TdT-mediated dUTP Nick End Labeling Research Articles

Dexmedetomidine alleviates the long-term neurodevelopmental toxicity induced by sevoflurane in the developing brain.

Sevoflurane is an extensively used anesthetic for pediatric patients, however, numerous studies showed that sevoflurane (SEVO) may cause long-term neurodevelopmental toxicity. Dexmedetomidine (DEX) has been shown to be protective against SEVO-induced neurotoxicity, but the mechanism remains unclear. The effects and mechanisms of different DEX administration routes on SEVO-induced neurotoxicity and long-term cognitive defects were determined and further investigated the role of sex in these processes. Male and female Sprague Dawley (SD) rats at postnatal day 7 (PND7) received an intraperitoneal injection of DEX (10 μg/kg) before or after exposure to 2.5% SEVO for 6 hours, or before and after SEVO exposure. The respiratory and mortality rates of the pups were recorded during anesthesia. Neuroapoptosis was evaluated by TdT-mediated dUTP Nick-End labeling (TUNEL) staining. Immunohistochemistry and immunofluorescence were employed to detect the expression of caspase-3 in neuronal cells and neurons. The expression of GSK-3β and DISC1 were determined by Western blotting or RT-qPCR. Morris Water Maze (MWM) test was used to evaluate the learning and memory ability of rats until they were 3 weeks and 5 weeks old. Compared with the control group, exposure to 2.5% SEVO resulted in increased neuroapoptosis, and decreased the expression of DISC1 at levels of mRNA and protein and phosphorylated GSK-3β in the developing brain. SEVO exposure during critical neurodevelopmental periods could cause persistent cognitive defects in adolescent male and female rats, and inhibited DISC1 and phosphorylated GSK-3β protein expression. The neurotoxic impacts of SEVO were lessened by the administration of DEX (10 μg/kg) before or after exposure. Our findings suggest that DEX (10 μg/kg) mitigates the neurotoxic effects of SEVO on the developing rat brain as well as postnatal cognitive defects by regulating the DISC1/GSK-3β signaling.

Kaixinsan regulates neuronal mitochondrial homeostasis to improve the cognitive function of Alzheimer's disease by activating CaMKKβ-AMPK-PGC-1α signaling axis

BackgroundAlzheimer's disease (AD) is a neurodegenerative disease primarily characterized by cognitive impairments. With the intensification of population aging, AD has become a major health issue faced by the elderly. Kaixinsan, a classical traditional Chinese prescription consists of Panax ginseng C. A. Mey., Polygala tenuifolia Willd., Poria cocos (Schw.) Wolf and Acori Tatarinowii rhizoma, is commonly used in clinical for treating memory decline. However, its mechanism remains unclear, which hinders its popularization and application. MethodMorris water maze (MWM) was performed to evaluate the effect of Kaixinsan on improving learning and memory ability in SAMP8 (senescence-accelerated mouse prone 8, an AD model mice) mice. Nissl staining, TdT-mediated dUTP Nick End Labeling (TUNEL) and western blotting (Bax and Bcl-2) were used to confirm the effect of Kaixinsan on the neuronal structure and apoptosis of SAMP8 mice. ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to identify the distribution components of the brain tissue after receiving Kaixinsan extraction. Based on the identified brain distribution components, the mechanism of Kaixinsan improving the cognitive function was predicted by network pharmacology. Then, using HSP60 as mitochondrial marker and RBFOX3 as neuron marker, immunofluorescence co-localization was used to confirm the effect of Kaixinsan on neuronal mitochondria quantity in SAMP8 mice. Using western blotting to detect the expression of predicted proteins (AMPK, CaMKKβ, PGC-1α and HSP90) affecting mitochondrial homeostasis. To further confirm the mechanism of Kaixinsa. The authors replicated SH-SY5Y cell injury model induced by Amyloid β - protein fragment 25-35 (Aβ25-35) and compared the effects of serum containing Kaixinsan on apoptosis of injured SH-SY5Y cells (detected by flow cytometer) and the expression level of apoptosis-associated protein (Bax and Bcl-2) and mitochondrial homeostasis related proteins (AMPK, CaMKKβ, PGC-1α and HSP90), with or without the addition of CaMKKβ inhibitor (STO-609). ResultsThe results indicate that Kaixinsan can improve the cognitive function of SAMP8 mice, alleviate the hippocampal tissue lesions and inhibit neuron apoptosis. Seventeen brain distribution components of Kaixinsan were identified. Based on the brain distribution components of Kaixinsan, the results of network pharmacology suggest that Kaixinsan may regulate mitochondrial homeostasis through the CaMKKβ-AMPK-PGC-1α signaling axis. In vivo experiments, The authors observed that Kaixinsan could reverse neuronal mitochondrial loss in SAMP8 mice by upregulating CaMKKβ, AMPK, HSP90 and PGC-1α to promote mitochondrial biogenesis and increase the number of neuronal mitochondria. Additionally, the in vitro experiments demonstrated that Kaixinsan can inhibit apoptosis of Aβ25-35 injured SH-SY5Y cells and upregulate mitochondrial homeostasis-related proteins CaMKKβ, AMPK and PGC-1α. However, in addition to CaMKKβ inhibitors, the neuroprotective effect disappeared. ConclusionThe results indicate that Kaixinsan can improve the cognitive function of SAMP8 mice by regulating CaMKKβ-AMPK-PGC-1α signaling axis to maintain mitochondrial homeostasis and inhibit neuronal apoptosis.

Roles and action mechanisms of NRIP1 in pre-eclampsia.

Pre-eclampsia (PE) is characterized by the onset of hypertension and proteinuria during pregnancy. Here, we aimed to explore the functions of nuclear receptor-interacting protein 1 (NRIP1) in PE mice and human placental JEG-3 cells. We evaluated its effects on JEG-3 cell proliferation, apoptosis, invasion, and inflammatory response and regulation of Wnt/β-catenin pathway. NRIP1 levels in human serum and placental tissues, JEG-3 cells, and mouse models were assessed via quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. JEG-3 cell growth, apoptosis, migration, and invasion were evaluated via 5-ethynyl-2'-deoxyuridine, flow cytometry, and transwell assays. Levels of the inflammatory factors, matrix metalloproteinase (MMP)-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, were determined via enzyme-linked immunosorbent assay. Wnt/β-catenin pathway was assessed via western blotting and qRT-PCR. Systolic blood pressure and proteinuria were measured using the non-invasive tail cuff method and Coomassie brilliant blue assay, respectively. TdT-mediated dUTP nick-end labeling assay was used to assess cell apoptosis in the placental tissues of PE mice. NRIP1 levels were upregulated in the serum and placental tissues of patients with PE. In vitro experiments revealed that NRIP1-small interfering RNA (siRNA) increased the cell viability, migration, and invasion and reduced the cell apoptosis compared to the control siRNA. Moreover, NRIP1-siRNA activated the Wnt/β-catenin signaling pathway, as indicated by the increased Wnt3a, β-catenin, p-glycogen synthase kinase-3β, c-Myc, and cyclin D1 levels. Levels of the inflammatory factors, IL-6, TNF-α, and MMP-2, were decreased in the NRIP1-siRNA-treated group. Notably, NRIP1 downregulation improved the PE-like symptoms, inhibited the inflammatory responses, and reduced apoptosis in PE mice. This study revealed the crucial roles of NRIP1 in PE. Our findings revealed that NRIP1 downregulation relieved PE symptoms by inhibiting cell proliferation, migration, and invasion via the Wnt/β-catenin pathway, thus providing a novel candidate for PE treatment.

Lipoxin A4 modulates the PKA/CREB and NF-κB signaling pathway to mitigate chondrocyte catabolism and apoptosis in temporomandibular joint osteoarthritis

Temporomandibular joint osteoarthritis (TMJ-OA) is characterized by the degradation of the extracellular matrix (ECM) in cartilage and the apoptosis of chondrocytes, which is caused by inflammation and disruptions of chondrocyte metabolism and inflammation. Lipoxin A4 (LXA4), a specialized pro-resolving mediator, has been shown to inhibit inflammation and regulate the balance between ECM synthesis and degradation. However, the therapeutic effects of LXA4 on TMJ-OA and its underlying mechanisms remain unclear. Interleukin-1 beta (IL-1β)-induced chondrocyte and surgically induced TMJ-OA rat models were established in this study. The viability of chondrocytes treated with LXA4 was evaluated with the cell counting kit-8 (CCK-8) assay, while protein levels were assessed by western blot analysis, and the apoptosis rate was evaluated with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) staining. Histological analysis was conducted to evaluate the impact of LXA4 on cartilage degradation in TMJ-OA rat models. In vitro, the qRT-PCR and western blot analysis demonstrated that LXA4 facilitated the upregulation of collagen proteins (Collagen II) and decreased expression of matrix metalloproteinases (MMP-3, and MMP-13) associated with ECM modulation. LXA4 enhanced the TMJ-OA chondrocyte viability and decreased apoptotic rate. In vivo, histology and immunohistochemistry (IHC) analysis revealed that intraperitoneal injection of LXA4 contributed to the amelioration of chondrocyte injuries and deceleration of TMJ-OA. Transcriptomic sequencing revealed that cAMP signaling pathway was up-regulated and NF-κB signaling pathway was down-regulated in LXA4 treated group. LXA4 inhibited the phosphorylation of P65 and inhibitor of nuclear factor kappa B (IκBα) proteins while enhancing the phosphorylation PKA and CREB. This study demonstrates the potential of LXA4 as a therapeutic agent for suppressing chondrocyte catabolism and apoptosis by increasing PKA/CREB activity and decreasing NF-κB signaling.

TCF4 as a potential prognostic biomarker and an anticancer target in gastric cancer.

Lymphoid enhancer-binding factor 1 (LEF1)/T cell factor (TCF) family members are key transcription factors in malignant tumors. In this study, the role of T cell factor 4 (TCF4) in the progression of gastric cancer (GC) cell migration and invasion was investigated. Fifty-five pairs of GC tissues and adjacent non-tumor tissues were collected for evaluating the expression of LEF1/TCF family members, which were also evaluated by the Gene Expression Profiling Interactive Analysis (GEPIA) database, an online analysis platform based on The Cancer Genome Atlas and Genotype-Tissue Expression databases. Through GEPIA online analysis and our experimental specimens, we found that TCF4 messenger RNA (mRNA) expression was significantly upregulated in GC tissues compared with normal non-tumor tissues. The findings from protein-protein interaction (PPI) analysis suggested that myocyte enhancer factor 2C (MEF2C) may function as a regulatory gene for TCF4 and play a role in the progression of GC. A significant increase in TCF4 mRNA expression was observed in the GC cell lines. Silencing of TCF4 led to significant inhibition of the proliferation, migration, and invasion of the MGC-803 and SGC-7901 cells. TdT-mediated dUTP nick end labeling (TUNEL)-positive staining cells were significantly increased after transfection with TCF4 small interfering (si)-RNA into GC cells. In addition, patients with GC with high TCF4 expression were associated with poor T stage, pathologic stages, histologic grade, overall survival, and recurrence-free survival, indicating that TCF4 may be a potential prognostic marker of GC. TCF4 potentially exerts a carcinogenic role in the progression of GC. TCF4 may serve as a prognostic indicator and therapeutic target for GC.

Brucine Suppresses Malignant Progression of Prostate Cancer by Decreasing Sarcosine Accumulation via Downregulation of GNMT in the Glycine/sarcosine Metabolic Pathway.

Prostate cancer (PCa) remains a leading cause of cancer-related incidence and mortality in men. Disruptions in amino acid (AA) metabolism contribute to the disease progression, with brucine, a glycine antagonist, exhibiting antitumor effects. This study explores the antitumor impact of brucine on PCa and investigates its mechanisms in regulating AA metabolic pathways. The study employed the PCa cell line DU-145, characterized by high sarcosine (Sar) levels, for various assays including Cell Counting Kit-8 (CCK8), wound healing, Transwell, 5-Ethynyl-2'-deoxyuridine (EDU), TdT mediated dUTP Nick End Labeling (TUNEL), flow cytometry, Western blot, and ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Network pharmacological analysis determined the anticancer mechanisms of brucine. Sar levels in DU-145 cells were significantly higher than in normal prostatic epithelial cells RWPE-1. Treatment with brucine resulted in a marked decrease in cell viability, proliferation, invasion, and migration, while promoting apoptosis in a dose-dependent manner. Sar levels decreased with increasing brucine concentration. Network pharmacology analysis linked brucine's anticancer effect to the AA metabolism and glycine N-methyltransferase (GNMT) pathways. GNMT expression in prostate cancer tissues and The Cancer Genome Atlas database was significantly elevated compared to controls. Treatment with brucine led to downregulation of GNMT expression in DU-145 cells without significant effect on sarcosine dehydrogenase (SARDH). Addition of recombinant GNMT partially reversed the inhibitory effects of brucine on DU-145 cells. Treatment with brucine downregulates GNMT expression in DU-145 cells, reducing Sar accumulation and inhibiting tumor progression. These findings provide new insights into the antitumor mechanisms of brucine in PCa.

RANKL/RANK contributes to the pathological process of type 2 diabetes mellitus through TRAF3 activation of NIK

Diabetic osteoporosis is a complication of diabetes mellitus (DM). Denosumab (DMB) is an effective anti-osteoporotic drug functions by inhibiting NF-κB ligand receptor-activating factor (RANKL). Previous study found that osteoprotegerin (OPG) regulated βcell homeostasis through the RNAK/RANKL pathway. The present study aimed to investigate the effect of RANKL/RANK on the pathological process of DM and the underlying mechanism. We used D-glucose-induced RINm5F cells to construct in vitro type 2 diabetes models (T2DM). A high-fat diet combined with intraperitoneal injection of streptozotocin (STZ) was used to establish a T2DM model in SD rats. The apoptosis of β-cells was determined by TdT-mediated dUTP nick-end labeling (TUNEL) analysis. qRT-PCR and western blotting assays were used to explore the mRNA and protein expression of the TRAF3 (Tumor necrosis factor receptor-associated factor)/NIK (NF-κB-inducible kinase) pathway. Furthermore, insulin expression was detected by ELISA and immunohistochemistry assay. The islet morphology was analyzed by H&E. In vivo experiments demonstrated that sRANKL-IN-3 down-regulated insulin secretion levels by significantly ameliorating pancreatic tissue damage and mitigating apoptosis of high glucose induced β-cells. Subsequently, sRANKL-IN-3, acting as an inhibitor of RANKL, mitigated functional decline in β-cells induced by high glucose, mainly manifested by the low expression of PDX-1 (pancreatic duodenal homeobox 1), BETA2 (beta-2 adrenoceptors), INS-1 (insulin 1), and INS-2 (insulin 2). Mechanistic studies revealed that deletion of TRAF3 combined with sRANKL-IN-3 administration reduced the activity of NIK, NF-κB2, and RelB in RINm5F cells. In addition, our study demonstrated that inhibition of either RANKL or TRAF3 had a protective effect on high glucose induced apoptosis. Moreover, the combined action of sRANKL-IN-3 and shTRAF3 had a more pronounced inhibitory effect on high glucose-induced apoptosis. In summary, RANKL/RANK deficiency may attenuate apoptosis of β-cells, a phenomenon associated with the TRAF3/NIK pathway. Therefore, RANKL/RANK could be regarded as a potential therapeutic strategy for DM.

Exploring the Effects and Potential Mechanisms of Hesperidin for the Treatment of CPT-11-Induced Diarrhea: Network Pharmacology, Molecular Docking, and Experimental Validation.

Chemotherapy-induced diarrhea (CID) is a potentially serious side effect that often occurs during anticancer therapy and is caused by the toxic effects of chemotherapeutic drugs on the gastrointestinal tract, resulting in increased frequency of bowel movements and fluid contents. Among these agents, irinotecan (CPT-11) is most commonly associated with CID. Hesperidin (HPD), a flavonoid glycoside found predominantly in citrus fruits, has anti-oxidation properties and anti-inflammation properties that may benefit CID management. Nevertheless, its potential mechanism is still uncertain. In this study, we firstly evaluated the pharmacodynamics of HPD for the treatment of CID in a mouse model, then used network pharmacology and molecular docking methods to excavate the mechanism of HPD in relieving CID, and finally further proved the predicted mechanism through molecular biology experiments. The results demonstrate that HPD significantly alleviated diarrhea, weight loss, colonic pathological damage, oxidative stress, and inflammation in CID mice. In addition, 74 potential targets for HPD intervention in CID were verified by network pharmacology, with the top 10 key targets being AKT1, CASP3, ALB, EGFR, HSP90AA1, MMP9, ESR1, ANXA5, PPARG, and IGF1. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the PI3K-Akt pathway, FoxO pathway, MAPK pathway, TNF pathway, and Ras pathway were most relevant to the HPD potential treatment of CID genes. The molecular docking results showed that HPD had good binding to seven apoptosis-related targets, including AKT1, ANXA5, CASP3, HSP90AA1, IGF1, MMP9, and PPARG. Moreover, we verified apoptosis by TdT-mediated dUTP nick-end labeling (TUNEL) staining and immunohistochemistry, and the hypothesis about the proteins above was further verified by Western blotting in vivo experiments. Overall, this study elucidates the potential and underlying mechanisms of HPD in alleviating CID.

TRPM2-AS promotes ovarian cancer cell proliferation and inhibits cell apoptosis by upregulating the nearby gene TRPM2 via miR-6764-5p

Ovarian cancer (OC) becomes a fatal gynecologic malignant cancer in females worldwide. Target therapy is a promising therapeutical choice for patients with OC, and identifying biomarkers and exploring molecular mechanisms are necessary. In this study, the functions and mechanism of long noncoding RNA transient receptor potential cation channel subfamily M member 2 antisense RNA (TRPM2-AS) in OC were explored. TRPM2-AS expression in OC cells was analyzed utilizing reverse transcription quantitative polymerase chain reaction (RT-qPCR). Cell counting kit-8 (CCK-8) and colony forming assays were carried out to explore the influence of TRPM2-AS on OC cell viability and proliferation. Cell apoptosis was detected using TdT-mediated dUTP Nick-End labeling (TUNEL) and flow cytometry analysis. Protein expression of apoptotic markers was subjected to western blotting. RNA pulldown or luciferase reporter assays were applied to explore the interaction between TRPM2-AS and miR-6764-5p or the binding of miR-6764-5p and TRPM2. The results showed that TRPM2-AS is highly expressed in OC cells and was mainly localized in cytoplasm. TRPM2-AS depletion suppressed OC cell viability and proliferation while increasing cell apoptotic rate. TRPM2 displayed a high level in OC cells and was positively regulated by TRPM2-AS. TRPM2-AS interacted with miR-6764-5p and thereby upregulated TRPM2 expression. In addition, TRPM2 overexpression reversed the repressive impact of TRPM2-AS depletion on malignant OC cellular process. In conclusion, TRPM2-AS promotes OC cell viability and proliferation while enhancing cell apoptosis through interaction with miR-6764-5p to regulate TRPM2 level.

Ligustilide alleviates oxidative stress during renal ischemia-reperfusion injury through maintaining Sirt3-dependent mitochondrial homeostasis

BackgroundRenal ischemia-reperfusion (I/R) injury is an inevitable complication during renal transplantation and is closely related to patient prognosis. Mitochondrial damage induced oxidative stress is the core link of renal I/R injury. Ligustilide (LIG), a natural compound extracted from ligusticum chuanxiong hort and angelica sinensis, has exhibited the potential to protect mitochondrial function. However, whether LIG can ameliorate renal I/R injury requires further investigation. Delving deeper into the precise targets and mechanisms of LIG's effect on renal I/R injury is crucial. PurposeThis study aimed to elucidate the specific mechanism of LIG's protective effect on renal I/R injury. MethodsIn this study, an in vivo model of renal ischemia-reperfusion (I/R) injury was developed in mice, along with an in vitro model of hypoxia-reoxygenation (H/R) using human proximal renal tubular epithelial cells (HK-2). To assess the impact of LIG on renal injury, various methods were employed, including serum creatinine (Cr) and blood urea nitrogen (BUN) testing, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) for kidney injury molecule-1 (KIM-1). The effects of LIG on oxidative stress were examined using fluorescent probes dihydroethidium (DHE) and dichlorodihydrofluorescein diacetate (DCFH-DA), TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and flow cytometry. Additionally, the influence of LIG on mitochondrial morphology and function was evaluated through transmission electron microscopy (TEM), Mito Tracker Red CMXRos staining, adenosine triphosphate (ATP) concentration assays, and JC-1 staining. The potential mechanism involving LIG and Sirt3 was explored by manipulating Sirt3 expression through cell transfection. ResultsThe results showed that LIG could provide protective function for mitochondria to alleviate oxidative stress induced by renal I/R. Further mechanistic studies indicated that LIG maintained mitochondrial homeostasis by targeting Sirt3. ConclusionOur findings demonstrated that LIG alleviated oxidative stress during renal I/R injury through maintaining Sirt3-dependent mitochondrial homeostasis. Overall, our data raised the possibility of LIG as a novel therapy for renal I/R injury.

Aspartic proteases gene family: Identification and expression profiles during stem vascular development in tobacco

Aspartic proteases (APs) constitute a large family in plants and are widely involved in diverse biological processes, like chloroplast metabolism, biotic and abiotic stress responses, and reproductive development. In this study, we focused on overall analysis of the APs genes in tobacco. Our analysis included the phylogeny and cis-elements in the cell wall-associated promoters of these genes. To characterize the expression patterns of APs genes in stem vascular development. The tissue expression analysis showed that NtAED3-like was preferentially expressed in the differentiating xylem and phloem cells of the vascular system. Based on histochemical staining analysis showed that the NtAED3-like gene was specifically expressed in stem vascular tissue, root vascular tissue, and petiole vascular tissue. The TdT-mediated dUTP nick-end labeling (TUNEL) assay illustrated a delayed progression of programmed cell death (PCD) within the xylem of the ko-ntaed3a-like mutant, relative to the wild type. The mutant ko-ntaed3a-like exhibited a phenotype of thinning stem circumference and changed in xylem structure and lignin content. In addition, the two-dimension heteronuclear single quantum coherent nuclear magnetic resonance (2D-HSQC) analysis of three milled wood lignins (MWLs) showed that the content of β-O-4 connection in ko-ntaed3a-like decreased slightly compared with wild type. In conclusion, this study provides our understanding of the regulation of vascular tissue development by the NtAED3-like gene in tobacco and provides a better basis for determining the molecular mechanism of the aspartic protease in secondary cell wall (SCW) development.

SEA Alleviates Hepatic Ischaemia-Reperfusion Injury by Promoting M2 Macrophage Polarisation.

Hepatic ischaemia-reperfusion (I/R) injury is a frequent and nearly inevitable pathophysiological process without widely accepted effective therapy. Soluble egg antigen (SEA) of Schistosoma japonicum (S. japonicum) is the main mediators capable of regulating immunological activities and has received increased attention in immune-mediated diseases. But its role in hepatic I/R injury has not been well defined. This study aimed to elucidate whether SEA protects liver against hepatic I/R injury and explore underlying mechanism. After intraperitoneal injecting SEA three times a week for 4 weeks, mice underwent 70% hepatic I/R injury. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), haematoxylin-eosin (HE) and TdT-mediated dUTP nick-end labelling (TUNEL) staining were used to evaluate liver injury. The severity related to the inflammatory response was also investigated. Furthermore, immunofluorescence was used to detect macrophage polarisation. Compared with the hepatic I/R injury group, SEA pretreatment significantly alleviated hepatic I/R injury induced liver damage, apoptosis and inflammatory. Interestingly, SEA enhanced the polarisation of macrophages towards M2 macrophages in vivo. We are the first to investigate the therapeutic efficacy of S. japonicum SEA in a hepatic I/R injury model in mice. We provided the first direct evidence that SEA attenuated hepatic I/R injury by promoting M2 macrophage polarisation.

AB045. E2F6 promotes temozolomide resistance in glioblastoma by enrichment in CARD6 and POSTN promoter region.

Glioblastoma (GBM) is the most aggressive primary malignant brain tumor. Temozolomide (TMZ) is the most used first-line chemotherapeutic agent for GBM after surgery, but acquired resistance to TMZ frequently leads to treatment failure and is a major challenge in the clinical treatment of GBM. Increasing evidence suggests that E2F transcription factor 6 (E2F6) is associated with a variety of tumor malignant biological behaviors and drug resistance, but its biological function and underlying molecular mechanisms in GBM are unknown. The study investigated the levels of E2F6 in both TMZ-sensitive and TMZ-resistant GBM cells and tissues using Western blotting and immunofluorescence assays. In vitro experiments were conducted to explore the impact of E2F6 on TMZ resistance and glioma stem cell stemness. These experiments included Western blotting, colony formation assay, flow cytometry assay, and TdT-mediated dUTP nick-end labeling (TUNEL) assay. Bioinformatic analyses were conducted to investigate the mechanism behind the high expression of E2F6 in TMZ-resistant cells and its correlation with caspase recruitment domain 6 (CARD6) and disulfide-linked cell adhesion protein (POSTN). The study employed bioinformatic analyses, messenger RNA (mRNA) sequencing, chromatin immunoprecipitation sequencing assay, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. To examine the function of E2F6, an intracranial xenograft tumor mouse model was used for in vivo experiments. It was found that CARD6 and POSTN were significantly associated with TMZ resistance and survival of GBM patients. E2F6 was up-regulated in TMZ-resistant cells and tissues. Knockdown of E2F6 down-regulated the expression of CARD6, promoted TMZ-induced apoptosis, and enhanced chemo-sensitivity, whereas its overexpression significantly increased TMZ resistance in vitro and in vivo. In addition, E2F6 can promote TMZ resistance through stem-like properties acquisition. We identified a signaling pathway related to E2F6 and POSTN, which maintains the self-renewal of GBM stem cells (GSCs). E2F6 concentrates in the promoter region of POSTN, thereby regulating the expression of GSCs-related genes cluster of differentiation 133 (CD133), Nestin, and sex-determining region Y-box 2 (SOX2), which may be involved in tumor metabolism and drug resistance processes. Down-regulation of E2F6 down-regulated the expression of POSTN and inhibited tumor growth in nude mice. These results suggest that the E2F6-CARD6/POSTN signaling axis regulates the malignant biological behaviors of GBM and TMZ resistance. These findings are expected to provide promising therapeutic targets for CARD6 overcoming GBM TMZ resistance.

Genistein inhibits Nlrp3/caspase-1 signalling to alleviate traumatic brain injury-induced anxiety-like behaviours in rats.

Traumatic brain injury (TBI)-induced anxiety is a common but under-investigated disorder, for which neuroinflammation is a significant contributor. Here we aim to investigate the protective effects of genistein, a plant-derived anti-inflammatory drug, against TBI-induced anxiety, and the underlying mechanisms. A rat model of TBI was constructed using the lateral fluid percussion injury method. Genistein at the doses of 5, 10, and 20 mg/kg were used to treat rats at 30 min, 12 h, 24 h, 48 h, and 72 h up to 14 days after TBI. The evaluation of neurological deficit was performed preoperatively, on days 1, 3, 7, and 14 after TBI. The elevated plus maze test was carried out to assess anxiety and explorative behaviours, and the open field test was performed to assess locomotive activities. Brain injury was assessed by measuring brain water content and TdT-mediated dUTP Nick-End Labeling staining. Inflammatory responses were examined using enzyme-linked immunosorbent assay. The mRNA and protein expression were analysed using real-time polymerase chain reaction and Western blot, respectively. In the behavioural level, genistein treatment alleviated TBI-induced anxiety behaviours and neurological deficit in rats. In the meanwhile, brain oedema was also reduced by genistein treatment, showing alleviating effects of genistein at the pathological level. TUNEL staining also showed reduced apoptosis in rats treated with genistein. Genistein also inhibited Nlrp3/caspase-1 signalling, unveiling the effects of genistein in altering molecular pathways in brains with TBI. Genistein alleviates anxiety-like behaviours in TBI rats, which may be mediated via inhibiting Nlrp/caspase-1 signalling pathway.

ATP-sensitive potassium channel opener, Nicorandil, inhibits NF-κB/AIM2/GSDMD pathway activation to protect against neuroinflammation in ischemic stroke

The absent in melanoma 2 (AIM2) inflammasome contributes to ischemic brain injury by inducing cell pyroptosis and inflammatory responses. Our research group has previously demonstrated that ATP-sensitive potassium channels (KATP channels) openers can modulate neuronal synaptic plasticity post-ischemic stroke for neuroprotection. However, the specific mechanisms of KATP channels in the inflammatory response following ischemic stroke remain unclear. Here, we assessed cellular damage by observing changes in BV-2 morphology and viability. 2,3,5-Triphenyl tetrazolium chloride (TTC) staining, mNSS scoring, Nissl staining, and TdT-mediated dUTP nick end labeling (TUNEL) staining were used to evaluate behavioral deficits, brain injury severity, and neuronal damage in mice subjected to middle cerebral artery occlusion (MCAO). Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were used to measure cell pyroptosis and nuclear factor-kappaB (NF-κB) activation in vivo and in vitro. We observed that AIM2 protein expression was upregulated and localized within the cytoplasm of BV-2 cells. Notably, low-dose Nicorandil treatment reduced inflammatory cytokine secretion and pyroptosis-related protein expression, including AIM2, cleaved cysteinyl aspartate-specific protease-1 (cleaved caspase-1), and Gasdermin D N-terminal (GSDMD-NT). Further investigations revealed that the KATP channel inhibitor 5-HD upregulated p-NF-κB p65, NF-κB p65, and p-IκBα expression, reversing Nicorandil's neuroprotective effect in vivo. In summary, our results suggest that Nicorandil may serve as a potential therapeutic option for ischemic stroke. Targeting AIM2 and NF-κB represents effective strategies for inhibiting neuroinflammation.

SMAD1 Regulates the Hippocampal Neuronal Death and Ferroptosis via Affecting the Transcription of PDCD4 in Cerebral Ischemia.

Results of previous studies suggested that programmed cell death 4 (PDCD4) was overexpressed in cerebral ischemia (CI), and mothers against decapentaplegic homolog 1 (SMAD1) is a transcription factor of PDCD4, and it is also elevated in CI; however, the regulatory mechanism of SMAD1/PDCD4 axis in CI remains unclear. The current work has been designed to explore the role and associated mechanisms of SMAD1/PDCD4 in CI. PDCD4 and SMAD1 expressions have been examined by real-time reverse transcription-polymerase chain reaction (RT-qPCR) method, and receiver operating characteristic (ROC) curve analysis has been performed to determine the potential diagnostic value of PDCD4 and SMAD1. An oxygen-glucose deprivation (OGD) model has been used to investigate the effects of PDCD4 and SMAD1 on CI in vitro. Cell apoptosis was evaluated using TdT-mediated dUTP nick end labeling (TUNEL) assays. The interaction between SMAD1 and PDCD4 axis has been confirmed by using dual-luciferase reporter as well as chromatin immunoprecipitation (Ch-IP) assays. Finally, the effects of SMAD1/PDCD4 axis on the ferroptosis of neuron cells have been examined. PDCD4 was overexpressed in blood samples of CI patients. ROC analysis showed the AUC for PDCD4 was 0.7478, and NIHSS and MRS scores were positively correlated with PDCD4 expression. Moreover, the cellular OGD model was established and knockdown of PDCD4 suppressed the apoptosis of neurons. Besides, knockdown of PDCD4 also inhibited ferroptosis of OGD-treated neuron cells in vitro. Additionally, SMAD1 was upregulated in blood samples of CI patients, NIHSS and MRS scores were positively correlated with SMAD1 expression, and SMAD1 is a transcriptional factor of PDCD4, and SMAD1 could transcriptionally regulate the expression of PDCD4. Finally, SMAD1 could regulate the ferroptosis of neuron cells through regulating the transcription of PDCD4. The SMAD1/PDCD4 axis regulates the growth, apoptosis, and ferroptosis of neuron cells, suggesting that targeting the SMAD1/PDCD4 axis may be a potential therapeutic method.

Analysis of tumor regression after concurrent radiochemotherapy for locally advanced hypopharyngeal carcinoma

Objective: To investigate the pathological characteristics of tumor regression and the expression level of chemoradiotherapy resistance-related molecular markers after preoperative concurrent radiochemotherapy in patients with locally advanced hypopharyngeal carcinoma. Methods: The clinical data of 44 patients with locally advanced hypopharyngeal carcinoma who underwent preoperative concurrent radiochemotherapy in the Department of Head and Neck Surgery of Shandong Otolaryngology Hospital from August 2016 to August 2020 were retrospectively analyzed. All patients received preoperative concurrent chemotherapy and radiotherapy. After radiochemotherapy, electronic laryngoscopy and imaging examination were performed to assess the tumor regression status. After 4 weeks, surgical resection was performed, and the specimens of the primary focus were processed as continuous pathological sections. After operation, HE staining and TdT-mediated dUTP nick-end labeling (TUNEL) method were used to detect the distribution characteristics and apoptosis of the remaining cancer focus, and immunohistochemistry was performed to determine the proliferation of the remaining cancer focus and the expression of radiation resistance-related molecular markers [signal transducer and activator of transcription 3 (STAT3), hypoxia-inducible factor-1alpha (HIF-1α), sex determining region Y-box 2 (SOX2), and P53]. Results: A total of 44 patients were included, all of whom were male, with a mean age of (58.3±3.5) years. There were 40 cases of pyriform sinus carcinoma and 4 cases of posterior pharyngeal wall carcinoma. Twenty-nine cases were in stage T3 and 15 cases were in stage T4. There were 6 stage Ⅲ cases and 38 stage Ⅳ cases. According to the response evaluation criteria in solid tumors (RECIST), 13 patients achieved complete response (CR), 22 patients had partial response (PR), and 9 patients achieved stable disease (SD) after concurrent radiochemotherapy. The primary lesion resection methods included 19 cases of hypopharyngeal circumferential resection and 2 cases of total laryngectomy and partial hypopharyngeal resection. Twenty-three cases underwent supracricoid cartilage subtotal laryngectomy cricoid tongue fixation (CHP). Among 22 patients with PR, 10 had large PR (remission rate ≥70%) and 12 had small PR (remission rate <70%). The residual tumor was found in 30 patients (68.2%) after resection of all primary lesions by HE staining of pathological sections, of which 3 patients (3/13) with CR had residual cancer, all of which were focal residues. In large PR patients, residual cancer was detected in 6 cases (6/10), scattered in 4 cases, and focal residual in 2 cases, respectively. Large residual tumors were detected in small PR and SD patients. TUNEL method did not show any sign of apoptosis in 30 specimens with residual cancer focus, and the positive expression rate of Ki-67 was less than 10%. The expression of STAT3 (3.40±2.49 vs 5.23±3.02, t=-2.932, P=0.007) in 19 cases (63.3%) and HIF-1α (3.73±2.66 vs 6.97±3.05, t=-4.45, P<0.001) in 22 cases (73.3%) of residual cancer were significantly higher than those before radiochemotherapy. Other molecular markers showed no significant changes. All patients were followed up for 3 years. The 2-year survival rate was 59.3%, and the 3-year survival rate was 54.1%. Conclusions: Preoperative radiochemotherapy can make some patients with locally advanced hypopharyngeal carcinoma achieve complete or significant remission in clinical evaluation, but pathological detection still shows some residual cancer lesions with enhanced anti-apoptosis ability and decreased proliferation activity.

Exosomal miR-17-92 Cluster from BMSCs Alleviates Apoptosis and Inflammation in Spinal Cord Injury.

Spinal cord injury (SCI) involves neuronal apoptosis and axonal disruption, leading to severe motor dysfunction. Studies indicate that exosomes transport microRNAs (miRNAs) and play a crucial role in intercellular communication. This study aimed to explore whether the bone marrow mesenchymal stem cell (BMSCs)-exosomal miR-17-92 cluster can protect against SCI and to explain the underlying mechanisms. In vivo and in vitro SCI models were established and treated with control exosomes (con-exo) or exosomes derived from BMSCs transfected with miR-17-92 cluster plasmid (miR-17-92-exo). Rat BMSCs were isolated and positive markers were identified by flow cytometry. BMSC-derived exosomes were extracted and verified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. The expression of the miR-17-92 cluster was validated by quantitative reverse transcription PCR (qRT-PCR). Spinal cord function, histopathological changes, apoptotic cells, and inflammatory cytokines release in spinal cord tissues were assessed using the Basso-Beattie-Bresnahan (BBB) score, hematoxylin and eosin (HE) staining, terminal deoxynucleotide transferase (TdT)-mediated dUTPnick-endlabeling(TUNEL)staining, enzyme-linkedimmunosorbentassay(ELISA), and qRT-PCR. In PC12 cells, cell proliferation, apoptosis, apoptosis-related proteins cleaved-Caspase3 expression, and inflammatory factors secretion were analyzed using a cell counting kit-8 (CCK8) assay, flow cytometry, western blotting, and ELISA. Our data revealed that the exosomes were successfully isolated from rat BMSCs. The BMSC-exosomal miR-17-92 cluster improved neural functional recovery after SCI, as evidenced by an increased BBB score, improved pathological damage, reduced neuronal apoptosis, and decreased inflammatory factors release. Additionally, miR-17-92-exo treatment significantly inhibited lipopolysaccharide (LPS)-induced reduction in cell viability, increase in cell apoptosis, and upregulation of inflammatory factors in PC12 cells. The exosomal miR-17-92 cluster derived from BMSCs improved functional recovery and exhibited neuroprotective effects in SCI by alleviating apoptosis and inflammation.

Propofol alleviates spinal cord ischemia-reperfusion injury by preserving PI3K/AKT/GIT1 axis.

Spinal cord ischemia-reperfusion injury (SCIRI) is a major contributor to neurological damage and mortality associated with spinal cord dysfunction. This study aims to explore the possible mechanism of Propofol and G-protein-coupled receptor-interacting protein 1 (GIT1) in regulating SCIRI in rat models. SCIRI rat models were established and injected with Propofol, over expression of GIT1 (OE-GIT1), or PI3K inhibitor (LY294002). The neurological function was assessed using Tarlov scoring system, and Hematoxylin & Eosin (H&E) staining was applied to observe morphology changes in spinal cord tissues. Cell apoptosis, blood-spinal cord barriers (BSCB) permeability, and inflammatory cytokines were determined by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, evans blue (EB) staining, and enzyme-linked immuno sorbent assay (ELISA), respectively. Reverse transcription-quantitative polymerase chain reaction and western blot were used to detect the expression levels of GIT1, endothelial nitric oxide synthase (eNOS), PI3K/AKT signal pathway and apoptosis-related proteins. SCIRI rats had decreased expressions of GIT1 and PI3K/AKT-related proteins, whose expressions can be elevated in response to Propofol treatment. LY294002 can also decrease GIT1 expression levels in SCIRI rats. Propofol can attenuate neurological dysfunction induced by SCIRI, decrease spinal cord tissue injury and BSCB permeability in addition to suppressing cell apoptosis and inflammatory cytokines, whereas further treatment by LY294002 can partially reverse the protective effect of Propofol on SCIRI. Propofol can activate PI3K/AKT signal pathway to increase GIT1 expression level, thus attenuating SCIRI in rat models.

Probiotics relieve perioperative postoperative cognitive dysfunction induced by cardiopulmonary bypass through the kynurenine metabolic pathway

Postoperative cognitive dysfunction (POCD) has become the popular critical post-operative consequences, especially cardiopulmonary bypass surgery, leading to an increased risk of mortality. However, no therapeutic effect about POCD. Probiotics are beneficial bacteria living in the gut and help to reduce the risk of POCD. However, the detailed mechanism is still not entirely known. Therefore, our research aims to uncover the effect and mechanism of probiotics in relieving POCD and to figure out the possible relationship between kynurenine metabolic pathway. 36 rats were grouped into three groups: sham operated group (S group, n = 12), Cardiopulmonary bypass group (CPB group, n = 12), and probiotics+CPB (P group, n = 12). After CPB model preparation, water maze test and Garcia score scale was performed to identify the neurological function. Immunofluorescence and Hematoxylin and eosin staining has been used for hippocampal neurons detection. Brain injury related proteins, oxidative stress factors, and inflammatory factors were detected using enzyme-linked immunosorbent assays (ELISA). Neuronal apoptosis was detected by TdT-mediated dUTP nick end-labeling (TUNEL) staining and western blot. High-performance liquid chromatography/mass spectrometry (HPLC/MS) was performed to detect the key factors of the kynurenine metabolic pathway. Our results demonstrated that probiotics improved neurological function of post-CPB rats. The administration of probiotics ameliorated memory and learning in spatial terms CPB rats (P < 0.05). Hematoxylin and eosin (H&E) staining data, S‐100β and neuron-specific enolase (NSE) data convinced that probiotics agonists reduced brain damage in CPB rats (P < 0.05). Moreover, probiotics regulated inflammatory factors, meanwhile attenuated hippocampal neuronal apoptosis. Probiotics alleviated POCD in rats with CPB through regulation of kynurenine metabolic signaling pathway.