RESUMO
Objective: To investigate the protective effects and mechanism of Naoxintong Capsules (NXT) on primary cultured neonate rat brain microvascular endothelial cells (rBMECs) induced by oxygen-glucose deprivation. Methods: The primary cultured rBMECs model was established and the identification of rabbit anti-rat VIII factor was carried out. MTT was used to screen out the concentration range of NXT intestinal absorption solution to pretect rBMEC in vitro, three doses were selected for experiment. The experimental groups were divided into control group, model group, nimodipine group (200 μg/mL, NXT intestinal absorption solution group (62.50 mg/L, 125.00 mg/L, and 250.00 mg/L), and NXT intestinal absorption solution (250.00 mg/L) and LY294002 (20 μmol/L, PI3K/Akt pathway inhibitor) co-administration group. The morphology of rBMECs was observed under inverted microscope. The expression of lactate dehydrogenase (LDH) and matrix metalloproteinase 9 (MMP-9) in the cell supernatant was detected by ELISA kit. The apoptosis was observed by Hoechst33342 staining fluorescence microscope. The early apoptotic rate of rBMECs in each group was detected by FCM, and the expression of PI3K/Akt signaling pathway key proteins was detected by Western blotting. Results: Compared with model group, the administration of NXT could significantly improve the morphology of rBMECs, decrease the intracellular levels of LDH and MMP-9, significantly reduce the number of apoptotic cells and early apoptotic rate of rBMECs, and inhibit the expression of p-Akt, Bcl-2 upregulation, decrease the expression of Bax, and inhibit caspase-3 activity. The addition of LY294002, a specific inhibitor of PI3K/Akt signaling pathway, blocked the signal transduction of this pathway and significantly reduced the protective effect of NXT. Conclusion: NXT have protective effects on rBMECs induced by oxygen-glucose deprivation, and its mechanism is related to the PI3K/Akt signaling pathway.
RESUMO
Objective: To study the protective effect of Danhong Injection (DI) on primary cultured neonate rat brain microvascular endothelial cells (rBMECs) injury. Methods: The primary cultured rBMEC model was established and the identification of rabbit anti rat VIII factor was carried out. The cells were divided into control, model, low-, mid-, and high-dose (25, 50, and 100 μL/mL) DI groups in hypoxic condition for 4 h after administration. The cell morphology was observed under microscope, the apoptosis rate and DNA content were determined by flow cytometry, and the lactate dehydrogenase (LDH) level in cultural supernatants and cell superoxide dismutase (SOD) activity were detected according to the kit methods. Results: DI (50 and 100 μL/mL) could alleviate the rBMEC damage induced by hypoxia remarkably, improve the cell morphology of rBMECs, decrease the apoptosis significantly, inhibit the blockage of rBMECs in G1/S phase and the leakage of LDH, and increase the SOD activity. Conclusion: DI plays a significant role in the protection on injured primary cultured rBMECs induced by hypoxia, and the mechanism may be related to the enhancement of cellular anti-oxidative capacity and the inhibition of apoptosis.