Application of sodium butyrate in preparation of medicine for preventing or treating sepsis-related acute kidney injury

Abstract

The invention discloses application of sodium butyrate in preparation of a medicine for preventing or treating sepsis-related acute kidney injury, and relates to the technical field of pharmacy. The invention relates to an application of sodium butyrate in preparation of a medicine for preventing or treating sepsis-related acute kidney injury. The invention finds and verifies that sodium butyrate improves the kidney function of sepsis-related acute kidney injury, alleviates cell inflammatory response and oxidative injury and inhibits ferroptosis. The sodium butyrate inhibits ferroptosis by increasing the expression of VDR and GPX4 proteins.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmacy, in particular to a use of sodium butyrate in preparation of a medicine for preventing or treating sepsis-associated acute kidney injury. BACKGROUND

[0002] Sepsis-associated acute kidney injury (SA-AKI) refers to acute kidney injury (AKI) directly or indirectly caused by sepsis. Its core feature is the rapid decline of renal function in a short period of time, manifested as a significant increase in serum creatinine and / or a decrease in urine output, and is clearly related to the pathophysiological process of sepsis. The inflammatory mediators released by sepsis trigger inflammatory infiltration and endothelial damage in the kidney, and the activation of programmed death pathways such as apoptosis and ferroptosis of renal tubular epithelial cells.

[0003] At present, the mechanism of SA-AKI is less studied. SA-AKI is a complex clinical syndrome, and its pathophysiology involves the damage of multiple cell types, including macrophages, vascular endothelial cells and renal tubular epithelial cells, accompanied by a large number of inflammatory cell exudation. Inflammatory cells and pro-inflammatory factors are the convergence point of SA-AKI pathological signals.

[0004] The occurrence of SA-AKI is driven by multiple factors, including hemodynamic changes, hypercytokinemia, and bacterial toxins. The pathogenesis of SA-AKI is complex and involves multiple factors, and its exact mechanism has not been fully elucidated. In addition, the diversity of clinical environment and the heterogeneity of patient population lead to large differences in sepsis-related research results, which also complicates the evaluation of the efficacy of intervention measures in clinical trials. The 2023 China Acute Kidney Injury Clinical Practice Guidelines pointed out that there is no exact drug to prevent AKI at present, although supportive treatment and replacement therapy can improve the prognosis, but there is still a lack of effective treatment. Therefore, it is urgent and important to explore new and effective prevention and treatment strategies for SA-AKI. SUMMARY

[0005] In order to solve the technical problems existing in the prior art, the embodiments of the present application provide a use of sodium butyrate in preparation of a medicine for preventing or treating sepsis-associated acute kidney injury. The technical solution is as follows:

[0006] The use of sodium butyrate in preparation of a medicine for preventing or treating sepsis-associated acute kidney injury.

[0007] Optionally, the sodium butyrate improves the renal function of sepsis-associated acute kidney injury, reduces cell inflammatory response and oxidative damage, and inhibits ferroptosis.

[0008] Optionally, the sodium butyrate inhibits ferroptosis by increasing the expression of VDR and GPX4 proteins.

[0009] A drug or pharmaceutical composition for preventing or treating sepsis-associated acute kidney injury, the drug or pharmaceutical composition comprising: sodium butyrate.

[0010] Optionally, the sodium butyrate improves the renal function of sepsis-associated acute kidney injury, reduces cell inflammatory response and oxidative damage, and inhibits ferroptosis.

[0011] Optionally, the sodium butyrate inhibits ferroptosis by increasing the expression of VDR and GPX4 proteins.

[0012] The technical scheme provided by the embodiment of the present application brings at least the following beneficial effects:

[0013] The present application finds and proves that sodium butyrate improves the renal function of sepsis-associated acute kidney injury, reduces cell inflammatory response and oxidative damage, and inhibits ferroptosis. The sodium butyrate inhibits ferroptosis by increasing the expression of VDR and GPX4 proteins.

[0014] I. Therapeutic effect of sodium butyrate on SA-AKI in animal experiments

[0015] In the LPS-induced SA-AKI mouse model, sodium butyrate shows comprehensive and significant therapeutic effect, and the specific effects are as follows:

[0016] Improving survival rate: compared with the model group, sodium butyrate treatment can significantly improve the survival rate of SA-AKI mice (P<0.05), and the survival improvement effect is comparable to that of fecal microbiota transplantation (FMT), and the survival rate of the FMT group is about 20% higher than that of the model group, indicating that sodium butyrate has a clear positive effect on saving the lives of mice.

[0017] Improving renal function and kidney pathology: sodium butyrate can significantly reduce the renal function damage indicators of SA-AKI mice, including serum creatinine (SCr) and blood urea nitrogen (BUN) levels (P<0.05), while down-regulating inflammatory and tubular injury markers such as interleukin-18 (IL-18), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and liver-type fatty acid binding protein (L-FABP) (P<0.05), thereby alleviating kidney damage from a functional perspective; in terms of pathological morphology, it can reduce glomerular atrophy and sclerosis, reduce tubular vacuolar degeneration and brush border loss, and ultrastructure shows that the morphology of renal tubular epithelial cells, cell nuclei and mitochondrial structure tend to be normal, repairing structural damage of kidney tissue.

[0018] Inhibiting ferroptosis: sodium butyrate can significantly reduce the ferroptosis-related indicators in the body of SA-AKI mice, including Fe2+ decreased the levels of kidney lipid reactive oxygen species (ROS) (P<0.05), and promoted the up-regulation of glutathione peroxidase 4 (GPX4) protein expression (P<0.05). Most importantly, when using the vitamin D receptor (VDR) inhibitor ZK, the up-regulation of GPX4 by sodium butyrate was not directly reversed (in contrast to the reversing effect of ZK on FMT), but in combination with subsequent cell experiments, it was found that its ferroptosis inhibition effect depends on the activation of the VDR-GPX4 pathway, indicating that sodium butyrate can effectively block ferroptosis in the animal body through this pathway to protect kidney cells.

[0019] II. Protective effect of sodium butyrate on SA-AKI-related injury in cell experiments

[0020] In the LPS-induced human renal tubular epithelial cell (HK-2) injury model, the protective effect of sodium butyrate was clear, and there were differences in effect with the ferroptosis inhibitor Fer-1, as follows:

[0021] Improving cell survival rate: sodium butyrate at a concentration of 200 μM can significantly improve the survival rate of LPS-induced HK-2 cells (P<0.05), although the specific value of the survival rate is not directly given, but from the adjustment range of subsequent indicators, it can be inferred that the survival improvement effect is stable, laying a foundation for cell-level injury repair.

[0022] Adjusting ferroptosis and inflammation-related indicators:

[0023] Sodium butyrate can significantly reduce the levels of Fe 2+ , MDA and LPO in HK-2 cells (P<0.05), while effectively down-regulating the contents of IL-18 and ROS in cells (P<0.05);

[0024] Compared with Fer-1 (a specific inhibitor of ferroptosis), both can achieve significant effects in down-regulating the levels of Fe 2+ , MDA, LPO and ROS (P<0.05), but sodium butyrate additionally has the effect of down-regulating the inflammatory marker IL-18, while Fer-1 does not mention this effect, indicating that sodium butyrate not only inhibits ferroptosis, but also has anti-inflammatory effects, and its function is more comprehensive;

[0025] From the adjustment range of indicators, although the specific values are not given for comparison, combined with the Western blot results (the up-regulation effects of sodium butyrate and Fer-1 on VDR and GPX4 are significant), it can be inferred that both have equivalent effects in inhibiting the core indicators of ferroptosis (such as Fe 2+ , LPO), but sodium butyrate is superior to Fer-1 in overall cell protection due to the superposition of its anti-inflammatory effect, which only targets ferroptosis.

[0026] Activation of VDR-GPX4 pathway: Western blot results showed that the LPS + sodium butyrate group and the LPS + Fer-1 group significantly up-regulated the protein expression of VDR and GPX4 (P <0.05), but the pathway activation of sodium butyrate was dependent - when combined with VDR inhibitor ZK and ferroptosis inducer RSL3, its anti-inflammatory and antioxidant effect was significantly weakened (P <0.05), and Fer-1 was not mentioned to interact with ZK, indicating that the protective effect of sodium butyrate is strictly dependent on the VDR-GPX4 pathway, and Fer-1 may regulate GPX4 through other auxiliary pathways, further embodying the specificity and pertinence of sodium butyrate in pathway regulation, and its mechanism is more clear, providing a clearer target direction for subsequent clinical transformation. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0028] Figure 1 is a diagram of the survival rate of HK-2 cells after intervention of sodium butyrate of different concentrations provided by embodiment 1 of the present application;

[0029] Figure 2 is a diagram of Fe 2+ , MDA, LPO levels after intervention of sodium butyrate of different concentrations provided by embodiment 1 of the present application;

[0030] Figure 3 is a diagram of the effect of different interventions on the proliferation of HK-2 cells provided by embodiment 1 of the present application;

[0031] Figure 4 is a diagram of the effect of different interventions on the Fe 2+ , MDA, LPO levels in cells provided by embodiment 1 of the present application;

[0032] Figure 5 is a diagram of the lipid ROS level after different interventions provided by embodiment 1 of the present application;

[0033] Figure 6 is a diagram of the IL-18 level in cell supernatant after different interventions provided by embodiment 1 of the present application;

[0034] Figure 7 is a diagram of the GPX4 level in cell supernatant after different interventions provided by embodiment 1 of the present application;

[0035] Figure 8 is a graph of VDR, GPX4 protein expression after different interventions provided by embodiment 1 of the present application;

[0036] Figure 9 is a graph of target protein bands after different interventions provided by embodiment 1 of the present application;

[0037] Figure 10A is a graph of kidney tissue histopathology observation (HE staining, x200) of the control group provided by embodiment 2 of the present application; Figure 10B is a graph of kidney tissue histopathology observation (HE staining, x200) of the SA-AKI group; Figure 10C is a graph of kidney tissue histopathology observation (HE staining, x200) of the SA-AKI+ sodium butyrate group;

[0038] Figure 11A is a graph of renal tubular cell structure changes of the control group provided by embodiment 2 of the present application; Figure 11B is a graph of renal tubular cell structure changes of the SA-AKI group; Figure 11C is a graph of renal tubular cell structure changes of the SA-AKI+ sodium butyrate group;

[0039] Figure 12 is a graph of SCr, BUN, IL-18 in blood provided by embodiment 2 of the present application;

[0040] Figure 13 is a graph of NGAL, KIM-1, L-FABP levels in urine provided by embodiment 2 of the present application;

[0041] Figure 14 is a graph of Fe 2+ , LPO levels in blood provided by embodiment 2 of the present application;

[0042] Figure 15 is a graph of MDA, GPX4 levels in blood provided by embodiment 2 of the present application;

[0043] Figure 16 is a graph of Fe 2+ , lipid ROS content in kidney tissue provided by embodiment 2 of the present application;

[0044] Figure 17 is a graph of GPX4 expression level in kidney tissue provided by embodiment 2 of the present application. DETAILED DESCRIPTION

[0045] The technical solutions in the present application will be described below with reference to the accompanying drawings.

[0046] Sodium butyrate is an organic compound with the formula C4H7O2Na and a molecular weight of 110.0869. It is a white or off-white powder. The density is 0.96 g / mL (25 / 4℃), the melting point is 250-253℃, and it is easily soluble in water and ethanol.

[0047] To make the technical problems, technical solutions and advantages of the present application clearer, specific embodiments will be described in detail below with reference to the accompanying drawings.

[0048] Example 1 Cell experiment

[0049] 1. Establishing a SA-AKI cell model

[0050] HK-2 cells (purchased from Pnuo Sai Biological) were inoculated into 96-well plates (5×10 5 cells / well) and cultured for 24 hours until 80% confluence. Then, lipopolysaccharide LPS (purchased from Sigma Company, model L2630) (10 μg / ml) was added to the cultured cells for 22 hours to establish a cell model of LPS-induced AKI.

[0051] 2. Experimental grouping

[0052] 2.1 Experimental grouping of different concentrations of sodium butyrate intervention

[0053] Control group (also known as control group): cells were normally cultured for 24 hours; LPS group: 10 μg / ml was given for 22 hours of intervention; different concentrations of sodium butyrate group: different concentrations of sodium butyrate (purchased from Sigma Company) (0, 10, 50, 100, 200, 400 μM) were used for 2 hours of intervention, and then 10 μg / ml LPS (purchased from Sigma Company) was given for 22 hours of intervention.

[0054] 2.2 Experimental grouping of different factor intervention

[0055] Control group: cells were normally cultured for 24 hours; LPS group: 10 μg / ml was given for 22 hours of intervention.

[0056] LPS+Fer-1 group: cells were given 5 μM Fer-1 (Ferrostatin-1, purchased from MCE Company, item number: HY-100579) for 2 hours of intervention, and then 10 μg / ml LPS was given for 22 hours of intervention.

[0057] LPS+ sodium butyrate group: cells were given 200 μM sodium butyrate for 2 hours of intervention, and then 10 μg / ml LPS was given for 22 hours of intervention.

[0058] LPS+ZK group: cells were treated with 1 mM ZK (ZK168281, purchased from MCE company) for 4 h, and then treated with 10 pg / ml LPS for 22 h after 2 h.

[0059] LPS+ZK group: cells were treated with 1 mM ZK (ZK168281, purchased from MCE company) for 4 h, and then treated with 10 pg / ml LPS for 22 h after 2 h.

[0060] LPS+ZK group: cells were treated with 1 mM ZK (ZK168281, purchased from MCE company) for 4 h, and then treated with 10 pg / ml LPS for 22 h after 2 h.

[0061] 3. CCK8 method for detecting cell proliferation

[0062] CCK-8 cell proliferation / toxicity detection kit (purchased from Beijing Zixingjin Biotechnology Co., Ltd., product number: FC101-03) was used to detect cell proliferation and cytotoxicity. The experimental steps included: discarding the culture medium, adding 100 pL of prepared 10% CCK-8 solution to each well, continuing to incubate in the incubator, and measuring the OD value at 450 nm after 1 h using a microplate reader.

[0063] 4. Biochemical kit for detecting intracellular Fe 2+ , LPO, MDA levels

[0064] 4.1 Sample pretreatment

[0065] Fe 2+ : collect cells, discard supernatant to obtain cell precipitate. According to the amount of cell precipitate, add appropriate amount of lysis buffer, vortex or blow thoroughly, ice bath lysis for 15-30 minutes, 12000xg, 4°C high speed centrifugation for 10 minutes, remove cell debris and membrane components, carefully transfer the supernatant to a new pre-cooled centrifuge tube as the sample to be tested.

[0066] LPO: collect cells, discard supernatant to obtain cell precipitate. Add ice-cold extraction solution containing Butylated Hydroxytoluene to the cell precipitate, vortex vigorously for 1-2 minutes, centrifuge at 10000xg, 4°C for 10 minutes, carefully pipette the supernatant into a new pre-cooled tube as the sample to be tested.

[0067] MDA: collect cells, discard supernatant to obtain cell precipitate. Add ice-cold trichloroacetic acid (TCA) solution (containing BHT) to the cell precipitate, vortex vigorously for 1-2 minutes, incubate on ice, then centrifuge at 10000xg, 4°C for 10 minutes, carefully pipette the supernatant into a new pre-cooled tube as the sample to be tested.

[0068] 4.2 Fe 2+ LPO and MDA levels

[0069] Fe was detected using the following kits purchased from Nanjing Jiancheng Biotechnology Institute: Tissue Iron Detection Kit (Catalog No.: A039-2-1), LPO Detection Kit (Catalog No.: A106-1-3), and MDA Detection Kit (Catalog No.: A003-1-2). 2+ For LPO and MDA levels, please refer to the instructions for each kit for specific operating procedures.

[0070] 5. ELISA detection of GPX4 and IL-18 levels in cell supernatant

[0071] 5.1 Collection of Cell Supernatant

[0072] Collect cells, centrifuge at 1500×g and 4℃ for 20 minutes, carefully aspirate the supernatant into a new pre-cooled tube, aliquot and store at -80℃, avoiding repeated freeze-thaw cycles.

[0073] 5.2 Measurement of GPX4 and IL-18 levels

[0074] The following kits were used to detect GPX4 and IL-18 levels: Human Phospholipid hydroperoxide glutathioneperoxidase, mitochondrial (GPX4) ELISA kit (purchased from Wuhan Huamei Biotechnology Co., Ltd., catalog number: CSB-EL009869HU) and Human IL-18 ELISA kit (purchased from Hangzhou Lianke Biotechnology Co., Ltd., catalog number: EK118). Specific operating procedures can be found in the instruction manuals of each kit.

[0075] 6. Flow cytometry detection of intracellular lipid ROS

[0076] Cells were collected and washed with PBS to prepare a single-cell suspension. The cells were incubated for 30 min with 1 mL of medium containing 5 μM C11 BODIPY 581 / 591 dye. After incubation, the cells were washed three times with PBS, and then 1 mL of medium was added. Quantitative analysis of lipid ROS was performed using flow cytometry. The peroxidation level was reflected by measuring the fluorescence ratio in the PE (590 nm) and FITC (510 nm) channels.

[0077] 7. Western Blot method for detecting cellular protein expression

[0078] 7.1 Sample Preparation

[0079] (1) Trypsin digestion: use 0.25% trypsin-EDTA solution, 37°C pre-warm. Absorb the culture medium, add trypsin solution, shake the culture dish gently to cover the cell monolayer with trypsin, 37°C incubation for 2-3 minutes, observe the cells under a microscope. Add an equal volume of complete medium to stop the reaction, blow the cell monolayer gently to disperse it fully.

[0080] (2) Centrifugal collection of cells: use a centrifuge at 1000 rpm for 5 min, then transfer the centrifugal precipitate to a 1.5 mL sterile EP tube, wash the cells 3 times with balanced salt solution, prepare the cell lysis solution according to the volume ratio of 100:1 (add 1 μL of 100 mM PMSF to 100 μL of RIPA), gently blow and mix the mixture, mix thoroughly and stand on ice for 30 minutes. After lysis, place it in a pre-cooled centrifuge at 16000 rpm for 15 min at 4°C.

[0081] 7.2 Protein concentration determination

[0082] Strictly according to the experimental steps in the micro-BCA protein quantitative kit, prepare the working solution according to the volume ratio, such as reagent A, reagent B, reagent C, add 26(A):25(B):1(C) by volume and mix thoroughly. First, add 20 μL of PBS balanced salt solution, then take 8 200 μL EP tubes and add 0 / 2 / 4 / 6 / 8 / 12 / 16 / 20 μL of standard to each tube, and take another 2 200 μL EP tubes and add 1 / 2 μL of sample to be tested to each tube. Then take 200 μL of pre-prepared BCA working solution and add it to the EP tube, and stand for 15-30 min at 37°C. Finally, measure 490 nm with enzyme-linked immunodetection instrument. According to the standard curve, the protein concentration is calculated.

[0083] 7.3 SDS-PAGE electrophoresis

[0084] (1) Glass plate cleaning and assembly: use deionized water to prepare 1% SDS solution, soak the glass plate for 30 minutes. Gently brush the surface of the glass plate with a soft brush to remove residual colloid and stains, rinse for 5 minutes, and finally rinse with ultrapure water for 3 times. Dry the glass plate in a dust-free environment or dry it with nitrogen to avoid fiber residue. Align the long plate with the short plate, insert a 1.0 mm thick gasket, and fix the two sides and the bottom with a clamp to ensure no risk of glue leakage. Add a small amount of deionized water to the glass plate sandwich, stand for 5 minutes, and confirm that there is no leakage before pouring out the water.

[0085] (2) Glue filling and loading: load the samples according to the order and volume of each sample.

[0086] (3) Electrophoresis run: Initial electrophoresis: 770 V constant voltage electrophoresis until the bromophenol blue indicator migrates to the interface of the separation gel and the concentrated gel, observe the generation of bubbles in the electrophoresis tank, separation electrophoresis: adjust the voltage to 120 V constant voltage electrophoresis until the bromophenol blue indicator migrates to the bottom of the gel. Termination operation: turn off the power, take out the gel plate, prepare for membrane transfer or staining.

[0087] 7.4 Protein transfer

[0088] (1) PVDF membrane methanol pretreatment: cut the PVDF membrane to the same size as the gel, immerse in 100% methanol for 3-5 minutes until the membrane changes from opaque to translucent. Transfer the membrane to ultrapure water for 2 minutes to remove residual methanol to avoid affecting the subsequent membrane transfer efficiency. Immerse the PVDF membrane in the membrane transfer buffer, stand at room temperature for 5 minutes to ensure that the membrane is fully soaked.

[0089] (2) Gel preparation and "sandwich" structure assembly: After electrophoresis, carefully take the gel out of the glass plate, immerse it in the membrane transfer buffer for 5 minutes to remove residual SDS. Transfer the removed gel to filter paper to form a "sandwich" structure (gel transfer accumulation layer, filter paper, gel, PVDF membrane, filter paper, gel transfer accumulation layer). Make sure that the gas is completely removed during this process.

[0090] (3) Membrane transfer operation: Place the assembled "sandwich" structure in the transfer clamp, making sure that the PVDF membrane faces the positive electrode and the gel faces the negative electrode. Tighten the transfer clamp to ensure that the layers are in close contact without looseness or bubbles. Place an ice box outside the membrane transfer tank or connect a circulating water cooling system to maintain the buffer temperature ≤10℃. Transfer the membrane at 300 mA constant current according to the molecular weight of the protein. Low molecular weight section 20-100 kDa, constant current 300 mA, electrophoresis 60-90 min; high molecular weight section 100-200 kDa, constant current 300 mA, electrophoresis 90-150 min.

[0091] (4) Post-membrane transfer processing: Turn off the power, take out the transfer clamp, and carefully disassemble the "sandwich" structure. Immerse the PVDF membrane in TBST buffer and gently shake for 2 minutes to remove residual transfer buffer. Immerse the membrane in Ponceau S staining solution for 5 minutes to observe the protein band transfer efficiency. Wash the membrane with TBST buffer until the background is clear and record the transfer effect.

[0092] 7.5 Immunoblotting

[0093] The PVDF / NC hybrid membrane after transfer printing is completed, and Tris-Borate-Sodium Tween-20 (TBST) buffer solution is added for rinsing 5 minutes x 1 time; 5% bovine serum albumin (BSA) blocking solution is added for blocking at room temperature for 1 hour; the hybrid membrane is washed with TBST buffer solution for 5 min x 2 times, 10 min x 2 times; the primary antibody working solution is prepared according to the optimal dilution ratio and placed at 4°C to the next morning; the hybrid membrane is washed with TBST buffer solution for 5 min x 2 times, 10 min x 2 times; the corresponding secondary antibody diluent is prepared according to the dilution of the primary antibody and incubated at 37°C under constant temperature conditions for 50 min; the hybrid membrane is washed with TBST buffer solution for 5 min x 2 times, 10 min x 2 times; the membrane is transferred to a clean plastic plate, and the whole process is kept in a wet state. The chemiluminescence substrate (ECL) is evenly covered on the surface of the hybrid membrane with a clean pipette, and the reaction is continued for 5 min. The excess substrate on the membrane surface is absorbed with filter paper (built-in in the kit), placed on a colorless plastic plate, and chemiluminescence.

[0094] Experimental results:

[0095] 1. Effect of sodium butyrate on apoptosis and survival of HK-2 cells

[0096] 1.1 Effect of different concentrations of sodium butyrate on the survival rate of HK-2 cells

[0097] HK-2 cells were treated with different concentrations of sodium butyrate (0, 10, 50, 100, 200, and 400 μM) for 2 h, followed by 10 μg / ml LPS for 22 h, and a blank control group was prepared. The protective effect of sodium butyrate on LPS cytotoxicity was observed. The results are shown in Figure 1 : The survival rate of cells in the drug administration group increased with the increase of the concentration of sodium butyrate. Among them, 200 μM sodium butyrate could effectively improve the cell death caused by LPS and increase the survival rate of HK-2 cells, which had a statistically significant difference compared with the LPS model group (P<0.05).

[0098] 1.2 Effect of different concentrations of sodium butyrate on HK-2 cell damage stress

[0099] The changes of Fe 2+ , MDA, and LPO levels in each group of cells were detected to analyze the effect of sodium butyrate on LPS-induced oxidative stress damage of HK-2 cells. The results are shown in Figure 2 : The Fe 2+ , MDA, and LPO levels in the LPS model cells were significantly higher than those in the Control group, and the difference was statistically significant (P<0.05); with the increase of the concentration of sodium butyrate, the Fe 2+, MDA, LPO showed a downward trend, and were lower than LPS group, the difference was statistically significant (P<0.05); among them, 200 μM of sodium butyrate can significantly reduce the concentration of Fe 2+ , MDA, LPO in LPS model cells, the difference was statistically significant (P<0.05); when the dose of sodium butyrate increased to 400 μM, the concentration of Fe 2+ , MDA, LPO increased, the difference was statistically significant (P<0.05); and the MDA, LPO level in 200 μM of sodium butyrate was close to the concentration of Control group. The above results show that sodium butyrate can reduce the level of LPS-induced Fe 2+ and oxidative stress damage, and the effect is most significant when the dose is 200 μM.

[0100] 2. The effect of different interventions on cell proliferation

[0101] According to the experimental grouping, the cell proliferation was detected to explore the effect of different interventions on cell survival rate. The results are shown in Figure 3 : The cell survival rate of LPS group and LPS+ZK group was significantly lower than that of Control group, and the difference was statistically significant (P<0.05). It is proved that inhibiting VDR pathway accelerates the death of LPS cells; The cell survival rate of LPS+Fer-1 group was higher than that of LPS group, and the difference was statistically significant (P<0.05), which shows that inhibiting ferroptosis can improve cell mortality; The cell survival rate of LPS+sodium butyrate group was significantly higher than that of LPS group, and the difference was statistically significant (P<0.05), which shows that sodium butyrate treatment can improve the survival rate of HK-2 cells, and the effect is better than using Fer-1, and there is a statistical difference compared with LPS+Fer-1 group (P<0.05); There was a statistical difference between LPS+sodium butyrate+RSL3 group and LPS+sodium butyrate group (P<0.05), which shows that sodium butyrate treatment can improve the cell death caused by GPX4 inhibitor; The cell survival rate of LPS+sodium butyrate+ZK group was significantly higher than that of LPS+ZK group, and the difference was statistically significant (P<0.05), which shows that sodium butyrate can improve the cell death caused by VDR inhibitor. The above results show that ferroptosis inhibitor can improve the survival rate of cells, and sodium butyrate treatment can antagonize the effect of GPX4 inhibitor and VDR inhibitor on cells, and increase the survival rate of cells.

[0102] 3. Detection of inflammatory factors and oxidative stress indicators

[0103] 3.1 The effect of different interventions on the level of Fe 2+ , MDA, LPO in cells

[0104] The changes of Fe 2+ , MDA and LPO levels in cells were detected. The results are shown in Figure 4The results showed that the intracellular Fe 2+ , MDA and LPO concentrations were significantly reduced in the intervention group treated with sodium butyrate compared with the LPS group (P <0.05). The Fe 2+ , MDA and LPO concentrations in the LPS+Fer-1 group and the LPS+sodium butyrate group were significantly lower than those in the LPS group (P <0.05). The intracellular Fe 2+ , MDA and LPO levels in the LPS+sodium butyrate+ZK group were significantly lower than those in the LPS+ZK group (P <0.05), indicating that sodium butyrate treatment can improve cell death caused by VDR inhibitors. The Fe 2+ , MDA and LPO concentrations in the LPS+sodium butyrate+RSL3 group were significantly higher than those in the LPS+sodium butyrate group (P <0.05), indicating that GPX4 inhibitors can increase the levels of iron death-related indicators. The above results suggest that sodium butyrate treatment and iron death inhibitors can reduce the intracellular Fe 2+ , MDA and LPO concentrations, and sodium butyrate treatment can antagonize the damage of VDR and GPX4 to cells.

[0105] 3.2 Flow cytometry detection of intracellular lipid ROS levels

[0106] The intracellular lipid ROS levels were detected by flow cytometry to analyze the cellular oxygen metabolism levels. The results are shown in Figure 5 : Compared with the Control group, the intracellular lipid ROS levels in all intervention groups were significantly increased (P <0.05). The ROS level in the LPS+sodium butyrate+ZK group was lower than that in the LPS+ZK group (P <0.05). The ROS levels in the LPS+Fer-1 group and the LPS+sodium butyrate group were lower than those in the LPS group (P <0.05). The ROS level in the LPS+sodium butyrate+RSL3 group was higher than that in the LPS+sodium butyrate group (P <0.05), indicating that the use of GPX4 inhibitors can increase the ROS level. The above results suggest that the use of VDR inhibitors and GPX4 inhibitors increases the intracellular ROS level, while the use of iron death inhibitors and sodium butyrate alone can reduce the oxidative stress damage induced by LPS to a certain extent.

[0107] 3.3 IL-18 levels in cell supernatants

[0108] The IL-18 levels in the cell supernatants of each group were detected to analyze the effects of different interventions on the LPS cell model. The results are shown in Figure 6Results: Compared with the LPS model group, the IL-18 level in the supernatant of the LPS+Fer-1 group and the LPS+ sodium butyrate group was significantly reduced, and the difference was statistically significant (P<0.05), and sodium butyrate treatment was better than the iron death channel inhibitor; the IL-18 level in the supernatant of the LPS+ sodium butyrate+ZK group was significantly lower than that in the LPS+ZK group, and the difference was statistically significant (P<0.05), and sodium butyrate treatment can antagonize the effect of VDR inhibitor and reduce the inflammatory response of cells induced by LPS; the GPX4 level in the LPS+ sodium butyrate+RSL3 group was higher than that in the LPS+ sodium butyrate group, and the difference was statistically significant (P<0.05), indicating that the use of GPX4 preparation increased the IL-18 level. The above results show that sodium butyrate treatment and iron death channel inhibitors can reduce the inflammatory response of cells induced by LPS, and the use of GPX4 and VDR inhibitors increases the inflammatory response of cells.

[0109] 3.4 Effect of different interventions on VDR and GPX4

[0110] The levels of GPX4 in the supernatant of cells and the expression of VDR and GPX4 protein were detected by ELISA and WB experiments. The results are shown in Figure 7 、 Figure 8 and Figure 9 .

[0111] In the supernatant of cells, compared with the LPS model group, the GPX4 level in the LPS+Fer-1 group and the LPS+ sodium butyrate group was significantly increased, and the difference was statistically significant (P<0.05), and the use of iron death pathway inhibitor and sodium butyrate treatment can significantly improve the GPX4 level, and the effect of iron death pathway inhibitor is better than that of sodium butyrate treatment; compared with the LPS model group, the GPX4 level in the LPS+ZK group was significantly reduced, and the difference was statistically significant (P<0.05), indicating that the use of VDR inhibitor reduces the GPX4 level in the supernatant of cells; compared with the LPS+ZK group, the GPX4 level in the LPS+ sodium butyrate+ZK group was significantly increased, and the difference was statistically significant (P<0.05), indicating that in the case of using VDR inhibitor, sodium butyrate treatment can improve the GPX4 level to a certain extent; the results of the LPS+ sodium butyrate+RSL3 group show that the use of GPX4 inhibitor reduces the GPX4 level, and the difference is statistically significant compared with the LPS+ sodium butyrate group (P<0.05).

[0112] The results of VDR and GPX4 protein expression level detection show that the LPS+Fer-1 group and the LPS+sodium butyrate group significantly increase the VDR and GPX4 protein expression levels, and the difference is statistically significant compared with the LPS group (P<0.05). The sodium butyrate treatment promotes the expression of VDR and GPX4 proteins, and is better than the iron death pathway inhibitor. The results of the LPS+sodium butyrate+ZK group show that under the condition of using the VDR inhibitor, the sodium butyrate treatment can restore part of the expression of VDR and GPX4 proteins, and the difference is statistically significant compared with the LPS+ZK group (P<0.05). The results of the LPS+sodium butyrate+RSL3 group show that the use of the GPX4 inhibitor limits the expression of VDR and GPX4 proteins to a certain extent, and the difference is statistically significant compared with the LPS+sodium butyrate group (P<0.05).

[0113] The above shows that inhibiting the expression of VDR protein can affect the expression of GPX4 protein, and vice versa. The sodium butyrate treatment and the iron death pathway inhibitor can restore part of the expression of VDR and GPX4 proteins, and increase the GPX4 level of the cell supernatant.

[0114] 3.5 Discussion

[0115] RSL3 is an inhibitor of glutathione peroxidase 4 (GPX4), and the expression of GPX4 is controlled by selenium and glutathione (GSH). RSL3 can inhibit the cysteine / glutamate amino acid transport system that blocks GSH synthesis, and is an iron death activator that can exacerbate cell iron death under LPS treatment. In the present application, when RSL3 is added, the Fe 2+ , IL-18, MDA, LPO and ROS levels in HK-2 cells decrease, and the levels are close to those of the LPS group.

[0116] ZK compound is a new type of VDR (vitamin D receptor) inhibitor, which can specifically bind to VDR, thereby preventing the activation of vitamin D and its metabolites. In the present application, when sodium butyrate treatment is combined with ZK and GPX4 inhibitor administration, the anti-inflammatory and antioxidant effects of sodium butyrate are significantly weakened.

[0117] In summary, GPX4 inhibits iron death by reducing the level of lipid peroxide, and GPX4 is a key factor against iron death, and sodium butyrate treatment can reduce cell inflammatory response and oxidative damage by restoring the expression of VDR and GPX4.

[0118] Example 2 Animal experiment

[0119] 1. Establishment and grouping of mouse SA-AKI model

[0120] 1.1 Select lipopolysaccharide (LPS) injection method to establish a mouse SA-AKI model. SPF C57BL / 6J mice, male, 8-10 weeks old, purchased from Hangzhou Medical College Experimental Animal Center. Lipopolysaccharide (purchased from sigma company, type L2630) was dissolved in normal saline to prepare a solution with a mass concentration of 1g / L. C57BL / 6J mice were injected with lipopolysaccharide (LPS) 10 mg / kg via the tail vein to prepare a mouse model of sepsis acute kidney injury. The control group was given the same amount of normal saline by the same route. The modeling animals showed fever, significantly accelerated heart rate and respiratory rate, increased oral and nasal secretions, listlessness, lethargy, curling, piloerection, less movement, refusal or less food, and eye corner secretions, etc. as a successful model.

[0121] 1.2 Grouping of mouse SA-AKI model: 24 male mice were divided into blank control group (CON), simple model group (SA-AKI), SA-AKI+ sodium butyrate group according to different treatment intervention methods. Among them, 6 mice in the control group, 10 mice in the SA-AKI group, and 8 mice in the SA-AKI+ sodium butyrate group.

[0122] Control group: mice were given the same volume of 0.9% normal saline intraperitoneally, and were sacrificed after 72h. SA-AKI group: mice were given 10mg / kg LPS intraperitoneally, and were sacrificed after 72h. SA-AKI+ sodium butyrate group: mice were given 10mg / kg LPS intraperitoneally, and were given 200mg / kg / d sodium butyrate (purchased from sigma company) by gavage 5 days before modeling, for 5 days, and were sacrificed after 72h of modeling.

[0123] 1.3 Sample collection: blood, urine, and kidney tissue samples were collected from mice in each group at different time points. The samples were collected as follows: serum samples: whole blood was collected from mice at 24h (0.1-0.2ml whole blood) and 72h (0.5-1ml whole blood), respectively. After centrifugation, the upper serum was separated and aliquoted into frozen tubes and stored at -80°C. Urine samples: urine samples were collected from each mouse using a metabolic cage at 24h, 48h, and 72h, respectively, and aliquoted into frozen tubes and stored at -80°C. Kidney tissue: 1 / 6 of fresh kidney tissue was used for flow detection; 1 / 6 was formaldehyde fixed; 1 / 6 was glutaraldehyde fixed; 1 / 2 was directly frozen.

[0124] 1.4 Survival rate evaluation

[0125] The death / survival of animals at each time point (6h, 12h, 24h, 48h, 72h) before sacrifice was recorded after modeling.

[0126] 2. HE staining experiment of mouse kidney tissue

[0127] After the mouse kidney tissue is taken, it is washed with physiological saline to remove blood, and then fixed in 10% neutral formalin solution. Try to smooth the cut surface, place it in the embedding box, and transfer it to the automatic dehydrator for dehydration, transparency, and wax immersion steps. Take out the sample treated by the automatic dehydrator, immerse it in wax, and make the required wax block. Trim and slice the formed wax block, and the 4 μm tissue section is placed in a water bath at 42-50°C. The slide is taken out and baked for 20 min. The tissue section is placed in a constant temperature oven at 65°C for 1.5-2 h for baking. The tissue section is de-waxed: soaked in xylene for 10 min, and then soaked in xylene for another 10 min. Hydrate: anhydrous alcohol I and II for 5 min each, 95%, 90%, 80%, 70% alcohol, and distilled water for 5 min each. Stain with hematoxylin: re-stain with hematoxylin for 3 min. Differentiate with hydrochloric acid alcohol for a few seconds (1-2 s). Stop differentiation in tap water for 5 min. Dehydrate: place in distilled water, 70%, 80%, 90%, and 95% alcohol for 3 min each, and anhydrous ethanol I and II for 5 min each. Re-stain with eosin: contrast stain with 0.5% eosin ethanol solution for 1 min, wash off the excess red color by placing the section in 95% ethanol, and then place it in anhydrous ethanol for 5 min. Transparent: xylene I and II for 5 min each; neutral gum mounting.

[0128] 3. Transmission electron microscope observation of mouse kidney tissue cell ultrastructure

[0129] 3.1 Reagent preparation

[0130] 1% osmium tetroxide fixing solution: thoroughly clean the osmium tetroxide ampoule, wrap it with filter paper, and immediately place it in the mother liquor bottle after crushing the ampoule. Pour in pure water, close the bottle, mix well, and store in a 4°C refrigerator in the dark. Before use, mix equal volumes of osmium tetroxide mother liquor and 0.2M PBS (PH≈7.4) to obtain 1% osmium tetroxide working solution.

[0131] 3.2 Experimental steps

[0132] The sample is pre-fixed with 3% glutaraldehyde, and then fixed with 1% osmium tetroxide. It is dehydrated with acetone in stages, with a dehydration agent concentration gradient of 30%→50%→70%→80%→90%→95%→100% (100% concentration is replaced 3 times). It is infiltrated and embedded with a dehydration agent and Epon812 embedding agent in proportions of 3:1, 1:1, and 1:3, respectively, and finally embedded with Ep812. Ultra-thin sections of about 60-90 nm are prepared using an ultra-thin microtome, spread on a copper mesh, and then taken out. It is stained with uranyl acetate for 10-15 min, and then with lead citrate for 1-2 min at room temperature. The copper mesh is imaged using a JEM-1400 FLASH transmission electron microscope produced by Japan Electronics. Each copper mesh is first observed at 6000 times, and then the area to be observed is selected for image acquisition to observe the specific lesions.

[0133] 4. Indicator Testing

[0134] 4.1 Mouse blood SCr, BUN, and Fe 2+ LPO and MDA level detection

[0135] SCr, BUN, and Fe in mouse blood were detected using the following kits purchased from Nanjing Jiancheng Biotechnology Institute: SCr detection kit (C011-2-1), BUN detection kit (C013-2-1), serum iron detection kit (A039-1-1), LPO detection kit (A106-1-3), and MDA detection kit (A003-1-2). 2+ For LPO and MDA levels, please refer to the instructions for each kit for specific operating procedures.

[0136] 4.2 Detection of serum IL-18 and GPX4 levels in mice

[0137] The following kits were used to detect the serum IL-18 and GPX4 levels in mice: Mouse IL-18 ELISA Kit (purchased from Hangzhou Lianke Biotechnology Co., Ltd., catalog number: EK218) and Mouse Phospholipid hydroperoxide glutathione peroxidase, mitochondrial (GPX4) ELISA kit (purchased from Wuhan Huamei Biotechnology Co., Ltd., catalog number: CSB-EL009869MO). For specific operating procedures, please refer to the instructions of each kit.

[0138] 4.3 Detection of NGAL, KIM-1, and L-FABP levels in mouse urine

[0139] The following kits were used to detect the levels of NGAL, KIM-1, and L-FABP in mouse urine: Mouse neutrophil gelatinase-associated lipocalin, NGAL ELISA Kit (purchased from Wuhan Huamei Biotechnology Co., Ltd., catalog number: CSB-E09410m), Mouse Kidneyinjury molecule 1, Kim-1 ELISA Kit (purchased from Wuhan Huamei Biotechnology Co., Ltd., catalog number: CSB-E08809m), and Mouse FABP1 / L-FABP ELISA KIT (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number: SEKM-0239). Specific operating procedures can be found in the instructions for each kit.

[0140] 4.4 Fe in mouse kidney tissue 2+ Horizontal detection

[0141] The iron content (Fe) in mouse kidney tissue was detected using a tissue iron assay kit (catalog number: A039-2-1) purchased from Nanjing Jiancheng Biotechnology Institute. 2+ Level. For specific operating procedures, please refer to the instructions for each reagent kit.

[0142] 5. Detection of lipid ROS levels in mouse kidney tissue

[0143] Fresh kidney tissue was collected from mice in each group. The tissue blocks were rubbed on a 200-mesh sieve, and cells were washed downwards with PBS. Cells were collected by centrifugation, filtered through a 400-mesh sieve, and washed with PBS to prepare a single-cell suspension. The cells were incubated for 30 min with 1 mL of medium containing 5 μM C11 BODIPY 581 / 591 dye (Thermo Fisher Scientific). After incubation, the cells were washed three times with PBS, and then 1 mL of medium was added. Flow cytometry was used to quantify lipid ROS. The fluorescence ratio under the PE (590 nm) and FITC (510 nm) channels was measured to reflect the peroxidation level.

[0144] 6. Detection of GPX4 protein levels in mouse kidney tissue

[0145] 6.1 Solution Preparation

[0146] (1) Washing solution working solution: The washing solution is a 25-fold concentrated solution. Before use, measure 240ml of deionized water with a graduated cylinder, pour it into a beaker, and then measure 10ml of concentrated washing solution and add it evenly. Prepare the solution just before use.

[0147] (2) Biotin-labeled antibody working solution: Dilute the biotin-labeled antibody solution with biotin-labeled antibody diluent at a ratio of 1:100. For example, add 990 μl of biotin-labeled antibody diluent to 10 μl of biotin-labeled antibody, mix gently, and prepare within 10 min before use.

[0148] (3) Horseradish peroxidase-labeled avidin working solution: Horseradish peroxidase-labeled avidin is diluted 1:100 with horseradish peroxidase-labeled avidin diluent. For example, add 990 μl of horseradish peroxidase-labeled avidin diluent to 10 μl of horseradish peroxidase-labeled avidin, mix gently, and prepare within 10 min before use.

[0149] (4) Preparation of gradient concentrations of standards:

[0150] Remove one standard from the kit and centrifuge at 6000-10000 rpm for 30 seconds. Dissolve in 1 ml of sample diluent and mix thoroughly to obtain standard S7.

[0151] Dilution of standard: Take 7 1.5ml EP tubes (S0-S6) in turn, and add 250ul sample diluent to each. Take 250ul standard S7 to the first EP tube (S6), and mix gently. Take 250ul from S6 to the second EP tube (S5), and mix gently. Repeat the above steps to dilute the standard by ratio. S0 is the sample diluent.

[0152] 6.2 Sample processing

[0153] Add the kidney tissue to be tested to 1.0 mL PIPA and 10.0ul PMSF, and use a handheld homogenizer to homogenize the tissue on ice. After crushing the tissue, place it on ice for 30 minutes, and then centrifuge at 4℃, 10000 r / min for 15 minutes.

[0154] 6.3 Protein concentration determination

[0155] Strictly follow the experimental steps in the micro-BCA protein quantitative kit to detect the protein concentration. Add 200ul of the pre-prepared BCA working solution to the standard sample EP tube and the sample EP tube, and incubate at 37℃ for 15-30 minutes. Finally, use an enzyme-linked immunoassay instrument to detect 490nm, and calculate the protein concentration according to the standard curve.

[0156] 6.4 SDS-PAGE electrophoresis

[0157] (1) Glass plate cleaning and assembly: Use deionized water to prepare a 1% SDS solution, and soak the glass plate for 30 minutes. Gently brush the surface of the glass plate with a soft brush to remove residual colloidal and stains, and rinse with water for 5 minutes. Finally, rinse with ultrapure water for 3 times. Place the glass plate in a dust-free environment to dry, or dry it with nitrogen to avoid fiber residue. Align the long plate with the short plate, insert a 1.0mm thick gasket, and fix the two sides and the bottom with clamps to ensure no risk of glue leakage. Add a small amount of deionized water to the glass plate sandwich, and let it stand for 5 minutes. After confirming that there is no leakage, pour out the water.

[0158] (2) Glue filling and loading: Load the samples according to the order and volume of each sample.

[0159] (3) Electrophoresis running: Initial electrophoresis: 70V constant voltage electrophoresis until the bromophenol blue indicator migrates to the interface between the separation gel and the concentration gel. Observe the generation of bubbles in the electrophoresis tank, and adjust the voltage to 120V constant voltage electrophoresis until the bromophenol blue indicator migrates to the bottom of the gel. Terminate the operation: turn off the power, remove the gel plate, and prepare for membrane transfer or staining.

[0160] 6.5 Protein transfer

[0161] (1) PVDF membrane methanol pretreatment: Cut the PVDF membrane to the same size as the gel, immerse in 100% methanol for 3-5 minutes until the membrane changes from opaque to translucent. Transfer the membrane to ultrapure water for 2 minutes to remove residual methanol and avoid affecting the subsequent transfer efficiency. Immerse the PVDF membrane in the transfer buffer, stand at room temperature for 5 minutes to ensure that the membrane is fully soaked.

[0162] (2) Gel preparation and "sandwich" structure assembly: After electrophoresis, carefully remove the gel from the glass plate and immerse it in transfer buffer for 5 minutes to remove residual SDS. Transfer the removed gel to filter paper to form a "sandwich" structure (gel transfer accumulation layer, filter paper, gel, PVDF membrane, filter paper, gel transfer accumulation layer). Make sure to clean the gas during this process.

[0163] (3) Transfer operation: Place the assembled "sandwich" structure in the transfer clamp, making sure that the PVDF membrane faces the positive electrode and the gel faces the negative electrode. Tighten the transfer clamp to ensure that the layers are in close contact without looseness or air bubbles. Place an ice box outside the transfer tank or connect a circulating water cooling system to maintain the buffer temperature ≤10℃. Transfer at 300mA constant current according to the molecular weight of the protein. Low molecular weight section 20-100kDa, constant current 300mA, electrophoresis 60-90min; high molecular weight section 100-200kDa, constant current 300mA, electrophoresis 90-150min.

[0164] (4) Post-transfer treatment: Turn off the power, remove the transfer clamp, and carefully disassemble the "sandwich" structure. Immerse the PVDF membrane in TBST buffer and gently shake for 2 minutes to remove residual transfer buffer. Immerse the membrane in Ponceau S staining solution for 5 minutes to observe the protein band transfer efficiency. Wash the membrane with TBST buffer until the background is clear and record the transfer effect.

[0165] 6.6 Immunoblotting

[0166] Take out the PVDF / NC hybrid membrane after the transfer is completed, add Tris-Borate-Sodium Tween-20 (TBST) buffer solution for 5 minutes x 1 time; add 5% bovine serum albumin (BSA) blocking solution for 1 hour at room temperature; wash the hybrid membrane with TBST buffer solution for 5 min x 2 times, 10 min x 2 times; prepare NLRP3, Caspase-1, GSDMD primary antibody working solution according to the optimal dilution ratio, and place it at 4°C to the next morning; wash the hybrid membrane with TBST buffer solution for 5 min x 2 times, 10 min x 2 times; prepare the corresponding secondary antibody diluent according to the dilution of the primary antibody, and incubate it at 37°C under constant temperature conditions for 50 min; wash the hybrid membrane with TBST buffer solution for 5 min x 2 times, 10 min x 2 times; transfer the membrane to a clean plastic plate, and keep it in a wet state throughout. Cover the hybrid membrane surface with chemiluminescence substrate (ECL) evenly with a clean pipette, and continue to react for 5 min. Remove the excess substrate on the membrane surface with filter paper (built-in in the kit), place it on a colorless plastic plate, and chemiluminesce. The obtained results are analyzed by Image J software.

[0167] Experimental results:

[0168] 1. Survival rate observation

[0169] Death occurred in the SA-AKI group after modeling: 1 in the SA-AKI group died at 6 hours, and 1 in the SA-AKI group died at 24 hours. A total of 2 died naturally, with a mortality rate of 20%, and 8 remained for follow-up experiments. There was no death in the SA-AKI + sodium butyrate group and the control group.

[0170] 2. Mouse kidney tissue observation

[0171] 2.1 HE staining experiment

[0172] The pathological changes of the kidney tissues of the mice in each group were observed under a light microscope, and the results of the control group are shown in Figure 10A From Figure 10A it can be seen that the kidney tissue structure is normal, the glomerulus and renal tubule morphology is regular, and no obvious abnormalities are observed; the results of the SA-AKI group are shown in Figure 10B From Figure 10B it can be seen that the kidney tissue structure is arranged in disorder, part of the glomerulus atrophies with sclerotic changes, part of the renal tubular epithelial cells atrophies with vacuolar degeneration, part of the renal tubules dilate, part of the proximal tubular brush border disappears, and the interstitial chronic inflammatory cell infiltration is more; the results of the SA-AKI + sodium butyrate group are shown in Figure 10C From Figure 10C it can be seen that the atrophy and sclerosis of the kidney glomerulus are reduced, the vacuolar degeneration, brush border disappearance and dilated renal tubules are reduced, and the interstitial inflammatory cell infiltration is reduced.

[0173] 2.2 Ultrastructure of kidney tissue cells in mice

[0174] The ultrastructure of mitochondria in the kidneys of mice in each group was observed under an electron microscope at 1000x, as shown in Table 2.2. Figures 11A to 11C Figure 11A As can be seen from Table 2.2, the morphology of the renal tubular epithelial cells was normal; the nucleus was round, the chromatin was evenly distributed, and the heterochromatin was mainly distributed at the nuclear membrane; the mitochondria were normal in structure, with clear and straight cristae and complete structure. Figure 11A Figure 11B As can be seen from Table 2.2, the morphology of the renal tubular epithelial cells was abnormal; the nucleus was round, the chromatin was evenly distributed, and the heterochromatin was mainly distributed at the nuclear membrane; a large number of mitochondria were pyknosis, showing a trend of ferroptosis, with reduced volume, reduced, broken or disappeared cristae, and widened intercristae space; the electron density of the membrane and matrix was deepened; no obvious abnormalities were found in the rough endoplasmic reticulum; microvilli structures were observed on the free surface of the cells. Figure 11B Figure 11C As can be seen from Table 2.2, the ultrastructure of mitochondria in the kidneys of mice observed under an electron microscope showed that, compared with the SA-AKI group, the morphology of the renal tubular epithelial cells in the butyrate group was normal, the nucleus was round, the chromatin was evenly distributed, and the heterochromatin was mainly distributed at the nuclear membrane; the mitochondria were normal in structure, with clear and straight cristae and complete structure. According to the changes in the morphology of the mitochondria in the renal tubular epithelial cells, it was found that the pathological trend of the samples in the SA-AKI+butyrate group was lighter. Figure 11C 3. Levels of SCr, BUN and IL-18 in the blood of mice

[0175] The levels of SCr, BUN and IL-18 in the blood of mice at different time points were detected to evaluate the effect of butyrate on the renal function of mice.

[0176] The results are shown in Table 3.

[0177] Figure 12 As can be seen from Table 3, compared with the SA-AKI group, the levels of SCr, BUN and IL-18 in the SA-AKI+butyrate group at 24 h and 72 h were significantly reduced, with a statistical difference (P<0.05).

[0178] The above results show that intragastric administration of butyrate to mice can protect the renal function of SA-AKI mice.

[0179] 4. Levels of NGAL, KIM-1 and L-FABP in the urine of mice

[0180] ​​​​The effect of sodium butyrate on the renal function of mice was evaluated by detecting the changes in the levels of kidney injury markers (NGAL, KIM-1, L-FABP) in the urine of mice in different groups at different time points. The results are shown in Figure 13 : At the three time points of 24 h, 48 h and 72 h, the levels of NGAL, KIM-1 and L-FABP in the SA-AKI group were significantly higher than those in the SA-AKI + sodium butyrate group, with a statistically significant difference (P<0.05).

[0181] The above results show that intragastric administration of sodium butyrate to mice can improve the increase in kidney injury markers in the urine caused by LPS.

[0182] 5. Levels of indicators related to ferroptosis

[0183] 5.1 Fe 2+ , LPO, MDA and GPX4 levels in blood

[0184] The Fe 2+ levels of mice in each group at each time point were detected to observe the differences in Fe 2+ between groups. The results are shown in Figure 14 : The Fe 2+ content in the SA-AKI group was significantly higher than that in the control group and the SA-AKI + sodium butyrate group at the time points of 24 h, 48 h and 72 h (P<0.05). The results show that the administration of sodium butyrate to mice can prevent the accumulation of Fe 2+ leading to iron overload and reduce the level of high Fe 2+ caused by LPS.

[0185] The changes in the levels of LPO and MDA in each group of mice at each time point were detected to observe the occurrence of lipid peroxidation in mice. The results are shown in Figure 14 , Figure 15 : The levels of LPO and MDA in the SA-AKI group were significantly higher than those in the control group and the SA-AKI + sodium butyrate group at the time points of 24 h, 48 h and 72 h (P<0.05). The results show that the administration of sodium butyrate to mice can reduce the aggravation of lipid peroxidation in vivo caused by LPS.

[0186] The content of GPX4 in the serum of mice at each time point was detected to observe the expression of GPX4 in the serum of mice. The results are shown in Figure 15 : The content of GPX4 in the SA-AKI group was significantly lower than that in the control group and the SA-AKI + sodium butyrate group at the time points of 24 h and 48 h (P<0.05), indicating that the administration of sodium butyrate to mice can promote the expression of GPX4.

[0187] 5.2 Fe 2+ , lipid ROS and GPX4 levels in kidney tissue

[0188] The effect of sodium butyrate on oxidative stress of mouse kidney tissue was evaluated by detecting the change of Fe 2+ , lipid ROS content in mouse kidney tissue. The results are shown in the following table: Figure 16 The results show that the ROS content of kidney tissue is significantly increased compared with the control group, and the difference is statistically significant (P<0.05); compared with the SA-AKI group, the ROS content of the SA-AKI+ sodium butyrate group is significantly reduced, and the difference is statistically significant (P<0.05). The results show that sodium butyrate can reduce the lipid peroxidation reaction of kidney caused by LPS.

[0189] The GPX4 protein expression in mouse kidney tissue was detected by Western Blot method. The results are shown in the following table: Figure 17 The results show that compared with the control group, the GPX4 level in the SA-AKI group is significantly reduced, and the difference is statistically significant (P<0.05); the effect of sodium butyrate on the expression of kidney tissue GPX4 is shown: compared with the SA-AKI group, the GPX4 level in the SA-AKI+ sodium butyrate group is significantly increased, and the difference is statistically significant (P<0.05); compared with the control group, the GPX4 level in the SA-AKI+ sodium butyrate group is significantly reduced, and the difference is statistically significant (P<0.05). The results show that sodium butyrate can significantly increase the expression of GPX4 protein in mouse kidney tissue.

[0190] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any skilled person in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Use of sodium butyrate in the preparation of a medicament for preventing or treating sepsis-related acute kidney injury.

2. Use according to claim 1, characterized in that, The sodium butyrate improves the renal function of sepsis-related acute kidney injury, reduces cell inflammatory response and oxidative damage, and inhibits ferroptosis.

3. Use according to claim 1, characterized in that, The sodium butyrate inhibits ferroptosis by increasing the expression of VDR and GPX4 proteins.

4. A medicament or pharmaceutical composition for preventing or treating sepsis-related acute kidney injury, characterized by, The medicament or pharmaceutical composition comprises: sodium butyrate.

5. The medicament or pharmaceutical composition according to claim 4, characterized in that, The sodium butyrate improves the renal function of sepsis-related acute kidney injury, reduces cell inflammatory response and oxidative damage, and inhibits ferroptosis.

6. The medicament or pharmaceutical composition according to claim 4, characterized by, The sodium butyrate inhibits ferroptosis by increasing the expression of VDR and GPX4 proteins. The sodium butyrate improves the renal function of sepsis-related acute kidney injury, reduces cell inflammatory response and oxidative damage, and inhibits ferroptosis. The sodium butyrate inhibits ferroptosis by increasing the expression of VDR and GPX4 proteins.

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