Extracellular vesicle encapsulated Homer1a as novel nanotherapeutics against intracerebral hemorrhage in a mouse model.

Homer1a and A2 astrocytes are involved in the regulation of inflammation induced by intracerebral hemorrhage (ICH). However, there is no anticipated treatment strategy based on the anti-inflammatory effect of Homer1a and A2 astrocytes. Here, we successfully induced A2 astrocytes in vitro, and then we report an efficient method to prepare Homer1a+ EVs derived from A2 astrocytes which making it more stable, safe, and targetable to injured neurons. Homer1a+ EVs promotes the conversion of A1 to A2 astrocytes in ICH mice. Homer1a+ EVs inhibits activation and nuclear translocation of NF-κB, thereby regulating transcription of IL-17A in neurons. Homer1a+ EVs inhibits the RAGE/NF-κB/IL-17 signaling pathway and the binding ability of IL-17A: IL17-AR and RAGE: DIAPH1. In addition, Homer1a+ EVs ameliorates the pathology, behavior, and survival rate in GFAPCreHomer1fl/-Homer1a± and NestinCreRAGEfl/fl ICH mice. Our study provides a novel insight and potential for the clinical translation of Homer1a+ EVs in the treatment of ICH.

Serum Homer1 is a Novel Biomarker for Predicting the Clinical Outcomes of Acute Ischemic Stroke Patients.

Purpose: We aim to explore the relationship between Homer1 and the outcomes of AIS patients at 3 months. Patients and Methods: This prospective cohort study was conducted from May 2022 to March 2023. In this study, we investigated the association between serum Homer1 levels by enzyme-linked immunosorbent assay at admission and functional outcomes of patients at 3 months after AIS. Results: Overall, 89 AIS patients (48 good outcomes and 41 poor outcomes) and 83 healthy controls were included. The median serum Homer1 level of patients at admission with poor outcomes was significantly higher than that of patients with good outcomes (39.33 vs 33.15, P<0.001). Serum Homer1 levels at admission were positively correlated with the severity of AIS (r = 0.488, P<0.001). The optimal cutoff of serum Homer1 level as an indicator for an auxiliary diagnosis of 3 months functional outcomes was 35.07 pg/mL, with a sensitivity of 75.0% and a specificity of 92.7% (AUC 0.837; 95% CI [0.744-0.907]; P<0 0.001). The odds ratio of MRS > 2 predicted by the level of serum Homer1 after 3 months was 1.665 (1.306-2.122; P<0.001). Conclusion: Serum concentrations of Homer1 have a high predictive value for neurobehavioral outcomes after acute ischemic stroke. Higher serum Homer1 levels (>35.07 pg/mL) were positively associated with poor functional outcomes of patients 3 months post-stroke.

Homer1 ameliorates ischemic stroke by inhibiting necroptosis-induced neuronal damage and neuroinflammation.

OBJECTIVE: Proinflammatory necroptosis is the main pathological mechanism of ischemic stroke. Homer scaffolding protein 1 (Homer1) is a postsynaptic scaffolding protein that exerts anti-inflammatory effects in most central nervous system diseases. However, the relationship between Homer1 and proinflammatory necroptosis in ischemic stroke remains unclear. AIM: This study aimed to investigate the role of Homer1 in ischemia-induced necroptosis. METHODS: C57BL/6 mice were used to establish a model of permanent middle cerebral artery occlusion model (pMCAO). Homer1 knockdown mice were generated using adeno-associated virus (AAV) infection to explore the role of Homer1 and its impact on necroptosis in pMCAO. Finally, Homer1 protein was stereotaxically injected into the ischemic cortex of Homer1flox/flox/Nestin-Cre +/- mice, and the efficacy of Homer1 was investigated using behavioral assays and molecular biological assays to explore potential mechanisms. RESULTS: Homer1 expression peaked at 8 h in the ischemic penumbral cortex after pMCAO and colocalized with neurons. Homer1 knockdown promoted neuronal death by enhancing necroptotic signaling pathways and aggravating ischemic brain damage in mice. Furthermore, the knockdown of Homer1 enhanced the expression of proinflammatory cytokines. Moreover, injection of Homer1 protein reduced necroptosis-induced brain injury inhibited the expression of proinflammatory factors, and ameliorated the outcomes in the Homer1flox/flox/Nestin-Cre+/- mice after pMCAO. CONCLUSIONS: Homer1 ameliorates ischemic stroke by inhibiting necroptosis-induced neuronal damage and neuroinflammation. These data suggested that Homer1 is a novel regulator of neuronal death and neuroinflammation.

A novel multimode biosensor for sensitive detection of AFB1 in food based on Mxenes nano enzymes.

In this work, Ti3C2 nano-enzymes (Ti3C2 NEs) materials with simulated peroxidase activity and fluorescence quenching properties were prepared. Then Ti3C2 NEs was functionalized using 6-carboxyfluorescein (FAM) labeled Aflatoxin B1 (AFB1) aptamers to construct a novel multimode nano enzyme biosensor for the detection of AFB1 in peanuts. Based on the fluorescence quenching characteristics and the superior simulated peroxidase activity of Ti3C2 NES and the specific binding of the aptamer to AFB1, the sensitive and rapid fluorescence/colorimetric/smart phone detection of AFB1 have been achieved, with detection limits of 0.09 ng mL-1, 0.61 ng mL-1 and 0.96 ng mL-1, respectively. The analytical method provided can not only detect AFB1 in multiple modes, but also has a wider detection range, lower limit of detection (LOD) and better recovery rate, and can achieve on-site accurate detection of AFB1 content in peanuts, which has great application potential in the field of food quality testing.

TRIM22 promotes the proliferation of glioblastoma cells by activating MAPK signaling and accelerating the degradation of Raf-1.

The tripartite motif (TRIM) 22 and mitogen-activated protein kinase (MAPK) signaling pathways play critical roles in the growth of glioblastoma (GBM). However, the molecular mechanism underlying the relationship between TRIM22 and MAPK signaling remains unclear. Here, we found that TRIM22 binds to exon 2 of the sphingosine kinase 2 (SPHK2) gene. An ERK1/2-driven luciferase reporter construct identified TRIM22 as a potential activator of MAPK signaling. Knockout and overexpression of TRIM22 regulate the inhibition and activation of MAPK signaling through the RING-finger domain. TRIM22 binds to Raf-1, a negative regulator of MAPK signaling, and accelerates its degradation by inducing K48-linked ubiquitination, which is related to the CC and SPRY domains of TRIM22 and the C1D domain of Raf-1. In vitro and in vivo, an SPHK2 inhibitor (K145), an ERK1/2 inhibitor (selumetinib), and the nonphosphorylated mutant Raf-1S338A inhibited GBM growth. In addition, deletion of the RING domain and the nuclear localization sequence of TRIM22 significantly inhibited TRIM22-induced proliferation of GBM cells in vivo and in vitro. In conclusion, our study showed that TRIM22 regulates SPHK2 transcription and activates MAPK signaling through posttranslational modification of two critical regulators of MAPK signaling in GBM cells.

3D bioprinted autologous bone particle scaffolds for cranioplasty promote bone regeneration with both implanted and native BMSCs.

Although autologous bone (AB) grafting is considered to be the gold standard for cranioplasty, unresolved problems remain, such as surgical-site infections and bone flap absorption. In this study, an AB scaffold was constructed via three-dimensional (3D) bedside-bioprinting technology and used for cranioplasty. To simulate the skull structure, a polycaprolactone shell was designed as an external lamina, and 3D-printed AB and a bone marrow-derived mesenchymal stem cell (BMSC) hydrogel was used to mimic cancellous bone for bone regeneration. Ourin vitroresults showed that the scaffold exhibited excellent cellular affinity and promoted osteogenic differentiation of BMSCs in both two-dimensional and 3D culture systems. The scaffold was implanted in beagle dog cranial defects for up to 9 months, and the scaffold promoted new bone and osteoid formation. Furtherin vivostudies indicated that transplanted BMSCs differentiated into vascular endothelium, cartilage, and bone tissues, whereas native BMSCs were recruited into the defect. The results of this study provide a method for bedside bioprinting of a cranioplasty scaffold for bone regeneration, which opens up another window for clinical applications of 3D printing in the future.

Knockdown of NADPH oxidase 4 reduces mitochondrial oxidative stress and neuronal pyroptosis following intracerebral hemorrhage.

Intracerebral hemorrhage is often accompanied by oxidative stress induced by reactive oxygen species, which causes abnormal mitochondrial function and secondary reactive oxygen species generation. This creates a vicious cycle leading to reactive oxygen species accumulation, resulting in progression of the pathological process. Therefore, breaking the cycle to inhibit reactive oxygen species accumulation is critical for reducing neuronal death after intracerebral hemorrhage. Our previous study found that increased expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (NADPH oxidase 4, NOX4) led to neuronal apoptosis and damage to the blood-brain barrier after intracerebral hemorrhage. The purpose of this study was to investigate the role of NOX4 in the circle involving the neuronal tolerance to oxidative stress, mitochondrial reactive oxygen species and modes of neuronal death other than apoptosis after intracerebral hemorrhage. We found that NOX4 knockdown by adeno-associated virus (AAV-NOX4) in rats enhanced neuronal tolerance to oxidative stress, enabling them to better resist the oxidative stress caused by intracerebral hemorrhage. Knockdown of NOX4 also reduced the production of reactive oxygen species in the mitochondria, relieved mitochondrial damage, prevented secondary reactive oxygen species accumulation, reduced neuronal pyroptosis and contributed to relieving secondary brain injury after intracerebral hemorrhage in rats. Finally, we used a mitochondria-targeted superoxide dismutase mimetic to explore the relationship between reactive oxygen species and NOX4. The mitochondria-targeted superoxide dismutase mimetic inhibited the expression of NOX4 and neuronal pyroptosis, which is similar to the effect of AAV-NOX4. This indicates that NOX4 is likely to be an important target for inhibiting mitochondrial reactive oxygen species production, and NOX4 inhibitors can be used to alleviate oxidative stress response induced by intracerebral hemorrhage.

Monitored anesthesia care and asleep-awake-asleep techniques combined with multiple monitoring for resection of gliomas in eloquent brain areas: a retrospective analysis of 225 patients.

BACKGROUND: Awake craniotomy (AC) has become gold standard in surgical resection of gliomas located in eloquent areas. The conscious sedation techniques in AC include both monitored anesthesia care (MAC) and asleep-awake-asleep (AAA). The choice of optimal anesthetic method depends on the preferences of the surgical team (mainly anesthesiologist and neurosurgeon). The aim of this study was to compare the difference in physiological and blood gas data, dosage of different drugs, the probability of switching to endotracheal intubation, and extent of tumor resection and dysfunction after operation between AAA and MAC anesthetic management for resection of gliomas in eloquent brain areas. METHODS: Two-hundred and twenty-five patients with super-tentorial tumor located in eloquent areas underwent AC from 2009 to 2021 in Xijing Hospital. Forty-one patients underwent AAA technique, and the rest one-hundred eighty-four patients underwent MAC technique. Anesthetic management, dosage of different drugs, intraoperative complications, postoperative outcomes, adverse events, extent of resection and motor, and sensory and language dysfunction after operation were compared between MAC and AAA. RESULT: There was no significant difference in gender, KPS score, MMSE score, glioma grade, type, and growth site between the patients in the two groups, except the older age of patients in MAC group than that in AAA group. During the whole process of operation, there were greater pulse pressure difference (P = 0.046), shorter operation time (P = 0.039), less dosage of remifentanil (P = 0.000), more dosage of dexmedetomidine (P = 0.013), more use of antiemetics (81%, P = 0.0067), lower use of vasoactive agent (45.1%, P = 0.010), and lower probability of conversion to general anesthesia (GA, P = 0.027) in MAC group than that in AAA group. Blood gas analysis showed that PetCO2 (P = 0.000), Glu concentration (P = 0.000), and PaCO2 (P = 0.000) were higher, but SPO2 (P = 0.002) and PaO2 (P = 0.000) were lower in MAC group than that in AAA group. In the postoperative recovery stage, compared with that of AAA group, the probability of dysfunction in MAC group at 1, 3, 5, and 7 days after operation was lower, which were 27.8% vs 53.6% (P = 0.003), 31% vs 68.3% (P = 0.000), 28.8% vs 63.4% (P = 0.000), and 25.6% vs 58.5% (P = 0.000), respectively. CONCLUSION: Compared with AAA, it seems that MAC has more advantages in the management for resection of gliomas in eloquent brain areas, and MAC combined with multiple monitoring such as cerebral cortical mapping, neuronavigation, and ultrasonic detection is worthy of popularization for the resection of gliomas in eloquent brain areas.

LncRNA GAS5 represses stemness and malignancy of gliomas via elevating the SPACA6-miR-125a/let-7e Axis.

Glioblastoma (GBM) is a highly invasive neurological malignancy with poor prognosis. LncRNA-GAS5 (growth arrest-specific transcript 5) is a tumor suppressor involved in multiple cancers. In this study, we explored the clinical significance, biological function, and underlying mechanisms of GAS5 in GBM. We showed that lncRNA-GAS5 expression decreased in high-grade glioma tissues and cells, which might be associated with poor prognosis. GAS5 overexpression lowered cell viability, suppressed GBM cell migration and invasion, and impaired the stemness and proliferation of glioma stem cells (GSCs). We further discovered that GAS5 inhibited the viability of glioma cells through miR-let-7e and miR-125a by protecting SPACA6 from degradation. Moreover, GAS5 played an anti-oncogenic role in GBM through the combined involvement of let-7e and miR-125a in vivo and in vitro. Notably, these two miRNAs block the IL-6/STAT3 pathway in tumor tissues extracted from a xenograft model. Taken together, our study provides evidence for an important role of GAS5 in GBM by affecting the proliferation and migration of GSCs, thus providing a new potential prognostic biomarker and treatment strategy for GBM.

TRIM22 orchestrates the proliferation of GBMs and the benefits of TMZ by coordinating the modification and degradation of RIG-I.

Tripartite motif 22 (TRIM22) is an agonist of nuclear factor κB (NF-κB) that plays an important role in the proliferation and drug sensitivity of glioblastoma (GBM). However, the molecular mechanism underlying the protein network between TRIM22 and nuclear factor κB (NF-κB) in GBM remains unclear. Here, we found that knockout of TRIM22 effectively inhibited tumor proliferation and increased the sensitivity of GBM cells to temozolomide (TMZ) in vivo and in vitro. Moreover, TRIM22 forms a complex with cytosolic purine 5-nucleotidase (NT5C2) in GBM and regulates the ubiquitination of retinoic acid-inducible gene-I (RIG-I). TRIM22 promotes the K63-linked ubiquitination of RIG-I, while NT5C2 is responsible for K48-linked ubiquitination. This regulation directly affects the RIG-I/NF-κB/cell division cycle and apoptosis regulator protein 1 (CCAR1) signaling axis. Ubiquitin modification inhibitor of RIG-I restores the inhibition of tumor growth induced by TRIM22 knockout. The follow-up results showed that compared with patients with high TRIM22 expression, patients with low TRIM22 expression had a longer survival time and were more sensitive to treatment with TMZ. Our results revealed that the TRIM22-NT5C2 complex orchestrates the proliferation of GBM and benefits of TMZ through post-translational modification of RIG-I and the regulation of the RIG-I/NF-κB/CCAR1 pathway and is a promising target for single-pathway multi-target therapy.

Prediction of Cancer-Specific Survival of Brainstem Glioma in Children Based on Risk Stratification Model.

Objective: To develop and authenticate a risk stratification framework and nomogram for ascertaining cancer-specific survival (CSS) among the pediatric brainstem gliomas. Methods: For patients less than 12 years, according to Surveillance, Epidemiology, and End Results (SEER), information from 1998 to 2016 is found in their databases. The survival outcomes, treatments, and demographic clinicopathologic conditions are scrutinized per the database validation, and training cohorts are divided and validated using multivariate Cox regression analysis. A nomogram was designed, and predominantly, the risk stratification conceptualization engaged selected tenets according to the multivariate analysis. The model's authenticity was substantiated through C-index measure and calibration curves. Results: There are 806 pediatric concerns of histologically concluded brainstem glioma in the research. According to multivariate analysis, age, grade, radiotherapy, and race (with P value < 0.05) depicted independent prognostic variations of the pediatric gliomas. The nomogram's C-index was approximately 0.75 and an accompanied predictive capability for CSS. Conclusion: The nomogram constructed in this glioma's context is the primary predictor of using risk stratification. A combination of nomograms with the risk stratification mechanism assists clinicians in monitoring high-risk individuals and engage targeted accessory treatment.

TLR4 Deletion Improves Cognitive Brain Function and Structure in Aged Mice.

Toll-like receptor-4 (TLR4), a member of the TLR family, plays a key role in inflammation-related diseases of the nervous system. TLR4 knockout mice are widely used in various neurological disease studies, and there is a clear correlation between inflammation and behavior. Therefore, elucidating the effect of TLR4 on neurobehavioral function is essential, and the related mechanisms need to be explored. Male TLR4 knockout (TLR4-/-) and wild-type (TLR4+/+) mice of different ages (4, 8, and 16 months) were used for behavioral experiments. Synaptic spine, blood-brain barrier (BBB) integrity, memory regulatory proteins, cortical blood flow, and inflammatory factor examinations were also conducted to explore the possible mechanism by which TLR4 works. Here, we found that compared with 16-m-old TLR4+/+ mice, age-matched TLR4-/- mice had better learning and memory abilities, increased expression of neuronal synaptic spines, and increased memory-related regulatory proteins in the hippocampus. TLR4 knockout significantly attenuated the fear response in 16-m-old mice. The TLR4-/- mice also had better blood-brain barrier integrity, increased expression of tight junction-associated proteins, increased cerebral cortical blood flow and reduced proinflammatory cytokine expression in the cortex and cerebrospinal fluid. Our results suggest that TLR4 deletion ameliorates significant neurobehavioral dysfunction during the aging stage, as well as multiple abnormalities in brain function and structure due to alterations in tight junction-associated proteins and inflammatory factors.

Homer1 promotes the conversion of A1 astrocytes to A2 astrocytes and improves the recovery of transgenic mice after intracerebral hemorrhage.

BACKGROUND: Inflammation induced by intracerebral hemorrhage (ICH) is one of the main causes of the high mortality and poor prognosis of patients with ICH. A1 astrocytes are closely associated with neuroinflammation and neurotoxicity, whereas A2 astrocytes are neuroprotective. Homer scaffolding protein 1 (Homer1) plays a protective role in ischemic encephalopathy and neurodegenerative diseases. However, the role of Homer1 in ICH-induced inflammation and the effect of Homer1 on the phenotypic conversion of astrocytes remain unknown. METHODS: Femoral artery autologous blood from C57BL/6 mice was used to create an ICH model. We use the A1 phenotype marker C3 and A2 phenotype marker S100A10 to detect astrocyte conversion after ICH. Homer1 overexpression/knock-down mice were constructed by adeno-associated virus (AAV) infection to explore the role of Homer1 and its mechanism of action after ICH. Finally, Homer1 protein and selumetinib were injected into in situ hemorrhage sites in the brains of Homer1flox/flox/Nestin-Cre+/- mice to study the efficacy of Homer1 in the treatment of ICH by using a mouse cytokine array to explore the potential mechanism. RESULTS: The expression of Homer1 peaked on the third day after ICH and colocalized with astrocytes. Homer1 promotes A1 phenotypic conversion in astrocytes in vivo and in vitro. Overexpression of Homer1 inhibits the activation of MAPK signaling, whereas Homer1 knock-down increases the expression of pathway-related proteins. The Homer1 protein and selumetinib, a non-ATP competitive MEK1/2 inhibitor, improved the outcome in ICH in Homer1flox/flox/Nestin-Cre+/- mice. The efficacy of Homer1 in the treatment of ICH is associated with reduced expression of the inflammatory factor TNFSF10 and increased expression of the anti-inflammatory factors activin A, persephin, and TWEAK. CONCLUSIONS: Homer1 plays an important role in inhibiting inflammation after ICH by suppressing the A1 phenotype conversion in astrocytes. In situ injection of Homer1 protein may be a novel and effective method for the treatment of inflammation after ICH.

The Neuroprotective Effect of Increased PINK1 Expression Following Glutamate Excitotoxicity in Neuronal Cells.

Ischemic injury in patients with stroke often leads to neuronal damage and mitochondrial dysfunction. Neuronal injury caused by ischemia can be partly attributed to glutamate (L-Glu) excitotoxicity. Previous studies have shown that PTEN-induced kinase 1 (PINK1) plays a neuroprotective role in ischemic brain injury by regulating mitochondrial integrity and function. However, there are few reports on the expression of PINK1 in L-Glu excitotoxicity models, its effect on neuronal survival, and whether PINK1 plays a protective role in stroke by regulating mitophagy. In the present study, different concentrations of L-Glu inhibited the viability of neurons. After L-Glu treatment at different times, the mRNA level, protein level, and cellular fluorescence intensity of PINK1 first increased and then decreased. Compared with normal cells, cells with low PINK1 expression enhanced the inhibitory effect of L-Glu on neuronal activity, while those with high PINK1 expression showed a protective effect on neurons by alleviating mitochondrial membrane potential loss. In addition, RAP (an autophagy activator) could increase the co-localization of the mitophagy-related proteins light chain 3 (LC3) and Tom20, whereas 3-MA (an autophagy inhibitor) exerted the opposite effect. Finally, we found that L-Glu could induce the expression of PINK1/Parkin/ LC3 in neurons at both mRNA and protein levels, while RAP could further increase their expression, and 3-MA decreased their expression. Taken together, PINK1 protects against L-Glu-induced neuronal injury by protecting mitochondrial function, and the potential protective mechanism may be closely related to the enhancement of mitophagy mediated by the PINK1/Parkin signaling pathway.

Integrative Studies of Human Cord Blood Derived Mononuclear Cells and Umbilical Cord Derived Mesenchyme Stem Cells in Ameliorating Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a common pulmonary complication observed in preterm infants that is composed of multifactorial pathogenesis. Current strategies, albeit successful in moderately reducing morbidity and mortality of BPD, failed to draw overall satisfactory conclusion. Here, using a typical mouse model mimicking hallmarks of BPD, we revealed that both cord blood-derived mononuclear cells (CB-MNCs) and umbilical cord-derived mesenchymal stem cells (UC-MSCs) are efficient in alleviating BPD. Notably, infusion of CB-MNCs has more prominent effects in preventing alveolar simplification and pulmonary vessel loss, restoring pulmonary respiratory functions and balancing inflammatory responses. To further elucidate the underlying mechanisms within the divergent therapeutic effects of UC-MSC and CB-MNC, we systematically investigated the long noncoding RNA (lncRNA)-microRNA (miRNA)-messenger RNA (mRNA) and circular RNA (circRNA)-miRNA-mRNA networks by whole-transcriptome sequencing. Importantly, pathway analysis integrating Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG)/gene set enrichment analysis (GSEA) method indicates that the competing endogenous RNA (ceRNA) network is mainly related to the regulation of GTPase activity (GO: 0043087), extracellular signal-regulated kinase 1 (ERK1) and ERK2 signal cascade (GO: 0070371), chromosome regulation (GO: 0007059), and cell cycle control (GO: 0044770). Through rigorous selection of the lncRNA/circRNA-based ceRNA network, we demonstrated that the hub genes reside in UC-MSC- and CB-MNC-infused networks directed to the function of cell adhesion, motor transportation (Cdk13, Lrrn2), immune homeostasis balance, and autophagy (Homer3, Prkcd) relatively. Our studies illustrate the first comprehensive mRNA-miRNA-lncRNA and mRNA-miRNA-circRNA networks in stem cell-infused BPD model, which will be valuable in identifying reliable biomarkers or therapeutic targets for BPD pathogenesis and shed new light in the priming and conditioning of UC-MSCs or CB-MNCs in the treatment of neonatal lung injury.

Arctigenin Inhibits Glioblastoma Proliferation through the AKT/mTOR Pathway and Induces Autophagy.

PURPOSE: Arctigenin (ARG) is a natural lignan compound extracted from Arctium lappa and has displayed anticancer function and therapeutic effect in a variety of cancers. Arctigenin is mainly from Arctium lappa extract. It has been shown to induce autophagy in various cancers. However, as for whether arctigenin induces autophagy in gliomas or not, the specific mechanism is still worth exploring. METHODS: Using CCK8, the monoclonal experiment was made to detect the proliferation ability. The scratch experiment and the transwell experiment were applied to the migration and invasion ability. PI/RNase and FITC-conjugated anti-annexin V were used to detect the cell cycle and apoptosis. Western blotting was used to determine the specified protein level, and constructed LC3B-GFP plasmid was used for analysis of autophagy. RESULTS: Our research showed that ARG inhibited the growth and proliferation and invasion and migration of glioma cells in a dose-dependent manner (U87MG and T98G) and arrested the cell cycle and induced apoptosis. Interestingly, ARG induced autophagy in a dose-dependent manner. We applied Western blotting to measure the increase in the key autophagy protein LC3B, as well as some other autophagy-related proteins (increase in Beclin-1 and decrease in P62). In order to further explore the mechanism that ARG passed initiating autophagy to inhibit cell growth, we further found by Western blotting that AKT and mTOR phosphorylation proteins (P-AKT, P-mTOR) were reduced after ARG treatment, and we used AKT agonists to rescue, and the phosphorylated proteins of AKT and mTOR increased, and we found that the autophagy-related proteins were also reversed. And interestingly, the protein of apoptosis was also reversed along with autophagy. CONCLUSIONS: We thought ARG inhibited the proliferation of glioma cells by inducing autophagy and apoptosis through the AKT/mTOR pathway.

A low MW inhibitor of CD44 dimerization for the treatment of glioblastoma.

BACKGROUND AND PURPOSE: As a hallmark of glioblastoma multiforme (GBM), CD44 plays a crucial role in promoting glioblastoma stem cell (GSC) stemness phenotypes and multiple drug resistance. The therapeutic potential of CD44 has been validated by the clinical successes of several CD44 inhibitors, including antibodies and hyaluronan-related drugs. EXPERIMENTAL APPROACH: We used systemsDock software to predict verbascoside as a candidate CD44 inhibitor. Microscale thermophoresis was used to confirm the interaction between CD44 and verbascoside. Four glioblastoma cell lines and a patient-derived glioblastoma cell line were used to test the influences of verbascoside on glioblastoma. CD44-overexpressing and CD44-knockout cell lines were also used. Real-time quantitative PCR and western blot analyses were performed. A xenograft mouse model was used to test verbascoside. KEY RESULTS: Verbascoside bound to CD44 and suppressed its dimerization. By inhibiting CD44 dimerization, verbascoside decreased the release of the CD44 intracellular domain (CD44ICD) and suppressed the expression of CD44 downstream genes. Verbascoside treatment suppressed the stemness phenotypes of cells with high CD44 expression. In a mouse model of glioma, verbascoside treatment highly reduced the growth of intracranial tumours and inhibited CD44ICD release. Both stem cell marker and mesenchymal GBM subtype marker genes were down-regulated in verbascoside-treated mice. CONCLUSION AND IMPLICATIONS: Verbascoside suppressed growth of glioblastoma cells by inhibiting CD44 dimerization. Stem cell-like cell properties and tumour cell growth were also suppressed by verbascoside, both in vitro and in vivo. Verbascoside significantly prolonged survival of xenografted mice.

The role of Toll-like receptor 4 in apoptosis of brain tissue after induction of intracerebral hemorrhage.

BACKGROUND: Inflammation and apoptosis caused by intracerebral hemorrhage (ICH) are two important factors that affect patient prognosis and survival. Toll-like receptor 4 (TLR4) triggers activation of the inflammatory pathway, causing synthesis and release of inflammatory factors. The inflammatory environment also causes neuronal apoptosis. However, no studies have reported the role of TLR4 in inflammation and apoptosis. METHODS: We performed survival curve analysis and behavioral scores on TLR4 knockout mice and wild-type mice after inducing ICH. We used TLR4 knockout mice and wild-type mice to make ICH models with type VII collagenase and explored the link between TLR4 in inflammation and apoptosis. We used Western blot to detect the expression of apoptosis-related proteins, inflammatory factors, and their receptors at different time points after ICH induction. The effects of TLR4 on apoptosis were observed by TUNEL, Hoechst, and HE staining techniques. The association with TLR4 in inflammation and apoptosis was explored using IL-1ß and TNF-α antagonists. Data conforming to a normal distribution are expressed as mean ± standard deviation. Grade and quantitative data were compared with rank sum test and t test between two groups. P < 0.05 was considered statistically significant. RESULTS: TLR4 knockout significantly increased the survival rate of ICH mice. The scores of TLR4 knockout mice were significantly lower than those of wild-type mice. We found that TLR4 knockout mice significantly inhibited apoptosis and the expression of inflammatory factors after the induction of ICH. The apoptosis of ICH-induced mice was significantly improved after injecting IL-1ß and TNF-α antagonists. Moreover, the anti-apoptotic effect of the antagonist in wild-type mice is more pronounced. A single injection of the antagonist failed to improve apoptosis in TLR4 knockout mice. CONCLUSIONS: We conclude that TLR4-induced inflammation after ICH promotes neuronal apoptosis. IL-1ß and TNF-α antagonists attenuate this apoptotic effect. Therefore, targeting TLR4 in patients with clinical ICH may attenuate inflammatory response, thereby attenuating apoptosis and improving prognosis.

Eupatilin attenuates the inflammatory response induced by intracerebral hemorrhage through the TLR4/MyD88 pathway.

BACKGROUND: Intracranial hemorrhage (ICH) is one of the most common brain traumas, and inflammation caused by ICH seriously affects the quality of life and prognosis of patients. Eupatilin has been shown to have anti-inflammatory effects in various diseases. However, only one paper has reported that Eupatilin has a therapeutic effect on the inflammatory response caused by ICH and the underlying mechanism needs to be studied. METHODS: We used erythrocyte lysis stimulation (ELS) to induce mouse microglia BV2 as the inflammation model. CCK-8 and Transwell assays were used to detect cell viability and migration. RT-PCR, western blotting, and ELISA were used to detect the secretion of inflammatory factors and the expression of related mechanism proteins. HE staining was used to detect cell edema and death. RESULT: We found that ELS significantly increased protein and mRNA levels and secretion of inflammatory factors IL-1ß and TNF-α, which Eupatilin attenuated through the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) pathway. The anti-inflammatory effect of Eupatilin was significantly attenuated after siRNA was used to reduce TLR4 expression. The experimental results and mechanism were also verified in TLR4 knockout mice in vivo. CONCLUSION: Eupatilin has a therapeutic effect on inflammation caused by ICH. The underlying mechanism may be related to TLR4/MyD88, which brings new hope for clinical patients to improve symptoms and prognosis.