Buyang Huanwu Decoction restores the balance of mitochondrial dynamics after cerebral ischemia-reperfusion through calcium overload reduction by the PKCε-Nampt-Sirt5 axis.

Stroke is a common condition that poses a significant threat to human health. Buyang Huanwu Decoction (BYHWD) is a traditional treatment used for stroke management. However, the exact mechanism by which BYHWD mitigates cerebral ischemia-reperfusion by regulating calcium overload and restoring mitochondrial function is not yet fully understood. The objective of this research was to examine the neuroprotective properties of BYHWD in reducing the damage produced by cerebral ischemia/reperfusion (I/R) injury via the modulation of calcium overload and mitochondrial dynamics (MD). MCAO/R model success was evaluated via PSI laser scatter flowmetry. The neurological function scores were assessed. The cerebral infarct (CI) volume was detected via TTC staining. NeuN expression was detected via immunohistochemistry, and degenerated neurons were observed via FJC staining. The mitochondrial permeability transition pore (mPTP), the mitochondrial membrane potential (MMP), and ATP were detected. The reactive oxygen species (ROS) content and the NAD+/NADH ratio were determined. The glutamate (Glu) and glutamine (Gln) contents as well as the Ca2+ concentration were determined. The expression of PKCε, p-PKCε, namely, Sirt5, GLS, Drp1, p-Drp1 616, Fis1, Opa1, and Mfn2 was determined via Western blotting. Immunohistochemistry was used to detect p-PKCε, which is expressed at high levels. Immunofluorescence was used to detect p-Drp1 616, Opa1 and Sirt5 fluorescence intensity. BYHWD treatment enhanced neurological function, decreased the amount of CI, mitigated neuronal damage, decreased mPTP opening, restored the MMP, increased ATP synthesis, and decreased the ROS content after brain I/R. It also increased PKCε, p-PKCε, Sirt5, GLS, Opa1 and Mfn2 expression; downregulated p-Drp1 616, Drp1 and Fis1 expression; elevated the NAD+/NADH ratio and Gln content; and decreased the Glu content and Ca2+ concentration. The effects of BYHWD were reversed by the administration of the PKCε inhibitor εV1-2. BYHWD administration led to increased PKCε mRNA expression. BYHWD modulates MD by diminishing calcium overload through the PKCε-Nampt-Sirt5 axis, which restores mitochondrial function and mitigates brain I/R damage.

Liu Z ，Yin M ，Li J ，Wang J ，Jin X ，Zhou X ，Gao W ... -  《-》

Piezo1 Modulates Neuronal Autophagy and Apoptosis in Cerebral Ischemia-Reperfusion Injury Through the AMPK-mTOR Signaling Pathway.

Cerebral ischemia-reperfusion (I/R) injury is a complex pathophysiological process involving multiple mechanisms, including apoptosis and autophagy, which can lead to significant neuronal damage. PIEZO1, a stretch-activated ion channel, has recently emerged as a potential regulator of cellular responses to ischemic conditions. However, its role in neuronal cell survival and death during ischemic events is not well elucidated. This study aimed to ascertain the regulatory function of PIEZO1 in neuronal cell apoptosis and autophagy in an in vitro model of hypoxia-reoxygenation and an in vivo model of brain I/R injury. HT22 hippocampal neuronal cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate ischemic conditions, with subsequent reoxygenation. In vitro, PIEZO1 expression was silenced using small interfering RNA (si-RNA) transfection. The effects on cell viability, apoptosis, and autophagy were assessed using CCK-8 assays, PI-Annexin/V staining combined with flow cytometry, and Western blot analysis. Additionally, intracellular Ca2+ levels in HT22 cells were measured using a Ca2+ probe. The involvement of the AMPK-mTOR pathway was investigated using rapamycin. For in vivo validation, middle cerebral artery occlusion/reperfusion (MCAO/R) in rats was employed. To determine the neuroprotective role of PIEZO1 silencing, sh-PIEZO1 adeno-associated virus was stereotaxically injected into the cerebral ventricle, and neurological and histological outcomes were assessed using neurological scoring, TTC staining, H&E staining, Nissl staining, and immunofluorescence. In HT22 cells, OGD/R injury notably upregulated PIEZO1 expression and intracellular Ca2+ levels. Silencing PIEZO1 significantly diminished OGD/R-induced Ca2+ influx, apoptosis, and autophagy, as indicated by lower levels of pro-apoptotic and autophagy-related proteins and improved cell viability. Additionally, PIEZO1 modulated the AMPK-mTOR signaling pathway, an effect that was counteracted by rapamycin treatment, implying its regulatory role. In vivo, PIEZO1 silencing ameliorated brain I/R injury in MCAO/R rats, demonstrated by improved neurological function scores and reduced neuronal apoptosis and autophagy. However, these neuroprotective effects were reversed through rapamycin treatment. Our findings indicate that PIEZO1 is upregulated following ischemic injury and facilitates Ca2+ influx, apoptosis, and autophagy via the AMPK-mTOR pathway. Silencing PIEZO1 confers neuroprotection against I/R injury both in vitro and in vivo, highlighting its potential as a therapeutic target for stroke management.

Yue Y ，Chen P ，Ren C 《-》

Investigating the possible mechanism of Cornus officinalis in the therapy of ischemic stroke by UHPLC-Q-TOF-MS, network pharmacology, molecular docking, and experimental verification.

Cornus officinalis is a conventional Chinese medicine for tonifying liver and kidney in ancient China. The active ingredients from Cornus officinalis can delay the progression of cerebral aneurysms, alleviate experimental autoimmune encephalomyelitis, and show a good intervention effect on brain diseases. Loganin, the active ingredient of Cornus officinalis, has a neuroprotective effect on cerebral ischemia-reperfusion injury in mice. It is yet unknown, nevertheless, how Cornus officinalis works to treat ischemic stroke. Based on ultra-high performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), network pharmacology and molecular docking, Cornus officinalis's mechanism of intervention in ischemic stroke is explored and verified by experiments. To examine the chemical components of Cornus officinalis, UHPLC-Q-TOF-MS was used. The network pharmacology was used to construct the "active ingredient-core target-main pathway" network of Cornus officinalis. Then, the link between the main active components and the key protein targets, as determined by network pharmacology, was verified through the application of molecular docking. The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model used in this study was created using the suture technique. The pharmacological effects of Cornus officinalis were explored by neurological function score, behavior, TTC staining, ultrasound and flow cytometry. Western blot and qPCR were used to confirm the core target. The outcomes of the investigation demonstrated that Cornus officinalis had a potent anti-ischemic stroke effect. UHPLC-Q-TOF-MS method was used to determine 24 chemical constituents in Cornus officinalis, of which 22 components had a close relationship with protein targets relevant to ischemic stroke. The 27 protein targets screened by "active ingredient-core target-main pathway" may be the possible targets of Cornus officinalis in the therapy of ischemic stroke. Most of the 27 protein targets had to do with the inflammatory response, apoptosis and energy metabolism. KEGG enrichment analysis showed that AGE/RAGE ranked high and was closely related to inflammatory response. Molecular docking predicted that the top 10 components in the network diagram had good binding with inflammatory factors IL6, IL-1β and TNF-α protein targets. Western blot research outcomes stated that Cornus officinalis could firmly impede the production of AGE, RAGE, and P-NFκB P65. Cornus officinalis had the potential to prevent ischemic stroke by drastically inhibiting the production of TNF-α, IL-1β, and IL-6, according to the results of qPCR study. This study found that Cornus officinalis can improve the brain injury, motor ability and blood flow velocity of MCAO/R rats and suppress the inflammatory reaction through the AGE/RAGE/NFκB pathway to exert the therapeutic effect on ischemic stroke.

Zhang Y ，Yuan PP ，Li PY ，Zheng YJ ，Li SF ，Zhao LR ，Ma QY ，Cheng JL ，Ma JS ，Feng WS ，Zheng XK ... -  《-》

[Sulforaphane regulates mitochondrial homeostasis through adenosine monophosphate-activated protein kinase signaling to treat acute carbon monoxide poisoning induced brain injury in rats].

Yue A ，Song H ，Zhou X ，Han W ，Li Q ... -  《-》

Edaravone Dexborneol protects against blood-brain barrier disruption following cerebral ischemia/reperfusion by upregulating pericyte coverage via vitronectin-integrin and PDGFB/PDGFR-β signaling.

Recent advancements in brain cytoprotection therapies following cerebral ischemia-reperfusion (I/R) injury have become an emerging interest. Pericytes were vulnerable during the early stages of ischemia. This study aims to explore the protective effects of Edaravone dexborneol (Eda.B) on pericyte loss, as well as and the underlying mechanisms, given its potential in alleviating I/R injury. The rat transient middle cerebral artery occlusion (tMCAO) model was established. Rats were randomly divided into Sham group (Sham, n = 24), tMCAO group (tMCAO, n = 24), Edaravone group (Eda, n = 24), Dexborneol group (Dexborneol, n = 24), and Eda.B group (Eda.B, n = 24). Neurological function recovery, infarct volume, and blood-brain barrier (BBB) disruption were assessed using Zea-Longa scoring, TTC staining, and Evans Blue extravasation, respectively. Alterations in Basement membrane (BM) and pericyte coverage were assessed by transmission electron microscopy (TEM). The expression levels of pericyte marker NG2 and PDGFR-β in the ischemic region, as well as BBB transcellular transport-related proteins vitronectin (VTN), α5 and PDGFB were detected by western blotting. Furthermore, a specific inhibitor of PDGFB, MOR8457, was employed (Eda.B + MOR8457, n = 8) to explore the protective effects of Eda.B on pericyte injury via PDGFB/PDGFR-β. Eda.B significantly reduced cerebral infarct volume and promoted neurological function recovery in comparison to the tMCAO, Eda and Dexborneol groups. Additionally, Eda.B significantly ameliorated BBB leakage, mitigated the decrease in pericyte coverage, and reduced vesicle density in endothelial cells and BM thickness following I/R. Mechanically, Eda.B inhibited the downregulation of NG2, PDGFB/PDGFR-β, VTN, while preventing upregulation of α5 protein expression in tMCAO rats. Blocking PDGFB with MOR8457 demonstrated that Eda.B improved pericyte loss and BBB permeability by activating PDGFB/PDGFR-β signaling. We elucidated that vitronectin-integrin and PDGFB/PDGFR-β signaling contributed to Eda.B's protective effects against pericyte loss and BBB permeability following I/R injury, unraveling new insights into mechanisms of pericyte as a promising therapeutic target.

Sun Z ，Zhao H ，Yang S ，Liu R ，Yi L ，Gao J ，Liu S ，Chen Y ，Zhang Z ... -  《-》