Muscone inhibits opening of mPTP to alleviate OGD/R-induced injury of HT22 cells / 中国中药杂志

This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.

Protective Effect of Cerebrospinal Fluid Containing Tongqiao Huoxuetang on Brain Microvascular Endothelial Cells from Rats with Oxygen Glucose Deprivation/Reoxygenation Injury / 中国实验方剂学杂志

Objective:To investigate the protective effect of cerebrospinal fluid containing Tongqiao Huoxuetang (TQHXT) on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced brain microvascular endothelial cells (BMECs), in order to explore the underlying mechanisms. Method:Primary BMECs were extracted by enzymatic digestion, and the cells were randomly divided into six groups: the normal control group, the OGD/R group, the TQHXT group（20%）, the nimodipine（NMDP） group (10 μmol·L-1), the cabozanix group (1 μmol·L-1) and the combination group. Except for the normal control group, the cells in the other groups were rapidly reoxygenated for 24 h after 2 h of oxygen-glucose deprivation, the OGD/R modeling was performed, and the rats were administered with drugs by groups. BMECs were identified by cell immunofluorescence staining, morphological and ultrastructural changes of OGD/R-induced BMECs were observed, and changes in cell transmembrane resistance (TEER) were detected. The levels of nitric oxide (NO), the activity of lactate dehydrogenase (LDH), the fluorescence intensity of reactive oxygen species (ROS) and the content of tissue-type plasminogen activator (tPA) were measured with kits. Intracellular Ca2+ concentration and cell apoptosis were detected by flow cytometry, and the expression of CD34 was observed. The protein expressions of zonula occluden-1 (ZO-1), vascular endothelial growth factor (VEGF), adhesion kinase (FAK), and Paxillin were detected by Western blot. Result:Compared with the normal control group, the cells in the OGD/R group were shrinking and rounded, TEER value and ZO-1 protein expression in cells were significantly decreased, the contents of NO, LDH and ROS in cells were significantly increased, the content of tPA was significantly decreased, the concentration of Ca2+ and the apoptosis in the cells were significantly increased, CD34 was expressed in cells, and the protein expressions of VEGF, FAK and Paxillin were significantly increased (P<0.01). Compared with the OGD/R group, cell damage in the TQHXT group was significantly improved, the TEER value and ZO-1 protein expression in cells were significantly increased, the contents of NO, LDH and ROS in cells were significantly reduced, the content of tPA was significantly increased, the concentration of Ca2+ and the apoptosis in the cells were significantly reduced, CD34 expression increased in cells, and the protein expressions of VEGF, FAK and Paxillin were significantly increased (P<0.05，P<0.01). Conclusion:CSF containing TQHXT protects BMECs from OGD/R injury possibly by promoting angiogenesis through the VEGF-VEGFR2/FAK/Paxillin signaling pathway.