Previous findings have shown that non-muscle myosin heavy-chain IIA (NMMHC IIA) is involved in autophagy induction triggered by starvation in D. melanogaster; however, its functional contribution to neuronal autophagy remains unclear. The aim of this study is to explore the function of NMMHC IIA in cerebral ischemia-induced neuronal autophagy and the underlying mechanism related to autophagy-related gene 9A (ATG9A) trafficking. Functional assays and molecular mechanism studies were used to investigate the role of NMMHC IIA in cerebral ischemia-induced neuronal autophagy in vivo and in vitro. A middle cerebral artery occlusion (MCAO) model in mice was used to evaluate the therapeutic effect of blebbistatin, a myosin II ATPase inhibitor. Herein, either depletion or knockdown of NMMHC IIA led to increased cell viability in both primary cultured cortical neurons and pheochromocytoma (PC12) cells exposed to oxygen–glucose deprivation/reoxygenation (OGD/R). In addition, NMMHC IIA and autophagic marker LC3B were upregulated by OGD/R, and inhibition of NMMHC IIA significantly reduced OGD-induced neuronal autophagy. Furthermore, NMMHC IIA-induced autophagy is through its interactions with F-actin and ATG9A in response to OGD/R. The NMMHC IIA–actin interaction contributes to ATG9A trafficking and autophagosome formation. Inhibition of the NMMHC IIA–actin interaction using blebbistatin and the F-actin polymerization inhibitor cytochalasin D significantly suppressed ATG9A trafficking and autophagy induction. Furthermore, blebbistatin significantly improved neurological deficits and infarct volume after ischemic attack in mice, accompanied by ATG9A trafficking and autophagy inhibition. These findings demonstrate neuroprotective effects of NMMHC IIA inhibition on regulating ATG9A trafficking-dependent autophagy activation in the context of cerebral ischemia/reperfusion.

Introduction: Traditional Chinese medicine (TCM) provides unique advantages for treatment of ischemic stroke, an aging-related vascular disease. Shengmai powder (GRS) is composed of three active components, specifically, ginsenoside Rb1, ruscogenin and schisandrin A, at a ratio of 6:0.75:6. The main objective of this study was to evaluate the effects of GRS on blood-brain barrier (BBB) dysfunction under conditions of middle cerebral artery occlusion/reperfusion (MCAO/R). Methods: C57BL/6J mice subjected to MCAO/R were used as a model to assess the protective effects of varying doses of GRS (6.4, 12.8, and 19.2 mg/kg) on BBB dysfunction. Results: GRS reduced cerebral infarct volume and degree of brain tissue damage, improved behavioral scores, decreased water content and BBB permeability, and restored cerebral blood flow. Moreover, GRS promoted expression of zona occludens-1 (ZO-1) and claudin-5 while inhibiting matrix metalloproteinase 2/9 (MMP-2/9) expression and myosin light chain (MLC) phosphorylation. In vitro, GRS (1, 10, and 100 ng/mL) enhanced the viability of bEnd.3 cells subjected to oxygen glucose deprivation/reoxygenation (OGD/R) and decreased sodium fluorescein permeability. Conclusion: Consistent with in vivo findings, ZO-1 and claudin-5 were significantly upregulated by GRS in bEnd.3 cells under OGD/R and MMP-2/9 levels and MLC phosphorylation reduced through the Rho-associated coil-forming protein kinase (ROCK)/cofilin signaling pathway. Based on the collective findings, we propose that the TCM compound, GRS, plays a protective role against I/R-induced BBB dysfunction.

BackgroundIschemic stroke causes brain inflammation and multi-organ injury, in which the peroxisome proliferator-activated receptor-gamma (PPARγ) signaling pathway plays a key role. Studies have indicated that ginsenoside Rb1 (GRb1) can protect blood–brain barrier integrity after stroke. Here, we aimed to determine whether GRb1 can ameliorate brain/lung/intestinal barrier damage in mice with acute cerebral ischemia through the PPARγ signaling pathway. MethodAdult male ICR (Institute of Cancer Research) mice underwent right middle cerebral artery occlusion for 1 h with subsequent 24 h reperfusion to produce ischemia/reperfusion(I/R) injury, following which the mice were administered GRb1 (20 mg/kg) intraperitoneally. The integrity of the brain, pulmonary, and intestinal barriers was assessed using an Evans blue leakage assay and connexin immunofluorescence staining. The serum levels of tumor necrosis factor-α (TNF-α) and interferon-gamma (IFN-γ) were measured by enzyme-linked immunosorbent assay (ELISA), and the expression of PPARγ and phospho-nuclear factor kappa B (NF-κB) p65 was determined by western blot. ResultGRb1 significantly mitigated multi-organ injury and increased the expression of cerebral microvascular, pulmonary vascular, and intestinal epithelial connexins (claudin-5, occludin, and zona occludens 1 (ZO-1). In brain, lung, and intestinal tissues, GRb1 activated PPARγ, decreased phospho-NF-kB p65 levels, and inhibited the production of proinflammatory cytokines, thereby maintaining barrier permeability. However, cotreatment with GRb1 and the PPARγ antagonist GW9662 reversed the barrier-protective effect of GRb1.ConclusionThe results showed that GRb1 can improve brain/lung/intestinal barrier damage post-stroke through the PPARγ pathway.

Background : Cerebral ischemia-reperfusion (I/R) injury as a serious threat to human health is characterized by cerebral endothelial leakage, as a result of the damage of blood-brain barrier (BBB). It is thus quite attractive to realize real-time monitoring of BBB damage for therapeutic surveillance. Methods : In this study, a radioactive probe is constructed by conjugating ruscogenin (Rus), a neuroprotectants, to technetium-99m (Tc 99m) to assess the damage of cerebral endothelial in BBB. Results : In vitro study proves that the probe can penetrate more e ciently in damaged BBB. Then, longitudinal nuclear imaging distinguishes mice with BBB leakage from normal ones, which is validated by evans blue staining of brain tissue. Higher nuclear signal also correlates with poorer blood circulation in brain. Further, by visualizing brain signal during drug treatment, the probe nds that the most obvious protective e cacy of Rus occurs at 12 h post administration, which is superior than edaravone (Edara). Conclusion : Altogether, the probe is promising to monitor I/R injury real-time by radioactive-imaging of BBB integrity. Importantly, Rus as a neuroprotectants may serve as a potential theranostic agent for I/R treatment.