Paeoniflorin Attenuates Cerebral Ischemia-Induced Injury by Regulating Ca2+/CaMKII/CREB Signaling Pathway

Abstract

Paeoniflorin (PF) is an active ingredient of Paeoniae Radix which possesses the neuroprotective effect. However, so far, the neuroprotective mechanism of PF has still not been fully uncovered. The Ca²⁺/Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)/cAMP response element-binding (CREB) signaling pathway plays an important role in the intracellular signal transduction pathway involved in cell proliferation, cell survival, inflammation and metabolism. Herein, the neuroprotective roles of PF in the models of middle cerebral artery occlusion (MCAO) followed by reperfusion in rats and N-methyl-d-aspartic acid (NMDA)-induced excitotoxicity in primary hippocampal neurons were investigated. Moreover, we attempted to confirm the hypothesis that its protection effect is via the modulation of the Ca²⁺/CaMKI)/CREB signaling pathway. In this study, PF not only significantly decreased neurological deficit scores and infarct volume in vivo, but also improved neurons' cell viability, and inhibited neurons' apoptosis and intracellular Ca²⁺ concentration in vitro. Furthermore, PF significantly up-regulated p-CREB and p-CaMKII, and down-regulated calmodulin (CaM) in vivo and in vitro. The results indicate that the protective effect of PF on cerebral ischemia reperfusion injury is possible through regulating the Ca²⁺/CaMKII/CREB signaling pathway.

Keywords: CREB; CaMKII; ischemic stroke; middle cerebral artery occlusion; paeoniflorin.

Figure 1

Effects of paeoniflorin (PF) on neurological deficit scores and cerebral infarct. (A) The neurological score of middle cerebral artery occlusion (MCAO) groups and PF groups (n = 12); (B) Representative photographs showing the cerebral infarct of rat brain slices measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Black arrow indicated ‘‘infarcted area’’; (C) The infarct volume of MCAO groups and PF groups. All data were expressed as mean ± SD. ^## p < 0.01 vs. sham, ** p < 0.01 vs. MCAO.

Figure 2

Effects of PF on the expression in the Ca²⁺/Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)/cAMP response element-binding (CREB) signaling pathway in ischemic penumbra after MCAO. (A) Western blot and (B) the relative optical densities analysis of the level of CaM, CaMKII, p-CaMKII, CREB, p-CREB. β-actin were used as the internal controls. All data were presented as mean ± SD. ^## p < 0.01 vs. sham, * p < 0.05 and ** p < 0.01 vs. MCAO.

Figure 3

Effects of PF on N-methyl-d-aspartic acid (NMDA)-induced excitotoxicity in neurons as assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT). NMDA stimulation decreased cell viability in neurons. PF promoted cell survival. ^## p < 0.01 vs. control; ** p < 0.01 vs. NMDA. Data are mean ± SD (n = 8).

Figure 4

Effect of PF in NMDA-induced neurons apoptosis by annexin V/propidium iodide (PI) staining (flow cytometry analysis). NMDA stimulation increased cell apoptosis in neurons. PF promoted cell survival.

Figure 5

Effect of PF on intracellular Ca²⁺ concentration in NMDA-induced PC12 cells. Intracellular Ca²⁺ concentration was measured by the Fura-2/AM fluorescent technique. All data were presented as mean ± SD (n = 6). ^## p < 0.01 vs. control; and ** p < 0.01 vs. NMDA.

Figure 6

Effects of PF on the expression in the Ca²⁺/CaMKII/CREB signaling pathway in neurons after NMDA-induced excitotoxicity. (A) Western blot and (B) the relative optical densities analysis of the level of CaM, CaMKII, p-CaMKII, CREB, p-CREB. β-actin was used as the internal controls. All data were presented as mean ± SD. ^## p < 0.01 vs. control;, ** p < 0.01 vs. NMDA.

Figure 7

Chemical constitution of paeoniflorin (PF).