Nephrotoxicity assessment of podophyllotoxin-induced rats by regulating PI3K/Akt/mTOR-Nrf2/HO1 pathway in view of toxicological evidence chain (TEC) concept.

Adverse reactions to traditional Chinese medicine have hindered the healthy development and internationalization process of the traditional Chinese medicine industry. The critical issue that needs to be solved urgently is to evaluate the safety of traditional Chinese medicine systematically and effectively. Podophyllotoxin (PPT) is a highly active compound extracted from plants of the genus Podophyllum such as Dysosma versipellis (DV). However, its high toxicity and toxicity to multiple target organs affect the clinical application, such as the liver and kidney. Based on the concurrent effects of PPT's medicinal activity and toxicity, it would be a good example to conduct a systematic review of its safety. Therefore, this study revolves around the Toxicological Evidence Chain (TEC) concept. Based on PPT as the main toxic constituent in DV, observe the objective toxicity impairment phenotype of animals. Evaluate the serum biochemical indicators and pathological tissue sections for substantial toxic damage results. Using metabolomics, lipidomics, and network toxicology to evaluate the nephrotoxicity of PPT from multiple perspectives systematically. The results showed that PPT-induced nephrotoxicity manifested as renal tubular damage, mainly affecting metabolic pathways such as glycerophospholipid metabolism and sphingolipid metabolism. PPT inhibits the autophagy process of kidney cells through the PI3K/Akt/mTOR and Nrf2/HO1 pathways and induces the activation of oxidative stress in the body, thereby causing nephrotoxic injury. This study fully verified the feasibility of the TEC concept for the safety and toxicity evaluation of traditional Chinese medicine. Provide a research template for systematically evaluating the safety of traditional Chinese medicine.

Analysis of differential metabolites in lung cancer patients based on metabolomics and bioinformatics.

Aim: Based on metabonomics, the metabolic markers of lung cancer patients were analyzed, combined with bioinformatics to explore the underlying disease mechanism. Materials & methods: Based on case-control design, using UPLC-Q-TOF/MS, urine metabolites were detected in discovery and validation set. Multivariate statistical analysis were performed to identify potential markers for lung cancer. A network analysis was constructed to integrate lung cancer disease targets with the above metabolic markers, and its possible mechanism and biological significance were explained. Results: A total of 35 potential markers were identified, 11 of which overlapped. Five key markers have a good linear correlation with serum biochemical indicators. Conclusion: The occurrence and development of lung cancer are closely related to disturbance of D-Glutamine and D-glutamate metabolism, amino acid imbalance. This test was registered on China clinical trial registration center (www.chictr.org.cn/index.aspx), registration number was ChiCTR1900025543.

Exendin-4 Plays a Protective Role in a Rat Model of Spinal Cord Injury Through SERCA2.

BACKGROUND/AIMS: Current therapies for spinal cord injury (SCI) have limited efficacy, and identifying a therapeutic target is a pressing need. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) plays an important role in regulating calcium homeostasis, which has been shown to inhibit apoptosis. Exendin-4 has been shown to inhibit the apoptosis of nerve cells in SCI, which can also improve SERCA2 expression. In this study, we sought to determine whether exendin-4 plays a protective role in a rat model of SCI via SERCA2. METHODS: To investigate the effects of exendin-4 on SCI, a rat model of SCI was induced by a modified version of Allen's method. Spinal cord tissue sections from rats and western blot analysis were used to examine SERCA2 expression after treatment with the long-acting glucagon-like peptide 1 receptor exendin-4 or the SERCA2 antagonist 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CE). Locomotor function was evaluated using the Basso Beattie Bresnahan locomotor rating scale and slanting board test. RESULTS: Cell apoptosis was increased with CE treatment and decreased with exendin-4 treatment. Upregulation of SERCA2 in female rats with SCI resulted in an improvement of motor function scores and histological changes. CONCLUSION: These findings suggest that exendin-4 plays a protective role in a rat model of SCI through SERCA2 via inhibition of apoptosis. Existing drugs targeting SERCA2 may be an effective therapeutic strategy for the treatment of SCI.

Exploring immune-related pathogenesis in lung injury: Providing new insights Into ALI/ARDS.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a significant global burden of morbidity and mortality, with lung injury being the primary cause of death in affected patients. The pathogenesis of lung injury, however, remains a complex issue. In recent years, the role of the immune system in lung injury has attracted extensive attention worldwide. Despite advancements in our understanding of various lung injury subtypes, significant limitations persist in both prevention and treatment. This review investigates the immunopathogenesis of ALI/ARDS, aiming to elucidate the pathological processes of lung injury mediated by dendritic cells (DCs), natural killer (NK) cells, phagocytes, and neutrophils. Furthermore, the article expounds on the critical contributions of gut microbiota, inflammatory pathways, and cytokine storms in the development of ALI/ARDS.

HSV: The scout and assault for digestive system tumors.

More than 25% of all malignant tumors are digestive system tumors (DSTs), which mostly include esophageal cancer, gastric cancer, pancreatic cancer, liver cancer, gallbladder cancer and cholangiocarcinoma, and colorectal cancer. DSTs have emerged as one of the prominent reasons of morbidity and death in many nations and areas around the world, posing a serious threat to human life and health. General treatments such as radiotherapy, chemotherapy, and surgical resection can poorly cure the patients and have a bad prognosis. A type of immunotherapy known as oncolytic virus therapy, have recently shown extraordinary anti-tumor effectiveness. One of the viruses that has been the subject of the greatest research in this field, the herpes simplex virus (HSV), has shown excellent potential in DSTs. With a discussion of HSV-1 based on recent studies, we outline the therapeutic effects of HSV on a number of DSTs in this review. Additionally, the critical function of HSV in the detection of cancers is discussed, and some HSV future possibilities are shown.

A new strategy for treating colorectal cancer: Regulating the influence of intestinal flora and oncolytic virus on interferon.

Colorectal cancer (CRC) has the third highest incidence and the second highest mortality in the world, which seriously affects human health, while current treatments methods for CRC, including systemic therapy, preoperative radiotherapy, and surgical local excision, still have poor survival rates for patients with metastatic disease, making it critical to develop new strategies for treating CRC. In this article, we found that the gut microbiota can modulate the signaling pathways of cancer cells through direct contact with tumor cells, generate inflammatory responses and oxidative stress through interactions between the innate and adaptive immune systems, and produce diverse metabolic combinations to trigger specific immune responses and promote the initiation of systemic type I interferon (IFN-I) and anti-viral immunity. In addition, oncolytic virus-mediated immunotherapy for regulating oncolytic virus can directly lyse tumor cells, induce the immune activity of the body, interact with interferon, inhibit the anti-viral effect of IFN-I, and enhance the anti-tumor effect of IFN-II. Interferon plays an important role in the anti-tumor process. We put forward that exploring the effects of intestinal flora and oncolytic virus on interferon to treat CRC is a promising therapeutic option.

The γδ T cells dual function and crosstalk with intestinal flora in treating colorectal cancer is a promising area of study.

The occurrence of colorectal cancer (CRC) is highly prevalent and severely affects human health, with the third-greatest occurrence and the second-greatest rate of death globally. Current CRC treatments, including surgery, radiotherapy, and chemotherapy, do not significantly improve CRC patients' survival rate and quality of life, so it is essential to develop new treatment strategies. Adoptive cell therapy and other immunotherapy came into being. Currently, there has been an especially significant emphasis on γδ T cells as being the primary recipient of adoptive cell therapy. The present investigation found that γδ T cells possess the capability to trigger cytotoxicity in CRC cells, secrete cytokines, recruit immune cells for the purpose of destroying cancer cells, and inhibit the progress of CRC indirectly. Nevertheless, It is possible for γδ T cells to initiate a storm of inflammatory factors and inhibit the immune response to promote the advancement of CRC. This review demonstrates a close association between the γδ T cell initiation pathway and their close association with the intestinal flora. It has been observed that the intestinal flora performs a vital function in facilitating the stimulation and functioning of γδ T cells. The tumor-fighting effect is mainly regulated by desulphurizing Vibrio and lactic acid bacteria. In contrast, the regulation of tumor-promoting impact is closely related to Clostridia and ETBF. This review systematically combs γδ T cell dual function and their relationship to intestinal flora, which offers a conceptual framework for the γδ T cell application for CRC therapies.

The gamble between oncolytic virus therapy and IFN.

Various studies are being conducted on oncolytic virotherapy which one of the mechanisms is mediating interferon (IFN) production by it exerts antitumor effects. The antiviral effect of IFN itself has a negative impact on the inhibition of oncolytic virus or tumor eradication. Therefore, it is very critical to understand the mechanism of IFN regulation by oncolytic viruses, and to define its mechanism is of great significance for improving the antitumor effect of oncolytic viruses. This review focuses on the regulatory mechanisms of IFNs by various oncolytic viruses and their combination therapies. In addition, the exerting and the producing pathways of IFNs are briefly summarized, and some current issues are put forward.

Mechanism of COVID-19 Causing ARDS: Exploring the Possibility of Preventing and Treating SARS-CoV-2.

Novel coronavirus pneumonia (COVID-19) is spreading worldwide, causing great harm and stress to humans. Since patients with novel coronavirus (SARS-CoV-2) have a high probability of developing acute respiratory distress syndrome (ARDS) in severe cases, the pathways through which SARS-CoV-2 causes lung injury have become a major concern in the scientific field. In this paper, we investigate the relationship between SARS-CoV-2 and lung injury and explore the possible mechanisms of COVID-19 in ARDS from the perspectives of angiotensin-converting enzyme 2 protein, cytokine storm, activation of the immune response, triggering of Fas/FasL signaling pathway to promote apoptosis, JAK/STAT pathway, NF-κB pathway, type I interferon, vitamin D, and explore the possibility of prevention and treatment of COVID-19. To explore the possibility of SARS-CoV-2, and to provide new ideas to stop the development of ARDS in COVID-19 patients.

Oncolytic Viruses: Immunotherapy Drugs for Gastrointestinal Malignant Tumors.

Oncolytic virus therapy has advanced rapidly in recent years. Natural or transgenic viruses can target tumor cells and inhibit tumor growth and metastasis in various ways without interfering with normal cell and tissue function. Oncolytic viruses have a high level of specificity and are relatively safe. Malignant tumors in the digestive system continue to have a high incidence and mortality rate. Although existing treatment methods have achieved some curative effects, they still require further improvement due to side effects and a lack of specificity. Many studies have shown that oncolytic viruses can kill various tumor cells, including malignant tumors in the digestive system. This review discusses how oncolytic virus therapy improves malignant tumors in the digestive system from the point-of-view of basic and clinical studies. Also, the oncolytic virus anti-tumor mechanisms underpinning the therapeutic potential of oncolytic viruses are expounded. In all, we argue that oncolytic viruses might eventually provide therapeutic solutions to malignant tumors in the digestive system.

Oncolytic viruses combined with immune checkpoint therapy for colorectal cancer is a promising treatment option.

Immunotherapy is one of the promising strategies in the treatment of oncology. Immune checkpoint inhibitors, as a type of immunotherapy, have no significant efficacy in the clinical treatment of patients with pMMR/MSS/MSI-L mCRC alone. Therefore, there is an urgent need to find combination therapies that can improve the response rate of immune checkpoint inhibitors. Oncolytic viruses are a new class of cancer drugs that, in addition to directly lysing tumor cells, can facilitate the action of immune checkpoint inhibitors by modulating the tumor microenvironment and transforming "cold" tumors into "hot" ones. The combination of oncolytic viruses and immune checkpoint inhibitors is currently being used in several primary and clinical studies to treat tumors with exciting results. The combination of genetically modified "armed" OV with ICIs is expected to be one of the treatment options for pMMR/MSS/MSI-L mCRC. In this paper, we will analyze the current status of oncolytic viruses and ICIs available for the treatment of CRC. The feasibility of OV in combination with ICI for CRC will be discussed in terms of the mechanism of action of OV in treating tumors.

COVID-19 pandemic: A multidisciplinary perspective on the pathogenesis of a novel coronavirus from infection, immunity and pathological responses.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus2 (SARS-CoV-2), has spread to more than 200 countries and regions, having a huge impact on human health, hygiene, and economic activities. The epidemiological and clinical phenotypes of COVID-19 have increased since the onset of the epidemic era, and studies into its pathogenic mechanisms have played an essential role in clinical treatment, drug development, and prognosis prevention. This paper reviews the research progress on the pathogenesis of the novel coronavirus (SARS-CoV-2), focusing on the pathogenic characteristics, loci of action, and pathogenic mechanisms leading to immune response malfunction of SARS-CoV-2, as well as summarizing the pathological damage and pathological manifestations it causes. This will update researchers on the latest SARS-CoV-2 research and provide directions for future therapeutic drug development.

The role of metabolic reprogramming in cancer metastasis and potential mechanism of traditional Chinese medicine intervention.

Metabolic reprogramming is one of the most prominent features underlying cancer cells progression and metastasis.Traditional Chinese medicine (TCM) has been widely used in the clinical treatment of cancer, with the advantages of multi-pathway, multi-target, multi-component anti-tumor pharmacological effects and low risk of adverse effects. However, the mechanisms underlying the anti-tumor effects of TCM are not fully understood, especially on cellular metabolic reprogramming. In this review, we summarize the role of glucose, lipid and amino acid metabolism in cancer metastasis, which is key in cancer cells and tumor micro-environment (TME) cell metabolism. Furthermore, we reviewed the potential mechanisms by which, most bioactive TCM compounds suppress cancer metastasis by regulating metabolic reprogramming and the possibility of sensitizing other anti-tumor drugs. TCM and its bioactive compounds have huge prospects for clinical application in the treatment of cancer metastasis. Unfortunately, little is currently known about the regulatory effects of Chinese herbal medicines and their bioactive compounds on the metabolic reprogramming of cancer cells and the combination therapy for cancers. This review provides novel insights into the regulation of metabolic reprogramming by TCM in combination with other anti-tumor drugs against cancer metastasis and the possibility of becoming sensitizers for other anti-tumor drugs.

A combination of PD­1/PD­L1 inhibitors: The prospect of overcoming the weakness of tumor immunotherapy (Review).

Programmed cell death protein­1 (PD­1)/programmed death protein ligand­1 (PD­L1) inhibitors for treatment of a various types of cancers have revolutionized cancer immunotherapy. However, PD­1/PD­L1 inhibitors are associated with a low response rate and are only effective on a small number of patients with cancer. Development of an anti­PD­1/PD­L1 sensitizer for improving response rate and effectiveness of immunotherapy is a challenge. The present study reviews the synergistic effects of PD­1/PD­L1 inhibitor with oncolytic virus, tumor vaccine, molecular targeted drugs, immunotherapy, chemotherapy, radiotherapy, intestinal flora and traditional Chinese medicine, to provide information for development of effective combination therapies.

Lentinan combined with cisplatin for the treatment of non-small cell lung cancer.

BACKGROUND: A growing number of studies suggest that lentinan combined with cisplatin thoracic injection for the treatment of lung cancer is an effective combination of traditional Chinese and Western medicine, which has a continuous and beneficial effect on eliminating clinical symptoms and improving cachexia in lung cancer patients. However, whether this treatment is effective and safe for lung cancer patients or not, evidence supporting the effectiveness and safety of this treatment is still incomplete. Besides, there is lack of systematic review to assess the detailed situation (including risk of bias and methodology) of current related clinical studies. OBJECTIVE: This study aimed to evaluate the effectiveness and safety of lentinan combined with cisplatin thoracic injection in the treatment of lung cancer. METHODS: The major databases (Embase, PubMed, the Cochrane Library, China National Knowledge Infrastructure, Chinese Scientific Journals Database [VIP] Database, Chinese Biomedical Literature Service System [SinoMed], and Wanfang Database) were searched from inception to March 1, 2020. Randomized controlled trials (RCTs) of lentinan combined with cisplatin chest injection on patients with non-small cell lung cancer (NSCLC) were identified. Two assessors reviewed each trial independently. The methodological quality of the eligible studies was evaluated according to the Cochrane Collaboration's tool for assessing risk of bias. Both the data extraction and the literature quality screening evaluation were conducted independently by 2 researchers. RESULTS: Totally 17 clinical RCTs were included in this study, involving 1390 patients. Meta-analysis results showed that the clinical efficacy (risk ratio [RR] = 1.34, 95% confidence interval [CI] 1.21-1.48), effective subgroup analysis (RR = 1.51, 95% CI 1.3-1.77), and quality of life (RR = 1.48, 95% CI 1.27-1.72), the differences are statistically significant. In terms of adverse reactions, mainly related to gastrointestinal reactions and bone marrow suppression, the incidence and degree of adverse reactions of lentinan combined with cisplatin thoracic injection group were lower than those of cisplatin thoracic injection group alone. CONCLUSIONS: The current evidence prompted that Lentinan combined with cisplatin in thoracic injection might benefit patients with NSCLC on a certain extent; this systematic review revealed some definite conclusions about the application of Lentinan combined with cisplatin in thoracic injection for NSCLC. Due to the low methodological quality, high risk of bias, and inadequate reporting on clinical data, these results still require verification by a large number of well designed, heterogeneous RCT studies. More rigorous, multicenter, sufficient-sample, and double-blind RCTs are warranted.

Combination of UPLC-Q-TOF/MS and Network Pharmacology to Reveal the Mechanism of Qizhen Decoction in the Treatment of Colon Cancer.

Traditional Chinese medicine (TCM) has been utilized for the treatment of colon cancer. Qizhen decoction (QZD), a potential compound prescription of TCM, possesses multiple biological activities. It has been proven clinically effective in the treatment of colon cancer. However, the molecular mechanism of anticolon cancer activity is still not clear. This study aimed to identify the chemical composition of QZD. Furthermore, a collaborative analysis strategy of network pharmacology and cell biology was used to further explore the critical signaling pathway of QZD anticancer activity. First, ultraperformance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) was performed to identify the chemical composition of QZD. Then, the chemical composition database of QZD was constructed based on a systematic literature search and review of chemical constituents. Moreover, the common and indirect targets of chemical components of QZD and colon cancer were searched by multiple databases. A protein-protein interaction (PPI) network was constructed using the String database (https://www.string-db.org/). All of the targets were analyzed by Gene Oncology (GO) bioanalysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and the visual network topology diagram of "Prescription-TCM-Chemical composition-Direct target-Indirect target-Pathway" was constructed by Cytoscape software (v3.7.1). The top molecular pathway ranked by statistical significance was further verified by molecular biology methods. The results of UPLC-Q-TOF/MS showed that QZD had 111 kinds of chemical components, of which 103 were unique components and 8 were common components. Ten pivotal targets of QZD in the treatment of colon cancer were screened by the PPI network. Targets of QZD involve many biological processes, such as the signaling pathway, immune system, gene expression, and so on. QZD may interfere with biological pathways such as cell replication, oxygen-containing compounds, or organic matter by protein binding, regulation of signal receptors or enzyme binding, and affect cytoplasm and membrane-bound organelles. The main antitumor core pathways were the apoptosis metabolic pathway, the PI3K-Akt signal pathway, and so on. Expression of the PI3K-Akt signal pathway was significantly downregulated after the intervention of QZD, which was closely related to the inhibition of proliferation and migration of colon cancer cells by cell biology methods. The present work may facilitate a better understanding of the effective components, therapeutic targets, biological processes, and signaling pathways of QZD in the treatment of colon cancer and provide useful information about the utilization of QZD.

Exendin-4 promotes actin cytoskeleton rearrangement and protects cells from Nogo-A-Δ20 mediated spreading inhibition and growth cone collapse by down-regulating RhoA expression and activation via the PI3K pathway.

Exendin-4 is a protein of the GLP-1 family currently used to treat diabetes. Recently, a greater number of biological properties have been associated with the GLP-1 family. Our data shows that exendin-4 treatment significantly increases the cytoskeleton rearrangement, which leads to an increasingly differentiated phenotype and reduced cell migration. We also found that exendin-4 could prevent SH-SY5Y and PC12 cells from Nogo-A-Δ20 mediated spreading inhibition and neurite collapse. Western blot analysis indicated that exendin-4 treatment both reduced the expression and activation of RhoA via the PI3K signaling pathway. These data suggest that exendin-4 may protect nerve regeneration by preventing the inhibition of Nogo-A via down-regulating RhoA expression and activation.

Saikosaponin a increases interleukin-10 expression and inhibits scar formation after sciatic nerve injury.

Nerve scarring after peripheral nerve injury can severely hamper nerve regeneration and functional recovery. Further, the anti-inflammatory cytokine, interleukin-10, can inhibit nerve scar formation. Saikosaponin a (SSa) is a monomer molecule extracted from the Chinese medicine, Bupleurum. SSa can exert anti-inflammatory effects in spinal cord injury and traumatic brain injury. However, it has not been shown whether SSa can play a role in peripheral nerve injury. In this study, rats were randomly assigned to three groups. In the sham group, the left sciatic nerve was directly sutured after exposure. In the sciatic nerve injury (SNI) + SSa and SNI groups, the left sciatic nerve was sutured and continuously injected daily with SSa (10 mg/kg) or an equivalent volume of saline for 7 days. Enzyme linked immunosorbent assay results demonstrated that at 7 days after injury, interleukin-10 level was considerably higher in the SNI + SSa group than in the SNI group. Masson staining and western blot assay demonstrated that at 8 weeks after injury, type I and III collagen content was lower and nerve scar formation was visibly less in the SNI + SSa group compared with the SNI group. Simultaneously, sciatic functional index and nerve conduction velocity were improved in the SNI + SSa group compared with the SNI group. These results confirm that SSa can increase the expression of the anti-inflammatory factor, interleukin-10, and reduce nerve scar formation to promote functional recovery of injured sciatic nerve.

A systematic investigation on animal models of cyclosporine A combined with Escherichia coli to simulate the immunosuppressive status of sepsis patients before onset.

Immunosuppression is an important mechanism for the development of sepsis pathology, and is the key to the high mortality of sepsis. However, patients appear to be immunocompromised before sepsis onset due to lack of enough attention. Present sepsis models cannot fully mimic the onset of sepsis in patients. Hence, effective treatments in animal experiments could not be transformed into clinical application. In the present study, we improved the animal model of sepsis and used cyclosporine A immunosuppressive mice to make it closer to immune status before the onset of sepsis, followed by the intraperitoneal injection of Escherichia coli (E. coli) CMCC (B) 44,102 standard strain to produce the immunocompromised sepsis model. This trial systematically evaluates the new immunosuppressive sepsis model. Compared with routine sepsis models, the release of inflammatory factors in the new sepsis model was insufficient, blood bacteria were more cultured, diffuse intravascular coagulation (DIC) was more severe, lung, liver and kidney damage were heavier, and mortality rate was higher. In conclusion, the new sepsis model can mimic the patient's pre-onset immunocompromised state, is suitable for the development and evaluation of new methods of sepsis, and solves the controversy of sepsis treatment, providing new ideas and direction.

Exendin-4 inhibits high-altitude cerebral edema by protecting against neurobiological dysfunction.

The anti-inflammatory and antioxidant effects of exendin-4 (Ex-4) have been reported previously. However, whether (Ex-4) has anti-inflammatory and antioxidant effects on high-altitude cerebral edema (HACE) remains poorly understood. In this study, two rat models of HACE were established by placing rats in a hypoxic environment with a simulated altitude of either 6000- or 7000-m above sea level (MASL) for 72 hours. An altitude of 7000 MASL with 72-hours of hypoxia was found to be the optimized experimental paradigm for establishing HACE models. Then, in rats where a model of HACE was established by introducing them to a 7000 MASL environment with 72-hours of hypoxia treatment, 2, 10 and, 100 µg of Ex-4 was intraperitoneally administrated. The open field test and tail suspension test were used to test animal behavior. Routine methods were used to detect change in inflammatory cells. Hematoxylin-eosin staining was performed to determine pathological changes to brain tissue. Wet/dry weight ratios were used to measure brain water content. Evans blue leakage was used to determine blood-brain barrier integrity. Enzyme-linked immunosorbent assay (ELISA) was performed to measure markers of inflammation and oxidative stress including superoxide dismutase, glutathione, and malonaldehyde values, as well as interleukin-6, tumor necrosis factor-alpha, cyclic adenosine monophosphate levels in the brain tissue. Western blot analysis was performed to determine the levels of occludin, ZO-1, SOCS-3, vascular endothelial growth factor, EPAC1, nuclear factor-kappa B, and aquaporin-4. Our results demonstrate that Ex-4 preconditioning decreased brain water content, inhibited inflammation and oxidative stress, alleviated brain tissue injury, maintain blood-brain barrier integrity, and effectively improved motor function in rat models of HACE. These findings suggest that Ex-4 exhibits therapeutic potential in the treatment of HACE.