gene in AML12 cells was significantly reduced under treatment with 10 μmol / L AFB1. P <0.05), while the expression levels of PINK1, Parkin, PGAM5, and LC3 genes were significantly increased ( P <0.05). In L. plantarum Pretreatment with SCS7 cell-free extract for 3 h reversed the effect of AFB1 on mitophagy-related genes, specifically, the expression level of the P62 gene was significantly increased compared to the AFB1-treated group. P <0.05), the expression levels of PINK1, Parkin, PGAM5, and LC3 genes decreased significantly ( P <0.05).

[0051] 2.5 Effects of Lactobacillus plantarum SCS7 cell-free extract and AFB1 on the expression of mitochondrial autophagy-related proteins in AML12 cells like Figure 6As shown in the figure (ag), compared with the control group, the relative expression levels of PINK1, Parkin, PGAM5, and LC3 proteins were significantly increased under AFB1 treatment, while the expression level of P62 was significantly decreased. P <0.05), indicating that AFB1 can activate PINK1 / Parkin-mediated mitophagy. Compared with the AFB1-treated group, the protein expression levels of PINK1, Parkin, PGAM5, and LC3 were significantly decreased, while the protein expression level of P62 was relatively low. L. plantarum The SCS7 cell-free extract pretreatment group significantly increased ( P <0.05). The expression trends of mitophagy-related proteins were consistent with those of gene expression. These results suggest that... L. plantarum Pretreatment with SCS7 cell-free extract can inhibit AFB1-induced excessive activation of mitophagy, thereby protecting AML12 cells.

[0052] 2.6 Effects of cell-free extract of Lactobacillus plantarum SCS7 and AFB1 on liver coefficient like Figure 7 As shown, compared with the control group, the liver coefficient of the AFB1 treatment group was significantly increased ( P <0.05, indicating that the mouse liver was damaged, congested, and enlarged under AFB1 stimulation. Compared with the AFB1-treated group, the liver coefficient of the cell-free extract pretreatment group was significantly reduced ( P <0.05), at different concentrations L. plantarum Pretreatment with SCS7 cell-free extract alleviated AFB1-induced liver damage in mice to some extent, indicating that the cell-free extract can inhibit AFB1-induced liver damage and protect the liver in mice.

[0053] 2.7 Effects of Lactobacillus plantarum SCS7 cell-free extract and AFB1 on mouse liver microstructure like Figure 8 As shown, compared with the control group, the liver tissue pathological sections of mice in the AFB1-treated group showed localized, single-point necrosis of hepatocytes. Single or multiple hepatocytes exhibited blurred structure, pyknosis, fragmentation, and dissolution of the nuclei. Inflammatory cell infiltration, predominantly rod-shaped neutrophils, was observed in the necrotic areas and surrounding sinusoids. Compared with the AFB1-treated group, it was found that after treatment with different concentrations of... L. plantarum Pretreatment with SCS7 cell-free extract reduced AFB1-induced hepatocyte inflammatory infiltration and decreased liver inflammation.

[0054] 2.8 Effects of cell-free extract of Lactobacillus plantarum SCS7 and AFB1 on serum biochemical parameters in mice ALT, AST, and ALP are important biochemical indicators reflecting the degree of liver damage. Figure 9As shown in Figure (ac), compared with the control group, the serum ALT, AST, and ALP enzyme activities in the AFB1-treated group were significantly increased. P <0.05%, AFB1 stimulation caused liver damage in mice. Compared with the AFB1-treated group, the serum ALT, AST, and ALP enzyme activities in the cell-free extract pretreatment group all showed a decreasing trend, but the ALT and AST enzyme activities in the low-dose cell-free extract pretreatment group were significantly lower than those in the AFB1-treated group. P <0.05%, AST enzyme activity was significantly lower in the high-dose cell-free extract pretreatment group than in the AFB1 treatment group ( P <0.05). The above results indicate that... L. plantarum Pretreatment of mice with SCS7 cell-free extract can effectively prevent AFB1 liver damage.

[0055] 2.9 Effects of Lactobacillus plantarum SCS7 cell-free extract and AFB1 on ROS levels in mouse liver like Figure 10 As shown, compared with the control group, the ROS content in the liver of mice treated with AFB1 was significantly increased, and AFB1 induced oxidative damage in the liver of mice. Compared with the AFB1-treated group, the ROS content in the liver of mice pretreated with different concentrations of cell-free extract was significantly reduced. P <0.05). The above results indicate that... L. plantarum Pretreatment with SCS7 cell-free extract effectively inhibited oxidative damage caused by AFB1.

[0056] 2.10 Effects of cell-free extracts of Lactobacillus plantarum SCS7 and AFB1 on the expression of mitochondrial autophagy-related proteins in mouse liver like Figure 11 In the AFB1-treated group (ag), compared with the control group, the relative expression levels of PINK1, Parkin, PGAM5, and LC3 proteins were significantly increased, while the relative expression level of P62 protein was significantly decreased. P <0.05 indicates that the PINK1 / Parkin-mediated mitophagy pathway is activated by AFB1. Compared with the AFB1-treated group, the cell-free extract pretreatment group effectively reversed the trend of mitophagy-related proteins in the AFB1-treated group ( P <0.05), bringing it closer to normal values, and this result is consistent with the results for AML12 cells, indicating that... L. plantarum SCS7 cell-free extract has a certain protective effect against AFB1-induced liver damage in mice.

[0057] 2.11 Effects of Lactobacillus plantarum SCS7 cell-free extract and AFB1 on mouse gut microbiota like Figure 12The study analyzed the changes in the species composition of the top 10 most abundant species in the gut microbiota at both the genus and phylum levels. At the genus level, the most abundant genus was Porphyromonas. Porphyromonas ), genus *Hengartella* Hungalella ), Akkermansia spp. Akkermansia ) and Clostridium ( Faecallbaculum Compared with the control group, the AFB1 treatment group had significantly higher levels of *Thomas* spp. (…). Thomasclavelia The abundance of AFB1 was significantly increased, suggesting that AFB1 may disrupt the balance of the gut microbiota in mice by increasing the abundance of *Thomas*. The HLP+A and LLP+A groups were... L. plantarum Pretreatment with SCS7 cell-free extract significantly increased the abundance of Akkermansia compared to the AFB1 treatment group, indicating that... L. plantarum The intervention effect of cell-free extracts from SCS7 may be achieved by increasing the abundance of Akkermansia. Additionally, such as Figure 12 As shown in (c), at the phylum level, compared with the control group, the ratio of Firmicutes to Bacteroidetes in the AFB1-treated group showed an increasing trend; compared with the AFB1-treated group, the ratios of LLP+A and HLP+A were significantly decreased. P <0.05), indicating that in L. plantarum SCS7 cell-free extract can maintain gut microbiota homeostasis in mice by regulating the ratio of Firmicutes to Bacteroidetes. Meanwhile, Verrucomicrobiota are beneficial gut bacteria, such as... Figure 12 As shown in (d), compared to the AFB1 treatment group, L. plantarum The SCS7 cell-free extract pretreatment group showed an increasing trend, and the low-dose cell-free extract pretreatment group showed a significant increase. P The value <0.05 indicates that the cell-free extract may reduce the damage caused by AFB1 by increasing gut probiotics such as Verrucous microbes.

[0058] Diversity assessment was performed on the sequencing data, and the results were primarily represented by alpha diversity and beta diversity indices. Alpha diversity represents the species diversity present in the community. The Chao1 index reveals community richness, i.e., the estimated number of species / traits per sample. Figure 13 As shown in (a), compared with the control group, the Chao1 value of the AFB1-treated group showed a decreasing trend; compared with the AFB1-treated group, the Chao1 value of the pretreated groups with different concentrations of cell-free extract all showed an increasing trend. Figure 13 As shown in (b), the Shannon value represents diversity, taking into account both the abundance and evenness of existing species, and its trend is consistent with that of the Chao1 value. Additionally, as... Figure 13As shown in (cd), PCoA analysis revealed that the gut microbiota structure of the AFB1-treated group was significantly different from that of the control group and... L. plantarum SCS7 cell-free extract pretreatment group. This indicates that AFB1 exposure leads to alterations in the richness and diversity of the mouse gut microbiota, while L. plantarum Pretreatment of the cell-free extract of SCS7 can reverse this change.

[0059] Linear discriminant analysis (LDA) effect size (LEfSe) was used to analyze the differences in community composition between different groups, with the LDA set to 2. For example... Figure 14 LEfSe analysis in (a) showed that, compared with the control group, the abundance of two genera of AFB1 was significantly different. *Hassaviridae* ( Hathewaya The significant increase in AFB1 suggests that its damaging effects on the body may be mediated through increased abundance of *Haemaphysalis*. The bacteria showing significant differences in the control group were *Verrucomicrobiota*, comprising *Verrucomicrobiae* and *Verrucomicrobiales*. These bacteria are beneficial gut bacteria in healthy individuals, maintaining gut microbiota homeostasis. Figure 14 As shown in (b), the abundance of three bacterial genera differed significantly in the AFB1 treatment group, namely, the genus *Alkaliophilus* (…). Alkaliphilus ), Desulfurosporium ( Desulfosporosinus ), Anaerobic myobacteria ( Anaerotruncus Desulfospora, belonging to the family Desulfitobacteriaceae, is a harmful intestinal bacterium. The damaging effects of AFB1 may be achieved through a significant increase in Desulfospora. Anaerotruncus It is also considered a harmful gut bacterium, playing a key role in the progression of inflammation, obesity, and metabolic diseases. Notably, in the low-dose cell-free extract pretreatment group, *Vibrio spp.* (…) Oscillospira (This is a dominant bacterial species.) Studies have shown that... Oscillospira These are beneficial gut bacteria that produce short-chain fatty acids such as butyrate. These substances offer various health benefits to the host, including providing energy, maintaining intestinal barrier function, and regulating immune responses. Low doses L. plantarum SCS7 cell-free extract may reduce AFB1-induced damage by increasing gut vibrio spp.

[0060] Meanwhile, as shown in Table 3, compared with the control group, the total content of short-chain fatty acids (SCFAs) in the AFB1-treated group was significantly reduced, indicating that AFB1 disrupts the balance of short-chain fatty acids in the intestine. Compared with the AFB1-treated group, different concentrations of... L. plantarumThe total SCFA content in the SCS7 cell-free extract pretreatment group showed an increasing trend. Simultaneously, the trends of individual fatty acids were consistent with the trend of the total SCFA content, indicating that low and high doses... L. plantarum Pretreatment of SCS7 cell-free extract helps regulate the total amount of short-chain fatty acids inhibited by AFB1.

[0061] Table 3 Short-chain fatty acid content Note: Results are expressed as mean ± standard deviation, n=3. * indicates that the difference between the data groups is statistically significant compared with the ND group. P <0.05); ND: 0.1% DMSO treatment group, MA: 300 μg / kg(bw) AFB1 treatment group, LLP+A: 1 mg / mL L. plantarum After pretreatment with SCS7 cell-free extract, the group was then treated with 300 μg / kg (bw) AFB1, HLP+A: 3 mg / mL. L. plantarum After pretreatment with SCS7 cell-free extract, the group was then treated with 300 μg / kg(bw) AFB1.

[0062] 3. Conclusion Cell-free extract of *Lactobacillus plantarum* SCS7 inhibited AFB1-induced mitophagy by upregulating p62 and downregulating the mRNA transcription and protein expression levels of PINK1, Parkin, PGAM5, and LC3, thus improving oxidative damage and mitochondrial dysfunction in AML12 cells. ROS levels in mouse liver tissue and the activities of enzymes such as ALT and AST in serum were also significantly reduced. P <0.05). Simultaneously, it improved intestinal microecological dysbiosis in mice by increasing the abundance of beneficial bacteria such as Akkermansia spp., inhibiting the proliferation of harmful bacteria such as Thomas A. spp., and increasing the content of short-chain fatty acids. These results indicate that the cell-free extract of *Lactobacillus plantarum* SCS7 can improve AFB1-induced liver injury by maintaining gut-hepatic axis homeostasis.

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Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum ( Lactobacillus plantarum Lactobacillus plantarum SCS7 was deposited on January 13, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33379.

2. The use of the cell-free extract of Lactobacillus plantarum SCS7 as described in claim 1 in the preparation of a drug to alleviate aflatoxin B1-induced liver injury.

3. The application according to claim 2, characterized in that, The cell-free extract of *Lactobacillus plantarum* SCS7 alleviates aflatoxin B1-induced liver injury by regulating mitophagy mediated by the PINK1 / Parkin signaling pathway.

4. The application according to claim 2, characterized in that, The cell-free extract of *Lactobacillus plantarum* SCS7 alleviates aflatoxin B1-induced liver damage by regulating the intestinal flora.

5. The application according to claim 2, characterized in that, Aflatoxin B1-induced liver damage can be alleviated by increasing the abundance of beneficial bacteria and decreasing the abundance of pathogenic bacteria. The beneficial bacteria include *Vibrio spp.* Oscillospira Akkermania ( ) Akkermansia Phylum Verrucomicrobiota, etc. The pathogenic bacteria include *Thorax* spp. ( Thomasclavella ).

6. The application according to claim 2, characterized in that, The cell-free extract of *Lactobacillus plantarum* SCS7 alleviates aflatoxin B1-induced liver damage by increasing the content of short-chain fatty acids.

7. The application according to claim 2, characterized in that, The short-chain fatty acids include one or more of acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and isobutyric acid.

8. The application according to claim 2, characterized in that, The method for preparing the cell-free extract of Lactobacillus plantarum SCS7 includes the following steps: 1) Mix the Lactobacillus plantarum SCS7 with PBS buffer and sonicate for 30 min under ice bath conditions to obtain the sonicated fragment; The conditions for ultrasonic fragmentation include: 4℃, 400 W, 5 s ultrasound, and 10 s interval. 2) The ultrasonically broken material obtained in step 1) is centrifuged at 4°C and 10,000 r / min for 10 min. The resulting supernatant is passed through a 0.22 µm sterile filter membrane. The filtrate is a cell-free extract of Lactobacillus plantarum SCS7.

9. The application according to claim 2 or 8, characterized in that, The concentration of protein in the cell-free extract of *Lactobacillus plantarum* SCS7 is 2–3.5 mg / mL.

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