Electroacupuncture of Baihui and Shenting ameliorates cognitive deficits via Pten/Akt pathway in a rat cerebral ischemia injury model

Abstract

Cerebral ischemic stroke is a huge threat to the health and life of many people. Electroacupuncture (EA) at Baihui (GV20) and Shenting (GV24) acupoints can notably alleviate cerebral ischemia/reperfusion injury (CIRI). However, the molecular basis underlying the effectiveness of EA at the GV20 and GV24 acupoints for CIRI remains largely unknown. Our present study demonstrated that EA treatment at the GV20 and GV24 acupoints markedly alleviated middle cerebral artery occlusion/reperfusion (MCAO/R)-induced cognitive deficits and cerebral infarction in rats. Proteomics analysis revealed that 195 and 218 proteins were dysregulated in rat hippocampal tissues in the MCAO/R vs. sham group and thhhe EA vs. MCAO/R group, respectively. Moreover, 62 proteins with converse alteration trends in MCAO/R vs. sham and EA vs. MCAO/R groups were identified. These proteins might be implicated in the EA-mediated protective effect against MCAO/R-induced cerebral injury. GO enrichment analysis showed that 39 dysregulated proteins in the MCAO/R vs. sham group and 40 dysregulated proteins in the EA vs. MCAO/R group were related to brain and nerve development. Protein-protein interaction analysis of the abovementioned dysregulated proteins associated with brain and nerve development suggested that Pten/Akt pathway-related proteins might play major roles in regulating EA-mediated protective effects against MCAO/R-induced brain and nerve injury. Western blot assays demonstrated that Pak4, Akt3, and Efnb2 were expressed at low levels in the MCAO/R group vs. the sham group but at high levels in the EA group vs. the MCAO/R group. In conclusion, multiple proteins related to the protective effect of EA at the GV20 and GV24 acupoints against CIRI were identified in our study.

Keywords: Baihui; Shenting; acupuncture; cerebral ischemia stroke; proteomics.

Figure 1

Schematic diagram of Baihui (GV20) and Shenting (GV24) acupoints.

Figure 2

EA treatment at the GV20 and GV24 acupoints markedly improved the spatial learning and memory abilities of MCAO/R rats. (A) The time that the rats spent finding the escape platform was examined by the MWM assay at 9–13 days after EA treatment. (B) The escape platform was removed on day 14 after EA treatment. Next, the number of times the rats passed through the platform position within 90 s was recorded. There were 12, 15, 15, and 15 rats in the control, sham, MCAO/R, and EA group, respectively. Compared with the sham group, ***P < 0.001; compared with the MCAO/R group, #P < 0.05, ##P < 0.01, ###P < 0.001.

Figure 3

EA treatment at the GV20 and GV24 alleviated MCAO/R-induced rat cerebral infarction. On Day 14 after EA treatment, the whole brains of rats were isolated and then stained with TTC. The cerebral infarction areas were stained white. Each group contained three brain samples from three rats, and five coronal sections were obtained from each brain. Compared with the sham group, ***P < 0.001; compared with the MCAO/R group, #P < 0.05, ##P < 0.01, ###P < 0.001.

Figure 4

Identification of dysregulated proteins and biological processes related to MCAO/R-induced brain injury in rats. (A) The volcano plot of differentially expressed proteins in the MCAO/R vs. sham group. (B) The volcano plot of differentially expressed proteins in the EA vs. MCAO/R group. Each group contained nine hippocampal tissue samples isolated from nine rats, and three samples per group were mixed into a proteomics specimen.

Figure 5

Identification of proteins with adverse alteration trends in the MCAO/R vs. sham and EA vs. MCAO/R groups. (A) Jvenn analysis of upregulated proteins in the MCAO/R vs. sham group and downregulated proteins in the EA vs. MCAO/R group. (B) K-means clustering plot of the expression profiles of proteins that were upregulated in the MCAO/R group vs. the sham group and downregulated in the EA group vs. the MCAO/R group. (C) Jvenn analysis of downregulated proteins in the MCAO/R vs. sham group and upregulated proteins in the EA vs. MCAO/R group. (D) K-means clustering plot of the expression profiles of proteins that were downregulated in the MCAO/R vs. sham group and upregulated in the EA vs. MCAO/R group. (E) Heatmap of 62 proteins with converse alteration trends in the MCAO/R vs. sham group and the EA vs. MCAO/R group. Each group contained nine hippocampal tissue samples isolated from nine rats and three samples per group were mixed into a proteomics specimen.

Figure 6

Identification of crucial proteins related to brain and nerve development in response to MCAO/R or EA treatment. (A) PPI networks of dysregulated proteins related to brain and nerve development in the MCAO/R vs. sham groups. (B) PPI networks of dysregulated proteins related to brain and nerve development in the EA vs. MCAO/R groups. (C) Histogram of the proteomics outcomes of Scrib, Pak4, Akt3, Hdac2, Pten, Msh2, Nrp2, Efnb2, and Sema4d in the sham, MCAO/R, and EA groups. (D) Jvenn analysis of dysregulated proteins related to brain and nerve development in the MCAO/R vs. sham and EA vs. MCAO/R groups. Each group contained nine hippocampal tissue samples isolated from nine rats, and three samples per group were mixed into a proteomics specimen. Compared with the sham group, *P < 0.05, **P < 0.01; compared with the MCAO/R group, #P < 0.05, ##P < 0.01, ###P < 0.001.

Figure 7

Western blot validation of Pak4, Akt3, and Efnb2 protein levels in hippocampal tissues of sham, MCAO/R, and EA rats. Protein levels of Pak4, Akt3, and Efnb2 were measured by western blot assay in hippocampal tissues of sham (n = 3), MCAO/R (n = 3), and EA (n = 3) rats. Compared with the sham group, ***P < 0.001; compared with the MCAO/R group, ###P < 0.001.