Lactobacillus helveticus CCFM1440 for regulating and controlling secretion of IL-10 (interleukin-10) in host kidney and improving kidney health and metagen of lactobacillus helveticus CCFM1440

Abstract

The invention discloses lactobacillus helveticus CCFM1440 for regulating and controlling secretion of IL-10 (interleukin-10) in a host kidney and improving kidney health and a metagen of the lactobacillus helveticus CCFM1440, and belongs to the technical field of microorganisms. The screened Lactobacillus helveticus CCFM1440 and the metagen thereof have the capacity of increasing secretion of IL-10 in the kidney of a host so as to assist in relieving kidney injury, can remarkably improve acute nephritis and kidney injury, reduce the content of creatinine and urea nitrogen in serum of an individual suffering from acute kidney injury, reduce the level of inflammatory markers in kidney tissue of the individual suffering from acute kidney injury, and can be used for treating acute nephritis and kidney injury. The oxidative stress injury of an acute kidney injury individual is relieved, and the application prospect is huge.

Description

Technical Field

[0001] The present invention relates to a Lactobacillus helveticus CCFM1440 that regulates host kidney IL-10 secretion to improve kidney health and a postbiotic thereof, belonging to the technical field of microorganisms. Background Art

[0002] The pathogenesis of acute kidney injury (AKI) and its complications is complex, with local or systemic renal inflammatory responses being a key driver of progressive renal function deterioration. The inflammatory microenvironment accelerates glomerulosclerosis and renal interstitial fibrosis by activating macrophage polarization, the sustained release of proinflammatory cytokines (such as TNF-α and IL-6), and oxidative stress and fibrotic pathways. In recent years, the emergence of the gut-kidney axis has further revealed the association between gut microbiota imbalance and renal inflammation. For example, gut-derived uremic toxins (such as trimethylamine oxide) can exacerbate renal inflammatory responses by activating the NF-κB pathway. However, while traditional anti-inflammatory drugs (such as glucocorticoids) can suppress inflammation in the short term, long-term use may aggravate metabolic disorders and immunosuppression and fail to target and regulate the inflammatory microenvironment.

[0003] As a key anti-inflammatory cytokine, IL-10 can inhibit the release of pro-inflammatory mediators (such as TNF-α and IL-1β) by macrophages, downregulate NF-κB activity, and reshape the immune balance by regulating T cell differentiation (such as inhibiting Th17 and promoting Treg cells). Studies have shown that IL-10 is significantly elevated in patients with chronic renal failure, and it delays the progression of kidney damage by antagonizing oxidative stress and fibrosis pathways (such as TGF-β / Smad). However, exogenous IL-10 delivery faces limitations such as short half-life and poor targeting, and there is an urgent need to develop interventions that can continuously induce endogenous IL-10 secretion.

[0004] Probiotics (such as Lactobacillus) have been shown to improve systemic inflammation by regulating the intestinal microbiota, enhancing barrier function, and through the immunomodulatory effects of their metabolites (such as short-chain fatty acids). Postbiotics (inactivated bacteria and their metabolites) have become a research hotspot as an alternative to live bacteria due to their high stability and strong tolerance. However, their application in renal inflammation is largely limited to indirect effects (such as through regulation of the intestinal microbiota), and research directly targeting strains that promote renal IL-10 secretion is lacking. Summary of the Invention

[0005] The present invention provides the use of Lactobacillus helveticus CCFM1440 and its postbiotics in a product for increasing host kidney IL-10 secretion and alleviating kidney inflammation.

[0006] The present invention provides a strain of Lactobacillus helveticus CCFM1440, which was deposited in Guangdong Provincial Microbiological Culture Collection on September 12, 2024, with a deposit number of GDMCC No. 65124, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] In one embodiment, the Lactobacillus helveticus CCFM1440 has the following characteristics:

[0008] The colonies on MRS culture medium are smooth, white, with small round protrusions. It is a Gram-positive bacterium, facultative anaerobic, and thermophilic. The optimal growth temperature is 35-40°C and the optimal growth pH is 6.0-7.0.

[0009] The present invention also provides a composition containing the Lactobacillus helveticus CCFM1440.

[0010] In one embodiment, the composition contains Lactobacillus helveticus CCFM1440 and a nutritional enhancer.

[0011] In one embodiment, the composition includes, but is not limited to, a microbial preparation.

[0012] The present invention also provides a postbiotic prepared by applying the Lactobacillus helveticus CCFM1440.

[0013] In one embodiment, the postbiotics include bacterial lysate, fermentation supernatant, or any of the above powders prepared by drying.

[0014] In one embodiment, the bacterial lysate is prepared by inoculating the above-mentioned Lactobacillus helveticus CCFM1440 into a fermentation medium to obtain a bacterial solution, and then heat-treating and high-pressure homogenizing the bacterial solution and then centrifuging it to obtain the bacterial lysate.

[0015] In one embodiment, the heat treatment is performed at 60-70° C. for 25-35 minutes.

[0016] In one embodiment, the heat treatment is at 65°C for 30 minutes.

[0017] In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging the above bacterial liquid.

[0018] In one embodiment, the postbiotics are prepared as a powder or liquid preparation.

[0019] In one embodiment, the powder is a solid powder prepared by drying the above-mentioned postbiotics.

[0020] In one embodiment, the drying method includes spray drying, vacuum freeze drying, fluidized bed drying or vacuum drying.

[0021] The present invention provides use of the Lactobacillus helveticus CCFM1440 and / or its postbiotics in preparing a medicine for alleviating kidney inflammation.

[0022] In one embodiment, the renal inflammation includes but is not limited to acute nephritis.

[0023] In one embodiment, the renal inflammation is drug-induced acute kidney injury; the drugs include but are not limited to chemotherapy drugs.

[0024] In one embodiment, the drug contains Lactobacillus helveticus CCFM1440 and pharmaceutical excipients.

[0025] In one embodiment, the excipients include fillers, flavoring agents, binders, disintegrants, lubricants, and antacids.

[0026] In one embodiment, the drug comprises the above composition, a drug carrier and / or a pharmaceutical excipient.

[0027] In one embodiment, the pharmaceutical excipient comprises an excipient and an additive.

[0028] In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and release retardants.

[0029] In one embodiment, the drug comprises at least one of the following effects:

[0030] (1) Increase the secretion of IL-10 in macrophages;

[0031] (2) Increase the level of IL-10, an inflammatory factor in the kidneys of individuals;

[0032] (3) reduce the signs of cisplatin-induced acute nephritis in individuals;

[0033] (4) Alleviate cisplatin-induced inflammatory response in individual kidney tissues.

[0034] In one embodiment, the symptoms of acute kidney injury include, but are not limited to, pathological changes in renal tissue, increased levels of creatinine and urea nitrogen, increased levels of the inflammatory marker TNF-α, and decreased levels of IL-10.

[0035] In one embodiment, the drug is administered orally.

[0036] Beneficial effects:

[0037] The Lactobacillus helveticus CCFM1440 and its postbiotics of the present invention have the ability to assist in alleviating kidney damage, which is specifically reflected in:

[0038] (1) Increased IL-10 secretion in macrophages (RAW264.7) and abnormal macrophage GPR43 mRNA expression caused by lipopolysaccharide;

[0039] (2) reduce serum creatinine and urea nitrogen levels in individuals with acute kidney injury;

[0040] (3) Increased levels of the anti-inflammatory factor IL-10 in kidney tissue of individuals with acute kidney injury;

[0041] (4) Alleviate the damage of oxidative stress in individuals with acute kidney injury;

[0042] (5) Reduce the content of proteins in inflammation and oxidative stress-related pathways in individuals with acute kidney injury.

[0043] Therefore, Lactobacillus helveticus CCFM1440 and its postbiotics have great application prospects in products that increase the body's secretion of the anti-inflammatory factor IL-10 and assist in alleviating kidney damage.

[0044] Biomaterial Deposit

[0045] The present invention provides a strain of Lactobacillus helveticus CCFM1440, which is classified and named Lactobacillus helveticus. It was deposited in Guangdong Provincial Microbiological Culture Collection on September 12, 2024, with a deposit number of GDMCC No: 65124, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Effects of postbiotics prepared for Lactobacillus helveticus CCFM1440 on the secretion of anti-inflammatory factor IL-10 in RAW264.7 cells.

[0047] Figure 2Effects of postbiotics prepared for Lactobacillus helveticus CCFM1440 on GPR43 gene expression in RAW264.7 cells.

[0048] Figure 3 Diagram of the experimental design for animal experiments.

[0049] Figure 4 Effects of postbiotics prepared for Lactobacillus helveticus CCFM1440 on body weight and food intake in mice.

[0050] Figure 5 This study investigates the effects of live Lactobacillus helveticus CCFM1440 and the postbiotics prepared therefrom on H&E-stained renal tissue sections of mice with acute kidney injury.

[0051] Figure 6 The purpose is to investigate the effects of live Lactobacillus helveticus CCFM1440 and its postbiotics on serum creatinine (Cr) and urea nitrogen (BUN) levels in mice with acute kidney injury.

[0052] Figure 7 The purpose of this study is to investigate the effects of live Lactobacillus helveticus CCFM1440 and its postbiotics on the levels of cytokines TNF-α and IL-10 in the kidney tissue of mice with acute kidney injury.

[0053] Figure 8 This study investigates the effects of live Lactobacillus helveticus CCFM1440 and its postbiotics on the oxidative stress level in renal tissue of mice with acute kidney injury.

[0054] Figure 9 This is the effect of live Lactobacillus helveticus CCFM1440 and its postbiotics on alleviating the expression of related genes in mice with acute kidney injury.

[0055] “*” indicates statistical difference compared with the model group (P<0.05), and “**” indicates significant statistical difference compared with the model group (P<0.01). DETAILED DESCRIPTION

[0056] The cells and animals involved in the following examples are as follows:

[0057] Mouse macrophages (RAW264.7) were purchased from Shanghai Cell Bank.

[0058] SPF grade C57BL / 6J male mice, 6 weeks old, weighing 16-18 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (production license number SCXK (Beijing) 2012-0001).

[0059] The culture medium involved in the following examples is as follows:

[0060] PBS buffer solution (per L): sodium chloride 8.0 g, potassium chloride 0.2 g, sodium phosphate dibasic 1.44 g, potassium phosphate monobasic 0.24 g, adjust pH to 7.4. High pressure sterilization at 121 ℃ for 20 min.

[0061] Cell culture medium: 89% (v / v) DMEM medium, 10% (v / v) fetal bovine serum, 1 x 10000 U / L penicillin, 10 mg / L streptomycin.

[0062] MRS liquid medium (per L): glucose 20.0 g, beef extract 10.0 g, proteose peptone 10.0 g, yeast powder 5.0 g, potassium phosphate dibasic trihydrate 2.6 g, sodium acetate anhydrous 2.0 g, diammonium citrate 2.0 g, magnesium sulfate heptahydrate 0.5 g, manganese sulfate monohydrate 0.25 g, Tween 80 1.0 mL. High pressure sterilization at 121 ℃ for 20 min.

[0063] MRS solid medium is added with 1.5% agar to the prepared liquid medium, and high pressure sterilization at 121 ℃ for 20 min.

[0064] Example 1: Screening and identification of Lactobacillus helveticus

[0065] The sample is derived from fermented dairy products, and after pretreatment, the sample is stored in 30% glycerol in a -80℃ refrigerator. After being taken out and thawed, 0.2 mL of the sample is taken in 1.8 mL of sterile normal saline to obtain 10 -1 dilution solution, then 0.5 mL of 10 -1 dilution solution is taken in 4.5 mL of normal saline to obtain 10 -2 dilution solution. In this way, dilution solutions of different concentrations are obtained in sequence; select appropriate gradient dilution solutions to be plated on MRS solid medium and cultured at 37℃ for 48 h, pick typical colonies of Lactobacillus helveticus on MRS solid medium for streaking and purification, pick single colonies to be transferred to MRS liquid medium and cultured at 37℃ for 18 h, preserved in 30% glycerol, and identified by 16s strain gene to obtain Lactobacillus helveticus CCFM1440; wherein the typical colonies of Lactobacillus helveticus are generally white and convex, round, smooth and moist on the surface, and the edges are neat.

[0066] The strain is subjected to 16S rDNA amplification and sequencing (determined by Suzhou Jinyuizhi Biological Technology Co., Ltd.), and the obtained 16S rDNA amplification nucleotide sequence of the strain is subjected to nucleic acid sequence alignment in NCBI, and the results show that the strain is Lactobacillus helveticus, which is named Lactobacillus helveticus CCFM1440.

[0067] Example 2: Preparation of postbiotic of Lactobacillus helveticus CCFM1440

[0068] 1. Preparation of Lactobacillus helveticus CCFM1440 bacterial solution:

[0069] (1) Preparation of seed solution:

[0070] Use a sterile inoculating loop to dip a small amount of Lactobacillus helveticus CCFM1440 culture into MRS solid medium for activation and incubate at 37°C for 48 hours. Then, pick a single colony and inoculate it into MRS liquid medium and incubate it in a 37°C incubator for 18 hours to prepare the seed solution.

[0071] (2) The prepared seed liquid was inoculated into MRS liquid medium at a 2% (v / v) inoculation rate and cultured in a 37°C incubator for 18 h. The bacteria were collected by centrifugation and concentrated, and the colonies were counted. 30% glycerol was stored in a -80°C refrigerator for later use. Before intervention, the glycerol was removed by centrifugation and the concentration of the bacterial suspension was adjusted to 1×10 11 CFU / mL.

[0072] 2. Preparation of postbiotics:

[0073] Lactobacillus helveticus CCFM1440 bacterial lysate postbiotics:

[0074] Prepare bacterial suspension according to the above step 1, centrifuge to obtain bacterial slurry, and then resuspend in sterile saline to adjust the bacterial suspension concentration to 1×10 11 CFU / mL, and homogenized in a high-pressure homogenizer (800-1200 MPa) 10 times, and then filtered through a 0.22 μm filter membrane to obtain bacterial lysate.

[0075] Lactobacillus helveticus CCFM1440 supernatant postbiotics:

[0076] Prepare bacterial solution according to the above step 1, centrifuge to obtain 1×10 11 CFU / mL Lactobacillus reuteri CCFM1439 fermentation supernatant, adjust the pH value to 7, filter through 0.22 μm filter membrane for sterilization, and store at -20°C.

[0077] Example 3: Lactobacillus helveticus CCFM1440 inhibits the release of inflammatory factors from macrophages

[0078] 100 μL of mouse macrophages (RAW264.7 cells) in the logarithmic growth phase were taken and 1×10 4Cells were seeded in a 96-well plate at a concentration of 10 cells / well. The outermost circle was filled with PBS solution to prevent edge effects. After 24 hours of culture, cells were attached to the plate. A control group and a strain sample treatment group were set up. The control group was treated with cell culture medium containing 6% PBS; the strain sample treatment group was treated with cell culture medium containing 6% of the postbiotics (bacterial lysate or supernatant) prepared in Example 2. Six replicate wells were set up for each group. After 24 hours, the cell supernatant was collected and the IL-10 and TNF-α levels in the supernatant were measured according to the ELISA kit instructions.

[0079] The results of the effect of postbiotics on macrophages showed that compared with the model group, the bacterial lysate and supernatant of Lactobacillus helveticus CCFM1440 could significantly downregulate the content of TNF-α in cells (P < 0.01), and the TNF-α content decreased from 103.15 pg / mL in the model group to 79.03 pg / mL and 72.56 pg / mL, respectively. The bacterial lysate and supernatant of Lactobacillus helveticus CCFM1440 could significantly upregulate the content of anti-inflammatory factor IL-10 in mouse macrophages (P < 0.01), and compared with the blank control group, the IL-10 content increased from 65.7 pg / mL in the blank control group to 95.67 pg / mL and 76.96 pg / mL, respectively. Figure 1 ).

[0080] Example 4: Lactobacillus helveticus CCFM1440 promotes macrophage GPR43 gene expression

[0081] Mouse macrophage RAW264.7 cells were revived and passaged three times, and the cell density was adjusted to 5×10 5 100 cells / mL, 2 mL of this concentration of cell suspension was inoculated into a 6-well cell culture plate and incubated at 5% CO2 and 37°C for 24 hours before adding the sample intervention. A control group, a model group, and a strain sample treatment group were set up. The control group was added with a cell culture medium containing 6% PBS; the model group was added with a cell culture medium containing 6% modeling agent (GPR43 inhibitor); and the strain sample treatment group was added with a cell culture medium containing 6% of the postbiotics prepared in Example 2 (bacterial lysate or supernatant). Three replicates were set up for each group. After 24 hours, the supernatant was aspirated and the cells at the bottom of the dish were retained. The cells in the 6-well plate were washed with PBS for 3 times, and 1 mL of cell lysis buffer was added to each well. The cells were pipetted repeatedly, and the cell lysate was aspirated to extract RNA. The RNA was reverse transcribed into cDNA using an RT-PCR reverse transcription kit. The gene expression in RAW264.7 cells was detected by real-time fluorescence quantitative analysis, and the expression level of GPR43 mRNA was calculated using the 2-△△Ct formula. The internal reference was GAPDH. The primers are described in Table 1 below. The results are shown in Table 1.

[0082] Table 1 Primer sequences

[0083]

[0084] GPR43 is a receptor protein that binds to short-chain fatty acids. During kidney damage, the expression of GPR43 will decrease significantly. However, it has a certain effect on alleviating kidney damage. For example, short-chain fatty acids bind to the GPR43 receptor protein, which will inhibit M1 macrophages, thereby reducing the release of inflammatory factors. In addition, short-chain fatty acids inhibit neutrophil crystallization by binding to the GPR43 receptor protein on immune cells, thereby alleviating renal fibrosis. Therefore, the increase in the secretion of GPR43 receptor protein can alleviate kidney damage to a certain extent. GPR43 expression results are as follows: Figure 2 As shown in the results, compared with the model group, both the cell lysate and supernatant of Lactobacillus helveticus CCFM1440 significantly upregulated the expression of GPR43 mRNA in macrophages (P < 0.01), by 78.93% and 103.37%, respectively. Therefore, at the cellular level, Lactobacillus helveticus CCFM1440 and its postbiotics can promote the expression of GPR43 in mouse macrophages.

[0085] Example 4: Use of Lactobacillus helveticus CCFM1440 in assisting the alleviation of kidney damage

[0086] The preparation method of the postbiotics of Lactobacillus helveticus CCFM1440 involved in the following examples is the same as that in Example 2.

[0087] Figure 3 This is the animal experiment process. Before the start of the experiment, all C57BL / 6J mice were randomly divided into groups and placed in an environment with a light-dark cycle of 22°C-24°C and 12 hours for one week. All groups of mice were raised normally throughout the experiment. Except for the blank group, which was injected with normal saline on days 8-14, the mice in other groups were injected with 10 mg / kg of cisplatin for acute kidney injury modeling. The blank group and the model group used normal saline instead of probiotics for intervention on days 8-21, and sham modeling was performed by intraperitoneal injection of normal saline on days 15-21.

[0088] The specific steps are as follows:

[0089] (1) Intervention experiment: Days 0-14, as follows:

[0090] Blank control group: 200 μL of sterile saline was drawn up by an 8-gauge elbow gavage needle for gavage once a day.

[0091] Model group mice: 200 μL of sterile saline was drawn up by gavage with a No. 8 elbow gavage needle, once a day.

[0092] Positive group mice: 200 μL of berberine solution was drawn up with a No. 8 elbow gavage needle and gavage was performed at a dose of 50 mg / kg, once a day.

[0093] Lactobacillus helveticus CCFM1440 live bacteria group: 200 μL of Lactobacillus helveticus CCFM1440 live bacteria suspension was drawn with an 8-gauge elbow gavage needle, and 5×10 9 Each mouse was gavaged with CFU once a day.

[0094] Lactobacillus helveticus CCFM1440 lysate group: 200 μL of Lactobacillus helveticus CCFM1440 cell lysate was drawn with an 8-gauge elbow gavage needle and 5×10 9 CFU bacterial lysate was administered orally once a day.

[0095] Lactobacillus helveticus CCFM1440 supernatant group: 200 μL of Lactobacillus helveticus CCFM1440 supernatant was drawn with an 8-gauge elbow gavage needle for gavage, with a daily gavage dose of 5 × 10 9 The supernatant obtained from CFU live bacteria fermentation, once a day.

[0096] The specific operation method of all gavage interventions was gavage with a No. 8 gavage needle (the bacterial solution concentration of the bacterial lysate group and the supernatant group was the same as that of the live bacteria, and was inactivated at 65°C for 30 minutes, and the inactivation effect was checked by plate coating).

[0097] (2) Modeling experiment: Intraperitoneal injection of 200 μL of cisplatin reagent was performed on days 8-14 in all groups except the blank group; the blank group received the same dose of normal saline for intervention.

[0098] After the intervention, the kidney tissues of the mice were sampled for subsequent analysis of the mouse weight and food intake, HE tissue pathology analysis, detection of serum creatinine and urea nitrogen levels, detection of inflammatory factors (TNF-α) and anti-inflammatory factors (IL-10) in kidney tissue, detection of oxidative stress (MDA, SOD, GSH-PX) expression, and detection of inflammatory and oxidative stress gene expression.

[0099] 3. Experimental results:

[0100] (1) Changes in mouse body weight and food intake

[0101] Cisplatin-induced acute kidney injury can cause mice to have anorexia, which reduces food intake and leads to rapid weight loss. Therefore, body weight and food intake are one of the important bases for indirectly evaluating kidney damage in mice during the experiment. We recorded the changes in body weight and food intake of mice during the experiment. Figure 4As shown in the data, compared with the model group, the weight and food intake of the mice in the three experimental groups (live Lactobacillus helveticus group, bacterial lysate group and supernatant group) increased after intervention. The weight of the live bacteria group was 19.7g and the food intake was 18.3g; the weight of the bacterial lysate group was 20.1g and the food intake was 18.6g; the weight of the supernatant group was 19.8g and the food intake was 18.4g.

[0102] (2) Histopathological analysis of mouse kidneys

[0103] After the intervention, the transverse sections of the mouse kidney tissue were observed. Figure 5 As shown, the model group showed significant dilation and deformation of the renal tubules, exposed nuclei of the tubular epithelial cells, and infiltration of the renal interstitium by numerous inflammatory cells, with cell swelling and increased cytoplasmic staining. Compared with the model group, kidney sections from the live bacteria group, supernatant group, and bacterial slurry group all showed varying degrees of improvement.

[0104] Compared with the model group, the pathological sections of the mouse kidney tissues treated with live Lactobacillus helveticus CCFM1440, its lysate, and its supernatant showed significant improvement. The rough surface of the renal tubules became smoother, the number of inflammatory cells in the renal interstitium decreased, and pathological damage was restored. This indicates that live Lactobacillus helveticus CCFM1440, its lysate, and its supernatant can effectively alleviate renal damage and, to a certain extent, reduce the damage caused by cisplatin.

[0105] (3) Creatinine and urea nitrogen levels in mouse serum

[0106] Creatinine and urea nitrogen in serum are metabolic products in the body. When kidney function is impaired, the excretion of these metabolites will be hindered, resulting in a rapid increase in serum creatinine and urea nitrogen levels. Therefore, they are one of the important indicators for evaluating kidney function. Figure 6 As shown in the results, compared with the model group, serum creatinine and urea nitrogen levels decreased in the three experimental groups after intervention. The creatinine levels in the live Lactobacillus helveticus group, bacterial lysate group, and supernatant group were 150.92 μmol / L, 143.16 μmol / L, and 141.62 μmol / L, respectively; and the urea nitrogen levels were 26.52 mmol / L, 31.64 mmol / L, and 22.98 mmol / L, respectively (P < 0.01).

[0107] (4) Content of inflammatory factors in mouse kidney tissue

[0108] Acute kidney injury is closely related to inflammatory responses. Cisplatin-induced acute kidney injury activates inflammatory signaling pathways in the kidneys. When renal tubular epithelial cells, endothelial cells, and immune cells are damaged, they release inflammatory factors such as TNF-α and IL-1β. These inflammatory factors further activate immune cells, including macrophages, monocytes, and neutrophils, through signaling cascade effects, thereby triggering more inflammatory responses. As immune cells infiltrate renal tissue, local inflammation intensifies, and more inflammatory mediators are released, forming a vicious cycle and aggravating renal damage. Continuous inflammatory responses not only lead to acute damage but may also lead to the development of chronic kidney disease.

[0109] like Figure 7 As shown in the results, compared with the model group, the live bacteria group, bacterial lysate group and supernatant group all reduced the content of TNF-α in kidney tissue to varying degrees (P < 0.01), decreasing by 21.41%, 10.35% and 28.83% respectively. At the same time, the IL-10 content of the three experimental groups was significantly increased compared with the model group (P < 0.01), increasing by 36.29%, 13.52% and 63.93% respectively. Therefore, Lactobacillus helveticus CCFM1440 and its postbiotics can effectively inhibit the secretion of inflammatory factor TNF-α, while upregulating the content of anti-inflammatory factor IL-10, thereby effectively alleviating inflammatory damage in mice.

[0110] (5) Expression of MDA, SOD, and GSH-PX in mouse kidney tissue

[0111] The pathogenesis of acute kidney injury is closely related to oxidative stress. Therefore, evaluating the level of antioxidant activity in the body can be used to infer whether the body has been damaged by oxidative stress and its ability to resist oxidative stress. Superoxide dismutase (SOD) catalyzes superoxide to produce oxygen and hydrogen peroxide through dismutation, playing a vital role in protecting biological cells from oxidative stress. Glutathione peroxidase (GSH-PX) is an overall evaluation index of antioxidant capacity, comprehensively reflecting the compensatory capacity of the antioxidant and non-enzymatic systems to external stimuli and their ability to scavenge free radicals. Malondialdehyde (MDA) is a product of cell membrane lipid peroxidation and can indirectly indicate the extent of oxidative stress damage to the body.

[0112] According to the results of determination of oxidative stress indexes in kidney tissue ( Figure 8) showed that compared with the model group, the live bacteria group, bacterial lysate group, and supernatant group all significantly reduced the MDA content. At the same time, the activities of SOD and GSH-PX increased after intervention. SOD activity increased from 18.75U / mg in the model group to 36.54U / mg in the live bacteria group, 31.48U / mg in the bacterial lysate group, and 37.26U / mg in the supernatant group, respectively. GSH-PX activity increased from 98.79U / mg in the model group to 184.68U / mg in the live bacteria group, 169.32U / mg in the bacterial lysate group, and 173.49U / mg in the supernatant group, respectively. Therefore, Lactobacillus helveticus CCFM1440 and its postbiotics can effectively enhance the activity of antioxidant enzymes and reduce oxidative damage in the body.

[0113] (6) Expression levels of GPR43, HMGB1, Colla1, and α-SMA genes in mouse kidney tissue

[0114] GPR43 is a coupled receptor protein that binds to short-chain fatty acids to inhibit the release of proinflammatory cytokines while promoting the secretion of anti-inflammatory factors, thereby reducing the inflammatory response. Furthermore, during acute kidney injury, GPR43 can regulate the phenotype of immune cells and reduce renal inflammatory damage. HMGB1 is a key gene that reflects inflammatory status in the body, and inhibiting its expression can effectively ameliorate inflammatory damage. Colla1 is a key marker of fibrosis. Increased expression of Colla1 typically indicates excessive deposition of type I collagen, which heralds the progression of renal fibrosis and may lead to irreversible renal damage. α-SMA is a marker of myofibroblasts. Its expression is upregulated after acute kidney injury, signaling myofibroblast activation and the onset of renal fibrosis. As AKI heals, persistently high expression of α-SMA and Colla1 suggests that renal injury repair may favor fibrosis over normal tissue repair, ultimately leading to the development of chronic kidney disease. Therefore, increased expression of Colla1 and α-SMA in acute kidney injury is considered a key indicator of renal fibrosis.

[0115] Table 2 Primer sequences

[0116]

[0117] Depend on Figure 9It was shown that oral administration of Lactobacillus helveticus altered the expression of the aforementioned genes in mice. Compared with the model group, the live bacteria group, bacterial lysate group, and supernatant group all downregulated the expression of HMGB1, Colla1, and a-SMA genes to varying degrees. The HMGB1 gene was downregulated from 3 in the model group to 1.61, 1.91, and 1.57, respectively; the Colla1 gene was downregulated from 5.23 in the model group to 2.81, 3.05, and 2.82, respectively; the a-SMA gene was downregulated from 5.87 in the model group to 3.72, 3.34, and 4.37, respectively; and the GPR43 gene was upregulated from 0.63 in the model group to 1.67, 1.47, and 1.88, respectively. These results indicate that Lactobacillus helveticus CCFM1440 and its postbiotics can effectively inhibit the expression of genes implicated in renal inflammation and renal fibrosis, alleviating acute kidney injury.

[0118] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Lactobacillus helveticus CCFM1440 was deposited in Guangdong Provincial Microbiological Culture Collection on September 12, 2024, with the deposit number GDMCC No: 65124.

2. A composition comprising the Lactobacillus helveticus CCFM1440 of claim 1.

3. A microbial preparation containing the Lactobacillus helveticus CCFM1440 according to claim 1.

4. A postbiotic prepared using the Lactobacillus helveticus CCFM1440 of claim 1, characterized in that: The postbiotics include bacterial lysate, fermentation supernatant, or any of the above powders prepared by drying.

5. The postbiotic according to claim 4, characterized in that The preparation method of the bacterial lysate comprises the following steps: culturing the Lactobacillus helveticus CCFM1440 in a fermentation medium, collecting the bacterial liquid, performing heat treatment and high-pressure homogenization, and then centrifuging to obtain the bacterial lysate.

6. The postbiotic according to claim 4, characterized in that The fermentation supernatant is the supernatant obtained by centrifuging the bacterial solution after the fermentation of Lactobacillus helveticus CCFM1440.

7. A medicine containing the Lactobacillus helveticus CCFM1440 according to claim 1 and / or its postbiotics.

8. Use of the Lactobacillus helveticus CCFM1440 and / or its postbiotics according to claim 1 in the preparation of a medicament for alleviating kidney inflammation.

9. The use according to claim 8, characterized in that The medicine contains the Lactobacillus helveticus CCFM1440 and / or its postbiotics, and pharmaceutical excipients.

10. The use according to claim 8 or 9, characterized in that: The drug has at least one of the following effects: (1) Increase the secretion of IL-10 in macrophages; (2) Increase the level of IL-10, an inflammatory factor in the kidneys of individuals; (3) reduce the signs of damage in acute nephritis; (4) Relieve the inflammatory response of acute nephritis.

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