Ruscogenin, an important steroid sapogenin derived from Ophiopogon japonicus, has been shown to inhibit cerebral ischemic injury. However, its potential molecular action on blood-brain barrier (BBB) dysfunction after stroke remains unclear. This study aimed to investigate the effects of ruscogenin on BBB dysfunction and the underlying mechanisms in middle cerebral artery occlusion/reperfusion (MCAO/R)-injured mice and oxygen–glucose deprivation/reoxygenation (OGD/R)-injured mouse brain microvascular endothelial cells (bEnd.3). The results demonstrated that administration of ruscogenin (10 mg/kg) decreased the brain infarction and edema, improved neurological deficits, increased cerebral brain flow (CBF), ameliorated histopathological damage, reduced evans blue (EB) leakage and upregulated the expression of tight junctions (TJs) in MCAO/R-injured mice. Meanwhile, ruscogenin (0.1–10 µM) treatment increased cell viability and trans-endothelial electrical resistance (TEER) value, decreased sodium fluorescein leakage, and modulated the TJs expression in OGD/R-induced bEnd.3 cells. Moreover, ruscogenin also inhibited the expression of interleukin-1β (IL-1β) and caspase-1, and markedly suppressed the expression of Nucleotide-binding domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) and thiredoxin-interactive protein (TXNIP) in vivo and in vitro. Furthermore, ruscogenin decreased reactive oxygen species (ROS) generation and inhibited the mitogen-activated protein kinase (MAPK) pathway in OGD/R-induced bEnd.3 cells. Our findings provide some new insights into its potential application for the prevention and treatment of ischemic stroke.
The neuroprotective role of schizandrin (SA) in cerebral ischemia-reperfusion (I/R) was recently highlighted. However, whether SA plays a regulatory role on autophagy in cerebral I/R injury is still unclear. This study aimed to explore whether the neuroprotective mechanisms of SA were linked to its regulation of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/autophagy pathway in vivo and in vitro. The present study confirmed that SA significantly improved oxygen-glucose deprivation/re-oxygenation (OGD/R)-induced PC12 cells injury. The results of immunoblotting and confocal microscope showed that SA decreased autophagy in OGD/R-injured PC12 cells, which was reflected by the decreased Beclin-1 and LC3-II expression, autophagy flux level, and LC3 puncta formation. In addition, the autophagy inducer rapamycin partially prevented the effects of SA on cell viability and autophagy after OGD/R, whereas the autophagy inhibitor 3-methyladenine (3-MA) exerted the opposite effect. The results of Western blotting showed that SA markedly decreased the phosphorylation of AMPK (p-AMPK), whereas the phosphor-mTOR (p-mTOR) levels increased in the presence of OGD/R insult. Furthermore, pretreatment with the AMPK inducer AICAR partially reversed the protective effects and autophagy inhibition of SA. However, AMPK inhibitor Compound C pretreatment further promoted the inhibition of SA on autophagy induction and cell damage induced by OGD/R. Taken together, these findings demonstrate that SA protects against OGD/R insult by inhibiting autophagy through the regulation of the AMPK-mTOR pathway and that SA may have therapeutic value for protecting neurons from cerebral ischemia.
Muscone is the main chemical ingredient in Musk which is main crude drug in Tongqiaohuoxue decoction (TQHXD), and TQHXD has a protective effect on damaged neurons, so we hypothesize that muscone can alter blood-brain barrier (BBB) permeability via the modulation of P-glycoprotein (P-gp) and matrix metalloproteinase-9 (MMP-9) expression. In this study, astrocytes (AC) and human umbilical vein endothelial cells (ECV304) were co-cultured to simulate the BBB model in vitro. Leak testing, transmembrane resistance experiments, and BBB-specific enzyme testing were used to test whether the model was successful. Different concentrations of muscone permeating the BBB were detected by gas chromatography (GC). The change of the transendothelial electrical resistance (TEER) on the BBB in vitro after treating with muscone was detected by Millicell-ERS. The protein expression of P-gp, MMP-9 in normal, and oxygen/glucose deprivation (OGD) BBB model was determined by western blotting to inquire that the mechanism of muscone penetrates the BBB model in vitro. The results show that muscone was detected in the lower medium of the BBB model by GC; the values of TEER were no significant difference before and after muscone (8 μM) was added to the BBB model; the expression of P-gp significantly decreased after the BBB model treatment with muscone (4, 8, and 16 μM) for 24 h; the expression of P-gp and MMP-9 in different concentrations of muscone groups had different degrees of reduction compared with the BBB in the state of OGD. In conclusion, muscone could permeate the BBB model, and it was associated with the inhibition of P-gp and MMP-9 expression. An understanding of the mechanisms of muscone across the BBB is crucial to the development of therapeutic modalities for cerebral vascular diseases.
Waltonitone (WA), an ursane-type pentacyclic triterpene extracted from Gentiana waltonii Burkill, was recently appeared to exert anti-tumor effect. However, the biological underpinnings underlying the role of WA in hepatocellular carcinoma (HCC) cells have not been completely elucidated. Our previous report indicated that the FXR-regulated miR-22-CCNA2 pathway contributed to the progression and development of HCC. Besides, a wide spectrum of microRNAs (miRNAs) could be up- or down-regulated upon WA treatment, including miR-22. Hence, we aimed to determine whether WA inhibited HCC cell proliferation via the FXR-miR-22-CCNA2 axis. In this study, we observed a significant downregulation of FXR and miR-22, along with upregulation of CCNA2 in 80 paired tumors relative to adjacent normal tissues of HCC subjects, which were obtained from the available GEO database in NCBI (GSE22058). Furthermore, we validated the expression patterns of these three targets in another set of HCC samples and found the highly correlation within each other. Additionally, our data demonstrated that WA induced miR-22 and repressed CCNA2 in HCC cells, which contributed to the cell proliferation arrest. In addition, evidence suggested that either miR-22 silencing or FXR knockdown reversed the diminished CCNA2 expression as well as cell proliferation inhibition caused by WA treatment and WA inhibited tumor masses in vivo in a subcutaneous xenograft mouse model of HCC. Overall, our data indicated that WA inhibited HCC cell proliferation and tumorigenesis through miR-22-regulated CCNA2 repression, which was at least partially through FXR modulation.
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