Involvement of P2X7R-mediated microglia polarization and neuroinflammation in the response to electroacupuncture on post-stroke memory impairment

Abstract

Purpose

Post-stroke cognitive impairment (PSCI) is a common complication of ischemic stroke episodes. Memory impairment is an important component of the poststroke cognitive syndrome. Microglial activation plays a critical role in stroke-induced neuroinflammation. Previous studies have reported that electroacupuncture (EA) provides neuroprotective effects by reducing the expression levels of the Purinergic receptor P2X ligand-gated ion channel 7 (P2X7) and inhibiting neuroinflammation in rat model of ischemic stroke. Further understanding of the role and connections between P2X7R and microglial activation in EA-induced anti-inflammatory can reveal novel targets for post-stroke memory impairment treatment.

Methods

A Middle cerebral artery occlusion and reperfusion (MCAO/R) model was established. We used 2’(3’)-O-(4-benzoyl) benzoyl ATP (BzATP) as a P2X7R agonist. Following MCAO/R injury, the rats underwent EA therapy at the Baihui (DU20) and Shenting (DU24) acupoints for seven consecutive days. The Barnes maze test was used to evaluate memory function. Following intervention, a T2 weighted images (T2WI) scan was performed to identify changes in cerebral infarction volume in MCAO/R rats. The levels of Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and Interleukin-4 (IL-4), Interleukin-10 (IL-10) in the peri-infarct hippocampal were examined by ELISA. Immunofluorescence was employed to evaluate Iba-1⁺ / P2X7R⁺, Iba-1⁺/ iNOS⁺ and Iba-1⁺/ Arg-1⁺ cell populations in the peri-infarct hippocampal DG area. The protein expression of P2X7R, Nuclear factor E2-related factor 2 (Nrf2), Recombinant nlr family, pyrin domain containing protein 3 (NLRP3), Inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg-1) in the peri-infarct hippocampal were investigated using western blot assays. Besides, we also measured the levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA).

Results

We found EA treatment reduced inflammation and oxidative stress, which is consistent with a decrease in P2X7R expression and improved learning and memory functions. In contrast, we found BzATP enhanced inflammation and oxidative stress. Moreover, our results showed EA treatment up-regulated Nrf2, down-regulated NLRP3, and promoted microglia M2 polarization. Finally, EA-mediated positive effects were reversed by intracerebroventricular injection of BzATP, which is consistent with an increase in P2X7R expression.

Conclusion

EA ameliorates memory impairment in a rat model of ischemic stroke by reducing inflammation and ROS through the inhibition of P2X7R expression. In turn, this mechanism regulates Nrf2 and NLRP3 expression, suggesting EA is beneficial for ischemic stroke treatment using P2X7R as target.