Mechanism of electroacupuncture at “Zusanli”(ST36) in improving cognitive impairment in mice with systemic inflammation based on the α7 nicotinic acetylcholine receptor pathway of hippocampal microglia

To observe the effect of low-intensity electroacupuncture (EA) at “Zusanli”(ST36) on the hippocampal cholinergic anti-inflammatory pathway and α7 nicotinic acetylcholine receptor (α7nAChR) of hippocampal microglia in mice with systemic inflammatory cognitive impairment, so as to investigate the central mechanism of EA in improving neuroinflammatory response and cognitive function.

C57BL/6 mice were randomly divided into blank, model and EA groups (6 mice/group); another set of C57BL/6 mice were randomly divided into blank, model, EA and EA+antagonist groups (6 mice/group); Cx3Cr1-Cre/ERT2 mice and α7nAChR^fl/fl mice were mated to construct transgenic mice with microglial α7nAChR knockout (EA+ knockout group, 6 mice), with the same litter negative mice used as non-knockout mice and randomly divided into blank, model+non-knockout and EA+non-knockout groups (6 mice/group). The systemic inflammatory cognitive impairment model was established by intraperitoneal injection of lipopolysaccharide (LPS, 1 mg/kg). Mice in the EA group received EA at bilateral ST36 (dense-sparse wave, 1 Hz/20 Hz, 0.5 mA), for 30 minutes each time; mice in the EA+antagonist group received injection of α7nAChR antagonist methyllycaconitine citrate (MLA) into hippocampus. The episodic memory behavioral tests were used to observe the abilities of mice in new object exploration, object location change recognition and object temporal order change memory. Nuclear magnetic resonance spectroscopy was used to observe the choline level in the hippocampal tissue. Western blot was used to detect the protein expression of α7nAChR in the hippocampus. Immunofluorescence staining was used to detect the expression of α7nAChR in hippocampal microglia and the activation of microglia. Real-time quantitative PCR was used to detect the mRNA expression of tumor necrosis factor-α (TNF-α) in hippocampus, and ELISA was used to detect the contents of α7nAChR, inflammatory factors and microglial phenotypic markers in hippocampal tissue.

Compared with the blank group, the abilities of new object exploration, location change recognition and object temporal order change memory, the choline level in the hippocampus, the protein expression level and content of α7nAChR and its co-expression with microglial markers, and the contents of anti-inflammatory factor interleukin (IL)-10 and M2 macrophage marker arginase 1 (Arg1) of mice in the model group were decreased (P<0.001, P<0.01); while the expression of hippocampal ionized calcium-binding adapter molecule 1 (Iba-1), the contents of M1 macrophage marker CD206, inducible nitric oxide synthase (iNOS), the content of pro-inflammatory factor IL-1β and the mRNA expression of TNF-α were increased (P<0.001, P<0.01). Compared with the model group, the above indexes in the EA group were reversed except for the ability of location change recognition (P<0.05, P<0.01, P<0.001). Compared with the EA group, the abilities of new object exploration and object temporal order change memory in the EA+antagonist group were decreased (P<0.05), and the mRNA expression of TNF-α was increased (P<0.01); the abilities of new object exploration and object temporal order change memory in the EA+knockout group were decreased (P<0.05), and the mRNA expression of TNF-α was increased (P<0.01).

EA at ST36 with low-intensity may improve neuroinflammatory response and systemic inflammatory cognitive dysfunction by increasing the choline level in the hippocampus and reducing the release of inflammatory factors through up-regulating the expression of α7nAChR in hippocampal microglia.