Effects of electroacupuncture intervention at different time points on cuproptosis-related proteins and copper homeostasis in rats with cerebral ischemia-reperfusion injury

To observe the effects of electroacupuncture (EA) intervention at different time points on cuproptosis-related proteins and Cu²⁺ levels in the ischemic penumbra of rats with cerebral ischemia-reperfusion injury (CIRI) after acute ischemic stroke (IS), so as to explore the mechanism by which acupuncture inhibits cuproptosis to improve neurological function in CIRI rats, as well as the therapeutic efficacy of intervention administered at different time windows.

SD rats were randomly divided into sham operation group, model group, 0 h acupuncture group, 24 h acupuncture group and 48 h acupuncture group, with 12 rats in each group. The middle cerebral artery occlusion/reperfusion (MCAO/R) model was established by Longa filament occlusion method. Rats in each EA group received 30 min of EA at “Baihui” (GV20) and “Fengfu” (GV16) at 0 h, 24 h and 48 h after reperfusion, respectively. Modified neurological severity score (mNSS) was used to evaluate the recovery of neurological deficit. 2,3,5-triphenyltetrazolium chloride (TTC) staining was adopted to detect cerebral infarct volume. Colorimetric assay was performed to measure Cu²⁺ content in brain tissues of the ischemic penumbra. Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression of ferredoxin 1 (FDX1), dihydrolipoamide S-acetyltransferase (DLAT), lipoic acid synthetase (LIAS), and solute carrier family 31 member 1 (SLC31A1) in the ischemic penumbra.

Compared with the sham operation group, the model group exhibited significantly elevated mNSS score, cerebral infarct volume, Cu²⁺ content in ischemic penumbra tissues, as well as increased mRNA and protein expression of DLAT and SLC31A1 (P<0.01)； meanwhile, the mRNA and protein expression of FDX1 and LIAS were markedly decreased (P<0.01). Compared with the model group, all acupuncture groups presented reduced mNSS score, cerebral infarct volume, Cu²⁺ level, together with down-regulated mRNA and protein expression of DLAT and SLC31A1 (P<0.01, P<0.05), and up-regulated mRNA and protein expression of FDX1 and LIAS (P<0.01, P<0.05). Compared with the 24 h acupuncture group and 48 h acupuncture group, the 0 h acupuncture group showed distinctly lower mNSS score, cerebral infarct volume, Cu²⁺ content, and weaker mRNA and protein expression of DLAT and SLC31A1 (P<0.05, P<0.01), accompanied by remarkably higher mRNA and protein expression of FDX1 and LIAS (P<0.01, P<0.05). The 24 h acupuncture group achieved better outcomes in cerebral infarct volume, Cu²⁺ content, and mRNA expression of DLAT, SLC31A1, FDX1 and LIAS relative to the 48 h acupuncture group (P<0.01, P<0.05).

EA intervention can effectively suppress cuproptosis and alleviate cerebral injury in CIRI rats, and early EA intervention exerts superior efficacy in ameliorating neurological impairment after CIRI. The underlying mechanism may involve regulating the expression of FDX1, DLAT, LIAS and SLC31A1, reducing brain Cu²⁺ concentration, and thereby inhibiting neuronal cuproptosis.