Application of sodium butyrate in preparation of medicine for relieving brain injury caused by gas explosion

Abstract

The invention discloses application of sodium butyrate in preparation of a medicine for relieving brain injury caused by gas explosion, the sodium butyrate regulates nerve cell ferroptosis through a JNK / P38 MAPK pathway to relieve the brain injury caused by gas explosion, firstly, an animal model of rat brain injury caused by gas explosion is established, and the relation between NaB and brain tissue neuron injury is intervened and observed by applying NaB; secondly, a shock wave physiotherapy instrument is used for impacting a co-culture system of three cells of CTX, H19-7 and GMI-R to simulate explosion to construct an in-vitro experimental model, P38, ERK and Fer-1 inhibitors are adopted for intervention, iron metabolism, ferroptosis and MAPK signal channel factor changes are observed from the cell and molecular level, a mechanism for inhibiting ferroptosis through a related cascade signal channel mediated by NaB-mediated intestinal-brain axis regulation and control is clarified, and the effect of inhibiting ferroptosis is achieved. And a theoretical basis is provided for further explaining the action mechanism of the gas explosion brain injury.

Description

Technical Field

[0001] The invention belongs to the technical field of alleviating and treating brain damage caused by gas explosion and new medical applications of sodium butyrate, and particularly relates to application of sodium butyrate in preparing a drug for alleviating brain damage caused by gas explosion. Background Art

[0002] Gas explosions are common disasters in the coal industry and are considered a type of public health emergency. They often occur in mines or tunnels, causing a large number of casualties among miners. The threat of gas explosions to the health of miners has become a focus of public health attention. Due to the complexity of underground tunnel structures, gas explosion accidents often cause complex injuries to miners (LIJ, QINY, WANGZ, XINY. How to analyze the injury based on 24 Model: a case study of coal mine gas explosion injury [J]. Inj Prev, 2021, 27(6): 542-53; DICKSTEIN DL, DE GASPERIR, GAMA SOSAMA, et al. Brain and blood biomarkers of tauopathy and neuronal injury in humans and rats with neurobehavioral syndromes following blast exposure [J]. Mol Psychiatry, 2021, 26(10): 5940-54.). Although innovations in mining technology have led to a downward trend in the mortality rate of miners, the disability rate remains high, and the prognosis of patients is generally poor. Studies have shown that brain tissue is the main target organ of gas explosion shock waves. The mechanical shock generated by the explosion can cause brain tissue damage and ultimately lead to traumatic brain injury (TBI) (LU LH, REID MW, TROYANSKAYAM, et al. Close proximity to blast: No long-term orlasting effect on cognitive performance in service members with and without TBI during blast exposure [J]. J Int Neuropsychol Soc, 2023, 29 (6): 551-60.). Cognitive dysfunction (CD) is an important complication of gas explosion injury in coal miners, with a high long-term morbidity rate. Neuronal and glial cell damage is the main cause of the patient's disease, and effective diagnosis and treatment methods are urgently needed. TBI caused by gas explosions has significant pathological complexity.Previous studies by our research group have clearly confirmed the pathogenic association between gas explosions and TBI (DONGX, DENGL, YAO S, et al. Protective effects of curcumin against thyroid hormone imbalance after gas explosion-induced traumatic brain injury via activation of the hypothalamic-pituitary-thyroid axis in malerats [J]. Environ Sci Pollut Res Int, 2022, 29(49): 74619-31.). Existing evidence shows that gas explosions are closely related to neurobehavioral abnormalities (SULLIVAND R, MILLERMW, WOLF EJ, et al. Cerebral perfusion is associated with blast exposure in military personnel without moderate orsevere TBI [J]. J Cereb Blood Flow Metab, 2021, 41(4): 886-900.), and this damaging effect is more significant on the developing brain. The reason is that the injury markers produced after trauma can penetrate into the brain through pathways such as the blood-brain barrier, thereby producing neurotoxic effects.

[0003] In recent years, a large number of clinical and basic studies have confirmed that TBI can trigger multiple pathophysiological changes including neuronal death, blood-brain barrier dysfunction, and neuroinflammatory response (PAVLOVIC D, PEKIC S, STOJANOVIC M, POPOVIC V. Traumatic brain injury: neuropathological, neurocognitive and neurobehavioral sequelae [J]. Pituitary, 2019, 22 (3): 270-82.). From the perspective of disease progression, TBI injury can be divided into two stages: primary injury and secondary injury. Among them, secondary injury refers to the pathological process characterized by inflammatory response, oxidative stress and cell death that occurs after primary mechanical injury (CORPS KN, ROTH TL, MCGAVERN DB. Inflammation and neuroprotection in traumatic brain injury [J]. JAMA Neurol, 2015, 72 (3): 355-62.). These changes are often the key factors leading to neurodegeneration and cognitive dysfunction. Ferroptosis, a newly discovered form of programmed cell death, has been shown to be involved in the secondary injury process after traumatic brain injury (TBI). Abnormal activation of the ferroptosis pathway can exacerbate oxidative damage in brain tissue through iron-dependent lipid peroxidation, thereby aggravating cognitive dysfunction and becoming an important factor affecting patients' clinical prognosis (FANG J, YUAN Q, DU Z, et al. Overexpression of GPX4 attenuates cognitive dysfunction through inhibiting hippocampus ferroptosis and neuroinflammation after traumatic brain injury [J]. Free Radic Biol Med, 2023, 204: 68-81). Studies have shown that obvious activation of ferroptosis signals can be observed after TBI, and inhibiting ferroptosis through drug or genetic means can effectively alleviate secondary brain injury (GE Y, WANG T, HU Q, et al. Adiponectin ameliorates traumatic brain injury-induced ferroptosis through AMPK-ACC1 signaling pathway[J]. Brain Behav Immun, 2025, 126: 160-75.).Since secondary injury occurs within hours to days after primary injury, there is a time window for intervention. Therefore, targeted intervention of secondary injury mechanisms such as ferroptosis may become a key therapeutic strategy to improve the prognosis of TBI patients.

[0004] Ferroptosis is a new type of iron-dependent programmed cell death, which is typically characterized by the abnormal accumulation of lipid peroxidation products in cells. Unlike other forms of cell death such as apoptosis, necrosis and autophagy, ferroptosis has a unique molecular mechanism. In mammalian cells, phospholipid membranes rich in polyunsaturated fatty acids are the main targets of lipid peroxidation during ferroptosis (JIANG X, STOCKWELLB R, CONRAD M. Ferroptosis: mechanisms, biology and role in disease [J]. Nat Rev Mol Cell Biol, 2021, 22 (4): 266-82.). Related studies (LIANG D, MINIKES AM, JIANG X. Ferroptosis at the intersection of lipid metabolism and cellular signaling [J]. Mol Cell, 2022, 82(12): 2215-27.) have shown that system Xc- (cystine / glutamate antiporter) and GPX4 (glutathione peroxidase 4) constitute the core molecular hubs regulating lipid peroxidation and ferroptosis. Neuronal membranes are rich in cholesterol and polyunsaturated fatty acids (PUFAs) and are highly sensitive to ROS-related peroxidation. In addition, the ability of neurons to clear reactive oxygen species in an autonomous manner is limited because the levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX4) in the brain are lower than in other types of tissues, and the levels of SOD and GPX4 in the late stages of oxidative metabolism are far from the amount required for the entire oxidative process (PARK E, CHUNG S W. ROS-mediated autophagy increases intracellular iron levels and ferroptosis by ferritin and transferrin receptor regulation [J]. Cell Death Dis, 2019, 10 (11): 822.).The above evidence indicates that neurons are particularly sensitive to damage caused by excessive iron. At the same time, studies have shown that sodium butyrate can inhibit ferroptosis and alleviate cell steatosis through the XCT / GPX4 / ROS pathway (CHENG X, HUY, YUX, et al. Sodium Butyrate Alleviates Free Fatty Acid-Induced Steatosis in Primary Chicken Hepatocytes via Regulating the ROS / GPX4 / Ferroptosis Pathway[J]. Antioxidants(Basel), 2024, 13(2).). However, it is still unclear whether brain tissue damage caused by gas explosions is associated with the ferroptosis process mediated by the JNK / P38 MAPK pathway.

[0005] A large number of studies (WANG Q, YANG Q, LIU X. The microbiota-gut-brain axis and neurodevelopmental disorders [J]. Protein Cell, 2023, 14 (10): 762-75.) have shown that the role of the gut microbiota-gut-brain axis in neurological diseases is increasingly being recognized. Short-chain fatty acids (SCFAs) produced by intestinal microbial fermentation of dietary fiber are a class of metabolites with important physiological functions, mainly composed of acetic acid, propionic acid, and butyric acid. As key substances for maintaining normal physiological functions of the colon and meeting the metabolic needs of the body, these three fatty acids play an indispensable role in the body (SILVAY P, BERNARDI A, FROZZA RL. The Role of Short-Chain Fatty Acids From Gut Microbiota in Gut-Brain Communication [J]. Front Endocrinol (Lausanne), 2020, 11: 25.). Among them, butyrate, due to its unique molecular structure and metabolic properties, exhibits more significant biological activity than acetate and propionate. As an important product of gut microbiota metabolism, short-chain fatty acids (SCFAs), especially sodium butyrate (NaB), have been shown to act on the central nervous system through multiple pathways: directly through the blood-brain barrier; indirectly through vagal afferents or by regulating immune cell function (WEI H, YU C, ZHANG C, et al. Butyrate ameliorates chronic alcoholic central nervous damage by suppressing microglia-mediated neuroinflammation and modulating the microbiome-gut-brain axis [J]. Biomed Pharmacother, 2023, 160: 114308.). As an important short-chain fatty acid produced by gut microbiota metabolism, sodium butyrate (NaB) has attracted much attention in recent years due to its multi-target neuroprotective effects.Studies (PATNALA R, ARUMUGAM TV, GUPTA N, DHEEN S T. HDACInhibitor Sodium Butyrate-Mediated Epigenetic Regulation Enhances Neuroprotective Function of Microglia During Ischemic Stroke [J]. Mol Neurobiol, 2017, 54 (8): 6391-411.) have shown that NaB can not only inhibit the activity of histone deacetylase (HDAC) and regulate epigenetic modification, but also has significant anti-inflammatory and anti-oxidative stress effects. Studies have shown through preliminary experiments that NaB pretreatment can significantly inhibit the activation of the JNK / p38 MAPK pathway in neurons in the oxygen glucose deprivation / reoxygenation (OGD / R) model, while reducing intracellular iron content and lipid ROS levels. These results strongly suggest that NaB may inhibit ferroptosis by regulating the JNK / p38 MAPK pathway, but its specific mechanism of action remains to be elucidated.

[0006] The signaling pathways of the MAPK family mainly include ERK (extracellular signaling regulated protein kinase), JNK / SAPK (c-JunNterminal kinase / stress-activated protein kinase), p38 MAPK and ERK5 / four pathways (CHEN X, NINGY, WANG B, et al. HET0016 inhibits neuronal pyroptosis in the immature brain post-TBI via the p38 MAPK signaling pathway[J]. Neuropharmacology, 2023, 239: 109687.). The sustained activation of JNK and p38 MAPK signaling pathways mediates neuronal apoptosis and inflammation in neurodegenerative diseases such as AD (LU H, TANA, ZHANGY, et al. Neuroprotective effects of Shenghui decoction via inhibition of the JNK / p38 MAPK signaling pathway in an AlCl(3)-induced zebrafish(Danio rerio)modelof Alzheimer's disease[J]. J Ethnopharmacol, 2024, 328: 117993.), while the ERK signaling pathway is involved in the process of tumorigenesis, including cancer cell proliferation, migration and invasion (ROBERTS PJ, DER CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer[J]. Oncogene, 2007, 26(22): 3291-310.). As a core regulator of cellular stress responses, the JNK / p38 MAPK signaling pathway plays a key role in oxidative stress and cell death. Activation of the p38 MAPK pathway is crucial for inflammatory responses and apoptosis in response to various stimuli. For example, various stress stimuli, such as hypoxia and ischemia, can phosphorylate p38 MAPK, which then acts as an upstream factor to induce apoptosis.Previous studies have also found that the p38 MAPK signaling pathway also induces ferroptosis in multiple myeloma cells (LI W, FU H, FANG L, et al. Andrographolide induced ferroptosis in multiple myeloma cells by regulating the P38 / Nrf2 / HO-1 pathway [J]. Arch Biochem Biophys, 2023, 742: 109622.). The JNK pathway also induces ferroptosis in colorectal cancer cells through NCOA4-mediated ferritinophagy (ZHAO L, MIAO H, QUAN M, et al. Beta-Lapachone induces ferroptosis of colorectal cancer cells via NCOA4-mediated ferritinophagy by activating the JNK pathway [J]. Chem Biol Interact, 2024, 389: 110866.). Another study (SUN C, GAO X, SHA S, et al. Berberine alleviates Alzheimer's disease by activating autophagy and inhibiting ferroptosis through the JNK-p38 MAPK signaling pathway [J]. Int Immunopharmacol, 2025, 155: 114550.) found that inhibiting the JNK-P38 MAPK signaling pathway activated autophagy and inhibited ferroptosis, thereby alleviating Alzheimer's disease symptoms in rats. This further reduced the deposition of Aβ brain plaques, suppressed the inflammatory response, and improved neuronal damage. However, whether the JNK / p38 MAPK pathway plays a role in NaB alleviating gas explosion-induced neuronal ferroptosis remains unclear and warrants further investigation. Summary of the Invention

[0007] The purpose of the present invention is to provide the use of sodium butyrate in the preparation of a drug for alleviating gas explosion-induced brain damage. By establishing a rat gas explosion brain damage model, the protective effect of sodium butyrate on gas explosion-induced brain damage and the regulatory effect of sodium butyrate on ferroptosis based on the JNK / P38 MAPK signaling pathway are explored.

[0008] To achieve the above-mentioned purpose, the present invention adopts the following technical solution: use of sodium butyrate in the preparation of a drug for alleviating brain damage caused by gas explosion, wherein sodium butyrate regulates ferroptosis of neurons through the JNK / P38 MAPK pathway to alleviate brain damage caused by gas explosion.

[0009] Furthermore, the sodium butyrate alleviates brain damage caused by gas explosion by reducing brain neuroinflammation induced by gas explosion.

[0010] Furthermore, the sodium butyrate inhibits ferroptosis of nerve cells by inhibiting the reduced expression of ferroptosis-related factors GPX4 and SLC7A11 caused by gas explosion, thereby alleviating brain damage caused by gas explosion.

[0011] Furthermore, the sodium butyrate alleviates brain damage caused by gas explosion by downregulating the activity of JNK and P38 MAPK signaling pathways in the hippocampus tissue of rats caused by gas explosion.

[0012] Furthermore, the sodium butyrate alleviates brain damage caused by gas explosion by improving cell migration and repair capabilities.

[0013] Furthermore, the sodium butyrate is used in combination with a pathway inhibitor to alleviate brain damage caused by gas explosion.

[0014] Furthermore, the pathway inhibitor is one or more of the JNK inhibitor SP600125, the P38 MAPK inhibitor SB203580 and the ferroptosis inhibitor Fer-1.

[0015] Furthermore, the sodium butyrate inhibits ferroptosis of nerve cells by negatively regulating the JNK / p38 MAPK signaling pathway, thereby alleviating brain damage caused by gas explosion.

[0016] The specific research process of the present invention is to use SPF-grade SD male rats as an in vivo experimental model and a co-culture system constructed with CTX, H19-7, and GMI-R cells as an in vitro experimental model. The experimental rats were randomly divided into four groups (n=8): a control group, a gas explosion model group, an antibiotic clearance group, and a sodium butyrate intervention group (1200 mg / kg). The experiment used a shock tube device to simulate the gas explosion, and the changes in the rats' weight, heart rate, and blood pressure were analyzed after the explosion. The mine experiment detected the total movement distance and average movement speed of the rats. HE staining and transmission electron microscopy were used to observe the pathological damage and mitochondrial microstructural changes in rat brain tissue. Immunofluorescence was used to detect the expression and distribution of p38 mitogen-activated protein kinase (p38MAPK), phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK), solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in rat hippocampus tissue. Immunohistochemistry was used to detect the expression and distribution of interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor α (TNF-α) in rat hippocampus tissue. 2+ ) and malondialdehyde (MDA) content; Western blotting and qPCR were used to detect the protein and mRNA expression levels of c-Jun amino-terminal kinase (JNK), phosphorylated c-Jun amino-terminal kinase (p-JNK), p38 MAPK, p-p38 MAPK, IL-6, IL-10, TNF-α, SLC7A11 and GPX4. The CCK8 method and wound wound test were used to detect the effects of gas explosion shock wave on cell proliferation rate and migration and repair ability; fluorescent probe was used to detect cell reactive oxygen species (ROS) level, and Fe 2+ , MDA, and SOD levels in the brain tissue. Immunofluorescence staining was used to detect the fluorescence expression and nuclear translocation of JNK and p-JNK in the cells. The JNK inhibitor SP600125, the P38 MAPK inhibitor Adezmapimod, and the ferroptosis inhibitor Fer-1 were used to investigate the relationship between gas explosion-induced JNK / P38 MAPK pathway activation and gas explosion-induced brain ferroptosis. After sodium butyrate intervention, the protective mechanism of sodium butyrate against gas explosion-induced brain damage was explored.

[0017] The present invention has the following advantages and beneficial effects: First, a stable gas explosion-induced brain injury model was established in rats using a shock tube biocidal system developed by our research group to simulate a gas explosion. NaB was then used as an intervention to observe the relationship between NaB and brain neuronal damage, clarifying the role of sodium butyrate in activating ferroptosis in CD neuronal injury. Second, a shock wave therapy device was used to simulate an explosion in a co-culture system of CTX, H19-7, and GMI-R cells to construct an in vitro experimental model. P38, ERK, and Fer-1 inhibitors were then used to intervene, observing changes in iron metabolism, ferroptosis, and MAPK signaling pathway factors at the cellular and molecular levels. This study elucidated the mechanism by which NaB mediates the gut-brain axis's regulation of MAPK-mediated signaling cascades to inhibit ferroptosis. This provides a theoretical basis for further elucidating the mechanism of gas explosion-induced brain injury and offers new insights and scientific evidence for the clinical diagnosis and treatment of CD in coal miners induced by gas explosions. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 2. Changes in body weight, heart rate, blood pressure, and exploratory behavior of rats during modeling and drug administration. A- Changes in body weight of rats in each group; B- Changes in heart rate and blood pressure of rats in each group; C- Total distance moved by rats in each group; D- Average moving speed of rats in each group. Data are expressed as mean ± SD (n = 8). Compared with the CON group, **P < 0.01; compared with the ABX group, ^P < 0.05. CON: Control group, no treatment; MOD: Model group, rats were shocked by gas explosion; ABX: Antibiotic removal group, before the gas explosion, the rats were treated with combined antibiotics to remove intestinal flora, and were kept normally after the gas explosion; CB group: Antibiotic removal was performed before the gas explosion, and sodium butyrate was administered orally at a dose of 1.2 g / kg after the explosion. Except for the CON group, rats in the other groups underwent open field test one day before the gas explosion and one day before sample collection. Body weight, heart rate, and blood pressure were measured on the day of the gas explosion.

[0019] Figure 2Sodium butyrate alleviates gas explosion-induced neuroinflammation. A-H&E staining was used to assess histological changes in the rat hippocampus; black arrows indicate inflammatory cell infiltration (x200). B- Transmission electron microscopy (TEM) was used to observe pathological changes in hippocampal tissue; black boxes indicate 2000x magnification, and red arrows indicate 8000x magnification. C- Immunohistochemistry was used to detect the expression of IL-6, IL-10, and TNF-α proteins in hippocampal tissue; DF- Changes in IL-6, IL-10, and TNF-α protein expression; GH- qPCR was used to detect the expression levels of inflammatory factor-related genes in hippocampal tissue: G- IL-6 gene expression level; H- IL-10 gene expression level; I- TNF-α gene expression level; KM- Western blot was used to detect the expression levels of inflammatory factor-related proteins in hippocampal tissue: L- IL-6 protein quantitative analysis; M- IL-10 protein quantitative analysis. Data are expressed as mean ± SD (n = 3). Compared with the CON group, **P<0.01, *P<0.05; compared with the MOD group, ## P < 0.01; compared with the ABX group, ^P < 0.05. CON: control group, no treatment; MOD: model group, rats subjected to gas explosion shock; ABX: antibiotic decongestion group, rats received combined antibiotics to decongest intestinal flora before the gas explosion and were housed normally after the gas explosion; CB: antibiotic decongestion group, rats received 1.2 g / kg sodium butyrate orally after the gas explosion.

[0020] Figure 3 Sodium butyrate reduces ferroptosis in hippocampal neurons of rats exposed to gas explosion. A- Immunofluorescence was used to detect the expression of ferroptosis core inhibitory protein GPX4 and ferroptosis regulatory factor SLC7A11; B- Ferrous ions (Fe 2+ ) content; C-malondialdehyde (MDA) content in hippocampal tissue; DF-western blot detection of GPX4 and SLC7A11 expression levels in hippocampal tissue; E-quantitative analysis of GPX4 protein; F-quantitative analysis of SLC7A11 protein; GH-qPCR detection of GPX4 and SLC7A11 expression levels in hippocampal tissue; G-GPX4 gene expression level; H-SLC7A11 gene expression level. Data are expressed as mean ± SD (n = 3). Compared with the CON group, **P < 0.01, *P < 0.05; compared with the MOD group, ##P < 0.01; compared with the ABX group, ^P < 0.05. CON: control group, no treatment; MOD: model group, rats subjected to gas explosion shock; ABX: antibiotic decongestion group, rats received combined antibiotics to decongest intestinal flora before the gas explosion and were housed normally after the gas explosion; CB: antibiotic decongestion group, rats received 1.2 g / kg sodium butyrate orally after the gas explosion.

[0021] Figure 4 Sodium butyrate significantly inhibited the abnormal activation of the JNK / P38 MAPK signaling pathway in the hippocampus of rats exposed to gas explosions. A- Immunofluorescence was used to detect the expression of P38 and p-P38; BD- western blot was used to detect the expression levels of P38, p-P38, JNK, and p-JNK in the hippocampus; C- quantitative analysis of p-P38 protein; D- quantitative analysis of p-JNK protein; EF- western blot was used to detect the expression levels of P38 and JNK in the hippocampus; E- GPX4 gene expression level; F- SLC7A11 gene expression level. Data are expressed as mean ± SD (n = 3). Compared with the CON group, **P < 0.01; compared with the MOD group, ## P < 0.01; compared with the ABX group, ^P < 0.05. CON: control group, no treatment; MOD: model group, rats subjected to gas explosion shock; ABX: antibiotic decongestion group, rats received combined antibiotics to decongest intestinal flora before the gas explosion and were maintained normally after the gas explosion; CB: antibiotic decongestion group, rats received 1.2 g / kg sodium butyrate orally after the gas explosion.

[0022] Figure 5Sodium butyrate improves the survival rate of neurons after shock wave induction. AD - Determination of modeling conditions for CTX, H19-7, and GMI-R cells in a co-culture system; A - Cell survival rate after 12 hours of culture at different shock times; B - Cell survival rate after 12 hours of culture at 1500 shocks at different energy levels; C - Cell survival rate after 24 hours of culture at 1500 shocks at different energy levels; D - Cell survival rate after 48 hours of culture at 1500 shocks at different energy levels; E - Evaluation of cell survival after establishment of an in vitro cerebral concussion injury model; FI - Determination of different drug intervention concentrations; F - Neuronal cell viability after treatment with different concentrations of NaB; G - Neuronal cell viability after treatment with different concentrations of SB203580 (P38 MAPK pathway inhibitor); H - Neuronal cell viability after treatment with different concentrations of SP600125 (JNK pathway inhibitor); I - Neuronal cell viability after treatment with different concentrations of Fer-1 (ferroptosis inhibitor); J - Effects of sodium butyrate on neuronal migration and repair capacity induced by shock waves; K - Analysis of neuronal cell migration after treatment with sodium butyrate. Data are expressed as mean ± SD (n = 3). Compared with the CON group, **P<0.01, *P<0.05. Mod: Cultured under defined shock modeling conditions for 12 h; NaB: Culture medium containing 1 mmol / L NaB for 12 h; SB203580: Culture medium containing 2.5 μmol / L SB203580 for 12 h; SP600125: Culture medium containing 2.5 μmol / L SP600125 for 12 h; Fer-1: Culture medium containing 1.25 μmol / L Fer-1 for 12 h. All groups received shock wave treatment except the CON group (untreated).

[0023] Figure 6 Sodium butyrate can alleviate the shock wave-induced ferroptosis of nerve cells. A-fluorescence intensity of ROS in different groups; B-quantitative analysis of ROS fluorescence intensity; C-ferrous ions (Fe 2+ ) content; D-malondialdehyde (MDA) content in different groups; E-superoxide dismutase (SOD) content in different groups. Data are expressed as mean ± SD (n = 3). **P < 0.01, compared with the Con group; ##P < 0.01, compared with the Mod group. Mod: cultured under defined shock modeling conditions for 12 h after shock; NaB: cultured with 1 mmol / L NaB for 12 h; SB203580: cultured with 2.5 μmol / L SB 203580 for 12 h; SP600125: cultured with 2.5 μmol / L SP600125 for 12 h; Fer-1: cultured with 1.25 μmol / L Fer-1 for 12 h; combined treatment groups (SB203580+NaB, SP600125+NaB, Fer-1+NaB) received 1 mmol / L NaB added to the corresponding inhibitor culture medium for 12 h. All groups except the CON group (untreated) received shock wave treatment.

[0024] Figure 7 Sodium butyrate negatively regulates the activity of the JNK / p38 MAPK signaling pathway to inhibit the ferroptosis process in neurons. AD-qPCR was used to detect the transcriptional levels of the JNK / p38 MAPK signaling pathway and ferroptosis-related factors; EI-Western blot was used to detect the expression levels of the JNK / p38 MAPK signaling pathway and ferroptosis proteins. Data are expressed as mean ± SD (n = 3). **P < 0.01, compared with the Con group. ## P<0.01, # P < 0.05, compared with the Mod group. Mod: cultured under the defined shock modeling conditions for 12 h; NaB: culture medium containing 1 mmol / L NaB, treated for 12 h; SB203580: culture medium containing 2.5 μmol / L SB 203580, treated for 12 h; SP600125: culture medium containing 2.5 μmol / L SP600125, treated for 12 h; Fer-1: culture medium containing 1.25 μmol / L Fer-1, treated for 12 h; combined treatment groups (SB203580+NaB, SP600125+NaB, Fer-1+NaB) were treated with 1 mmol / L NaB added to the corresponding inhibitor culture medium for 12 h. All groups except the CON group (untreated) received shock wave treatment.

[0025] Figure 8 Figure 2 is a potential mechanism of action of the present invention. Sodium butyrate can alleviate brain tissue damage and ferroptosis caused by gas explosions, and this effect is related to inhibiting the activation of the JNK / P38MAPK pathway in neurons. DETAILED DESCRIPTION

[0026] The above contents of the present invention are further described in detail below through examples, but this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above contents of the present invention fall within the scope of the present invention.

[0027] Example

[0028] 1. Materials and Methods

[0029] 1.1 Drugs, reagents and instruments

[0030] Neomycin sulfate (N109017), natamycin (P107822), and sodium butyrate (S102956) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Bacitracin (S17005) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. RNA extraction Total RNA extraction reagent (R401-01) was purchased from Nanjing Weizyme Biotechnology Co., Ltd. Protein quantification kit (BCA Assay) (KTD3001) was purchased from Abbkine (Wuhan) Science Co., Ltd. BeyoECL Star (ultra-high sensitivity ECL chemiluminescence kit) (P0018AS) was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. High glucose culture medium (DMEM, ZQ-100), penicillin (CSP006), streptomycin, 0.25% trypsin digestion solution (CSP087), phosphate buffer (PBS, ZQ1300) were purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. CCK-8 cell counting box (A311), RNA extraction reagent (R401), IIIRT 125Super Mix for q PCR(R323) and Ace Universal SYBR qPCR Master Mix (Q511), fetal bovine serum (FBS, F101-00), and PVDF membrane (E801-01) were purchased from Vazyme Biotech. xCT antibody (DF12509), JNK1 / 2 / 3 antibody (AF6318), and Phospho-JNK1 / 2 / 3 antibody (AF3318) were purchased from Jiangsu Qinke Biological Research Center Co., Ltd. Goat anti-rabbit IgG (H+L) (bs-0295), rabbit anti-β-Actin antibody (bs-0061R), P38 MAPK antibody (bs-0637R), Phospho-P38 MAPK antibody (bs-5476R), IL-10 antibody (bs-0698R), IL-6 antibody (bs-6309R), and TNF-a antibody (bs-10802R) were purchased from Beijing Biosen Biotechnology Co., Ltd., and GPX4 antibody (BM5231) was purchased from Wuhan Boster Biotechnology Co., Ltd. GMI-R1 rat microglia (HTX4068), H19-7 rat hippocampal neuronal cells (HTX3536), and CTX rat astrocytes (HTX2248) were purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd.; SB203580 (p38 MAPK inhibitor, HY-10256), SP600125 (JNK inhibitor, HY-12041), and Ferrostatin-1 (ferroptosis inhibitor, HY-100579) were purchased from MedChemexpress Biotechnology Inc.; and ferrous ions (Fe 2+ ) content detection kit (BC5415). Malondialdehyde (MDA) content detection kit (BC0025), reactive oxygen species (ROS) detection kit (red fluorescence, CA1420), and high-efficiency RIPA tissue / cell lysis buffer (R0010) were purchased from Beijing Solarbio Technology Co., Ltd. A protein quantification kit (BCA method, KTD3001) was purchased from Abbkine Scientific; (immunofluorescence secondary antibody) was purchased from Abbkine Scientific. All primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0031] 1.2 Animals and Experimental Design

[0032] This study was conducted in accordance with the international standards for the care and use of laboratory animals. Forty healthy male Sprague-Dawley rats, weighing (100 ± 5) g, were provided by Henan Skobes Biotechnology Co., Ltd. (China (License: SCXK (Yu)) 2020-0005). The SD rats were housed in standard cages and maintained under typical environmental conditions of temperature (21 ± 1°C) and relative humidity (50 ± 10%). A 12-hour light / 12-hour dark cycle was maintained regularly. All rats underwent a one-week adaptation period before the formal experiment.

[0033] The rats were randomly divided into 4 groups (n = 8 / group): The control group (CON) received no treatment; The GE model group (MOD): The shock wave tube gas explosion biological lethality detection experimental system designed by Northern Petrochemical Equipment Factory in Huludao, China (Utility model patent: CN 214150529U) was used for GE exposure. The rats were anesthetized with pentobarbital and fixed in an iron cage facing the explosion source. A methane mixture with a volume content of 10% and a shock wave of 23.6 J were used to simulate the GE model group. The rats were positioned 2.4 m from the explosion source; The antibiotic clearance + GE group (ABX): Neomycin sulfate (5 mg / mL), natamycin (1.25 μg / mL), and bacitracin (5 mg / mL) were dissolved in the drinking water of the rats to prepare a combined antibiotic drinking water for the rats to achieve the purpose of antibiotic clearance. The intestinal flora of the GE model group was cleared with the combined antibiotic before exposure, and the rats in the GE model group were fed normally after exposure. The sodium butyrate + ABX group (CB): Seven days before the gas explosion, the intestinal flora was cleared with the combined antibiotic. After the gas explosion, 1.2 g of sodium butyrate was required per 1000 g of body weight. For every 1.2 g of sodium butyrate, 10 mL of normal saline was used to make a sodium butyrate solution, which was intraperitoneally injected into the rats in this group. This study was approved by the Animal Medical Ethics Committee of Xinxiang Medical University (XYLL-2020007).

[0034] 1.3 Sample collection and processing

[0035] The body weight of the rats was recorded daily after gavage, and euthanasia was performed on the 7th day using 1 wt% sodium pentobarbital. Blood was collected from the abdominal aorta, incubated at 4°C for 2 hours, and centrifuged at 3000 rpm for 15 minutes. After centrifugation, the serum was separated and stored in a -80°C refrigerator. Then, the brain tissues were collected and stored at -80°C for future use. In addition to macroscopic examination of the collected tissues, the brain tissues (n = 3) were also subjected to hematoxylin-eosin staining (H&E) and transmission electron microscopy (TEM) examination. These operations were to facilitate the subsequent observation of histopathological changes and the evaluation of tissue damage. The remaining tissues were stored in a -80°C refrigerator for molecular biology experiments.

[0036] 1.4 Antibiotic clearance experiment

[0037] Neomycin sulfate (5 mg / mL), bacitracin (5 mg / mL) and natamycin (1.25 μg / mL) were co-dissolved in purified water to facilitate antibiotic clearance by rats through free drinking, and the remaining rats drank purified water.

[0038] 1.5 Blood pressure and heart rate test

[0039] Blood pressure and heart rate were measured before euthanasia. Four groups of rats (n = 8) were tested sequentially. Rats were positioned in the testing apparatus and tail-tip blood pressure was measured, with five replicates per rat. Data were collected using an intelligent noninvasive blood pressure monitor (Softron BP-2010 series, China) according to the operating manual, and statistical analysis was performed using Graphpad Prism 8.

[0040] 1.6 Open Field Test (OFT)

[0041] The apparatus consisted of an 80×80 black square base and a 50 cm long black wall. The base was divided into 16 equally spaced squares, with a central area consisting of 4 squares and the remaining 12 squares as peripheral areas. Each rat was gently placed in the center of the apparatus and allowed to explore freely for 3 minutes before being captured. Subsequently, all rats were observed in the open field box for 5 minutes using a video tracking system. After each training session, the open field box was cleaned with a 10% volume alcohol solution to mitigate any potential odor effects on subsequent rats. Locomotor activity, total distance moved, and average distance moved were assessed based on the distance covered across the entire area.

[0042] 1.7 Hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM)

[0043] After sample collection, histopathological examination was performed to assess GE-induced TBI. Brain tissue from each group of rats (n=3) was pre-fixed in 4 wt% paraformaldehyde, then embedded, sectioned, stained, and mounted. H&E-stained samples were then examined using an upright light microscope (Nikon Eclipse E100, Japan), and the resulting images were captured and analyzed using an imaging system (NIKON DS-U3, Japan).

[0044] For TEM analysis, the brain tissue of each group (n=3) of rats was fixed with 2.5wt% glutaraldehyde solution. Each sample was cut into 1mm^3 cubes and immersed in the fixative at 4°C for 4 hours. Subsequently, the tissue was fixed in 1wt% osmium tetroxide for 2 hours at room temperature and then dehydrated using a gradient alcohol series. The specimens were then embedded in resin and baked at 60°C for 48 hours for polymerization. Ultrathin sections (60nm) were made using an ultramicrotome. Finally, the tight junctions in the brain tissue were examined for ultrastructure using a transmission electron microscope (HITACHI, HT7700, Japan).

[0045] 1.8 Targeted short-chain fatty acid detection

[0046] Thaw the sample on ice and transfer an appropriate amount to a 2 mL centrifuge tube. Resuspend in 50 μL of 20 wt% phosphoric acid. Add 4-methylvaleric acid as an internal standard to a final concentration of 500 μM and vortex to mix for 2 minutes. Centrifuge at 14,000 g for 20 minutes. Remove the supernatant and transfer it to a sample injection vial for GC-MS analysis. Inject 1 μL of sample, split ratio 10:1, split injection. Chromatography-mass spectrometry conditions: 1) Chromatographic conditions: Samples were separated using an Agilent DB-FFAP capillary column (30 m x 250 μm x 0.25 μm) using a gas phase chromatography system. Temperature program: Initial temperature 90°C; increase to 160°C at 10°C / min; then increase to 240°C at 40°C / min and hold for 5 minutes. Carrier gas was helium at a flow rate of 1.0 mL / min. A QC sample was set every few experimental samples in the sample queue to test and evaluate system stability and reproducibility. 2) Mass Spectrometry Conditions: Mass spectrometry analysis was performed using an Agilent 5977B MSD mass spectrometer. The 5977B MSD conditions were as follows: inlet temperature, 250°C; ion source temperature, 230°C; transfer line temperature, 250°C; and quadrupole temperature, 150°C. Electron impact ionization (EI) source, electron energy, 70 eV; analytes were detected in SCAN / SIM mode.

[0047] 1.9 IHC staining

[0048] For IHC staining, brain tissue sections were incubated with IL-6, TNF-a, and IL-10 antibodies (dilution 1:500) at 4°C overnight. Subsequently, the sections were treated with secondary antibodies (dilution 1:500) at room temperature for 2 hours. Subsequently, the tissue sections were incubated in 0.003wt% hydrogen peroxide in 0.01M PBS and 0.05wt% DAB in the dark for 10 minutes to visualize the immune reaction; then counterstained with hematoxylin for 5 minutes. The localization and distribution of immunoreactive substances in the brain tissue were examined under a microscope. The results were quantified as the percentage of positive area.

[0049] 1.10 Immunofluorescence (IF)

[0050] Paraffin sections were dewaxed and hydrated, followed by antigen retrieval with sodium citrate, blocking with 5wt% goat serum for 30 minutes, and then incubated with an appropriate concentration of primary antibody at 4°C overnight. After washing with PBS, goat anti-rabbit IgG / HRP was added dropwise and incubated in the dark for 50 minutes. Cell nuclei were counterstained with DAPI, and finally, sections were mounted with anti-fluorescence quenching mounting solution. In situ fluorescence microscopy (NIKON ECLIPSE C1, Japan) was used for observation, with cell nuclei appearing blue and the target protein appearing red. Quantification was performed using mean fluorescence intensity.

[0051] 1.11 Cells and Experimental Design

[0052] An injury model was established in vitro using a co-culture system of rat astrocytes (CTX), neurons (H19-7), and microglia (GMI-R). The CCK8 assay was used to determine the appropriate parameters for the model. After measuring the concentrations of sodium butyrate, a JNK inhibitor (SP600125), a p38 MAPK inhibitor (Adezmapimod), and a ferroptosis inhibitor (Fer-1), the experimental group was divided into control (Con), model group (Mod), sodium butyrate group (NaB), a p38 MAPK inhibitor group (SB203580), a JNK inhibitor group (SP600125), a ferroptosis inhibitor group (Fer-1), a sodium butyrate + p38 MAPK inhibitor group (NaB + Adezmapimod), a sodium butyrate + JNK inhibitor group (NaB + SP600125), and a sodium butyrate + Fer-1 inhibitor group (NaB + Fer-1). The control group was cultured normally without any intervention; the sodium butyrate group received only sodium butyrate intervention; the model group used a shock wave therapy device to simulate the shock wave of a gas explosion to create a shock model. The three inhibitor groups were then cultured with the corresponding inhibitors after the shock wave. In the sodium butyrate + inhibitor group, sodium butyrate and the corresponding inhibitor were added after the shock wave for intervention culture.

[0053] 1.12 Cell culture and establishment of cell impact model

[0054] The cells were cultured in 10wt% fetal bovine serum H-DMEM medium (89wt% 215H-DMEM + 10wt% fetal bovine serum + 1wt% penicillin-streptomycin solution), and the cells were placed in a CO2 incubator with a volume fraction of 5% at 37°C. When the cells grew to approximately 80%, the cells were digested and passaged. The solution was replaced every 2 days, and the passage ratio was 1:3. During passage, 1mL of trypsin-EDTA was added to the culture flask, digested at 37°C for 1 minute, and then 2mL of culture medium was added to terminate the digestion. The cells were gently blown until completely detached and then transferred to a centrifuge tube.

[0055] Then the density was 1×105 / mL of 1mL cell suspension was placed in a 5mL centrifuge tube. A shock wave therapy device was used to simulate the shock wave of a gas explosion for impact modeling. After the centrifuge tube was sealed with a sterile sealing film, it was hung upside down on the top of the shock handle of the shock wave therapy device, and a coupling agent was applied to the joint. The shock wave therapy device mechanically strikes the massage head to impact the cell mixture in the centrifuge tube, thereby causing impact damage to the cells. The modeling conditions were then determined by CCK-8 experiment, the impact frequency was adjusted to 10Hz, and the number of impacts (1000, 1500, 2000, 2500, 3000 times) was determined in sequence. The energy level (1, 3, 5, 7, 9, 10bar), the culture time (24h, 48h), and 5000 cells per well were immediately inoculated into a 96-well plate after impact. After the corresponding incubation time, 10 μL of CCK8 was added to each well. After incubation in the incubator for 2 h, the absorbance at 450 nm was measured using a Bio Tek microplate reader to calculate the survival rate of each group of cells. The experiment was repeated 3 times for each condition to determine the survival rate of cells under different impact conditions.

[0056] 1.13 CCK8 Determination of Drug and Inhibitor Intervention Concentrations

[0057] The neural cells in the co-culture system were seeded in 96-well plates at a seeding density of 1×10 4 / well. The sodium butyrate stock solution was diluted with ultrapure water. After establishing the cell impact injury model, cells were incubated with sodium butyrate at concentrations of 0, 0.25, 0.5, 1, 2.5, and 5 mmol / L for 12 hours, and cell viability was measured by CCK8 assay. Similarly, cells were incubated with SB203580, SP600125, and Fer-1 (0, 1.25, 2.5, 5, 10, and 20 μmol / L) for a period of time, and cell viability was measured by CCK8 assay.

[0058] 1.14 Scratch test

[0059] Cells in the logarithmic growth phase were evenly seeded in 6-well culture plates. All groups, except the control group, were treated with shock wave modeling. After cells were fully attached, a 200 μL pipette tip was used to draw a straight line perpendicular to the bottom of the plate, ensuring uniform scratch width. Subsequently, the cells were gently rinsed 3–5 times with serum-free DMEM medium to remove detached cell debris. Complete culture medium containing the corresponding intervention drug or inhibitor was then added. The initial scratch state (0 h) was observed and photographed under a microscope, and the cells were then returned to a 37°C, 5% CO2 incubator for continued culture. Twelve hours later, the wound healing process was observed again in the same field of view, and images were recorded. Image J software was used to quantify the change in scratch area, and cell migration rate was calculated using the following formula: Cell migration rate (%) = (initial scratch area - 24-hour scratch area) / initial scratch area × 100%.

[0060] 1.15 ROS detection

[0061] The production of reactive oxygen species in neurons exposed to shock waves was assessed using a reactive oxygen species detection kit (Beijing Solarbio Science & Technology Co., Ltd., China). ROS production was observed by inverted fluorescence (AzioVert.Al) after cells were labeled with dihydroethidium (5 μM) for 30 minutes following drug treatment, and ROS levels were quantified using ImageJ 6.0.

[0062] 1.16Fe 2+ Determination

[0063] Iron ion detection kit (Solarbio, Beijing, China) was used to detect iron ion content in rat hippocampal tissue and cells in the co-culture system. The extract was added proportionally according to the number of cells, and after ultrasonication, the cells were centrifuged in an ice bath for detection, and serum was directly tested. The sample was added to a 1.5 mL centrifuge tube, which was divided into an assay tube, a standard tube, and a blank tube. The sample, standard, distilled water, and reagent 1 (tripyridyl triazine) were added to the tube, mixed, and incubated at 37°C for 10 minutes. Chloroform was then added, vortexed, and shaken for 5 minutes. The tube was then centrifuged at 12,000 g for 10 minutes at room temperature. 200 μL of the supernatant was pipetted at 593 nm, and the absorbance was measured.

[0064] 1.17 MDA assay

[0065] MDA levels in rat hippocampal tissue and cells cultured in the co-culture system were determined using an MDA assay kit (Solarbio, Beijing, China). Cell and serum pretreatment were the same as in Section 1.15. Different reagents (MDA assay working solution, distilled water, sample, and Reagent 3 (protein precipitation solution)) were added to the assay tube and a blank tube, respectively. The mixture was incubated in a 100°C water bath for 60 minutes, cooled in an ice bath, and then centrifuged at 10,000 g for 10 minutes. 200 μL of the supernatant was aspirated and placed in a 96-well plate. The absorbance of each sample was measured at 532 nm and 600 nm.

[0066] 1.18 Cellular SOD detection

[0067] Collect the cells from the treated co-culture system, disrupt them using an ultrasonic disruptor, and then centrifuge them at 8500 rpm and 4°C for 10 minutes. The supernatant is placed on ice for testing, and the protein concentration of each sample is measured using a BCA kit. Prepare the test according to the kit instructions. After adding samples in sequence to a 96-well plate, shake and mix them in a microplate reader. Incubate at 37°C for 30 minutes, and then measure the absorbance of each sample at 560nm. Calculate the inhibition rate using the absorbance value according to the kit instructions. Substitute the inhibition rate and protein concentration to calculate the final SOD content of each group of samples.

[0068] 1.19 RNA extraction and real-time fluorescence quantitative PCR analysis

[0069] Total RNA was extracted using TRIzol reagent (R401-01, Nanjing Novozyme Biotechnology Co., Ltd.), and the concentration was determined using a nucleic acid detector. Subsequently, 2 μL of total RNA was mixed with reverse transcriptase using a gradient PCR instrument to synthesize cDNA. The cDNA was diluted 10-fold before polymerase chain reaction (PCR). The reaction system consisted of 0.4 μL of forward primer, 0.4 μL of reverse primer, 7.2 μL of enzyme-free water, 2 μL of cDNA, and 10 μL of SYBR qPCR Master Mix in a total volume of 20 μL. After completion of the reaction, the cycle number for each gene was calculated using a multi-function microplate reader. Each sample was repeated three times to ensure the accuracy of the measurement. Results: Relative expression levels were calculated using the 2-ΔΔCt method, using β-actin as an internal reference. The genes and primer sequences used for mRNA detection are shown in Table 1.

[0070] Table 1 Gene primer sequences

[0071]

[0072] 1.20 Western blotting

[0073] According to the experimental groups, cells and rat hippocampal tissues were collected from each group, the hippocampal tissues were placed on the operating table, sliced ​​with a blade, and then immersed in RIPA solution. After homogenization, EP tubes were used for centrifugation to extract the supernatant. The collected cells were homogenized in 200 μL cell lysis buffer (RIPA, R0010, Solarbio), and 2 μL PMSF (100 mM, R0010, Solarbio) was added for Western blotting detection. The protein concentration was determined using a BCA protein assay kit (Abbott Gene Technologies Co., Ltd.). Equal amounts of protein were electrophoresed by 12 wt% SDS polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4°C with primary antibodies against β-Actin (bs-0061R, 1:3000), IL-6 (bs-6309R, 1:2000), IL-10 (bs-0698R, 1:3000), GPX4 (BM5231, 1:2000), SLC7A11 (DF12509, 1:2000), JNK1 / 2 / 3 antibody (AF6318, 1:2000), Phospho-JNK1 / 2 / 3 antibody (AF3318, 1:2000), P38 MAPK antibody (bs-0637R, 1:2000), and Phospho-P38 MAPK antibody (bs-5476R, 1:2000). The membrane was then incubated with HRP-conjugated secondary antibodies at 37°C for 1 h. The ECL reaction solution was used to visualize the proteins on the membrane. Films were scanned and archived using a scanner (Amersham ImageQuant 800), and colors were organized using Image J (Adobe Photoshop, Adobe). The optical density values ​​of target bands were analyzed using grayscale analysis software (Alpha Ease FC, Alpha Innotech).

[0074] 1.21 Statistical Analysis

[0075] All data are expressed as mean ± standard deviation (SD), and statistical graphs were generated using Graph Pad Software (Graph Pad Prism version 8, La Jolla, CA, USA). Statistical analysis was performed using SPSS 20.0 software. Differences between groups were assessed using one-way analysis of variance (ANOVA) and Turkey's multiple comparison test. Comparisons between two groups were performed using independent t-tests. Correlations between different experiments were determined using Spearman correlation analysis performed with R (V3.5.1). P < 0.05 was considered statistically significant.

[0076] 2. Results

[0077] 2.1 Sodium butyrate improves GE-induced brain damage

[0078] To investigate the potential effects of sodium butyrate on brain damage in rats with GE-induced traumatic brain injury (TBI), the ABX and CB groups were injected with antibiotics. Compared with the CON group, the rats showed abnormalities in body weight, heart rate, and blood pressure; however, these parameters were improved after sodium butyrate injection ( Figure 1 A and B). Open field test ( Figure 1 The results (C) showed that compared with the control group (CON), the movement distance and movement speed of animals in the model group (MOD) and the antibiotic treatment group (ABX) were significantly reduced. However, the sodium butyrate intervention group (CB) showed improved movement ability after gas explosion exposure, and the decline in movement distance and speed was significantly reduced ( Figure 1 D and E).

[0079] 2.2 Sodium butyrate alleviates gas explosion-induced neuroinflammation

[0080] To further investigate GE-induced brain damage and evaluate potential improvements after sodium butyrate treatment, brain damage was assessed using H&E staining and TEM scanning ( Figure 2 A and B). H&E results showed ( Figure 2 (A) Compared with CON, both the MOD and ABX groups showed obvious signs of tissue damage: significantly reduced cell density, disorganized tissue structure, and a large number of inflammatory cell infiltration. The CB group treated with sodium butyrate showed significant improvement, with increased cell density (more dark-stained nuclei) and a significant reduction in inflammatory cell infiltration. Transmission electron microscopy was used to observe changes in mitochondria in hippocampal tissue ( Figure 2 Middle B), compared with the CON group, the MOD group showed characteristic changes in mitochondrial morphology of ferroptosis, including mitochondrial shrinkage and reduced or disappeared mitochondrial cristae. Immunohistochemistry results showed ( Figure 2 Compared with CON, the IL-6 and TNF-a proteins in MOD and ABX groups ( Figure 2 The positive area ratio of D and G) increased significantly (P<0.01); the positive area of ​​IL-10 protein decreased significantly ( Figure 2 D). After the administration of sodium butyrate, these ratios showed an opposite trend in both the model group and the antibiotic group (P<0.05). In addition, in order to detect the expression of inflammatory-related factors at the mRNA level, their transcriptional levels were analyzed ( Figure 2 The expression of these three indicators is generally consistent with the above results. The expression of IL-6 and IL-10 proteins is consistent with the gene results ( Figure 2 L and M).

[0081] 2.3 Sodium butyrate inhibits gas explosions, leading to downregulation of GPX4 and SLC7A11 expression to suppress ferroptosis

[0082] Immunofluorescence was used to detect the expression of GPX4 and SLC7A11 in tissues. Figure 3 Middle A), compared with the CON group, the fluorescence intensity of GPX4 and SLC7A11 in the MOD group was significantly reduced, but the fluorescence signal in the CB group was stronger than that in the MOD group. 2+ The content of Fe in the ABX group was significantly higher than that in the CON group (P<0.01), while that in the ABX group was 2+ The level was further increased compared with the MOD group (P<0.01), while the CB group was significantly lower than the ABX group (P<0.05) ( Figure 3 In terms of oxidative stress indicators, the MDA content in the MOD group was significantly higher than that in the CON group (P<0.01), the MDA level in the ABX group maintained an upward trend, and the MDA level in the CB group was significantly lower than that in the ABX group (P<0.05) ( Figure 3 Middle C). Western blot analysis showed ( Figure 3 In the DF), the expression levels of GPX4 and SLC7A11 proteins in the MOD group were significantly lower than those in the CON group (P<0.01); the expression of GPX4 protein in the ABX group was further downregulated compared with the MOD group (P<0.01), and the level of SLC7A11 protein also decreased significantly. After sodium butyrate intervention, the expression of GPX4 and SLC7A11 proteins in the CB group was significantly increased compared with the ABX group (P<0.05) ( Figure 3 E and F). GPX4 and SLC7A11 mRNA expression was consistent with the protein results ( Figure 3 G and H).

[0083] 2.4 Sodium butyrate can effectively downregulate the overactivation of JNK and P38 MAPK signaling pathways in rat hippocampus induced by gas explosion

[0084] Immunofluorescence detection of p38 and p-p38 pathway proteins in hippocampus tissue ( Figure 4 The results showed that compared with CON, the p-p38 fluorescence expression intensity in the MOD group increased, and the p-p38 fluorescence expression intensity in the ABX group showed an upward trend compared with the MOD group. Compared with the ABX group, the p-p38 fluorescence intensity and nuclear expression in the CB group decreased. Western blot analysis of related protein expression showed that the expression of p-JNK and p-p38 MAPK in the MOD group was significantly increased compared with the CON group (P<0.01); p-p38 MAPK in the ABX group was further increased compared with the MOD group (P<0.01), while p-JNK was decreased (P<0.01); the expression of both proteins in the CB group was decreased compared with the ABX group (P<0.05) ( Figure 4 C and D). Detection of factors related to the JNK / p38 MAPK signaling pathway showed that compared with the CON group, the expression levels of JNK and P38 MAPK mRNA in the MOD group were increased (P<0.01). Compared with the MOD group, the ABX group showed an upward trend but no statistical significance. However, the CB group was significantly lower than the ABX group (P<0.05). Figure 4 E and F).

[0085] 2.5 Sodium butyrate improves the establishment of a model of nerve cell injury induced by shock wave exposure

[0086] A co-culture system constructed with CTX, H19-7, and GMI-R cells was established as a cell model. The model was cultured for 12 hours and 24 hours under different shock levels of 1000, 1500, 2000, 2500, and 3000 times and 1, 3, 5, 7, 9, and 10 bar. The corresponding survival rate was then measured using a CCK8 kit. It was found that the cell survival rate was lowest when the shock number was 1500 times, the energy level was 10 bar, and the culture time was 12 hours. Subsequently, increased expression of p-JNK and p-P38 proteins was detected, indicating that shock wave exposure activated the expression of JNK / P38 MAPK pathway proteins ( Figure 5 Then, the intervention concentrations of sodium butyrate, JNK inhibitor SP600125, P38 MAPK inhibitor SB203580, and ferroptosis inhibitor Fer-1 were determined, and the intervention concentrations of NaB, SP600125, SB203580, and Fer-1 were determined to be 1 mmol / L ( Figure 5 Medium F), 2.5 μmol / L ( Figure 5 Medium G), 2.5μmol / L( Figure 5 H) and 1.25 μmol / L ( Figure 5 Subsequently, the cells were divided into 9 groups for experiments: CON, MOD, NaB, JNK, SB203580, SP600125, Fer-1, SB203580+NaB, SP600125+NaB, and Fer-1+NaB. After determining the shock wave conditions, the intervention concentrations of sodium butyrate and inhibitors, the migration and repair ability of the nerve cells were observed ( Figure 5 The results showed that the migration and repair ability of cells in the Mod group was significantly reduced (P<0.01), accompanied by a decrease in cell density and connections ( Figure 5 Each intervention group (NaB, SB203580, SP600125, Fer-1) and the combination group were able to improve the cell migration and repair ability. Among them, the cell density increased more significantly in the SB203580+NaB, SP600125+NaB and Fer-1+NaB combination groups, and the cell status was significantly improved.

[0087] 2.6 Sodium butyrate improves shock wave-induced neuronal ferroptosis

[0088] The expression levels of ROS in cells of each group ( Figure 6 Middle A), compared with the CON group, the ROS level in the MOD group induced by shock wave was significantly increased (P<0.01). Compared with the MOD group, the ROS levels in the NaB, SB203580, SP600125 and Fer-1 intervention groups were significantly reduced, and the ROS level in the combined NaB group was more significantly reduced (P<0.01) ( Figure 6 (B) Fe in each group 2+ Expression level ( Figure 6 C), the MOD group was significantly higher than the CON group (P<0.01); compared with the MOD group, the NaB, SB203580, SP600125 and Fer-1 intervention groups all significantly reduced Fe 2+ (P<0.01), and the effect of the combined NaB group was more significant (P<0.01). Figure 6 The expression levels of SOD in each group ( Figure 6 In the middle E), the SOD in the MOD group was significantly lower than that in the CON group (P<0.01). Compared with the MOD group, the SOD in each intervention group increased, and that in the Fer-1+NaB group increased significantly (P<0.01).

[0089] 2.7 Sodium butyrate negatively regulates the JNK / p38 MAPK signaling pathway to inhibit neuronal ferroptosis

[0090] qPCR was used to detect the expression of JNK / P38 MAPK pathway and ferroptosis-related genes. The mRNA expression of GPX4 and SCLA711 was significantly decreased in the MOD group (P<0.01), and each intervention group increased their expression (P<0.01), and the effect of the combined NaB group was more significant (P<0.01). Figure 7 The expression of JNK and P38 MAPK mRNA in the MOD group was significantly higher than that in the CON group (P<0.01), while NaB, SB203580, SP600125, and Fer-1 intervention alone and in combination with NaB significantly inhibited their expression (P<0.01). Figure 7 C and D). Western Blotting results showed that ( Figure 7 Middle E), the protein expression of GPX4 and SLC7A11 was consistent with the gene expression results ( Figure 7F and G), the protein expressions of p-JNK and p-P38 MAPK were consistent with the results of JNK and P38 MAPK genes ( Figure 7 H and I).

[0091] 3. Discussion

[0092] In summary, the present invention reveals the key regulatory role of the JNK / p38 MAPK pathway in neuronal ferroptosis after GE-TBI, and confirms that NaB can alleviate gas explosion-induced neuronal ferroptosis and secondary nerve damage by inhibiting this pathway. These findings not only deepen the understanding of the molecular mechanism of GE-TBI, but also provide new ideas for the development of neuroprotective strategies targeting ferroptosis. Future studies should further explore the clinical application potential of NaB and other ferroptosis inhibitors to improve the prognosis of GE-TBI patients ( Figure 8 ).

[0093] The basic principles, main features and advantages of the present invention are shown and described above. Without departing from the spirit and scope of the present invention, the present invention may also be subject to various changes and improvements, which fall within the scope of the present invention to be protected.

Claims

1. The application of sodium butyrate in the preparation of drugs to alleviate brain damage caused by gas explosions, wherein sodium butyrate regulates ferroptosis of neurons through the JNK / P38 MAPK pathway to alleviate brain damage caused by gas explosions.

2. The use according to claim 1, characterized in that: The sodium butyrate alleviates brain damage caused by gas explosion by reducing brain nerve inflammation induced by gas explosion.

3. The use according to claim 1, characterized in that: The sodium butyrate inhibits the ferroptosis of nerve cells by inhibiting the reduced expression of ferroptosis-related factors GPX4 and SLC7A11 caused by gas explosion, thereby alleviating brain damage caused by gas explosion.

4. The use according to claim 1, wherein: The sodium butyrate alleviates brain damage caused by gas explosion by downregulating the activities of JNK and P38 MAPK signaling pathways in the hippocampus tissue of rats caused by gas explosion.

5. The use according to claim 1, characterized in that: The sodium butyrate alleviates brain damage caused by gas explosion by improving cell migration and repair capabilities.

6. The use according to claim 1, characterized in that: The sodium butyrate is used in combination with a pathway inhibitor to alleviate brain damage caused by gas explosion.

7. The use according to claim 6, characterized in that: The pathway inhibitor is one or more of the JNK inhibitor SP600125, the P38 MAPK inhibitor SB203580, and the ferroptosis inhibitor Fer-1.

8. The use according to claim 1, characterized in that: The sodium butyrate inhibits ferroptosis of nerve cells by negatively regulating the JNK / p38 MAPK signaling pathway, thereby alleviating brain damage caused by gas explosion.

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