Electroacupuncture attenuates retinal ischemia/reperfusion injury by protecting the outer blood-retina barrier via enkephalins activate delta opioid receptor

Abstract

Objectives

Retinal ischemia-reperfusion (RIR) injury results in irreversible visual impairments. The disruption of the outer blood-retinal barrier (OBRB) is a major ocular pathogenic process that RIR injury affects. Current clinical strategies are limited. This study aimed to elucidate how electroacupuncture (EA) protects the OBRB against RIR injury.

Methods

Male Wistar rats (7 weeks old, 250 g to 280 g) were used in this study. Three independent experiments were conducted. First, Opioid peptide levels were quantified using enzyme-linked immunosorbent assay (ELISA). 42 rats were randomly divided into 7 groups (n = 6/group): Control: No treatment; high intraocular pressure(HIOP): Acute intraocular pressure elevation-induced RIR injury; HIOP + SHAM EA: RIR injury + sham EA at Xinming (Extra acupoint) and Jingming (BL1) for 30 min (shallow needle insertion but without electric stimulation); HIOP + 2 Hz EA: RIR injury + 2 Hz EA at Xinming and BL1 for 30 min; HIOP +100 Hz: RIR injury + 100 Hz EA at Xinming and BL1 for 30 min; HIOP + 2/100 Hz EA: RIR injury + 2/100 Hz EA at Xinming and BL1 for 30 min; HIOP + 4/20 Hz EA: RIR injury + 4/20 Hz EA at Xinming and BL1 for 30 min. Second, retinal morphology was assessed by hematoxylin and eosin (HE) staining. 20 rats were randomly allocated into 4 groups (n = 5/group): Control: No treatment; HIOP: Acute intraocular pressure elevation-induced RIR injury; HIOP + SHAM EA: RIR injury + sham EA at Xinming and BL1 for 30 min (shallow needle insertion but without electric stimulation); HIOP + 2 Hz EA: RIR injury + 2 Hz EA at Xinming and BL1 for 30 min. Third, the permeability of OBRB was evaluated using the fluorescein isothiocyanate(FITC)-dextran leakage assay. 15 rats were randomly divided into 5 groups (n = 3/group): Control: No treatment; HIOP: Acute intraocular pressure elevation-induced RIR injury; HIOP + SHAM EA: RIR injury + sham EA at Xinming and BL1 for 30 min (shallow needle insertion but without electric stimulation); HIOP + 2 Hz EA: RIR injury + 2 Hz EA at Xinming and BL1 for 30 min; Nal + HIOP + 2 Hz EA: Intravitreal injection of δ-opioid receptor antagonist Naltridole (10µl, 100 nM) 30 min before RIR injury induction, followed by 2 Hz EA treatment at Xinming and BL1 for 30 min. In vitro studies examined enkephalins' effects on oxygen–glucose deprivation /reperfusion (OGD/R) induced injury in ARPE‐19 cells. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay, and morphological changes were recorded by Molecular Devices. Apoptosis was detected by Annexin V-FITC flow cytometry. Delta opioid receptor (DOR) expression in total protein and membrane protein were analyzed by western blotting (WB). Immunofluorescence (IF) staining and WB assessed ZO-1 and Claudin-19. For cell-based assays, n indicates the number of biologically independent replicates.

Results

It was found that 2 Hz EA treatment increased enkephalins (methionine-enkephalin and leucine-enkephalin) levels (P < 0.01), restoring the increased retinal thickness (P < 0.05) and mitigating RGCs loss (P < 0.05) post-RIR injury. FITC-dextran leakage in the outer retina was ameliorated by 2 Hz EA (P < 0.05), reversibly countered by Naltrindole(P < 0.05), a DOR antagonist. Treatment with 30 µM enkephalins enhanced ARPE-19 cell viability (P < 0.001, P < 0.0001) and inhibited apoptosis (P < 0.0001). Enkephalins elevated DOR levels in total protein (P < 0.05) and membrane protein fractions (P < 0.001, P < 0.0001), as well as elevated ZO-1 (P < 0.001, P < 0.01) and Claudin-19 (P < 0.0001, P < 0.001) levels following OGD/R, counteracted by Naltrindole.

Conclusion

It was found that 2 Hz EA inhibits the breakdown of OBRB via enkephalins activate DOR in RIR injury.