Identification and functional analysis of perforin 1 from largemouth bass (Micropterus salmoides).

In this study, we present the first cloning and identification of perforin (MsPRF1) in largemouth bass (Micropterus salmoides). The full-length cDNA of MsPRF1 spans 1572 base pairs, encoding a 58.88 kDa protein consisting of 523 amino acids. Notably, the protein contains MACPF and C2 structural domains. To evaluate the expression levels of MsPRF1 in various healthy largemouth bass tissues, real-time quantitative PCR was employed, revealing the highest expression in the liver and gut. After the largemouth bass were infected by Nocardia seriolae, the mRNA levels of MsPRF1 generally increased within 48 h. Remarkably, the recombinant protein MsPRF1 exhibits inhibitory effects against both Gram-negative and Gram-positive bacteria. Additionally, the largemouth bass showed a higher survival rate in the N. seriolae challenge following the intraperitoneal injection of rMsPRF1, with observed reductions in the tissue bacterial loads. Moreover, rMsPRF1 demonstrated a significant impact on the phagocytic and bactericidal activities of largemouth bass MO/MΦ cells, concurrently upregulating the expression of pro-inflammatory factors. These results demonstrate that MsPRF1 has a potential role in the immune response of largemouth bass against N. seriolae infection.

Effects of Lactobacillus plantarum Ep-M17 on growth, immunity and intestinal microbiota of Penaeus vannamei under Microcystin-LR stress.

Microcystins (MCs) are biologically active cyclic heptapeptide compounds released by cyanobacteria in water bodies, and MC-LR is one of the most widespread and toxic isoforms. It frequently poses a serious threat to Penaeus vannamei aquaculture. Our previous study revealed that the supplementation of Lactobacillus plantarum Ep-M17 has a probiotic effect on P. vannamei health and whether Ep-M17 can alleviate the stressful effects of MC-LR on shrimp remains unclear. Therefore, in the present work, shrimp were fed MC-LR alone or combined with Ep-M17 for six weeks, and then evaluated the effects on histology, enzyme activity, gene expression, and intestinal flora. The results showed that MC-LR stress lead to slow growth and reduced survival rates in shrimp. However, feeding Ep-M17 significantly increased both the growth rate and survival rate. Meanwhile, MC-LR stress caused severe tissue damage in the hepatopancreas and intestines of shrimp, but Ep-M17 significantly reduced the toxic effects and protected the integrity of these tissues. Additionally, Ep-M17 significantly enhanced the activities of antioxidant enzymes and digestive enzymes, and induced higher expression of immune-related genes, thereby promoting the digestive and immune responses in shrimp. Furthermore, MC-LR stress disrupted the intestinal flora in shrimp intestines, while the use of Ep-M17 significantly increased the abundance of immune- and metabolism-related bacteria and inhibited the growth of pathogenic bacteria to maintain intestinal flora balance and intestinal health. In conclusion, our results indicate that Ep-M17 can reduce the toxic effect of MC-LR on shrimp and has a positive function in the prevention and control of shrimp diseases caused by MC-LR.

Quantification of Isoniazid-Heteroresistant Mycobacterium tuberculosis Using Droplet Digital PCR.

The quantitative detection of drug-resistance mutations in Mycobacterium tuberculosis (MTB) is critical for determining the drug resistance status of a sample. We developed a drop-off droplet digital PCR (ddPCR) assay targeting all major isoniazid (INH)-resistant mutations. The ddPCR assay consisted of three reactions: reaction A detects mutations at katG S315; reaction B detects inhA promoter mutations; and reaction C detects ahpC promoter mutations. All reactions could quantify 1%-50% of mutants in the presence of the wild-type, ranging from 100 to 50,000 copies/reaction. Clinical evaluation with 338 clinical isolates yielded clinical sensitivity of 94.5% (95% confidence interval [CI] = 89.1%-97.3%) and clinical specificity of 97.6% (95% CI = 94.6%-99.0%) compared with the traditional drug susceptibility testing (DST). Further clinical evaluation using 194 nucleic acid-positive MTB sputum samples revealed clinical sensitivity of 87.8% (95% CI = 75.8%-94.3%) and clinical specificity of 96.5% (95% CI = 92.2%-98.5%) in comparison with DST. All the mutant and heteroresistant samples detected by the ddPCR assay but susceptible by DST were confirmed by combined molecular assays, including Sanger sequencing, mutant-enriched Sanger sequencing and a commercial melting curve analysis-based assay. Finally, the ddPCR assay was used to monitor longitudinally the INH-resistance status and the bacterial load in nine patients undergoing treatment. Overall, the developed ddPCR assay could be an indispensable tool for quantification of INH-resistant mutations in MTB and bacterial loads in patients.

Investigation of the effects of cup plant (Silphium perfoliatum L.) on the growth, immunity, gut microbiota and disease resistance of Penaeus vannamei.

To investigate the effects of adding different concentrations of cup plant (Silphium perfoliatum L.) to the feed on the growth performance, hepatopancreas and intestinal microstructure, gene expression, enzyme activity, as well as intestinal microorganisms and resistance to Vibrio parahaemolyticus E1 and White spot syndrome virus (WSSV) infection of the shrimp, cup plant was added to the basal feed at 1%, 3%, 5% and 7% respectively, and fed the shrimp for 6 weeks. It was found that the addition of different concentrations of cup plant could significantly improve the specific growth rate and survival rate of shrimp, reduce the feed conversion rate, and improve the resistance to V. parahaemolyticus E1 and WSSV in shrimp, with the best effect of 5% addition. The tissue sections observations showed that the addition of cup plant significantly improved the hepatopancreas and intestinal tissues of shrimp, especially in alleviating the tissue damage caused by V. parahaemolyticus E1 and WSSV infection, but too high an addition (7%) could also cause side effects on the shrimp intestinal tract. Meantime, the addition of cup plant can also increase the activity of immunodigestive-related enzymes in the hepatopancreas and intestinal tissues of shrimp, and can significantly induce the up-regulation of immune-related genes expression, and it is positively correlated with the amount of addition in a certain range. In addition, it was found that the addition of cup plant has a significant regulating effect on the intestinal flora of shrimp, which can significantly promote the growth of beneficial bacteria such as Haloferula sp., Algoriphagus sp. and Coccinimonas sp., and inhibit pathogenic bacteria Vibrio sp., such as the number of Vibrionaceae_Vibrio and Pseudoalteromonadaceae_Vibrio in the experimental group were significantly reduced, and the lowest level in the 5% addition group. In summary, the study shows that cup plant can promote the growth of shrimp, improve the resistance of shrimp to disease, and is a potential green environmental feed additive that can replace antibiotics.

Cell surface-associated protein elongation factor Tu interacts with fibronectin mediating the adhesion of Lactobacillus plantarum HC-2 to Penaeus vannamei intestinal epithelium and inhibiting the apoptosis induced by LPS and pathogen in Caco-2 cells.

The adherent colonization of lactic acid bacteria in the animal intestine is the basis for their probiotic effect, and their bacteria surface proteins play an important role in this process. Previous work has demonstrated that Lactobacillus plantarum HC-2 can adhere and colonize the intestine of Penaeus vannamei, modulate the intestinal immune response and microbial diversity, protect the intestinal tissues from pathogenic damage, and improve the protection rate of shrimp. The aim of this work was to identify adhesion molecules on the surface of HC-2 and its adhesion receptors in the intestinal epithelium of shrimp. The elongation factor Tu (EF-Tu) on the surface of HC-2 was found to interact with Fibronectin (Fib) in the shrimp intestine by immunoblotting and yeast two-hybrid assays, and this interaction relationship was verified by immunoprecipitation (Co-IP). The adhesion of HC-2 to Caco-2 cells could be blocked via EF-Tu antibody confinement, and the adhesion of Fib to HC-2 could be blocked by Fib antibody confinement. Expression of Fib on the surface of HEK293T cells revealed a significant increase in the adhesion rate of HC-2 to HEK293T cells. Using immunofluorescence, a significant reduction in HC-2 adhesion to the intestine of shrimp was observed after blocking the Fib site in the shrimp intestine, particularly in Vibrio parahaemolyticus E1-infected intestines. In addition, the recombinant protein rEF-Tu was found to promote the growth of Caco-2 cells in a certain concentration range and significantly inhibit the apoptosis induced by LPS, Staphylococcus aureus and V. parahaemolyticus E1. Our results indicate that EF-Tu might participate in gut immunity and homeostasis, through its binding to the shrimp intestinal cells and inhibiting apoptosis.

Molecular cloning and characteristic analysis of polymeric immunoglobulin receptor-like (plgRL) in large yellow croaker (Larimichthys crocea).

In the present study, the polyimmunoglobulin receptor-like (pIgRL) of large yellow croaker (Larimichthys crocea) was first cloned and characterized. LcpIgRL's full-length cDNA was 1610 bp, encoding 377 amino acids, and the protein's predicted molecular weight was 41.9 kDa, containing two immunoglobulin-like structural domains. The transcript levels of LcpIgRL in different tissues of healthy large yellow croaker were examined by real-time fluorescence quantitative PCR, and the results showed that the gills and head kidney had the highest levels. Within 36 h of the large yellow croaker being infected with Vibrio harveyi, pIgRL mRNA first increased and then decreased in all determined tissues, with the highest expression in the skin and hindgut. Furthermore, a recombinant protein of the extracellular region of LcpIgRL was expressed in E. coli BL21, and a murine rLcpIgRL polyclonal antibody was prepared, which could react specifically with the natural LcpIgRL in skin mucus, but no natural LcpIgRL was detected in serum. Meanwhile, it was found that the rLcpIgRL could bind to the recombinant IgM and the natural IgM, indicating that LcpIgRL could mediate the transport of IgM in mucus. In addition, rLcpIgRL binds to Aeromonas hydrophila and V. harveyi, as well as lipopolysaccharide (LPS) and various saccharides, and reduced binding to bacteria was observed under LPS treatment, suggesting that LcpIgRL can bind to bacteria to prevent infection and that saccharide binding is an important mechanism of interaction between pIgRL and bacteria.

Current status and development prospects of aquatic vaccines.

Diseases are a significant impediment to aquaculture's sustainable and healthy growth. The aquaculture industry is suffering significant financial losses as a result of the worsening water quality and increasing frequency of aquatic disease outbreaks caused by the expansion of aquaculture. Drug control, immunoprophylaxis, ecologically integrated control, etc. are the principal control strategies for fish infections. For a long time, the prevention and control of aquatic diseases have mainly relied on the use of various antibiotics and chemical drugs. However, long-term use of chemical inputs not only increases pathogenic bacteria resistance but also damages the fish and aquaculture environments, resulting in drug residues in aquatic products, severely impeding the development of the aquaculture industry. The development and use of aquatic vaccines are the safest and most effective ways to prevent aquatic animal diseases and preserve the health and sustainability of aquaculture. To give references for the development and implementation of aquatic vaccines, this study reviews the development history, types, inoculation techniques, mechanisms of action, development prospects, and challenges encountered with aquatic vaccines.

Development of Risk Prediction Model for Muscular Calf Vein Thrombosis with Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

Purpose: This study aims to establish a risk prediction model for muscular calf vein thrombosis (MCVT) in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Methods: The research sample consisted of 248 patients with AECOPD and all of them underwent vascular ultrasounds of both lower limbs in this retrospective study. Univariate analysis and multivariate logistic regression analysis were conducted on factors with significant group differences to screen for the independent risk factors of MCVT. A nomogram to predict the risk of MCVT was constructed and validated with bootstrap resampling. Results: According to the exclusion criteria, 240 patients were included for analysis, divided into the MCVT group (n = 81) and the non-MCVT group (n = 159). Multivariate logistic regression analyses showed that hypertension, elevated MPV, reduced albumin (ALB), elevated D-dimer and bed rest ≥3 days were independent risk factors for MCVT in AECOPD. A nomogram model for predicting AECOPD with MCVT was established based on them. The area under the curve (AUC) of receiver operating characteristic (ROC) curve for the prediction model and the simplified Wells score was 0.784 (95% CI: 0.722-0.847) and 0.659 (95% CI: 0.583-0.735), respectively. The cut-off value and Youden index of prediction model were 0.248 and 0.454, respectively. At the same time, the sensitivity, specificity, positive predictive value, and negative predictive value of the prediction model were 85.9%, 59.5%, 84.6%, and 77.4%, respectively. The sensitivity and specificity of the simplified Wells score were 67.9% and 56.3%, respectively. Validation by the use of bootstrap resampling revealed optimal discrimination and calibration, and the decision analysis curve (DAC) suggested that this prediction model involved high clinical practicability. Conclusion: We developed a nomogram that can predict the risk of MCVT for AECOPD patients. This model has the potential to assist clinicians in making treatment recommendations and formulating corresponding prevention measures.

Enhancement of growth, survival, immunity and disease resistance in Litopenaeus vannamei, by the probiotic, Lactobacillus plantarum Ep-M17.

Green ecological prevention and control technology is a hot spot for aquatic disease research in recent years, and lactic acid bacteria is an important type of probiotic widely used in aquaculture. In this study, a strain of Lactobacillus plantarum Ep-M17 was isolated from the intestine of healthy grouper, which showed good antibacterial activity in vitro. To investigate the application prospects of Ep-M17 as a probiotic, we added it to the diet and fed Litopenaeus vannamei, and then detected its influence on the growth performance, survival rate, disease resistance, intestinal tissue structure, gene transcription, and the flora in the gut of shrimp. The results showed that feeding Ep-M17 increased the specific growth rate, reduced the feed conversion rate, improved the survival rate, and achieved a 76.9% relative protection rate after Vibrio parahaemolyticus E1 infection in shrimp. Histological examination displayed that Ep-M17-fed shrimp had a thick intestinal villi layer, which enhanced the protection against pathogen damage. It was also found that Ep-M17 significantly increased the activity levels of immune and digestion-related enzymes SOD, CAT, TRY, AKP, LIP, and AMS in the gut of shrimp, especially after V. parahaemolyticus E1 infection, these enzymes increased significantly higher than that of control. Transcriptome analysis revealed that Ep-M17 activated significantly differential expression of genes in immune, nutritional, metabolic, and Signal Transduction-related pathways in the gut of shrimp. In addition, Ep-M17 enriched the bacterial diversity of the shrimp gut, with a significant increase in many low-abundance bacterial species, a significant decrease in the number of pathogenic bacteria like Vibrio, and a significant increase in the number of beneficial bacteria. The above results evaluated that Ep-M17 as a potential probiotic can promote the growth and improve the disease resistance of shrimp by regulating the nutritional immune response and flora of the intestine.

miR-3934 regulates the apoptosis and secretion of inflammatory cytokines of basophils via targeting RAGE in asthma.

BACKGROUND: Several miRNAs are now known to have clear connections to the pathogenesis of asthma. The present study focused on the potential role of miR-3934 during asthma development. METHODS: miR-3934 was detected as a down-regulated miRNA in basophils by sequencing analysis. Next, the expression levels of miR-3934 in peripheral blood mononuclear cells of 50 asthma patients and 50 healthy volunteers were examined by RT-qPCR methods. The basophils were then treated with AGEs and transfected with miR-3934 mimics. The apoptosis levels were examined by flow cytometry assay; and the expression levels of cytokines were detected using the ELISA kits. Finally, the Western blot was performed to examined the expression of key molecules in the TGF-ß/Smad signaling pathway. RESULTS: miR-3934 was down-regulated in the basophils of asthmatic patients. The expression of the pro-inflammatory cytokines IL-6, IL-8 and IL-33 was enhanced in basophils from asthmatic patients, and this effect was partially reversed by transfection of miR-3934 mimics. Furthermore, receiver operating characteristics analysis showed that miR-3934 levels can be used to distinguish asthma patients from healthy individuals. miR-3934 partially inhibited advanced glycation end products-induced increases in basophil apoptosis by suppressing expression of RAGE. CONCLUSION: Our results indicate that miR-3934 acts to mitigate the pathogenesis of asthma by targeting RAGE and suppressing TGF-ß/Smad signaling.

Clinical Application and Evaluation of Metagenomic Next-Generation Sequencing in Pulmonary Infection with Pleural Effusion.

Purpose: Metagenomic next-generation sequencing (mNGS) is a novel technique of pathogens detection that plays an increasingly important role in clinical practice. In this study, we explored the application value of mNGS in pulmonary infection combined with pleural effusion applied to samples of pleural effusion fluid. Patients and Methods: We reviewed 80 cases of pulmonary infection with pleural effusion between August 2020 and October 2021. Among them, 40 patients were placed in the mNGS group and underwent both culture and mNGS testing; the patients in the control group were only subjected to culture test. The effectiveness of mNGS was evaluated for microbial composition and diagnostic accuracy in every pleural effusion specimen type. Results: We found that the positive rate of mNGS was 70% (28/40). The comparison between mNGS and culture method resulted that the sensitivity was 100% (95% CI: 29.2-100%) and the specificity was 64.9% (95% CI: 47.5-79.8%). The positive predictive value of mNGS was 18.8% (95% CI, 13.0-26.3%), and the negative predictive value was 100%. The most commonly identified potential pathogens were bacteria, such as Streptococcus, Prevotella, Parvimonas, Porphyromonas and Gemella. The most detected fungal infection was Candida and Pneumocystis. A total of 11 patients were identified as mixed infection by mNGS. Treatment regimen adjustments were made according to mNGS results and the overall length of hospital stay in the mNGS group was shorter compared to that of the control group. Conclusion: In this study, mNGS produced higher positive rates than the culture method in detecting pathogens in the pleural effusion specimens. The technology performed satisfactorily, providing more diagnostic evidence and reducing the length of hospital stay.

Adhesion and Colonization of the Probiotic Lactobacillus plantarum HC-2 in the Intestine of Litopenaeus Vannamei Are Associated With Bacterial Surface Proteins.

Surface proteins are a type of proteins expressed on the surface of bacteria that play an important role in cell wall synthesis, maintenance of cell morphology, and signaling with the host. Our previous study showed that the probiotic Lactobacillus plantarum HC-2 improved the growth performance and immune response of Litopenaeus vannamei. To further investigate the probiotic mechanism, we determined the automatic aggregation ability of the bacteria and surface hydrophobicity of HC-2 after being treated with 5 M of lithium chloride (LiCl) and observed the morphology and adhesion of the bacteria to HCT116 cells. The results showed that with the removal of the HC-2 surface protein, the auto-aggregation ability and surface hydrophobicity of HC-2 decreased, and the crude mucus layer coated on the bacterial surface gradually dissociated. The adhesion rate of HC-2 to HCT116 cells decreased from 98.1 to 20.9%. Moreover, a total of 201 unique proteins were identified from the mixture of the surface proteins by mass spectrometry (MS). Several proteins are involved in transcription and translation, biosynthetic or metabolic process, cell cycle or division, cell wall synthesis, and emergency response. Meanwhile, a quantitative real-time PCR qPCR_ showed that HC-2 was mainly colonized in the midgut of shrimp, and the colonization numbers were 15 times higher than that in the foregut, while the colonization rate in the hindgut was lower. The adhesion activity measurement showed that the adhesion level of HC-2 to crude intestinal mucus of L. vannamei was higher than that of bovine serum albumin (BSA) and collagen, and the adhesion capacity of the bacterial cells decreased with the extension of LiCl-treatment time. Finally, we identified the elongation factor Tu, Type I glyceraldehyde-3-phosphate dehydrogenase, small heat shock protein, and 30S ribosomal protein from the surface proteins, which may be the adhesion proteins of HC-2 colonization in the shrimp intestine. The above results indicate that surface proteins play an important role in maintaining the cell structure stability and cell adhesion. Surface proteomics analysis contributes to describing potential protein-mediated probiotic-host interactions. The identification of some interacting proteins in this work may be beneficial to further understand the adhesion/colonization mechanism and probiotic properties of L. plantarum HC-2 in the shrimp intestine.

Metagenomic Approaches Reveal Strain Profiling and Genotyping of Klebsiella pneumoniae from Hospitalized Patients in China.

Klebsiella pneumoniae is a leading cause of highly drug-resistant infections in hospitals worldwide. Strain-level bacterial identification on the genetic determinants of multidrug resistance and high pathogenicity is critical for the surveillance and treatment of this clinically relevant pathogen. In this study, metagenomic next-generation sequencing was performed for specimens collected from August 2020 to May 2021 in Ruijin Hospital, Ningbo Women and Children's Hospital, and the Second Affiliated Hospital of Harbin Medical University. Genome biology of K. pneumoniae prevalent in China was characterized based on metagenomic data. Thirty K. pneumoniae strains derived from 14 sequence types were identified by multilocus sequence typing. The hypervirulent ST11 K. pneumoniae strains carrying the KL64 capsular locus were the most prevalent in the hospital population. The phylogenomic analyses revealed that the metagenome-reconstructed strains and public isolate genomes belonging to the same STs were closely related in the phylogenetic tree. Furthermore, the pangenome structure of the detected K. pneumoniae strains was analyzed, particularly focusing on the distribution of antimicrobial resistance genes and virulence genes across the strains. The genes encoding carbapenemases and extended-spectrum beta-lactamases were frequently detected in the strains of ST11 and ST15. The highest numbers of virulence genes were identified in the well-known hypervirulent strains affiliated to ST23 bearing the K1 capsule. In comparison to traditional cultivation and identification, strain-level metagenomics is advantageous to understand the mechanisms underlying resistance and virulence of K. pneumoniae directly from clinical specimens. Our findings should provide novel clues for future research into culture-independent metagenomic surveillance for bacterial pathogens. IMPORTANCE Routine culture and PCR-based molecular testing in the clinical microbiology laboratory are unable to recognize pathogens at the strain level and to detect strain-specific genetic determinants involved in virulence and resistance. To address this issue, we explored the strain-level profiling of K. pneumoniae prevalent in China based on metagenome-sequenced patient materials. Genome biology of the targeted bacterium can be well characterized through decoding sequence signatures and functional gene profiles at the single-strain resolution. The in-depth metagenomic analysis on strain profiling presented here shall provide a promising perspective for culture-free pathogen surveillance and molecular epidemiology of nosocomial infections.

[Effect of early mechanical ventilation on the expression of inflammatory factors and prognosis in patients with severe traumatic brain injury].

OBJECTIVE: To observe the effect of early mechanical ventilation on the expression of inflammatory factors and prognosis of patients with severe traumatic brain injury (sTBI). METHODS: From January 2017 to December 2020, 138 patients with sTBI admitted to the department of the emergency of Xinhua Hospital Chongming Branch were enrolled. Although some patients were admitted to hospital without acute respiratory failure, their Glasgow coma score (GCS) were less than 8, they bad risk of hypoxia, so early mechanical ventilation was required. According to the patient's condition and the willingness of family members, patients were divided into mechanical ventilation group (tracheal intubation mechanical ventilation) and conventional oxygen inhalation group (nasal catheter or mask oxygen inhalation) in the end. The arterial partial pressure of oxygen (PaO2), arterial partial pressure of carbon dioxide (PaCO2), oxygenation index (PaO2/FiO2), tumor necrosis factor-α (TNF-α), and interleukin (IL-6, IL-10) levels at admission, preoperation and 72 hours postoperation, as well as GCS before operation and 1 week after operation, the duration and number of patients successfully evacuated from the ventilator within 1 week after surgery were observed and analyzed. RESULTS: A total of 138 sTBI patients were enrolled in the study, including 72 cases in the mechanical ventilation group and 66 cases in the routine oxygen inhalation group. In the two groups, PaO2, PaO2/FiO2 and IL-10 were higher, and PaCO2, TNF-α and IL-6 were lower at 72 hours post operation than that before operation. Moreover, the changes in the mechanical ventilation group were more significant than those in the conventional oxygen inhalation group [PaO2 (1 mmHg = 0.133 kPa): 94.6±7.7 vs. 92.5±6.8, PaO2/FiO2 (mmHg): 351±94 vs. 319±89, IL-10 (ng/L): 8.2±2.7 vs. 7.4±1.8, PaCO2 (mmHg): 35.6±1.8 vs. 37.5±2.7, TNF-α(ng/L): 71.5±6.3 vs. 96.8±15.5, IL-6 (ng/L): 10.8±3.9 vs. 14.4±6.5, all P < 0.05]. There were 17 patients with severe respiratory insufficiency or failure in the conventional oxygen inhalation group before operation. Compared with the conventional oxygen inhalation group, the GCS score (11.7±3.1 vs. 9.1±4.6) and the proportion of successful weaning [62.5% (45/72) vs. 44.0% (29/66)] were significantly higher, and the duration of successful weaning (hours: 63.5±28.6 vs. 88.1±33.9) was significantly shorter in the mechanical ventilation group 1 week after operation. CONCLUSIONS: Early mechanical ventilation in sTBI patients can significantly improve oxygen supply, inhibit the release of pro-inflammatory factors, reduce secondary brain damage, and effectively improve the prognosis.