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Plant exosome-like nanoparticles (ELNs) have shown great potential in treating tumor and inflammatory diseases, but the neuroprotective effect of plant ELNs remains unknown. In the present study, we isolated and characterized novel ELNs from Momordica charantia (MC) and investigated their neuroprotective effects against cerebral ischemia-reperfusion injury. In the present study, MC-ELNs were isolated by ultracentrifugation and characterized. Male Sprague–Dawley rats were subjected to middle cerebral artery occlusion (MCAO) and MC-ELN injection intravenously. The integrity of the blood–brain barrier (BBB) was examined by Evans blue staining and with the expression of matrix metalloproteinase 9 (MMP-9), claudin-5, and ZO-1. Neuronal apoptosis was evaluated by TUNEL and the expression of apoptotic proteins including Bcl2, Bax, and cleaved caspase 3. The major discoveries include: 1) Dil-labeled MC-ELNs were identified in the infarct area; 2) MC-ELN treatment significantly ameliorated BBB disruption, decreased infarct sizes, and reduced neurological deficit scores; 3) MC-ELN treatment obviously downregulated the expression of MMP-9 and upregulated the expression of ZO-1 and claudin-5. Small RNA-sequencing revealed that MC-ELN-derived miRNA5266 reduced MMP-9 expression. Furthermore, MC-ELN treatment significantly upregulated the AKT/GSK3β signaling pathway and attenuated neuronal apoptosis in HT22 cells. Taken together, these findings indicate that MC-ELNs attenuate ischemia-reperfusion–induced damage to the BBB and inhibit neuronal apoptosis probably via the upregulation of the AKT/GSK3β signaling pathway.

Blood brain barrier dysfunction (BBB) causing edema and hemorrhagic transformation is one of the pathophysiological characteristics of stroke. Protection of BBB integrity have shown great potential in improving stroke outcome. Here, we assessed the efficacy of exosomes extracted from healthy rat serum in protection against ischemic stroke in vivo and in vitro. Exosomes were isolated by gradient centrifugation and ultracentrifugation, and exosomes were characterized by transmission electron microscope (TEM) and nanoparticle tracking video microscope. Exosomes were applied to middle cerebral artery occlusion (MCAO) rats or brain microvascular endothelial cell line (bEnd.3) subjected to oxygen-glucose deprivation (OGD) injury. Serum-derived exosomes were injected intravenously into adult male rats 2 hours after transient MCAO. Infarct volume and gross cognitive function were assessed 24 hours after reperfusion. Post-stroke rats treated with serum-derived exosomes exhibited significantly reduced infarct volumes and enhanced neurological function. Apoptosis was assessed via TdT-mediated dUTP nick end labeling (TUNEL) staining and the expression of Bcl-2, Bax and cleaved caspase-3 24 hours after injury. Our data showed that serum exosomes treatment strikingly decreased TUNEL+CD31+ cells in the striatum, enhanced the ratio of Bcl-2 to Bax and inhibited cleaved caspase-3 production in MCAO rats and OGD/reoxygenation insulted bEnd.3 cells. Under the consistent treatment, the expression of LC3B-II, LC3B-I and SQSTM1/p62 was detected by western blotting. Autolysosomes were observed via TEM. We found that serum exosomes reversed the ratio of LC3B-II to LC-3I, prevented SQSTM1/p62 degradation, autolysosome formation and autophagic flux. Together, these results indicated that exosomes isolated from healthy serum provided neuroprotection against experimental stroke partially via inhibition of endothelial cell apoptosis, and autophagy-mediated BBB breakdown. Intravenous serum-derived exosome treatment may therefore provide a novel clinical therapeutic strategy for ischemic stroke.

Depression is a common and debilitating condition for which effective treatments are needed. Lepidium meyenii Walp (Maca) is a plant with potential medicinal effects in treating depression. Recently, there has been growing interest in plant‐derived extracellular vesicles (EVs) due to their low toxicity and ability to transport to human cells. Targeting the gut–brain axis, a novel strategy for depression management, may be achieved through the use of Maca‐derived EVs (Maca‐EVs). In this study, we successfully isolated Maca‐EVs using gradient ultracentrifugation and characterized their shape, size, and markers (CD63 and TSG101). The in vivo imaging showed that the Dil‐labeled Maca‐EVs crossed the brain–blood barrier and accumulated in the brain. The behavioral tests revealed that Maca‐EVs dramatically recovered the depression‐like behaviors of unpredictable chronic mild stress (UCMS) mice. UCMS mice fecal were characterized by an elevated abundance of g_Enterococcus, g_Lactobacillus, and g_Escherichia_Shigella, which were significantly restored by administration of Maca‐EVs. The effects of Maca‐EVs on the altered microbial and fecal metabolites in UCMS mice were mapped to biotin, pyrimidine, and amino acid (tyrosine, alanine, aspartate, and glutamate) metabolisms, which were closely associated with the serotonin (5‐HT) production. Maca‐EVs were able to increase serum monoamine neurotransmitter levels in UCMS mice, with 5‐HT showing the most significant changes. We further demonstrated that 5‐HT improved the expression of brain‐derived neurotrophic factor, a key regulator of neuronal plasticity, and its subsequent activation of TrkB/p‐AKT signaling by regulating the GTP‐Cdc42/ERK pathway. These findings suggest that Maca‐EVs enhance 5‐HT release, possibly by modulating the gut–brain axis, to improve depression behavior. Our study sheds light on a novel approach to depression treatment using plant‐derived EVs. We successfully isolated Maca‐EVs using ultracentrifugation and tested its antidepressant effects in the unpredictable chronic mild stress mice. Maca‐EVs increased serum serotonin (5‐HT) levels probably via the gut microbiota and metabolism modulation. The antidepressant activity of Maca‐EVs may be attributed to the enhanced serum 5‐HT levels and subsequent activation of the GTP‐Cdc42/ERK and BDNF/TrkB/AKT signaling pathway. Highlights We successfully isolated Maca‐EVs using ultracentrifugation and tested its antidepressant effects in the unpredictable chronic mild stress mice. Maca‐EVs increased serum serotonin (5‐HT) levels probably via the gut microbiota and metabolism modulation. The antidepressant activity of Maca‐EVs may be attributed to the enhanced serum 5‐HT levels and subsequent activation of the GTP‐Cdc42/ERK and BDNF/TrkB/AKT signaling pathway.

Background Doxorubicin (DOX) is a first-line chemotherapeutic drug for various malignancies that causes cardiotoxicity. Plant-derived exosome-like nanovesicles (P-ELNs) are growing as novel therapeutic agents. Here, we investigated the protective effects in DOX cardiotoxicity of ELNs from Momordica charantia L. (MC-ELNs), a medicinal plant with antioxidant activity. Results We isolated MC-ELNs using ultracentrifugation and characterized them with canonical mammalian extracellular vesicles features. In vivo studies proved that MC-ELNs ameliorated DOX cardiotoxicity with enhanced cardiac function and myocardial structure. In vitro assays revealed that MC-ELNs promoted cell survival, diminished reactive oxygen species, and protected mitochondrial integrity in DOX-treated H9c2 cells. We found that DOX treatment decreased the protein level of p62 through ubiquitin-dependent degradation pathway in H9c2 and NRVM cells. However, MC-ELNs suppressed DOX-induced p62 ubiquitination degradation, and the recovered p62 bound with Keap1 promoting Nrf2 nuclear translocation and the expressions of downstream gene HO-1. Furthermore, both the knockdown of Nrf2 and the inhibition of p62-Keap1 interaction abrogated the cardioprotective effect of MC-ELNs. Conclusions Our findings demonstrated the therapeutic beneficials of MC-ELNs via increasing p62 protein stability, shedding light on preventive approaches for DOX cardiotoxicity.

Cerebral ischemia-reperfusion injury produces excessive reactive oxygen and nitrogen species, including superoxide, nitric oxide, and peroxynitrite (ONOO - ). We recently developed a new ONOO - -triggered metal-free carbon monoxide donor (PCOD585), exhibiting a notable neuroprotective outcome on the rat middle cerebral artery occlusion model and rendering an exciting intervention opportunity toward ischemia-induced brain injuries. However, its therapeutic mechanism still needs to be addressed. In the pharmacological study, we found PCOD585 inhibited neuronal Bcl2/Bax/caspase-3 apoptosis pathway in the peri-infarcted area of stroke by scavenging ONOO - . ONOO - scavenging further led to decreased Acyl-CoA synthetase long-chain family member 4 and increased glutathione peroxidase 4, to minimize lipoperoxidation. Additionally, the carbon monoxide release upon the ONOO - reaction with PCOD585 further inhibited the neuronal Iron-dependent ferroptosis associated with ischemia-reperfusion. Such a synergistic neuroprotective mechanism of PCOD585 yields as potent a neuroprotective effect as Edaravone. Additionally, PCOD585 penetrates the blood-brain barrier and reduces the degradation of zonula occludens-1 by inhibiting matrix metalloproteinase-9, thereby protecting the integrity of the blood-brain barrier. Our study provides a new perspective for developing multi-functional compounds to treat ischemic stroke.

Caspase-12 is a caspase family member for which functions in regulating cell death and inflammation have previously been suggested. In this study, we used caspase-12 lacZ reporter mice to elucidate the expression pattern of caspase-12 in order to obtain an idea about its possible in vivo function. Strikingly, these reporter mice showed that caspase-12 is expressed explicitly in Purkinje neurons of the cerebellum. As this observation suggested a function for caspase-12 in Purkinje neurons, we analyzed the brain and behavior of caspase-12 deficient mice in detail. Extensive histological analyses showed that caspase-12 was not crucial for establishing cerebellum structure or for maintaining Purkinje cell numbers. We then performed behavioral tests to investigate whether caspase-12 deficiency affects memory, motor, and psychiatric functions in mice. Interestingly, while the absence of caspase-12 did not affect memory and motor function, caspase-12 deficient mice showed depression and hyperactivity tendencies, together resembling manic behavior. Next, suggesting a possible molecular mechanistic explanation, we showed that caspase-12 deficient cerebella harbored diminished signaling through the brain-derived neurotrophic factor/tyrosine kinase receptor B/cyclic-AMP response binding protein axis, as well as strongly enhanced expression of the neuronal activity marker c-Fos. Thus, our study establishes caspase-12 expression in mouse Purkinje neurons and opens novel avenues of research to investigate the role of caspase-12 in regulating psychiatric behavior.

It has been demonstrated that Src could modulate NMDA receptor, and PAR1 could also affect NMDAR signaling. However, whether PAR1 could regulate NMDAR through Src under ICH has not yet been investigated. In this study, we demonstrated the role of Src-PSD95-GluN2A signaling cascades in rat ICH model and in vitro thrombin challenged model. Using the PAR1 agonist SFLLR, antagonist RLLFS and Src inhibitor PP2, electrophysiological analysis showed that PAR1 regulated NMDA-induced whole-cell currents ( I NMDA ) though Src in primary cultured neurons. Both in vivo and in vitro results showed the elevated phosphorylation of tyrosine in Src and GluN2A and enhanced interaction of the Src-PSD95-GluN2A under model conditions. Treatment with the PAR1 antagonist RLLFS, AS-PSD95 (Antisense oligonucleotide against PSD95) and Src inhibitor PP2 inhibited the interaction among Src-PSD95-GluN2A, and p-Src, p-GluN2A. Co-application of SFLLR and AS-PSD95, PP2, or MK801 (NMDAR inhibitor) abolished the effect of SF. In conclusion, our results demonstrated that activated thrombin receptor PAR1 induced Src activation, enhanced the interaction among Src-PSD95-GluN2A signaling modules, and up-regulated GluN2A phosphorylation after ICH injury. Elucidation of such signaling cascades would possibly provide novel targets for ICH treatment.

Introduction Neuroprotective agents for acute ischemic stroke often fall short in efficacy due to the blood–brain barrier challenges, lack of target specificity, and limited effectiveness. Recently, plant-derived extracellular vesicle-like particles (EVLP) have shown promise in their multifaceted functions. Objectives The neuroprotective advantages that EVLP produced from Houttuynia cordata Thunb against cerebral ischemia/reperfusion injury are investigated. Methods The extraction of HT-EVLP was performed using gradient centrifugation and ultracentrifugation, followed by identification of its particle size, morphology, and exosomal marker proteins. Using behavioral tests and a rat model of middle cerebral artery occlusion (MCAO), the neuroprotective attributes of HT-EVLP were assessed. To evaluate the effect of HT-EVLP on ferroptosis and cell survival, the oxygen–glucose deprivation/reoxygenation (OGD/R) induced HT22 cell model was used. Utilizing bioinformatics analysis and small RNA sequencing, the miRNA composition and downstream target genes of HT-EVLP were predicted. The dual-luciferase reporter gene assay was used to confirm that miR159a bound to long-chain acyl-coenzyme A synthase 4 (ACSL4). The impact of miR159a transfection on OGD/R-induced ferroptosis in HT22 cell was also observed. Results Using a MCAO model, we found that HT-EVLP preserved blood brain barrier integrity, naturally penetrated the infarct core area, reduced cerebral infarct volume, mitigated neuronal apoptosis and ferroptosis, and facilitated recovery of neuronal function. In vitro studies further revealed that HT-EVLP enhanced cell survival and suppressed ACSL4-mediated ferroptosis in OGD/R-treated HT22 cells. Small RNA sequencing indicated that HT-EVLP are rich in miRNAs, with miR159a, among the top 10, potentially regulating ferroptosis-related pathways and directly binding to the 3’UTR of ACSL4. Overexpression of miR159a reduced Erastin-induced ACSL4 expression and alleviated mitochondrial damage in HT22 cells without causing toxicity. Conclusions This study highlights the potential of HT-EVLP as carriers of endogenous miR159a, offering a promising strategy for ischemic brain injury therapy.

Aim： To investigate the expression of cyclooxygenase-2 （COX-2） and epidermal growth factor receptor （EGFR） and the possible mechanism in the development in androgen independent prostate cancer （AIPC）. Methods： Immunohistochemistry was performed on paraffin-embedded sections with goat polyclonal against COX-2 and mouse monoclonal antibody against EGFR in 30 AIPC and 18 androgen dependent prostate cancer （ADPC） specimens. The effect of epidermal growth factor （EGF） treatments on the expression of COX-2 and signal pathway in PC-3 and DU-145 cells was studied using reverse transcription-polymerase chain reaction （RT-PCR） and Western blot analysis. ELISA was used to measure prostaglandin E2 （PGE2） levels in the media of PC-3 and DU-145 incubated with EGF for 24 h. Results： COX-2 was positively expressed in AIPC and ADPC, which were predominantly in endochylema of prostate cancer （PCa） cells. Intense staining was seen in AIPC （80%） and in ADPC （55.5%）, but there was no significant association between the two groups. EGFR expression was also positive in the two groups （61.8% in ADPC and 90% in AIPC, P 〈 0.01）. A significant association was found between EGFR expression and a higher Gleason score （P 〈 0.05） or tumor stage （P 〈 0.05）. The expression of PGE2 was increased in PC-3 and DU-145 cells after being incubated with EGF. Both p38MAPK and PI-3K pathway were involved in the PC-3 cell COX-2 upregulation course. In DU- 145, only p38MAPK pathway was associated with COX-2 upregulation. Conclusion： EGFR activation induces COX-2 expression through PI-3K and/or p38MAPK pathways. COX-2 and EGFR inhibitors might have a cooperative anti-tumor effect in PCa.