A strain of Lactobacillus plantarum with antioxidant activity and its application in the preparation of ferroptosis inhibitors

Abstract

The present invention belongs to the field of biomedicine technology, and in particular to a strain of Lactobacillus plantarum with antioxidant properties and its application in the preparation of ferroptosis inhibitors. The Lactobacillus plantarum LP35 strain has been deposited in the China Center for Type Culture Collection on January 10, 2025, and its deposit number is CCTCC NO: M 2025078; the deposit address is Wuhan University. The strain has the effects of anti-lipid peroxidation, anti-inflammatory, and regulating iron metabolism. The strain and its exosomes can produce a significant inhibitory effect on ferroptosis, and can be used to prepare ferroptosis inhibitors or drugs for treating ferroptosis-related diseases. It has a wide range of application prospects in the prevention and treatment of ferroptosis-related diseases, and provides a new and natural treatment method for the prevention and treatment of ferroptosis-related diseases, which has important clinical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a strain of Lactobacillus plantarum with antioxidant properties and application thereof in the preparation of a ferroptosis inhibitor. Background Art

[0002] Ferroptosis is a form of programmed cell death caused by iron accumulation, characterized by excessive accumulation of ferrous ions in cells, leading to lipid peroxidation, which in turn causes cell membrane damage and cell death. Ferroptosis plays a key role in many diseases, especially in neurodegenerative diseases (such as Alzheimer's disease and Parkinson's disease), cardiovascular diseases (such as myocardial ischemia-reperfusion injury), and metabolic diseases (such as fatty liver and diabetes). The imbalance of iron metabolism and the occurrence of ferroptosis are closely related to disease progression.

[0003] Existing ferroptosis inhibitors are mostly small molecule compounds or specific protein substances, such as Fer-1. However, these inhibitors often have side effects. Therefore, the development of new, natural, safe and reliable ferroptosis inhibitors has become a research hotspot. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a strain of Lactobacillus plantarum with antioxidant properties and its application in the preparation of ferroptosis inhibitors. The Lactobacillus plantarum LP35 strain has the effects of anti-lipid peroxidation, anti-inflammatory, and regulating iron metabolism. The strain and its exosomes can effectively inhibit ferroptosis and can be used to prepare ferroptosis inhibitors and medicines for treating ferroptosis-related diseases.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a strain of Lactobacillus plantarum LP35 having antioxidant properties, which is classified as Lactiplantibacillus plantarum LP35 was deposited in the China Center for Type Culture Collection on January 10, 2025, and its deposit number is CCTCC NO: M 2025078; the deposit address is Wuhan University.

[0007] The Lactobacillus plantarum LP35 strain provided by the present invention is obtained by separation and screening from traditional yogurt in the Altay region. Studies have shown that the Lactobacillus plantarum LP35 and its exosomes have the potential to inhibit ferroptosis.

[0008] Ferroptosis is a type of programmed cell death that depends on iron and reactive oxygen species (ROS). Its occurrence is mainly related to abnormal iron metabolism, lipid peroxidation and dysregulation of the antioxidant defense system.

[0009] Glutathione peroxidase 4 (GPX4) is the only enzyme in the cell that can reduce lipid peroxides. It can reduce the peroxy bond (PLOOH) in lipid peroxides to hydroxyl (PLOH) to prevent the accumulation of peroxides, which is crucial for inhibiting ferroptosis. The activity of GPX4 depends on a cofactor with strong reducing properties - GSH (glutathione). The present invention has found through experimental research that Lactobacillus plantarum LP35 can increase the GSH level of body tissues, prevent and improve the decrease of GSH caused by increased iron content, and thus help reduce the accumulation of lipid peroxides, thereby helping to inhibit ferroptosis and improve the symptoms caused by ferroptosis.

[0010] During ferroptosis, lipid peroxidation in cells will lead to the generation of malondialdehyde (MDA). MDA is an important product of lipid peroxidation. Its accumulation in cells is an important sign of ferroptosis, and its content can evaluate the degree of ferroptosis. At the same time, the accumulation of MDA further aggravates cell damage and death. Some studies have found that by inhibiting lipid peroxidation or removing MDA, the degree of ferroptosis can be reduced and cells can be protected from damage. The present invention has found through experimental studies that Lactobacillus plantarum LP35 can reduce the MDA content of body tissues, prevent and improve the increase of MDA content caused by increased iron content, indicating that Lactobacillus plantarum LP35 can delay the process of ferroptosis and reduce cell damage.

[0011] In addition, ferroptosis can induce inflammation and further damage tissues. At the same time, inflammation can promote ferroptosis by affecting iron metabolism and antioxidant capacity. There is a complex causal relationship between ferroptosis and inflammation. Studies have shown that ferroptosis occurs during the development of intestinal inflammation. The present invention has found through animal experiments that Lactobacillus plantarum LP35 can reduce the levels of proinflammatory factors and inflammation-related enzymes, thereby helping to prevent and alleviate the inflammatory response caused by ferroptosis and improve the symptoms of related diseases.

[0012] However, although inhibiting lipid peroxidation or inflammatory response alone may partially inhibit ferroptosis, comprehensive inhibition of ferroptosis usually requires multi-target joint intervention, including the synergistic effects of regulating iron metabolism, inhibiting lipid peroxidation, and reducing inflammation. The specific reasons are: although inhibiting the production of lipid peroxides can help directly prevent the occurrence of ferroptosis, if the level of ferrous ions is too high or other oxidative stress persists, then inhibiting lipid peroxidation alone may not be enough to completely prevent ferroptosis; inflammatory response promotes ferroptosis by affecting iron metabolism and increasing oxidative stress. If inflammation is effectively inhibited, the inducing factors of ferroptosis (such as ROS and intracellular iron accumulation) can be reduced, but inflammation is usually the result of ferroptosis rather than the initiating cause, so inhibiting inflammation alone may not be able to completely prevent ferroptosis.

[0013] The present invention has found through experimental research that Lactobacillus plantarum LP35 can reduce the ferrous ion content in body tissues, and can increase the expression level of genes negatively correlated with ferroptosis (SLC40A1, FTH1, NFE2L2, GPX4), and reduce the expression level of genes positively correlated with ferroptosis (HSPB1). The results show that Lactobacillus plantarum LP35 specifically inhibits ferroptosis, and its mechanism of action in inhibiting ferroptosis does not rely solely on anti-oxidation and anti-inflammatory effects, but is more likely to reduce the accumulation of ferrous ions by regulating iron ion metabolism, thereby reducing the level of lipid peroxidation, alleviating inflammation, and further achieving the purpose of inhibiting ferroptosis.

[0014] A second aspect of the present invention provides a Lactobacillus plantarum LP35 exosome, which is prepared from the above-mentioned Lactobacillus plantarum LP35.

[0015] Preferably, the method for preparing the Lactobacillus plantarum LP35 exosomes comprises the following steps: culturing the activated Lactobacillus plantarum LP35 strain in an MRS liquid culture medium, collecting the supernatant, and centrifuging the supernatant at 3000 g and 4°C for 20 min to obtain supernatant I; centrifuging the supernatant I at 5000 g and 4°C for 20 min to obtain supernatant II; centrifuging the supernatant II at 10000 g and 4°C for 20 min to obtain supernatant III; centrifuging the supernatant III at 140,000 g and 4°C for 1 h, discarding the supernatant, resuspending the precipitate with 1×PBS, centrifuging the resuspended liquid at 200,000 g and 4°C for 1 h, discarding the supernatant, resuspending the precipitate with 1×PBS to 1 mL, filtering with a 0.22 μm water filter membrane, and retaining the filtrate to obtain the Lactobacillus plantarum LP35 exosomes.

[0016] The third aspect of the present invention provides the use of the above-mentioned Lactobacillus plantarum LP35 strain and / or Lactobacillus plantarum LP35 exosomes in the preparation of ferroptosis inhibitors, wherein the ferroptosis inhibitors include ferroptosis inhibitors for use in vivo and ferroptosis inhibitors for use in vitro. The former can be used to treat diseases related to ferroptosis, and the latter can be used to screen drugs that induce ferroptosis, or as a control drug for screening other ferroptosis inhibitors, or for in vitro investigation of the mechanism of inducing / inhibiting ferroptosis.

[0017] Preferably, the ferroptosis inhibitor is in the form of oral capsules, tablets, liquids and topical ointments to suit different treatment needs.

[0018] Preferably, when preparing the ferroptosis inhibitor, the Lactobacillus plantarum LP35 strain is added in the form of a live bacteria suspension, live bacteria mud or live bacteria powder.

[0019] Exemplarily, a live bacterial suspension of Lactobacillus plantarum LP35 strain and / or a solution of Lactobacillus plantarum LP35 exosomes are mixed with a thickener and a stabilizer to obtain a liquid ferroptosis inhibitor; live bacterial mud of Lactobacillus plantarum LP35 strain and / or Lactobacillus plantarum LP35 exosomes are mixed with a filler and pressed into a capsule shell using a soft capsule machine to obtain a soft capsule form of the ferroptosis inhibitor; live bacterial powder of Lactobacillus plantarum LP35 strain and / or Lactobacillus plantarum LP35 exosomes are mixed with a filler, a disintegrant, a glidant, and a binder, and tableted to obtain a tablet form of the ferroptosis inhibitor.

[0020] A fourth aspect of the present invention provides the use of the above-mentioned Lactobacillus plantarum LP35 strain and / or Lactobacillus plantarum LP35 exosomes in the preparation of medicines for treating ferroptosis-related diseases.

[0021] Preferably, the ferroptosis-related diseases include neurodegenerative diseases caused by ferroptosis, degenerative bone diseases, multiple sclerosis, tumors, myocardial ischemia-reperfusion injury, acute lung injury, acute kidney injury, non-alcoholic fatty liver disease, inflammatory bowel disease and diabetes.

[0022] Further preferably, the ferroptosis-related diseases do not include neurodegenerative diseases not caused by ferroptosis, degenerative bone diseases, multiple sclerosis, tumors, myocardial ischemia-reperfusion injury, acute lung injury, acute kidney injury, non-alcoholic fatty liver disease, inflammatory bowel disease and diabetes.

[0023] More preferably, the ferroptosis-related disease is inflammatory bowel disease.

[0024] Preferably, the medicine is in the form of oral capsules, tablets, liquids and external ointments to suit different treatment needs.

[0025] Preferably, when preparing the medicine, the Lactobacillus plantarum LP35 strain is added in the form of a live bacterial suspension, live bacterial mud or live bacterial powder. The live bacterial suspension, live bacterial mud or live bacterial powder of the Lactobacillus plantarum LP35 strain is mixed with a pharmaceutically acceptable excipient, and is made into oral capsules, tablets, liquids and external ointments according to a conventional preparation process.

[0026] Exemplarily, a live bacterial suspension of Lactobacillus plantarum LP35 strain and / or a solution of Lactobacillus plantarum LP35 exosomes are mixed with a thickener and a stabilizer to obtain a liquid preparation for treating ferroptosis-related diseases; live bacterial mud of Lactobacillus plantarum LP35 strain and / or Lactobacillus plantarum LP35 exosomes are mixed with a filler and pressed into a capsule shell using a soft capsule machine to obtain a soft capsule for treating ferroptosis-related diseases; live bacterial powder of Lactobacillus plantarum LP35 strain and / or Lactobacillus plantarum LP35 exosomes are mixed with a filler, a disintegrant, a glidant, and a binder, and tableted to obtain tablets for treating ferroptosis-related diseases; live bacterial mud of Lactobacillus plantarum LP35 strain and / or Lactobacillus plantarum LP35 exosomes are mixed with an ointment excipient, a thickener, and a moisturizer to obtain an external ointment for treating ferroptosis-related diseases.

[0027] Optionally, when the above-mentioned ferroptosis inhibitor or drug is prepared using a live bacterial suspension of Lactobacillus plantarum LP35 strain, the preparation method of the live bacterial suspension can adopt the following steps: culturing Lactobacillus plantarum LP35 in MRS liquid culture medium, centrifuging the bacteria, and then resuspending the obtained bacterial sediment in physiological saline to obtain the live bacterial suspension.

[0028] Optionally, when the above-mentioned ferroptosis inhibitor or medicine is prepared using live bacteria mud of Lactobacillus plantarum LP35 strain, the preparation method of the live bacteria mud can adopt the following steps: culturing Lactobacillus plantarum LP35 in MRS liquid culture medium, centrifuging and precipitating the bacteria to obtain the live bacteria mud.

[0029] Optionally, when the above-mentioned ferroptosis inhibitor or medicine is prepared using live bacterial powder of Lactobacillus plantarum LP35 strain, the preparation method of the live bacterial powder can adopt the following steps: culturing Lactobacillus plantarum LP35 in MRS liquid culture medium, centrifuging and precipitating the bacteria, and drying to obtain the live bacterial powder. The drying method can adopt freeze drying, low-temperature spray drying, etc. to ensure the activity and stability of the strain.

[0030] The beneficial effects of the present invention are:

[0031] The plant lactobacillus LP35 provided by the present invention has the effects of regulating iron metabolism, anti-lipid peroxidation, anti-inflammatory, etc., and can effectively inhibit ferroptosis. Experiments have shown that the plant lactobacillus LP35 and its exosomes can produce a significant inhibitory effect on ferroptosis. In addition, plant lactobacillus, as a natural probiotic that has been widely used in the field of food and health products, has the characteristics of being natural, safe, and having no side effects. Therefore, the plant lactobacillus LP35 and its exosomes can be used to prepare ferroptosis inhibitors or medicines for treating ferroptosis-related diseases, and have a wide range of application prospects in the prevention and treatment of diseases related to ferroptosis such as neurodegenerative diseases, cardiovascular diseases, and metabolic diseases. The invention provides a new and natural treatment method for the prevention and treatment of ferroptosis-related diseases, and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a Gram-stained micrograph of Lactobacillus plantarum LP35 in Example 1 of the present invention;

[0033] Figure 2 is the growth curve of Lactobacillus plantarum LP35 strain in Example 1 of the present invention;

[0034] Figure 3 This is a transmission electron microscope photograph of the exosomes of Lactobacillus plantarum LP35 in Example 2 of the present invention;

[0035] Figure 4 The alleviating effect of Lactobacillus plantarum LP35 exosomes on ferroptosis of HEK293T cells in Example 3 of the present invention;

[0036] Figure 5 This is a comparison of the inhibitory effects of Lactobacillus plantarum LP35 exosomes and the ferroptosis inhibitor Fer-1 on ferroptosis in HEK293T cells in Example 3 of the present invention;

[0037] Figure 6 The effect of Lactobacillus plantarum LP35 on body weight and disease activity index in Example 4 of the present invention;

[0038] Figure 7 The improvement effect of Lactobacillus plantarum LP35 on inflammatory factors and inflammation-related enzymes in Example 4 of the present invention;

[0039] Figure 8 The improvement effect of Lactobacillus plantarum LP35 on mitochondrial morphology in Example 4 of the present invention;

[0040] Fig. 9 The effect of Lactobacillus plantarum LP35 on the ferrous ion concentration, glutathione level and malondialdehyde content in colon in Example 4 of the present invention;

[0041] Fig.10 The effect of Lactobacillus plantarum LP35 in Example 4 of the present invention on the fragments per kilobase million (FPKM) of the key genes SLC40A1, FTH1, and NFE2L2 associated with ferroptosis;

[0042] Fig.11 The effects of Lactobacillus plantarum LP35 and the ferroptosis inhibitor Fer-1 on body weight and disease activity index in Example 5 of the present invention;

[0043] Fig.12 The improving effects of Lactobacillus plantarum LP35 and the ferroptosis inhibitor Fer-1 on inflammatory factors and inflammation-related enzymes in Example 5 of the present invention;

[0044] Fig.13 This is the improvement effect of Lactobacillus plantarum LP35 and the ferroptosis inhibitor Fer-1 on intestinal loss and ferroptosis indicators in Example 5 of the present invention. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work fall within the scope of protection of the present invention.

[0046] Ferroptosis is closely related to the occurrence and development of many diseases, such as neurodegenerative diseases, degenerative bone diseases, myocardial ischemia-reperfusion injury, inflammatory bowel disease, etc. The core mechanism of ferroptosis is the excessive accumulation of intracellular iron, which leads to lipid peroxidation, and then causes cell membrane damage and cell death.

[0047] The embodiment of the present invention provides a Lactobacillus plantarum LP35 strain with antioxidant properties.

[0048] The present invention proves through animal experiments that the Lactobacillus plantarum LP35 can inhibit ferroptosis through mechanisms such as anti-oxidation, anti-inflammation, and regulation of iron metabolism, and effectively alleviate the symptoms of diseases related to ferroptosis.

[0049] The embodiment of the present invention also provides Lactobacillus plantarum LP35 exosomes.

[0050] The scheme of the present invention is described below through specific embodiments.

[0051] The components of the culture medium used in the following examples are as follows:

[0052] MRS liquid culture medium: peptone: 1%wt, beef extract powder: 0.8%wt, yeast extract powder: 0.4%wt, glucose: 2%wt, dipotassium hydrogen phosphate (anhydrous): 0.2%wt, triammonium citrate (anhydrous): 0.2%wt, sodium acetate (containing trihydrate): 0.5%wt, magnesium sulfate (containing heptahydrate): 0.22%wt, manganese sulfate (containing tetrahydrate): 0.05%wt, Tween 80: 0.1%wt, and the rest is distilled water.

[0053] MRS solid medium: glucose 2%wt, peptone 1%wt, beef extract powder 1%wt, yeast extract powder 0.5%wt, sodium acetate 0.5%wt, Tween 80 0.1%wt, diammonium hydrogen citrate 0.1%wt, magnesium sulfate 0.02%wt, manganese sulfate 0.005%wt, agar powder 1.5%wt, and the balance is distilled water.

[0054] Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the materials, reagents, culture media, etc. used in the following examples can all be obtained from commercial channels. Example 1

[0055] This example provides the screening, isolation and identification of Lactobacillus plantarum LP35 strain.

[0056] Screening and Isolation Process of Lactobacillus plantarum LP35 Strain

[0057] The traditional yogurt samples from the Altay region were diluted with sterile saline in a 10-fold dilution gradient. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Dilution, dilute with sterile saline. Take 100 μL of each dilution solution and spread it on MRS solid medium, and culture at 37℃ for 48 h. Pick a single colony, further separate and purify the characteristic colonies by streak separation method, record the morphological characteristics of each colony and observe by Gram staining microscopy.

[0058] Colony morphological characteristics of LP35 strain: On MRS agar plates, the colonies are round, smooth and slightly convex, and light milky white in color.

[0059] 2. Identification of LP35 strain

[0060] 2.1 Morphology and general properties of Lactobacillus plantarum LP35

[0061] The morphological characteristics of LP35 strain are as follows: Gram-positive (G +), short rods, single or paired, no spores, no flagella, Gram staining micrographs such as Figure 1 shown.

[0062] 2.2 16S rDNA sequence determination of Lactobacillus plantarum LP35

[0063] The LP35 strain was inoculated in MRS liquid medium and cultured at 37°C for 24 h. The genomic DNA of the LP35 strain was extracted according to the instructions of the Ezup column bacterial genomic DNA extraction kit. The 16s rDNA was amplified using the genomic DNA as a template to obtain its 16s rDNA sequence (as shown in SEQ ID No.1). The 16s rDNA sequence of the LP35 strain was subjected to BLAST comparison analysis in the NCBI database. The results showed that the LP35 strain was similar to Lactobacillus plantarum ( Lactiplantibacillus plantarum ) with an Identity = 99% (>97%). Therefore, this strain was identified as Lactobacillus plantarum ( Lactiplantibacillus plantarum ), named it Lactobacillus plantarum LP35, and preserved it in China Center for Type Culture Collection (CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province), with the preservation number CCTCC M2025078 and the preservation date of January 10, 2025.

[0064] 3. Growth curve determination of Lactobacillus plantarum LP35

[0065] Select Lactobacillus plantarum and take LP35 colonies to inoculate in MRS liquid medium, and incubate at 37℃ for 24 hours to obtain seed solution. Inoculate the seed solution into MRS liquid medium at a 1% inoculum volume, take 200 μL and inoculate into a 96-well plate and incubate at 37℃ for 24 hours. The culture time is used as the horizontal axis and the OD is used as the horizontal axis. 600 As the vertical axis, draw the growth curve of the bacteria (such as Figure 2 as shown). Example 2

[0066] This example provides a method for preparing exosomes of Lactobacillus plantarum LP35.

[0067] (1) The frozen strain of Lactobacillus plantarum LP35 obtained in Example 1 was inoculated into MRS liquid culture medium for recovery, and the OD value of the strain was increased within 10-12 h of the second generation culture. 600 Adjust to 0.5, transfer to 300 mL MRS liquid medium at 2% inoculum, and culture in a constant temperature shaker at 37°C and 190 rpm for 48 h;

[0068] (2) High-speed centrifugation: Centrifuge the supernatant obtained from the culture at 3000 g and 4°C for 20 min to obtain supernatant I. Centrifuge the obtained supernatant I at 5000 g and 4°C for 20 min to obtain supernatant II. Centrifuge the obtained supernatant II at 10000 g and 4°C for 20 min to obtain supernatant III.

[0069] (3) Ultracentrifugation: The supernatant III obtained by high-speed centrifugation was centrifuged at 140,000 g and 4 °C for 1 h, the supernatant was discarded, and the precipitate was resuspended in 1× PBS. The resuspended solution was centrifuged at 200,000 g and 4 °C for 1 h, the supernatant was discarded, and the precipitate was resuspended in 1× PBS to 1 mL, filtered with a 0.22 μm water filter membrane, and the filtrate was retained. The filtrate obtained was the solution containing Lactobacillus plantarum LP35 exosomes. The results of the particle size, Zeta potential and protein content are shown in Table 1. The electron transmission microscopy photos of the exosomes are shown in Figure 3 shown.

[0070] The solution containing Lactobacillus plantarum LP35 exosomes was stored at -80°C for future use.

[0071] Table 1 Some basic indicators of exosomes of Lactobacillus plantarum LP35

[0072]

[0073] Example 3

[0074] This example investigates the inhibitory effect of the solution containing Lactobacillus plantarum LP35 exosomes prepared in Example 2 on ferroptosis.

[0075] The solution containing Lactobacillus plantarum LP35 exosomes stored at -80°C in Example 2 was thawed and the following experiment was performed.

[0076] 5 × 10 3 HEK293T cells were cultured in 200 μL GIBCO DMEM medium (Thermo Fisher Scientific (China) Co., Ltd.), and different concentrations of ferroptosis inducer (RSL3) were added to the culture wells. Then, 20 μL of PBS or the solution containing Lactobacillus plantarum LP35 exosomes prepared in Example 2 were added respectively. After 20 h, the cell viability was determined by CCK-8 kit method. The results are shown in Figure 4 As shown, PBS had no alleviating effect on RSL3-induced ferroptosis. As the concentration of RSL3 increased, cells died, while Lactobacillus plantarum LP35 exosomes could significantly improve cell death caused by RSL3, proving that Lactobacillus plantarum LP35 exosomes could inhibit ferroptosis.

[0077] 5 × 103 HEK293T cells were cultured in 200 μL GIBCO DMEM medium (Thermo Fisher Scientific (China) Co., Ltd.), 1 μM RSL3 was added to the culture wells, and the solution containing Lactobacillus plantarum LP35 exosomes prepared in Example 2 or 5 μM Fer-1, an inhibitor of ferroptosis, was added. Figure 5 As shown, Lactobacillus plantarum LP35 exosomes showed a ferroptosis inhibitory effect comparable to that of Fer-1. Example 4

[0078] This example investigates the inhibitory effect of Lactobacillus plantarum LP35 obtained in Example 1 on ferroptosis.

[0079] According to the inoculation amount of 1%, Lactobacillus plantarum LP35 was cultured in MRS liquid medium. After inoculation, it was cultured at 37°C for 24 h until the bacteria grew to an optical density (OD 600 ) is 1.2 (equivalent to a concentration of 4 × 10 11 CFU / mL), and then centrifuged at 5000 g at 4°C for 10 min to precipitate the bacteria. Subsequently, the bacteria were resuspended in sterile saline to obtain a live bacterial suspension. The live bacterial suspension was freshly prepared according to this method before each administration.

[0080] Male C57BL / 6J mice of 6 weeks old and weighing about 20 grams were selected. All mice were allowed to drink water and eat freely, and the feeding conditions were 21℃–24℃, 45%–60% humidity, and 12 hours of light / 12 hours of darkness. After 1 week of adaptive feeding, the mice were randomly divided into three groups, namely, blank control group (Con), model group (DSS) and experimental group (TD), with 6 mice in each group. DSS (dextran sulfate) with a molecular weight of 36–40 kDa was dissolved in the drinking water of each group of mice at a concentration of 2.5% (w / v), and the mice were given free drinking for 7 consecutive days to induce colitis.

[0081] Experimental methods: The mice in the TD group were gavaged with a live bacterial suspension of Lactobacillus plantarum LP35 for 16 consecutive days, once a day at a fixed time, 200 µL each time, and the Con group and DSS group were gavaged with an equal amount of normal saline. Starting from the 16th day (i.e., day 0 of modeling), the drinking water of the mice in each group was changed to a 2.5% (w / v) DSS aqueous solution, and each group of mice continued to be gavaged with 200 µL of normal saline or live bacterial suspension of Lactobacillus plantarum LP35 at a fixed time every day according to Table 2. The model was established for 7 consecutive days (i.e., the drinking water from day 0 to day 6 contained 2.5% DSS). 24 h after the last gavage (day 7), all mice were euthanized, the neck was broken, and plasma and colon tissue were collected.

[0082] Table 2 Animal groups

[0083]

[0084] During the modeling period, the body weight and disease activity index (DAI) of each group of mice were recorded. The results are shown in Table 3. Figure 6 As shown, oral administration of live bacterial suspension of Lactobacillus plantarum LP35 can effectively alleviate the symptoms of inflammatory mice, improve the degree of weight loss and reduce the disease activity index.

[0085] Table 3 DAI scoring criteria

[0086]

[0087] Plasma levels of proinflammatory cytokines (tumor necrosis factor-α, interleukin-6, and interleukin-1β) and inflammatory enzymes (such as myeloperoxidase) were measured. Figure 7 As shown, oral administration of live bacterial suspension of Lactobacillus plantarum LP35 can reduce the levels of typical pro-inflammatory cytokines and inflammation-related enzymes, help alleviate inflammatory responses and improve the symptoms of related diseases.

[0088] The mitochondrial morphology (one of the indicators of ferroptosis in the body) in the colon tissue cells of each group of mice was observed. Figure 8 As shown, the colon tissues of mice in the DSS group showed typical symptoms of ferroptosis, namely, reduction or disappearance of mitochondrial cristae and rupture of mitochondrial membranes. Oral administration of a live bacterial suspension of Lactobacillus plantarum LP35 can reduce the impact of ferroptosis of colon epithelial cells on mitochondria.

[0089] The ferroptosis indicators in the colon tissue of each group of mice were detected: ferrous ion content, glutathione (GSH) level and malondialdehyde (MDA) content. The results are as follows Fig. 9 As shown, oral administration of live bacterial suspension of Lactobacillus plantarum LP35 can significantly improve the body's ferroptosis indicators such as increased ferrous content, decreased GSH level and increased MDA.

[0090] Colon transcriptome analysis: Total RNA was extracted from colon tissue cells of each group of mice. Libraries were constructed using TruSeq Stranded Total RNA and Ribo-Zero Gold according to the instructions of the kit manufacturer. To deplete Ribo-Zero and fragment RNA, approximately 1 g of total RNA was treated with TruSeq Stranded Total RNA and Ribo-Zero Gold Kit. Afterwards, first-strand cDNA synthesis was treated with Act D Mix, followed by synthesis of second-strand cDNA. The 3' end of the cDNA was then adenylated, and adapters were ligated to the 3' adenylated end. PCR Primer Cocktail and PCR Master Mix were also used to enrich cDNA fragments. The size and purity of the samples were checked using an Agilent Technologies 2100 Bioanalyzer. Finally, the libraries were sequenced on an Illumina sequencing instrument (HiSeqTM 2,500 platform) to generate paired-end reads of 150 bp / 125 bp. Transcriptome sequencing showed that oral administration of live bacterial suspension of Lactobacillus plantarum LP35 could affect the expression of ferroptosis-related genes in colitis mice. Specifically, the expression levels of key ferroptosis-related genes SLC40A1, FTH1, and NFE2L2 were significantly increased after the intake of Lactobacillus plantarum LP35 (P<0.01), while the expression level of HSPB1 gene was significantly decreased after the intake of Lactobacillus plantarum LP35 (P<0.1). In addition, oral administration of Lactobacillus plantarum LP35 increased the expression level of GPX4 gene. Fig.10 shown. Example 5

[0091] This example uses existing ferroptosis inhibitors as a control and ferric citrate as an agent for inducing ferroptosis to investigate the therapeutic effect of Lactobacillus plantarum LP35 on ferroptosis obtained in Example 1. Ferric citrate can be actively transported into cells through specific transport proteins, resulting in an increase in intracellular iron content, thereby inducing lipid peroxidation and ferroptosis.

[0092] A live bacterial suspension of Lactobacillus plantarum LP35 was prepared according to the method of Example 4. A live bacterial suspension was freshly prepared according to the method before each administration.

[0093] Male C57BL / 6J mice of 6 weeks old and weighing about 20 g were selected. All mice were allowed to drink water and eat freely, and the feeding conditions were 21℃–24℃, 45%–60% humidity, and 12 h light / 12 h dark photoperiod. After 1 week of adaptive feeding, the mice were randomly divided into 5 groups, 6 mice in each group, and the specific treatment schemes of each group are shown in Table 4. Starting from day 0, the TFD group and the TRD group were gavaged with 200 μL of live bacterial suspension of Lactobacillus plantarum LP35 at a fixed time every day, while the DSS group, the FD group, and the RD group were gavaged with an equal volume of normal saline; at the same time, the FD group and the TFD group were injected with ferric citrate (15 mg / kg) through the tail vein once every two days for a total of three times to further aggravate ferroptosis, and the RD group was injected with the ferroptosis inhibitor Fer-1 (1 mg / kg) intraperitoneally, also once every two days for a total of three times.

[0094] Table 4 Animal groups

[0095]

[0096] The weight and disease activity index (DAI) of each group of mice were recorded during the experiment. The results are shown in Table 3 of Example 4. Fig.11 As shown, in the group supplemented with ferroptosis promoters (FD), the mice lost significant weight and the disease activity index increased significantly; on the contrary, in the group supplemented with Lactobacillus plantarum LP35 or ferroptosis inhibitor Fer-1, the symptoms of weight loss and increased disease activity index of mice were significantly alleviated.

[0097] 24 h after the last oral administration (day 6), all mice were euthanized by cervical dislocation, and plasma and colon tissues were collected.

[0098] The levels of typical pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6, and interleukin-1β) and inflammation-related enzymes (such as myeloperoxidase) in plasma are measured. Fig.12 As shown, in the group supplemented with ferroptosis promoters (FD), the levels of proinflammatory cytokines and inflammation-related enzymes in mice were significantly increased; while Lactobacillus plantarum LP35 and ferroptosis inhibitor Fer-1 significantly alleviated these inflammatory responses.

[0099] The mitochondrial morphology in the colon tissue and colon tissue cells of each group of mice was observed. Fig.13As shown, in the group supplemented with ferroptosis promoters (FD), the mice had more severe intestinal tissue destruction, such as colon ulcers, damaged crypts, and infiltration of neutrophils and monocytes; however, Lactobacillus plantarum LP35 and ferroptosis inhibitor Fer-1 significantly alleviated these symptoms. In addition, in the FD group, the mitochondrial cristae of the mouse colon tissue decreased or disappeared, and the mitochondrial membrane ruptured. Lactobacillus plantarum LP35 can effectively reduce the damage of mitochondria caused by cellular ferroptosis, with the same effect as ferroptosis inhibitors. Example 6

[0100] This embodiment provides a live bacterial suspension of Lactobacillus plantarum LP35 strain, and the preparation method thereof is as follows:

[0101] According to the inoculation amount of 1%, Lactobacillus plantarum LP35 was cultured in MRS liquid medium. After inoculation, it was cultured at 37°C for 24 h until the bacteria grew to an optical density (OD 600 ) is 1.2 (equivalent to a concentration of 4 × 10 11 CFU / mL), and then centrifuged at 5000 g at 4°C for 10 min to precipitate the bacteria. Subsequently, the bacteria were resuspended in sterile saline to obtain a live bacterial suspension. Example 7

[0102] This embodiment provides a live bacterial slurry of Lactobacillus plantarum LP35 strain, and the preparation method thereof is as follows:

[0103] According to the inoculation amount of 1%, Lactobacillus plantarum LP35 was cultured in MRS liquid medium. After inoculation, it was cultured at 37°C for 24 h until the bacteria grew to an optical density (OD 600 ) is 1.2 (equivalent to a concentration of 4 × 10 11 CFU / mL), and then centrifuged at 5000 g at 4 °C for 10 min to precipitate the bacteria, and the precipitate was collected as the live bacterial sludge. Example 8

[0104] This embodiment provides live bacterial powder of Lactobacillus plantarum LP35 strain, and the preparation method thereof is as follows:

[0105] According to the inoculation amount of 1%, Lactobacillus plantarum LP35 was cultured in MRS liquid medium. After inoculation, it was cultured at 37°C for 24 h until the bacteria grew to an optical density (OD 600 ) is 1.2 (equivalent to a concentration of 4 × 10 11 CFU / mL), and then centrifuge at 5000 g at 4°C for 10 min to precipitate the bacteria. Collect the precipitate, and freeze-dry or low-temperature spray-dry (inlet air temperature <45°C) to obtain live bacterial powder.

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum LP35 having antioxidant properties, characterized in that: The classification name is Lactiplantibacillus plantarum LP35 was deposited in the China Center for Type Culture Collection on January 10, 2025, and its deposit number is CCTCC NO: M 2025078; the deposit address is Wuhan University.

2. A Lactobacillus plantarum LP35 exosome, characterized in that Prepared by the Lactobacillus plantarum LP35 described in claim 1; the preparation method of the Lactobacillus plantarum LP35 exosomes comprises the following steps: culturing the activated Lactobacillus plantarum LP35 strain in MRS liquid culture medium, collecting the supernatant, centrifuging the supernatant at 3000 g, 4°C for 20 min to obtain supernatant I; centrifuging the supernatant I at 5000 g, 4°C for 20 min to obtain supernatant II; centrifuging the supernatant II at 10000 g, 4°C for 20 min to obtain supernatant III; centrifuging the supernatant III at 140,000 g, 4°C for 1 h, discarding the supernatant, resuspending the precipitate with 1×PBS, centrifuging the resuspended liquid at 200,000 g, 4°C for 1 h, discarding the supernatant, resuspending the precipitate with 1×PBS to 1 mL, filtering with a 0.22 μm water filter membrane, retaining the filtrate, and obtaining the Lactobacillus plantarum LP35 exosomes.

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