摘要
OBJECTIVE:To evaluate the anti-apoptotic efficacy of Qingnao Yizhi formula(清脑益智方,QNYZ)in cultured cerebral cortical neuronal cells(CNCs)and the regulation of the NogoA-Nogo receptor(NgR)/Rho-Rho kinase(ROCK)signaling pathway.METHODS:Primary cultured CNCs were randomly divided into the following groups:normal control group(N-C),hypoxia-reoxygenation group(H/R),high-dose QNYZ group(Q-H),low-dose QNYZ group(Q-L)butylphthalide(NBP)group,and Y-27632(a selective ROCK transduction pathway inhibiter)group.Except those in the N-C group,CNCs were placed in hypoxic conditions for 24 h and then in reoxygenation conditions for 24 h.Cell media was changed every 48 h,and various assays were performed on the 7 th day.Cell viability was evaluated by measuring mitochondrial dehydrogenase activity,using a CCK-8 assay,in triplicate.Synapsin(SYN)protein concentrations were evaluated by enzyme-linked immunosorbent assay.NogoA and RhoA protein expression were evaluated through Western blotting.The gene expression of NogoA,NgR,RhoA,and ROCK was evaluated by reverse transcription-polymerase chain reaction.Cell apoptosis was measured using a terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling assay.RESULTS:Compared with the N-C group,the cell viability of the H/R group decreased significantly(P<0.05).The cell viability values for the Q-H and Q-L groups increased compared with that for the H/R group,and the difference was significant for the Q-H group(P<0.05).The NogoA and RhoA protein levels and the NogoA,NgR,RhoA,and ROCK m RNA expression levels increased in the H/R group,compared with the N-C group,and decreased significantly in the Q-H and Q-L groups(P<0.05)and in the Y-27632 group(P<0.05)compared with the H/R group.The SYN levels in the Q-H,Q-L,and NBP groups significantly increased compared with that in the H/R group(P<0.05).Compared with the H/R group,the numbers of apoptotic cells in the Q-H,Q-L,and NBP groups significantly decreased(P<0.05).CONCLUSION:The presented study demonstrated that QNYZ exerted anti-apoptotic effects on H/R-induced CNCs,possibly through the modulation of the NogoA-NgR/Rho-ROCK signaling pathway and the promotion of synaptic plasticity in H/R CNCs.
基金
Supported by the National Natural Science Foundation of China(Study on the Mechanism of NogoA-NgR/Rho-ROCK in Regulating the Synaptic Remodeling of VD,No.81202653)
the China Postdoctoral Fund of Sciences(Study on the Mechanism of Qingnao Yizhi Formula Based on the PI3K-Akt-mTOR Signal Transduction Pathway in the Treatment of Vascular Dementia,No.20110490080)
Science and Innovation Commission of Shenzhen(Mechanism Study of Hydroxysafflor Yellow A Regulating Mitochondrial Autophagy through ROS Mediated PINK1/parkin Pathway in the Treatment of Acute Cerebral Infarction,JCYJ20180302173504891)
Science and Innovation Commission of Shenzhen(lncRNA Malat1 Mediates SDF1/CXCR4 Axis in Cerebral Angiogenesis after Acute Cerebral Infarction and the Intervention Mechanism of Hydroxysafflor Yellow A,JCYJ 20190812161807600)。
