Application of intestinal microbial flora in preparation of microecological preparation for treating gas explosion brain injury

Abstract

The invention discloses an application of intestinal microbial flora in preparation of a microecological preparation for treating gas explosion brain injury, and the neuroprotective effect of fecal microbial transplantation on GE-induced TBI rats, including neurological dysfunction, neuropathological change and neuroinflammation, is researched by adjusting a microflora-intestinal tract-brain axis. According to the invention, a model of intestinal flora imbalance and brain injury of male rats is established by using a fecal microbial transplantation technology, and then non-targeted metabonomics and microbial diversity analysis are carried out to explore bacteria with potential functions. Meanwhile, the intestinal microbiota composition, the intestinal and brain barrier functions and the number of regulatory T cells of the brain are evaluated. A potential anti-inflammatory mechanism determined by integrating microbiome and metabolome analysis relates to a peripheral immune pathway, and a promising strategy is provided for relieving GE-induced TBI.

Description

Technical Field

[0001] The invention belongs to the technical field of intestinal microecological flora regulation and gas explosion brain injury treatment, and specifically relates to the application of intestinal microbial flora in the preparation of microecological preparations for treating gas explosion brain injury. Background Art

[0002] As an important public health issue in the coal mining industry, gas explosion (GE) poses a major threat to the safety of male miners. Due to the complexity of the roads, the occurrence of GE often causes miners to suffer various injuries. Although there is evidence that the mortality rate of coal miners has decreased with the improvement of mining technology, the disability rate remains high and the prognosis is poor (Li Jing, Qin Yaru, Wang Zhen, Xin Yanli, How to analyse the injury based on 24 Model: a case study of coal mine gas explosion injury, Injury Prevention, 27 (2021) 542-553; Dickstein Dara L., De Gasperi Rita, Gama Sosa Miguel A., Perez-Garcia Georgina, Short Jennifer A., ​​Sosa Heidi, Perez Gissel M., Tschiffely Anna E., Dams-O'Connor Kristen, Pullman Mariel Y., Knesaurek Karin, Knutsen Andrew, Pham Dzung L., Soleimani Lale, Jordan Barry D., Gordon Wayne A., Delman Bradley N., Shumyatsky Gleb, Shahim Pashtun-Poh, DeKosky Steven T., Stone James R., Peskind Elaine, Blennow Kaj, Zetterberg Henrik, Chance Steven A., TorsoMario, Kostakoglu Lale, SanoMary, HofPatrick R., Ahlers Stephen T., Gandy Sam, Elder Gregory A., Brain and blood biomarkers of tauopathy and neuronalinjuryinhumansandratswithneurobehaviouralsyndromesfollowingblastexposure,MolecularPsychiatry,26(2020)5940-5954.).Brain tissue is an important target organ in GE patients. The shock wave generated during the explosion can cause brain tissue damage and secondary traumatic brain injury (TBI) (Dong X., Deng L., Yao S., Wu W., Cao J., Sun L., Bai Y., Li H., Weng X., Ren H., Ren W., Protective effects of curcumin against thyroid hormone imbalance after gas explosion-induced traumatic brain injury via activation of the hypothalamic-pituitary-thyroid axis in male rats, Environmental science and pollution research international, 29 (2022) 74619-74631.).In addition, TBI caused by GE has multiple complexities; previous studies of our research group have confirmed the association between GE and TBI (Dong X., Yao S., Wu W., Cao J., Weng X., Sun L., Li J., Li H., Li R., Ren W., [Short-term effect of gas explosion in real roadway environment on rats' brain neural behavior], Wei Sheng Yan Jiu, 49 (2020) 889-894.), while the current research mainly focuses on lung injury (Dong X. W., Yao SQ, Wu W. D., Cao J., Weng X. G., Sun L., Li J., Ren HC, Ren W. J., [Influences of gas explosion on acute blast lung injury and time phase changes of pulmonary function in rats under real roadway environment], Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi, 39 (2021) 137-142; Zhang M., Sun Y., Ding C., Hong S., Li N., Guan Y., Zhang L., Dong X., Cao J., Yao W., Ren W., Yao S., Metformin mitigates gas explosion-induced blast lung injuries through AMPK-mediated energy metabolism and NOX2-related oxidation pathway in rats, Exp Ther Med, 24 (2022) 529.). Among these findings, there is ample evidence that GE is associated with neurobehavioral abnormalities, and its effects on injury are more pronounced in the developing brain.This is because during the recovery period after injury, GE-induced injury markers can easily enter the brain through the blood-brain barrier or other pathways, causing adverse effects on the brain (Dong XW, Yao SQ, Wu WD, Cao J., Tian LQ, Ren WJ, [Study on serum metabolomics of combined injury induced by gas explosion in rats], Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi, 39 (2021) 808-814.). Accumulated evidence from epidemiological and experimental studies has confirmed the effects of TBI on neuronal damage, blood-brain barrier disruption, and other related effects (Thapak Pavan, Ying Zhe, Palafox-Sanchez Victoria, Zhang Guanglin, Yang Xia, Gomez-Pinilla Fernando, Human in ameliorates TBI-related cognitive impairment by attenuating mitochondrial dysfunction and inflammation, Biochimica et Biophysica Acta (BBA) -Molecular Basis of Disease, 1870 (2024).). TBI can be divided into primary injury and secondary injury (Barrett James P., Knoblach Susan M., Bhattacharya Surajit, Gordish-Dressman Heather, Stoica Bogdan A., Loane David J., Traumatic Brain Injury Induces cGAS Activation and Type I Interferon Signaling in Aged Mice, Frontiers in Immunology, 12 (2021).).Secondary brain injury refers to additional damage that occurs after the initial injury, such as pathological inflammation (Khellaf Abdelhakim, Khan Danyal Zaman, Helmy Adel, Recent advances intraumatic brain injury, Journal of Neurology, 266 (2019) 2878-2889.), which often leads to neurodegenerative diseases and cognitive impairment (Willis Emily F., MacDonald Kelli PA, Nguyen Quan H., Garrido Adahir Labrador, Gillespie Ellen R., Harley Samuel BR, Bartlett Perry F., Schroder Wayne A., Yates Abi G., Anthony Daniel C., Rose-John Stefan, Ruitenberg Marc J., Vukovic Jana, Repopulating Microglia Promote Brain Repair in an IL-6-Dependent Manner, Cell, 180 (2020) 833-846. e816.). Therefore, GE-induced TBI may lead to cognitive dysfunction in patients with poor prognosis, highlighting the urgent need to identify potential therapeutic targets to address cognitive impairment.

[0003] TBI-induced neuroinflammation and neurodegeneration affect intestinal function through bidirectional communication between intestinal and brain pathways (Jameson KG, Olson CA, Kazmi SA, Hsiao EY, Toward Understanding Microbiome-Neuronal Signaling, Molecular Cell, 78 (2020) 577-583.). At the same time, various metabolites produced by intestinal microorganisms affect brain tissue through multiple pathways (Cryan John F., O'Riordan Kenneth J., Cowan Caitlin SM, Sandhu Kiran V., Bastiaanssen Thomaz FS, Boehme Marcus, Codagnone Martin G., Cussotto Sofia, Fulling Christine, Golubeva Anna V., Guzzetta Katherine E., Jaggar Minal, Long-Smith Caitriona M., Lyte Joshua M., Martin Jason A., Molinero-Perez Alicia, Moloney Gerard, Morelli Emanuela, Morillas Enrique, O'Connor Rory, Cruz-Pereira Joana S., Peterson Veronica L., Rea Kieran, Ritz Nathaniel L., Sherwin Eoin, Spichak Simon, Teichman Emily M., van de Wouw Marcel, Ventura-Silva Ana Paula, Wallace-Fitzsimons, Shauna E., Hyland Niall, Clarke Gerard, Dinan Timothy G., The Microbiota-Gut-BrainAxis, PhysiologicalReviews, 99(2019)1877-2013.).This emphasizes the importance of maintaining gastrointestinal homeostasis as a potential target for the treatment of TBI (Hanscom Marie, Loane David J., Shea-Donohue Terez, Brain-gut axis dysfunction in the pathogenesis of traumatic brain injury, Journal of Clinical Investigation, 131 (2021).). At present, it is increasingly recognized that various strategies are used to regulate beneficial bacteria in the intestinal microbiota, such as fecal microbial transplantation as a clinical intervention to restore intestinal flora (Antushevich Hanna, Fecal microbiota transplantation in disease therapy, Clinica Chimica Acta, 503 (2020) 90-98.). It is used to solve the imbalance of intestinal flora and rebuild the intestinal microecological system (Ooijevaar R.E., Terveer EM, Verspaget HW, Kuijper EJ, Keller JJ, Clinical Application and Potential of Fecal Microbiota Transplantation, Annual Review of Medicine, 70 (2019) 335-351.). In addition, fecal microbiota transplantation involves the transfer of donor microbiota to the recipient, aiming to reshape the commensal microbial community to provide protection against TBI (Koszewicz M., Jaroch J., Brzecka A., Ejma M., Budraewicz S., Mikhaleva LM, Muresanu C., Schield P., Somasundaram SG, Kirkland CE, Avila-Rodriguez M., Aliev G., Dysbiosis is one of the risk factor for stroke and cognitive impairment and potential target for treatment, Pharmacol Res, 164 (2021) 105277.).Studies have shown that changes in the composition of the intestinal flora can disrupt homeostasis within the "gut-brain axis" and aggravate secondary damage after nervous system injury (Yuan Bin, Lu Xiao-jie, Wu Qi, Gut Microbiota and Acute Central Nervous System Injury: A New Target for Therapeutic Intervention, Frontiers in Immunology, 12 (2021).). Our recent research found that experimental rats exposed to GE had cognitive impairment and intestinal microbiota dysbiosis, emphasizing the need for further research on how fecal microbial transplantation can treat GE-induced TBI. In addition, intestinal bacteria and their metabolites can activate M2 microglia, reduce neuroinflammation (Mossad Omar, Erny Daniel, The microbiota–microglia axisin central nervous system disorders, Brain Pathology, 30 (2020) 1159-1177.), and enhance macrophage activity to enhance immune response while alleviating neurodegenerative diseases (Soriano Sirena, Curry Kristen, Wang Qi, Chow Elsbeth, Treangen Todd J., Villapol Sonia, Fecal Microbiota Transplantation Derived from Alzheimer's Disease Mice Worsens Brain Trauma Outcomes in Wild-Type Controls, International Journal of Molecular Sciences, 23 (2022).).Studies in humans and laboratory animal models have shown a strong link between the effectiveness of fecal microbiota transplantation in improving gut barrier function, microbiota composition, and levels of bacterial metabolites (Prochazkova P., Roubalova R., Dvorak J., Tlaskalova-Hogenova H., Cermakova M., Tomasova P., Sediva B., Kuzma M., Bulant J., Bilej M., Hrabak P., Meisnerova E., Lambertova A., Papezova H., Microbiota, Microbial Metabolites, and Barrier Function in A Patient with Anorexia Nervosa after Fecal Microbiota Transplantation, Microorganisms, 7 (2019); Ma S., Wang N., Zhang P., Wu W., Fu L., Fecal microbiota transplantation mitigates bone loss by improving gut microbiome composition and gut barrier function in aged rats, Peer J, 9 (2021) e12293.). However, there is a lack of knowledge about the long-term effects of fecal microbiota transplantation therapy during recovery from GE-induced TBI.

[0004] The precise mechanism between intestinal microbiota and brain injury effects has not yet been fully elucidated. It is currently believed that a stable intestinal microbiota has a positive regulatory effect on the long-term neurodegenerative process after brain injury through the microbiota-gut-brain axis (Chiu LS, Anderton RS, The role of the microbiota-gut-brain axis in long-term neurodegenerative processes following traumatic brain injury, Eur J Neurosci, 57 (2023) 400-418.). Changes in intestinal microbiota can be detected by Toll-like receptors (TLRs), among which TLR4 is particularly good at recognizing bacterial endotoxins such as lipopolysaccharide (LPS) and triggering the production of proinflammatory cytokines, as shown in previous studies by our group (Dong X., Deng L., Su Y., Han X., Yao S., Wu W., Cao J., Tian L., Bai Y., Wang G., Ren W., Curcumin alleviates traumatic brain injuryinduced by gas explosion through modulating gut microbiota and suppressing the LPS / TLR4 / MyD88 / NF-kappaB pathway, Environmental science and pollution research international, 31 (2024) 1094-1113.). Subsequently, LPS and proinflammatory cytokines further damage the intestinal barrier function, leading to increased permeability and entering the systemic circulation, causing systemic inflammation. Eventually, these substances may even break through the blood-brain barrier (BBB), affect the expression of regulatory T cell (Tregs)-related factors and cause neuronal death (Paudel YN, Angelopoulou E., Akyuz E., Piperi C., Othman I., Shaikh MF, Role of Innate Immune Receptor TLR4 and its endogenous ligands in epileptogenesis, Pharmacol Res, 160 (2020) 105172.).In a stress-induced depressive-like behavior mouse model, the results showed that fecal microbiota transplantation treatment improved depressive-like behavior, regulated intestinal flora imbalance, and alleviated intestinal inflammation, intestinal mucosal damage, and neuroinflammation. Given the intestinal flora imbalance observed after GE, further studies are necessary to evaluate the potential role of the microbiota-gut-brain axis in regulating the therapeutic effect of GE-induced TBI, thereby achieving neuroprotection against GE-induced TBI, which has not been reported so far. Summary of the invention

[0005] The technical problem solved by the present invention is to provide the application of intestinal microbial flora in the preparation of microecological preparations for the treatment of gas explosion brain injury, and to study the neuroprotective effect of fecal microbial transplantation on GE-induced TBI rats by regulating the microbiome-gut-brain axis, including neurological dysfunction, neuropathological changes and neuroinflammation. The present invention uses fecal microbial transplantation technology to establish a model of intestinal flora imbalance and brain injury in male rats, and then conducts non-targeted metabolomics and microbial diversity analysis to explore bacteria and their metabolites with potential functional effects. At the same time, the composition of the intestinal microbiome, intestinal and brain barrier function, and the number of regulatory T cells in the brain were evaluated. The potential anti-inflammatory mechanism determined by integrating microbiome and metabolome analysis involves peripheral immune pathways, providing a promising strategy for alleviating GE-induced TBI.

[0006] In order to solve the above technical problems, the present invention adopts the following technical scheme: application of intestinal microbial flora in the preparation of microecological preparations for treating brain damage caused by gas explosion, wherein the intestinal microbial flora is a normal intestinal microbial flora.

[0007] Furthermore, the gas explosion brain injury increased the levels of regulatory T cell (Tregs) related factors interleukin-10 (IL-10), programmed death 1 (PD-1) and forkhead box protein P3 (Foxp3) in brain tissue; normal intestinal microbial flora transplantation improved the intestinal structure, upregulated the expression of tight junction proteins claudin, Occludin and ZO-1, regulated the composition of intestinal microbial flora through the "gut-brain axis" and enhanced the intestinal and brain barrier function.

[0008] Furthermore, the microecological preparation is used to improve cognitive dysfunction and brain barrier dysfunction caused by gas explosion.

[0009] Furthermore, the microecological preparation is used to induce upregulation of regulatory T cell-related factor protein expression in the striatum, thereby achieving a protective effect on gas explosion-induced blood-brain barrier damage.

[0010] Furthermore, the microecological preparation is used to promote the improvement of gastrointestinal barrier damage caused by gas explosion, thereby improving the intestinal microbial flora to treat gas explosion brain damage.

[0011] Furthermore, the microecological preparation is used to improve intestinal inflammation caused by gas explosions and enhance the expression of regulatory T cell factors in the colon, thereby achieving a protective effect on intestinal barrier damage.

[0012] Furthermore, the microecological preparation is used to influence the composition of the intestinal microbiota caused by gas explosion, and then to treat brain damage caused by gas explosion.

[0013] The present invention shows that GE causes significant pathological damage and inflammatory response to rat brain tissue through hematoxylin-eosin staining and transmission electron microscopy analysis, and causes varying degrees of damage to neuronal mitochondria, which is associated with cognitive decline. In addition, GE significantly increased the levels of regulatory T cell (Tregs) related factors interleukin-10 (IL-10), programmed death 1 (PD-1), and forkhead box protein P3 (Foxp3) in rat brain tissue. Similar changes were observed in the rat colon, but these changes were reversed after the normal flora was transferred to GE rats. Combined microbiome and metabolome analysis showed that fatty acid synthesis was activated after fecal microbial transplantation, and the expression of Clostridium_T and Allobaculum was upregulated. Association network analysis indirectly showed that there was a causal relationship between fecal microbial transplantation and the alleviation of GE-induced brain damage. Fecal microbial transplantation improved intestinal structure and upregulated the expression of tight junction proteins claudin, Occludin, and ZO-1, which may contribute to its protective effect on the brain and intestine. Transplantation of the gut microbiota from healthy rats into male rats with gas explosion-induced brain injury improved cognitive function by regulating the composition of the gut microbiota and enhancing gut-brain barrier function through the "gut-brain axis". These findings provide a scientific basis for potential clinical intervention of fecal microbiota transplantation against GE-induced TBI.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: Based on the fact that GE induces dysbacteriosis in the intestinal flora of male rats with TBI, the present invention specifically supplements the intestines of male rats with GE-induced TBI with normal intestinal bacteria, which can not only alleviate acute brain injury and cognitive dysfunction, but also significantly improve the immune microenvironment and promote the recovery of tight connections between the colon and the brain after GE-induced TBI. This is further supported by fecal microbial transplantation intervention after surgery. In short, specific intestinal microorganisms such as Clostridium_T and Allobaculum and their metabolites stimulate the restorative activation of the "gut-brain axis" to protect the brain, thereby promoting the accelerated repair of the colon and brain and the recovery of cognitive function after GE-induced TBI. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Fecal microbiota transplantation helps improve cognitive impairment and blood-brain barrier damage caused by GE. The study included five groups: control group (CON), GE model group (MOD), GE+fecal microbiota transplantation group (FMT), antibiotic removal+GE group (ABX), and antibiotic removal+GE+fecal microbiota transplantation group (AF). (A) Flow chart of fecal microbiota transfer experiment. (B) Body weight changes of male rats in each group. (C) Heart rate and mean arterial pressure measurement results of male rats in each group. (D) Heat map of open field test trajectories of male rats in each group. (E, F) Total moving distance and average moving speed of male rats in the open field test in each group. (G) Brain tissue was stained with H&E and scanned by transmission electron microscopy (TEM). (H) Immunohistochemistry (IHC) was used to detect tight junction proteins in the striatum. (IK) Image analysis software was used to quantify the positive area of ​​tight junction proteins (Claudin5, Occludin, and ZO-1) in the striatum. Data are expressed as mean ± standard deviation, ns indicates no statistical significance, *p<0.05, **p<0.01, ***p<0.001. Black arrows point to inflammatory cells, blue arrows point to nuclear membrane structures, red arrows point to mitochondria, and yellow arrows point to endoplasmic reticulum.

[0016] Figure 2Fecal microbiota transplantation promotes the expression of Tregs-related factors in the striatum. The patients were divided into five groups: control group (CON), GE model group (MOD), GE+fecal microbiota transplantation group (FMT), antibiotic removal+GE group (ABX), and antibiotic removal+GE+fecal microbiota transplantation group (AF). (A) Tregs-related factor proteins in the striatum were detected by IHC (n=3). (BD) The positive area of ​​Tregs-related factor proteins (PD-1, Foxp3, and IL-10) in the striatum (n=3). (EJ) The relative expression of connexins (Claudin5, Occludin, and ZO-1) and Tregs-related factors (PD-1, Foxp3, and IL-10) at the mRNA level and β-actin in the striatum (n=6). Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001.

[0017] Figure 3 Fecal microbiota transplantation promoted the improvement of intestinal barrier damage caused by GE. The study included five groups: control group (CON), GE model group (MOD), GE+fecal microbiota transplantation group (FMT), antibiotic removal+GE group (ABX), and antibiotic removal+GE+fecal microbiota transplantation group (AF). (A) H&E staining and transmission electron microscopy scanning were performed on the colon. (B) Immunohistochemistry was used to detect the junction proteins of the colon (n=3). (CE) The H score of tight junction proteins including Claudin1, Occludin and ZO-1 in the colon was calculated (n=3). Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001. Yellow arrows point to intestinal glands, black arrows point to intestinal villus structures, and red arrows point to cell junctions.

[0018] Figure 4It is GE induced by intestinal inflammation that enhances the effect of fecal microbiota transplantation and promotes the expression of regulatory T cell factors in the colon. The study included 5 groups: control group (CON), GE model group (MOD), GE+fecal microbiota transplantation group (FMT), antibiotic removal+GE group (ABX), and antibiotic removal+GE+fecal microbiota transplantation group (AF). (AF) Relative protein levels of PD-1, Claudin-1, IL-10, IL-1β, and cleaved Foxp3 in the colon (n=5). (G) Protein expression of Tregs-related factors in the colon was detected by IHC (n=3). (HJ) Positive area of ​​proteins of Tregs-related factors (PD-1, Foxp3, and IL-10) in the colon (n=3). (KP) Relative expression of tight junction proteins (Claudin-1, Occludin, and ZO-1) and Tregs-related factor proteins (PD-1, Foxp3, and IL-10) at the mRNA level and β-actin in the colon (n=6). Data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

[0019] Figure 5 Fecal microbiota transplantation intervention affects the composition of the intestinal microbiota in GE rats. The study included five groups: control group (CON), GE model group (MOD), GE+fecal microbiota transplantation group (FMT), antibiotic removal+GE group (ABX), and antibiotic removal+GE+fecal microbiota transplantation group (AF). (A) Venn diagram of the overlap of microbial species in each group (n=5). (B) Rarefaction curve of species richness in each group (n=5). (C) Rank abundance curve of species diversity in each group (n=5). (D) Taxonomic composition analysis at the phylum level for each sample set (n=5). (E) Taxonomic composition analysis at the genus level for each sample set (n=5). (F) PCA plot showing species composition among different samples at the phylum level (n=5). (G) PCA plot showing species composition among different samples at the genus level (n=5). (H) Heat map showing the differences in species composition among groups (n=5).

[0020] Figure 6Fecal microbiota transplantation regulates the composition of intestinal microbial metabolites in GE rats. The rats were divided into 5 groups: control group (CON), GE model group (MOD), GE+fecal microbiota transplantation group (FMT), antibiotic removal+GE group (ABX), and antibiotic removal+GE+fecal microbiota transplantation group (AF). (A) Box plot of alpha diversity index (n=5). (B, C) LDA effect size analysis (n=5). (DG) Species difference composition analysis among sample groups (COM-MOD, CON-ABX, MOD-FMT, ABX-AF) (n=5). (H) Statistics of bacterial genus metabolic pathways (n=5).

[0021] Figure 7 Fecal microbiota transplantation regulates the composition of intestinal microbial metabolites in GE rats. There are five groups: control group (CON), GE model group (MOD), GE+fecal microbiota transplantation group (FMT), antibiotic removal+GE group (ABX), and antibiotic removal+GE+fecal microbiota transplantation group (AF). (A, B) Venn diagrams of each group in positive and negative ion modes (n=5). (C, D) Comparison of DEMs statistics in positive and negative ion modes of each group (n=5). (E, F) Proportion of differentially expressed metabolites in positive and negative ion modes of each group (n=5). (G, H) Partial least squares discriminant analysis in positive and negative ion modes (n=5). (IL) Metabolic pathway statistics between sample groups (COM-MOD, CON-ABX, MOD-FMT, ABX-AF) (n=5).

[0022] Figure 8 Correlation analysis between microbiome composition, differentially expressed metabolites (DEMs), and phenotypic variables was performed. (AD) The correlation between the top 20 DEMs and microbiome composition at the genus level was analyzed in the four groups (COM-MOD, CON-ABX, MOD-FMT, ABX-AF). (E) The correlation between the top 10 microbiome composition genera and tight junction proteins (Claudin1, Occludin, and ZO-1) and Tregs-related factor proteins (PD-1, Foxp3, and IL-10) in the striatum was analyzed. (FI) In addition, the correlation between the top 20 DEMs and tight junction proteins (Claudin1, Occludin, and ZO-1) and Tregs-related factor proteins (PD-1, Foxp3, and IL-10) in the striatum in the four groups was analyzed.

[0023] Fig. 9is a schematic diagram showing the restoration of colon and brain barrier function after GE-induced traumatic brain injury (TBI) through fecal microbiota transplantation intervention. GE causes gut microbiota dysbiosis in rats with TBI. Targeted supplementation of gut bacteria not only alleviates acute brain injury and cognitive dysfunction, but also significantly improves the immune microenvironment and promotes the recovery of colon and brain barrier function after GE-induced TBI. These findings were confirmed by the application of fecal microbiota transplantation intervention. In summary, these results indicate that the gut microbiota and its metabolites can promote the restorative activation of the "gut-brain axis" to protect neurons and promote accelerated repair of the colon and brain barrier after GE-induced TBI, as well as the recovery of cognitive function. DETAILED DESCRIPTION

[0024] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0025] Example

[0026] 1 Materials and methods

[0027] 1.1 Main materials and equipment

[0028] 1.1.1 Chemicals, reagents and instruments

[0029] Neomycin sulfate (N109017), natamycin (P107822), and sodium butyrate (S102956) were provided by Shanghai Aladdin Biochemical Technology Co., Ltd. Bacitracin (S17,005) was provided by Shanghai Yuanye Biotechnology Co., Ltd. RNA isolation total RNA extraction reagent (R401-01) was provided by Nanjing Huamei Biotechnology Co., Ltd. Protein quantification kit (BCA method) (KTD3001) was provided by Wuhan Yacoin Technology Co., Ltd. BeyoECL Star (Ultra-sensitive ECL Chemiluminescence Kit) (P0018AS) was provided by Shanghai Biyuntian Biotechnology Co., Ltd. Western blot and IHC showed the presence of β-Actin (bs-0061R), interleukin 10 (IL-10) (bs-0698R), programmed death 1 (PD-1) (bs-1867R), forkhead box protein P3 (Foxp3) (bs-10211R), tight junction protein 1 (ZO-1) (bs-1329R), Occludin (bs-10011R), Claudin-1 (bs-1428R) and Claudin-5 (bs-1241R) provided by Beijing Biosen Biotechnology Co., Ltd.

[0030] 1.1.2 Experimental animals

[0031] The study bred and used experimental animals in accordance with international standards. 40 healthy male Sprague-Dawley rats (100±5g) were provided by Henan Scobes Biotechnology Co., Ltd., China (License No.: SCXK(Yu)2020-0005). SD rats were kept in standard cages and maintained under temperature (21±1°C) and relative humidity (50±10%) conditions with a 12-hour light / 12-hour dark cycle. All rats underwent a one-week adaptation period before the formal experiment. This study has been approved by the Animal and Medical Ethics Committee of Xinxiang Medical College (No.: XYLL-2020007).

[0032] 1.2 Experimental methods

[0033] 1.2.1 Animal grouping and treatment

[0034] Rats were randomly divided into 5 groups (8 in each group): control group (CON) did not receive any treatment; GE model group (MOD): exposed to GE, using the bio-lethal gas explosion shock tube experimental system designed by Huludao North Petrochemical Equipment Factory (China Utility Model Patent Certificate: CN214150529U). Rats were anesthetized with pentobarbital and fixed in an iron cage facing the explosion source. GE was simulated using a 10wt% methane mixed gas and a 23.6 joule shock wave. The rats were placed 2.4 meters away from the explosion source; GE+fecal microbial transplantation group (FMT): after exposure to GE, fecal microbial suspension extracted from normal rat feces was administered by gavage; antibiotic removal+GE group (ABX): before exposure to GE, the rats received combined antibiotic treatment to clear the intestinal flora, and then resumed normal diet after exposure to GE; antibiotic removal+GE+fecal microbial transplantation group (AF): before exposure to GE, the rats received combined antibiotic treatment to clear the intestinal flora, and then after exposure to GE, fecal microbial suspension extracted from normal rat feces was administered by gavage. The impact tube device of GE was simulated at the Institute of Traumatology and Orthopedics of Xinxiang Medical College. After anesthesia, the rats were fixed in the explosion cage with their heads and faces facing the explosion source. Neomycin sulfate (5 mg / mL), natamycin (1.25 μg / mL) and bacitracin (5 mg / mL) were dissolved in rat drinking water to prepare combined antibiotic drinking water for rats to drink for the purpose of removing antibiotics (see previous studies (Bercik P., Denou E., Collins J., Jackson W., Lu J., Jury J., Deng Y., Blennerhassett P., Macri J., McCoy KD, Verdu EF, Collins SM, The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice, Gastroenterology, 141 (2011) 599-609, 609e591-593.). Fresh feces of mice in the control group were collected and dissolved in physiological saline to prepare fecal microbial suspension. Feces were collected at a volume / weight ratio of 1:5.

[0035] 1.2.2 Sample collection and processing

[0036] Fecal samples from each rat were collected on the 21st day after administration and stored at -80°C for subsequent 16SrRNA gene sequencing. Subsequently, the weight of the rats was recorded and euthanized using 1wt% sodium pentobarbital. Blood was drawn from the abdominal aorta, incubated at 4°C for 2 hours, and centrifuged at 3000rpm for 15 minutes. After centrifugation, serum was separated and stored in a -80°C refrigerator. Brain and colon tissues were then collected, and the intestinal contents were removed at the same time when the colon tissues were collected, and they were also stored at -80°C for future use. In addition to macroscopic observation of the collected tissues, brain and colon tissues (n=3) were also subjected to hematoxylin-eosin staining (H&E) and transmission electron microscopy (TEM) examinations. These procedures are intended to facilitate the subsequent observation of histopathological changes and the assessment of tissue damage. The remaining tissues were stored in a -80°C freezer for molecular biology experiments.

[0037] 1.2.3 Fecal microbiota transplantation

[0038] Fecal microbial transplantation is performed according to a set of established procedures (Chang CJ, Lin CS, Lu CC, Martel J., Ko YF, Ojcius DM, Tseng SF, Wu TR, Chen YY, Young JD, Lai H.C., Ganoderma lucidum reduces obesity in mice by modulating the composition of the gut microbiota, Nat Commun, 6 (2015) 7489.). In short, fresh fecal samples were obtained from male rats in the control group. Feces were collected in sterile test tubes and weighed. Then, a 0.9wt% sodium chloride solution was added to the weighed feces at a weight-to-weight ratio of 1:5, and the mixture was stirred with a sterile cotton swab and centrifuged at 1500 rpm for 5 minutes. The precipitate formed was then resuspended in a 0.9wt% sodium chloride solution to achieve a microbial concentration of 2×10^9CFU per milliliter for fecal microbial transplantation testing. Each gastrointestinal model rat was given 1 ml / 100 g of fecal bacterial suspension by oral gavage daily for 28 consecutive days.

[0039] 1.2.4 Antibiotic clearance experiment

[0040] In the previous study, neomycin sulfate (5 mg / mL), bacitracin (5 mg / mL), and natamycin (1.25 μg / mL) were dissolved in purified water to promote the elimination of antibiotics by free ingestion. The remaining experimental mice drank normal water.

[0041] 1.2.5 Blood pressure and heart rate test

[0042] Blood pressure and heart rate were measured before euthanasia. Five groups of rats (8 rats in each group) were tested sequentially. The rats were placed in the detection device and the tail was tested 5 times for each rat. Data were collected using a non-invasive intelligent blood pressure monitor (Sofron BP-2010 series, China) according to the operating manual, and statistical analysis was performed using GraphpadPrism 8.

[0043] 1.2.6 Open Field Test (OFT)

[0044] The open field test (OFT) apparatus consisted of a black checkered floor measuring 80 × 80 and a 50-cm-high black wall. The floor was divided into 16 equally spaced squares, with a central area consisting of 4 squares and the remaining 12 squares designated as peripheral areas. Each rat was gently placed in the center of the apparatus and then allowed to explore freely for 1 min before being captured. Subsequently, all rats were observed in the open box for 5 min using a video tracking system. After each trial, the open box was cleaned with a 10% alcohol solution to mitigate any potential odor effects on subsequent rats. The motor activity of the rats was assessed based on the distance they walked throughout the area. The total distance moved and the average movement speed of the rats were used to assess neurobehavioral changes. The data collected during the experiment were recorded and analyzed using an animal track tracking system (Etho Vision XT16.0, Noldus).

[0045] 1.2.7 Hematoxylin-eosin (H&E) staining and transmission electron microscopy (TEM) analysis

[0046] After collecting the samples, histopathological examination was performed to evaluate TBI caused by GE. Brain and colon tissues from each group of rats (n=3) were first fixed with 4wt% paraformaldehyde, then embedded, sectioned, stained, and mounted. Subsequently, H&E-stained samples were observed using a Nikon Eclipse E100 (Japan) inverted microscope, and the resulting images were captured and analyzed using a NIKON DS-U3 (Japan) imaging system.

[0047] For TEM analysis, brain and colon tissues from each group of rats (n=3) were fixed with 2.5 wt% glutaraldehyde solution. Each sample was cut into 1 mm 3The cubes were immersed in fixative at 4°C for 4 hours. Subsequently, the tissues were fixed in 1% hydrogen peroxide for 2 hours at room temperature and then dehydrated with a gradient alcohol series. The specimens were embedded in resin and baked at 60°C for 48 hours to polymerize. Ultrathin sections were then prepared using an ultramicrotome (60 nm). Finally, the tight junction structures of brain and colon tissues were observed using a transmission electron microscope (HT7700 model produced by Hitachi, Japan).

[0048] 1.2.8IHC staining and Western blot detection

[0049] IHC staining: Brain and colon tissue sections were immersed in Claudin, Occludin, ZO-1, PD-1, Foxp3 and IL-10 antibodies (dilution ratio of 1:500) at 4°C overnight. Subsequently, the sections were treated with secondary antibodies diluted 1:500 for 2 hours at room temperature. Next, 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 develop the immune reaction; then stained with magenta for 5 minutes. The localization and distribution of immunoreactive substances in brain and colon tissues were observed under a microscope. The results are expressed as the percentage of positive areas.

[0050] Western blot analysis: Brain and colon tissues were placed on the operating table, sliced ​​with a blade, and then immersed in RIPA solution. After homogenization, the supernatant was extracted by centrifugation using an EP tube, and the protein concentration was determined using a BCA kit. Subsequently, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. The membrane was incubated overnight at 4°C with primary antibodies (1:2000) such as β-Actin, Claudin, Occludin, ZO-1, PD-1, Foxp3, and IL-10. Then, HRP-labeled anti-rabbit secondary antibodies (1:50000) were applied for incubation for 1 hour, washed with TBST, and developed with an ECL kit. The film was developed using the Amersham ImageQua system, and density analysis was performed using ImageJ software. The results are expressed as the gray value of the target protein relative to the internal reference protein.

[0051] 1.2.9 RNA extraction and real-time quantitative PCR (RT-PCR)

[0052] Total RNA was extracted using TRIzol reagent (R401-01, Nanjing Huajie Biotechnology Co., Ltd., Nanjing, China), and the extraction concentration was determined using a nucleic acid detector. Subsequently, 2 μL of total RNA was mixed with reverse transcriptase to synthesize cDNA in a gradient PCR device. After the cDNA was diluted 10 times, polymerase chain reaction was performed. The reaction system included 0.4 μL forward primer, 0.4 μL reverse primer, 7.2 μL enzyme-free water, 2 μL cDNA, and 10 μL SYBR qPCR Master Mix in a total volume of 20 μL. After the reaction, the number of cycles for each gene was obtained using a multifunctional microplate reader. Each sample was tested three times to ensure the accuracy of determining the relative expression of the target gene in the sample. Results: The relative expression was obtained using the 2-△△Ct method with β-Actin as a reference. The primer names and sequences used for mRNA detection are shown in Table S1.

[0053] Table S1 Primer names and sequences for RT-PCR detection of mRNA

[0054]

[0055] 1.2.10 DNA extraction from intestinal contents, 16S rRNA gene sequencing and bioinformatics analysis

[0056] A total of 25 qualified intestinal content samples from 5 groups (n = 5) were used for DNA extraction. Genomic DNA was extracted using the OMEGA Soil DNA Kit (M5635-02) from Omega Bio-Tek (Norcross, GA, USA). Subsequently, the purity and concentration of the DNA were evaluated using a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. After this, an appropriate amount of sample DNA was placed in a centrifuge tube, diluted to 1 ng / μL with sterile water, and then PCR amplification was performed. The bacterial 16S rRNA gene V3-V4 region was amplified by PCR using forward primer 338F (5'-ACTCCTACGGGAGGCAGC-3') and reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The obtained 16S rRNA sequencing data were analyzed as previously described (Ma S., Qin J., Hao Y., Fu L., Association of gut microbiota composition and function with an agedrat model of senile osteoporosis using 16S rRNA and metagenomic sequencing analysis, Aging (Albany NY), 12 (2020) 10795-10808.). After completing the individual quantification step, the amplicons were evenly mixed together and then subjected to paired-end 2×250 bp sequencing (NovaSeq 6000SP Reagent Kit, 500 cycles) on the Illlumina NovaSeq platform of Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).

[0057] The QIIME2 2022.11 version was used and the official tutorial (https: / / docs.qiime2.org / 2022.11 / tutorials / ) was used for microecological group bioinformatics analysis. Sequence data were mainly processed using QIIME2 and R packages (v3.2.0). Diversity indices at the level of amplicon sequence variation (ASV), such as Chao1 richness estimator, observed number of species, Shannon diversity index, and Simpson index, were calculated using the ASV table in QIIME2 and presented in the form of box plots. Abundance ranking curves at the ASV level were generated to compare the richness and uniformity of ASVs in different samples. Beta diversity analysis was performed using the Bray-Curtis indicator to explore the structural differences of microbial communities between samples and visualized by principal coordinate analysis (PCoA). In addition, principal component analysis (PCA) was performed based on the composition profile at the genus level. PERMANOVA (PERmutational MANOVA) was used in QIIME2 to evaluate the significant differences in microbial community structure between different groups. In addition, we used the linear discriminant analysis effect size (LEfSe) with default parameters to identify differentially abundant species between groups. We used PICRUSt2 (inferred state reconstruction of communities based on MetaCyc (https: / / metacyc.org / ) and KEGG (https: / / www.kegg.jp / ) databases) to predict microbial functions.

[0058] 1.2.11 Non-targeted metabolomics analysis of feces

[0059] Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) was used to perform non-targeted metabolomics analysis of fecal samples of rats in different treatment groups (5 rats per group) at Shanghai PersonalbioTechnology Co., Ltd., as described previously (Li S., Yang H., Li L., Wang W., Tan H.Y., Qu Y., Wang D., The involvement of gut microbiota in the anti-tumor effect of carnosic acid via IL-17 suppression in colorectal cancer, Chem Biol Interact, 365 (2022) 110080.). The processed data were evaluated for overall differences using Pareto-scaling principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Metabolites with variable importance projection (VIP) values ​​> 1 and P values ​​< 0.05 (two-tailed Student's t-test) were considered significant and potentially relevant. The metabolic and signal transduction pathways associated with the significantly differentially expressed metabolites in each treatment group were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. This study was conducted by Shanghai Personal Biotechnology Co., Ltd. in Shanghai, China.

[0060] 1.2.12 Statistical analysis

[0061] All data were expressed as “mean ± standard deviation (SD)”, and statistical graphs were created using GraphPad software (GraphPadPrism version 8, La Jolla, CA). Statistical analyses were performed using SPSS software (version 20.0). Differences between groups were assessed by one-way analysis of variance and two-way analysis of variance (ANOVA), and further comparisons were performed using Turkey's multiple comparison test. Independent t-tests were used to compare the two groups of data. Spearman correlation analysis was performed using R (V3.5.1) software to determine the correlation between different experiments. Results with a P value of less than 0.05 were considered statistically significant.

[0062] 2 Results

[0063] 2.1 Fecal microbiota transplantation helps improve cognitive dysfunction and brain barrier dysfunction caused by GE

[0064] To investigate the role of intestinal microbiota in ameliorating brain damage (induced by gastric aspiration) in rats, antibiotics were administered to the ABX and AF groups, whereas fecal bacteria from the CON rats were transplanted into the FMT and AF groups (e.g. Figure 1 After the gas explosion, abnormal body weight, heart rate, and blood pressure were observed in the experimental group rats compared with the control group rats; however, these parameters improved after receiving fecal microbiota transplantation (as shown in Figure 2A). Figure 1 The results of the open field test (shown in B and C) Figure 1 Figures DF (in Figure 5) showed that the movement distance and speed of rats in the MOD and ABX groups were reduced compared with those in the CON group, while these impairments were alleviated in the FMT and AF groups that received fecal microbiota transplantation. These findings suggest that intestinal microbiota may play a role in alleviating cognitive dysfunction caused by gas explosions.

[0065] To further investigate GE-induced brain damage and evaluate the possible improvement brought about by fecal microbiota transplantation, H&E staining and transmission electron microscopy scanning (eg Figure 1 Brain injury was assessed by hematoxylin-eosin staining (shown in G). Hematoxylin-eosin staining showed a decrease in the number of cells and loose arrangement in the MOD and ABX groups. After fecal microbiota transplantation, an increase in the number of cells and a darker color were observed in the FMT and AF groups. Transmission electron microscopy scanning showed a decrease in the number of cells, swollen mitochondria, vacuolation, and dilation of the endoplasmic reticulum in the MOD and ABX groups. After fecal microbiota transplantation, an intact nuclear membrane structure, a decrease in the number of mitochondria with visible cristae, and a continuous dilation of the endoplasmic reticulum were observed, which was observed in both the FMT and AF groups.

[0066] To further evaluate striatal damage, we observed changes in the expression of tight junction proteins in the striatum ( Figure 1 Compared with the CON group, the positive area ratios of Claudin5 and Occludin proteins in the MOD and ABX groups were significantly reduced (P<0.001) ( Figure 1 After fecal microbiota transplantation, these ratios increased in the FMT group (P < 0.01), whereas no significant changes were observed in the AF group ( Figure 1 Middle K). There was no significant difference in the expression level of ZO-1 protein ( Figure 1 The results suggest that gas explosions can lead to damage to the blood-brain barrier, and fecal microbiota transplantation treatment can alleviate this damage, thereby playing a protective role.

[0067] 2.2 Fecal microbiota transplantation induced upregulation of regulatory T cell-related factor protein expression in the striatum

[0068] To investigate whether the protective effect of fecal microbiota transplantation on GE-induced blood-brain barrier damage is related to in vivo immune system regulation, the immunohistochemical expression changes of regulatory T cell (Tregs)-related proteins in the striatum were evaluated ( Figure 2 Compared with the CON group, the positive area ratios of PD-1, Foxp3, and IL-10 proteins in the MOD and ABX groups were significantly increased ( Figure 2 After fecal microbiota transplantation, these ratios decreased in the FMT group and the AF group (P<0.001). In addition, in order to investigate the expression of tight junction proteins and Tregs-related factors at the mRNA level, their transcriptional levels were analyzed ( Figure 2 The expression patterns of these six indicators were basically consistent with those observed at the protein level.

[0069] 2.3 Fecal microbiota transplantation promotes the improvement of gastrointestinal barrier damage caused by GE

[0070] To evaluate the effects of GE on intestinal health and investigate the direct impact of the intestinal microbiota, H&E staining and transmission electron microscopy scanning ( Figure 3 Middle A) Histological evaluation of colonic injury. In the MOD and ABX groups, the number of cells was reduced, the intestinal glands showed irregular curvature and disordered arrangement, and lacked intact connective tissue. After fecal microbiota transplantation treatment, the cell structure and number of the FMT group were restored; however, the recovery of the intestinal glands and connective tissue was not obvious. In the AF group, the cell structure and number were improved, but the epithelial tissue damage was limited, and the recovery of the intestinal glands and connective tissue was also limited. Transmission electron microscopy scanning showed that the intestinal villi in the MOD and ABX groups were flaked, the borders were damaged, and the tight junctions of the cells were severely damaged. After fecal microbiota transplantation treatment, the intestinal villi in both the FMT and AF groups were regenerated, and the cell-cell connections were relatively intact; however, these connections still appeared somewhat blurred.

[0071] To further confirm intestinal damage, changes in tight junction protein expression in intestinal cells were evaluated ( Figure 3 The results showed that the H-scores of Claudin5, Occludin and ZO-1 proteins in the MOD and ABX groups were significantly lower than those in the CON group (P<0.001) ( Figure 3 CE). After receiving fecal microbiota transplantation, the expression levels of the FMT group and the AF group showed different degrees of improvement. Our experiments proved that intestinal endotoxemia can lead to intestinal barrier damage, and fecal microbiota transplantation can directly improve this barrier.

[0072] 2.4 Fecal microbiota transplantation improves GE-induced intestinal inflammation and enhances the expression of regulatory T cell factors in the colon

[0073] To investigate whether the protective effect of fecal microbiota transplantation on GE-induced intestinal barrier damage is related to the regulation of the intestinal immune system, the protein expression changes of Tregs-related factors in the colon were evaluated ( Figure 4 Compared with the CON group, the expression of PD-1, Foxp3, and IL-10 in the MOD and ABX groups was significantly increased (P<0.001). After receiving fecal microbiota transplantation, the expression of PD-1, Foxp3, and IL-10 in the FMT group decreased to varying degrees; no significant changes were observed in the AF group ( Figure 4 Similarly, similar changes in the inflammatory factor IL-1β were observed. In addition, the expression of Claudin1 was decreased in the MOD and ABX groups ( Figure 4 C), but increased after receiving fecal microbiota transplantation (P < 0.05).

[0074] The expression of Tregs-related protein factors in the colon was evaluated by immunohistochemistry ( Figure 4 The results were consistent with those of Western blot analysis. Subsequently, the mRNA levels of tight junction proteins and Tregs-related protein factors were investigated, and their expression patterns were consistent with the above findings.

[0075] 2.5 Fecal microbiota transplantation affects the composition of the intestinal microbiota in GE rats

[0076] To evaluate the effect of fecal microbiota transplantation on the regulation of intestinal microbiota, 16S rDNA gene sequencing was performed to analyze the bacterial composition of rats after microbial treatment. A total of 25 samples from five groups of rats (n = 5) were used to generate 16S rRNA gene profiles. The ASV / OTU numbers of CON, MOD, FMT, ABX, and AF groups were 750, 626, 802, 724, and 739, respectively ( Figure 5 When the sequencing depth of all samples exceeded 5000, a plateau appeared, indicating that the samples had sufficient diversity ( Figure 5 In addition, when the sample abundance exceeds 200, the flat trend line indicates a high consistency between samples ( Figure 5 Alpha diversity assessment showed that there were no significant differences in Chao1 and Shannon index among the five groups (P>0.05), which reflects the richness and diversity of the microbial community ( Figure 6 A and B). To assess community similarity, Bray-Curtis PCoA was used to evaluate β diversity, and it was found that principal components PCo1, PCo2, and PCo3 explained 44.1%, 10.41%, and 8% of the variation, respectively ( Figure 5At the phylum level, Firmicutes, Proteobacteria, and Actinobacteria were the main bacterial phyla ( Figure 5 At the genus level, Akkermansia, Romboutsia_B, and Allobaculum showed a dominant position ( Figure 5 The heatmap shows the abundance of species and the correlation between groups before reaching the 20-species threshold ( Figure 5 Middle H).

[0077] Table S2 Average proportions of the top 10 different plant groups at the genus level

[0078]

[0079] Note: Mean CON: average proportion of bacterial genera in the control group; Mean MOD: average proportion of bacterial genera in the gas explosion model group; Mean FMT: average proportion of bacterial genera in the fecal microbiota transplantation group; Mean ABX: average proportion of bacterial genera in the antibiotic elimination group; Mean AF: average proportion of bacterial genera in the antibiotic elimination + fecal microbiota transplantation group.

[0080] To identify specific genera within each group, LEfSe and LDA were used to identify the core genera most likely responsible for the observed differences between groups, such as Figure 6 As shown in Figures B and C. The abundance of Firmicutes was significantly higher in the ABX group compared with the CON group (P = 0.025, LDA = 3.268). The abundance of Clostridium_T was higher in the FMT group (P = 0.042, LDA = 4.438), while the abundance of Faecousia, Onthebecus, and Choladousin was higher in the AF group (Faecousia, P = 0.011, LDA = 4.384; Onthebecus, P = 0.042, LDA = 4.297; Choladousin, P = 0.006, LDA = 3.886). Subsequently, Metagenome Seq analysis was performed between the two groups to identify differential bacterial communities (such as Figure 6(DG in Figure 2A). Compared with the CON group, the MOD group showed a dominance of the genus Actinomycetes (Firmicutes) at the genus level, including Closttridium_T (LogFC=3.445), Closttridium_Q (LogFC=2.482), and Lachnospiraceae (LogFC=3.3). In contrast, in the ABX group, Firmicutes and Proteobacteria dominated, with Closttridium_T (LogFC=5.404), Ligilacttobacillus (LogFC=5.338), and Eubacterium_F (LogFC=4.498) dominating at the genus level. Compared with the MOD group, Firmicutes dominated in the FMT group, with Closttridium_T (LogFC=4.095), Ruminococcus_C (LogFC=3.908), and Lawsonibacter (LogFC=3.277) dominating at the genus level. Compared with the ABX group, Firmicutes and Bacteroidetes dominated in the AF group, among which Blautia_A (LogFC=7.73), Faecousia (LogFC=6.379) and Choladousia (LogFC=4.877) dominated at the genus level. In the metabolic pathway statistics, more differential genera were enriched in amino acid synthesis, nucleotide and nucleotide synthesis, and fatty acid and lipid synthesis ( Figure 6 Middle H).

[0081] 2.6 Fecal microbiota transplantation can regulate the composition of intestinal microbial metabolites in transgenic mice

[0082] To investigate the effects of intestinal microbes on the host through metabolites, metabolomics analysis was performed on 25 samples from 5 groups (n = 5). The Venn diagram showed the obvious metabolic changes caused by different treatments ( Figure 7 A and B). Figure 7 Figures C and D show the specific number of unique metabolites in positive and negative ion modes. Based on the screening criteria of VIP>1.0 and P<0.05, a total of 1,381 significantly different metabolites were identified. These metabolites were comprehensively classified in positive and negative ion modes ( Figure 7 E and F). To further elucidate the differences between the groups, an in-depth analysis was performed using a partial least squares discriminant analysis (PLS-DA) model. The PLS-DA model revealed limited separation between the five groups, with quality parameters R2X = 0.241, R2Y = 0.376, and Q2 = 0.117, indicating poor reliability and predictive power of the model ( Figure 7J, G, and H). Subsequently, KEGG enrichment analysis was performed on the differential metabolites between the two groups. The top 20 pathways with the lowest P values ​​(representing the most significant enrichment) were selected for display (see Figure 2). Figure 7 Compared with the CON group, the differentially expressed metabolites (DEMs) in the MOD group (Table S3) were enriched in cysteine ​​and methionine metabolism, metabolic pathways, and purine metabolism (e.g. Figure 7 The DEMs of the ABX group (Table S4) were enriched in arachidonic acid metabolism, glutathione metabolism, and amino acid biosynthesis metabolic pathways (as shown in Figure 1). Figure 7 Compared with the MOD group, the DEMs of the FMT group (Table S5) were enriched in taurine and hypotaurine metabolism, glycine, serine and threonine metabolism, and D-amino acid metabolism (e.g. Figure 7 Compared with the ABX group, the DEMs of the AF group (Table S6) were enriched in phenylalanine, tyrosine, and tryptophan biosynthesis as well as metabolic and amino acid biosynthesis pathways (as shown in Figure 5K). Figure 7 (shown in L).

[0083] Table S3 Screening of the top 20 differentially expressed metabolites in positive and negative ion modes between the gas explosion model group and the control group

[0084]

[0085] Note: VIP: variable importance projection; Mean CON: average expression of differentially expressed metabolites in the control group; MeanMOD: average expression of differentially expressed metabolites in the gas explosion model group; pos: positive ion mode; neg: negative ion mode.

[0086] Table S4 Screening of the top 20 differentially expressed metabolites in positive and negative ion modes between the antibiotic removal group and the control group

[0087]

[0088] Note: VIP: variable importance projection; Mean CON: average expression of differentially expressed metabolites in the control group; Mean ABX: average expression of differentially expressed metabolites in the antibiotic clearance group; pos: positive ion mode; neg: negative ion mode.

[0089] Table S5 Screening of the top 20 differentially expressed metabolites in positive and negative ion modes between the fecal microbiota transplantation group and the gas explosion model group

[0090]

[0091] Note: VIP: variable importance projection; Mean MOD: mean expression of differentially expressed metabolites in the gas explosion model group; Mean FMT: mean expression of differentially expressed metabolites in the fecal microbiota transplantation group; pos: positive ion mode; neg: negative ion mode. Table S6 Screening of the top 20 differentially expressed metabolites in positive and negative ion modes between the antibiotic clearance + fecal microbiota transplantation group and the antibiotic clearance group

[0092]

[0093] Note: VIP: variable importance projection; MeanABX: average expression level of differentially expressed metabolites in the antibiotic clearance group; MeanAF: average expression level of differentially expressed metabolites in the antibiotic clearance + fecal microbiota transplantation group; pos: positive ion mode; neg: negative ion mode.

[0094] 2.7 Correlations between microbial community composition, differentially expressed metabolites, and phenotypic variables

[0095] To further investigate the mutual influences between microbiota composition, differentially expressed metabolites (DEMs), and phenotypic variables, we performed pairwise correlation analyses involving two or three variables. In addition, we performed correlation analyses to explore the interactions between microbiota composition, DEMs, and phenotypic variables by comparing the pairwise relationships between the three variables. We focused on the top 10 bacterial genera with the most significant differences at the genus level (Table S2). We selected the top 20 metabolites with the most significant differences in various comparison groups (MOD vs CON: Table S3; ABX vs CON: Table S4; FMT vs MOD: Table S5; AF vs ABX: Table S6) for correlation analysis ( Figure 8 Compared with the control group, Allobaculum and Ligilactobacillus were significantly correlated with differentially expressed metabolites (DEMs) in the MOD group ( Figure 8 In the ABX group, Allobaculum, Clostridium_T, etc. were correlated with DEMs ( Figure 8 Compared with the MOD group, Marvinbryantia and Blautia_A in the FMT group were correlated with DEMs ( Figure 8 In contrast, Marvinbryantia and Faecousia were associated with DEMs in the AF group compared with the ABX group ( Figure 8 There was no strong correlation between microbes and metabolites; therefore, we also performed correlation analysis between microbes and tight junction proteins and Tregs-related factors ( Figure 8E). The results showed that the expression of tight junction proteins was positively correlated with Ligilactobacillus and Blautia_A. In contrast, Tregs-related factors were negatively correlated with Ligilactobacillus and Allobaculum. In addition, we also investigated the correlation between differentially expressed genes (DEGs) and the expression of IL-10, Foxp3, PD-1, Occludin, Claudin1, and ZO-1 ( Figure 8 The results showed that, compared with the CON group, in the MOD group, poncirin and trihexyphenidyl were positively correlated with the index expression. In contrast, perphenazine and sulfoacetic acid were negatively correlated with the index expression ( Figure 8 In the ABX group, hc-toxin was positively correlated with index expression, while zectran and cinchonidine were negatively correlated with index expression ( Figure 8 In addition, the FMT group showed positive correlations for substances such as trihexyphenidyl and simazine compared with the MOD group; however, oxybutynin showed a negative correlation ( Figure 8 Finally, compared with the ABX group, the AF group showed a positive correlation to hc-toxin, but a negative correlation to zectran and pilocapine ( Figure 8 Middle I).

[0096] 3. Conclusion

[0097] Gas explosions (GE) increase the risk of traumatic brain injury (TBI) in miners. Currently, there is limited research on the pathogenesis of GE-induced TBI. However, our study provides new insights into addressing these unknowns. In short, GE induced dysbiosis in the gut microbiota of male rats with TBI. Targeted supplementation of intestinal bacteria not only alleviated acute brain injury and cognitive dysfunction, but also significantly improved the immune microenvironment and promoted the recovery of tight junctions in the colon and brain after GE-induced TBI. These findings were further supported by fecal microbiota transplantation (FMT) intervention after surgery. In summary, specific intestinal microorganisms, such as Clostridium_T and Allobaculum, and their metabolites, stimulate the reparative activation of the "gut-brain axis" to protect the brain and promote accelerated repair of the colon and brain and recovery of cognitive function after GE-induced TBI ( Fig. 9 ).

[0098] 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 all fall within the scope of the present invention to be protected.

Claims

1. Application of intestinal microbial flora in the preparation of microecological preparations for treating brain damage caused by gas explosion, wherein the intestinal microbial flora is a normal intestinal microbial flora.

2. The use according to claim 1, characterized in that: The gas explosion brain injury increased the levels of regulatory T cell-related factors interleukin-10, programmed death 1 and forkhead box protein P3 in brain tissue; normal intestinal microbial flora transplantation improved intestinal structure, upregulated the expression of tight junction proteins claudin, occludin and ZO-1, regulated the composition of intestinal microbial flora through the "gut-brain axis" and enhanced the intestinal and brain barrier function.

3. The use according to claim 1, characterized in that: The microecological preparation is used for improving cognitive dysfunction and brain barrier dysfunction caused by gas explosion.

4. The use according to claim 1, characterized in that: The microecological preparation is used to induce up-regulation of the expression of regulatory T cell-related factor proteins in the striatum, thereby achieving a protective effect on gas explosion-induced blood-brain barrier damage.

5. The use according to claim 1, characterized in that: The microecological preparation is used to promote the improvement of gastrointestinal barrier damage caused by gas explosion, and then improve the intestinal microbial flora to treat gas explosion brain.

6. The use according to claim 1, characterized in that: The microecological preparation is used to improve intestinal inflammation caused by gas explosions and enhance the expression of regulatory T cell factors in the colon, thereby achieving a protective effect on intestinal barrier damage.

7. The use according to claim 1, characterized in that: The microecological preparation is used to influence the composition of gas explosion intestinal microbiota, and further used to treat gas explosion brain damage.

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