Anti-apoptotic efficacy of Qingnao Yizhi formula in hypoxia/reoxygenation primary cortical neurons through the promotion of synaptic plasticity by modulating the NogoA-Nogo receptor/Rho-Rho kinase signaling pathway.

OBJECTIVE

To evaluate the anti-apoptotic efficacy of Qingnao Yizhi formula (，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 7th 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-dUTP 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 mRNA 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.