Benign Regulation of the Astrocytic Phospholipase A2-Arachidonic Acid Pathway: The Underlying Mechanism of the Beneficial Effects of Manual Acupuncture on CBF

Abstract

Background: The astrocytic phospholipase A₂ (PLA₂)-arachidonic acid (AA) pathway is crucial in understanding the reduction of cerebral blood flow (CBF) prior to cognitive deterioration. In complementary and alternative medicine, manual acupuncture (MA) is used as one of the most important therapies for Alzheimer's disease (AD). The beneficial effects of MA on CBF were reported in our previous study. However, the underlying molecular mechanism remains largely elusive.

Objective: To investigate the effect of MA on the astrocytic PLA₂-AA pathway in SAMP8 mice hippocampi.

Methods: SAMP8 mice were divided into the SAMP8 control (Pc) group, the SAMP8 MA (Pm) group and the SAMP8 donepezil (Pd) group. SAMR1 mice were used as the SAMRl control (Rc) group. Mice in the Pd group were treated with donepezil hydrochloride at 0.65 μg/g. In the Pm group, MA was applied at Baihui (GV20) and Yintang (GV29) for 20 min. The above treatments were administered once a day for 26 consecutive days. The Morris water maze was applied to assess spatial learning and memory. Immunofluorescence staining, western blot and liquid chromatography-tandem mass spectrometry were used to investigate the expression of related proteins and measure the contents of the metabolic intermediates of the PLA₂-AA pathway.

Results: Compared with that in the Rc group, the escape latency in the Pc group significantly increased (p < 0.01); whereas, the platform crossover number and percentage of time and swimming distance in the platform quadrant decreased (p < 0.01). The hippocampal expression of PLA₂, cyclooxygenase-1, cytochrome P450 proteins 2C23 and the levels of AA, prostaglandin E₂ and epoxyeicosatrienoic acids of the Pc group was drastically higher than that in the Rc group (p < 0.01). These changes were reversed by MA and donepezil (p < 0.01 or p < 0.05).

Conclusion: MA can effectively improve the learning and memory abilities of SAMP8 mice and has a negative regulatory effect on the PLA₂-AA pathway. We propose that the increase of the arterial tone, which is induced by the inhibition of vasodilatory pathway, may be a reason for the beneficial effect of MA on CBF.

Keywords: Alzheimer’s disease; arachidonic acid; astrocyte; cerebral blood flow; hippocampus; manual acupuncture; phospholipase A2.

FIGURE 1

Timeline of experimental design and mouse acupoint locations. (A) The timeline of experimental design. (B) Illustrated diagram of GV29 and GV20 in the mouse. GV20 is at the midpoint between the auricular apices, and GV29 is located midway between the medial ends of the two eyebrows.

FIGURE 2

Results of the hidden platform trial in each group (n = 10, mean ± SD). (A,B) Comparison between the swimming speed and escape latency of all groups. A two-way ANOVA with repeated measures was used. LSD-t was presented in Supplementary Table 1. ^◆◆P < 0.01, ^◆P < 0.05 compared with the Rc group. ^◆◆P < 0.01, ^◆P < 0.05 compared with the Pc group.

FIGURE 3

Results of the probe trial in each group (n = 10, mean ± SD). (A–D) Comparison between the platform crossover numbers, the percentage of time and swimming distances in the SW quadrant, and the swimming speed of all groups. A one-way ANOVA followed by the LSD multiple-range test was used with an exception when comparing the platform crossover numbers, which was analyzed by the Kruskal–Wallis test. LSD-t and Chi-Square are presented in Supplementary Tables 2, 3. ^◆◆P < 0.01, ^◆◆P < 0.01.

FIGURE 4

Representative images of IF staining of GFAP (green) and co-expressed PLA₂ (red) in each group. GFAP is labeled with green fluorescence and the coexpressed PLA₂ with red fluorescence. The nucleus is labeled with blue fluorescence. The astrocytes showed a polygonal nucleus. In the hippocampus, the positively stained cells are shown with white arrows. Scale bar is 20 μm.

FIGURE 5

Representative images of IF staining of GFAP (green) and co-expressed COX-1 (red) in each group. GFAP is labeled with green fluorescence and the coexpressed COX-1 with red fluorescence. The nucleus is labeled with blue fluorescence. The astrocytes showed a polygonal nucleus. In the hippocampus, the positively stained cells are shown with white arrows. Scale bar is 20 μm.

FIGURE 6

Representative images of IF staining of GFAP (green) and co-expressed CYP2C23 (red) in each group. GFAP is labeled with green fluorescence and the coexpressed CYP2C23 with red fluorescence. The nucleus is labeled with blue fluorescence. The astrocytes showed a polygonal nucleus. In the hippocampus, the positively stained cells are shown with white arrows. Scale bar is 20 μm.

FIGURE 7

Comparison of the expression of astrocytic PLA₂, COX-1 and CYP2C23 in the hippocampus between groups (n = 10, mean ± SD). (A–C) Comparison between the mean optical density of PLA₂, COX-1 and CYP2C23 of all groups. A one-way ANOVA followed by LSD multiple-range test was used with an exception when comparing COX-1, which was analyzed by the Kruskal–Wallis test. LSD-t and Chi-Square are presented in Supplementary Tables 2, 4. ^◆◆P < 0.01, ^◆◆P < 0.01.

FIGURE 8

WB results in each group. (A–C) Comparison between the relative expression of PLA₂, COX-1 and CYP2C23 in the hippocampus (n = 8, mean ± SD). A one-way ANOVA followed by LSD multiple-range test was used with an exception when comparing PLA₂, which was analyzed by Kruskal–Wallis test. LSD-t and Chi-Square are presented in Supplementary Tables 2, 5. ^◆◆P < 0.01, ^◆P < 0.05 compared with the Rc group. ^◆◆P < 0.01, ^◆P < 0.05 compared with the Pc group.

FIGURE 9

The LC-MS/MS results. (A–H) Chromatographs of AA, PGE₂, 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, 8,9-EET-d₁₁ and PGE2-d₄.

FIGURE 10

Comparison between the contents of AA, PGE₂ and EETs between groups (n = 8, mean ± SD). (A–H) Comparison between the contents of AA, PLA₂, 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET in the hippocampus. A one-way ANOVA followed by LSD multiple-range test was used with an exception when comparing PGE₂, which was analyzed by Kruskal–Wallis test. LSD-t and Chi-Square are presented in Supplementary Tables 2, 6. ^◆◆P < 0.01, ^◆P < 0.05 compared with the Rc group. ^◆◆P < 0.01, ^◆P < 0.05 compared with the Pc group.