电针通过脑啡肽激活阿片受体保护外血-视网膜屏障，减轻视网膜缺血再灌注损伤

IF 1.3 4区医学 Q4 INTEGRATIVE & COMPLEMENTARY MEDICINE

World Journal of Acupuncture-Moxibustion Pub Date : 2025-07-01 DOI:10.1016/j.wjam.2025.05.001

Ping CHEN (陈平) , Xian-dan ZHU (朱娴丹) , Run-jie GUO (郭润杰) , Yong XIA (夏勇) , Jing-ling JIN (金京玲) , Nan-ge JIN (金南革) , Ying-min GU (谷颖敏) , Xue-song TIAN (田雪松)

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First, Opioid peptide levels were quantified using enzyme-linked immunosorbent assay (ELISA). 42 rats were randomly divided into 7 groups (<em>n</em> = 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 (<em>n</em> = 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 (<em>n</em> = 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. 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引用次数: 0

摘要

目的视网膜缺血再灌注损伤可导致不可逆的视力损害。外血-视网膜屏障（OBRB）的破坏是RIR损伤影响的一个主要的眼部致病过程。目前的临床策略有限。本研究旨在阐明电针（EA）如何保护OBRB免受RIR损伤。方法采用7周龄Wistar小鼠，250 ~ 280 g。进行了三个独立的实验。首先，使用酶联免疫吸附法（ELISA）定量测定阿片肽水平。42只大鼠随机分为7组（n = 6/组）：对照组：不治疗；高眼压（HIOP）：急性眼压升高引起的RIR损伤；HIOP + SHAM EA: RIR损伤+假性EA在新明（Extra穴）、敬明（BL1穴）30 min（浅针插入，无电刺激）；HIOP + 2 Hz EA: RIR损伤+ 2 Hz EA在新明和BL1处持续30 min；HIOP +100 Hz: RIR损伤+新明和BL1处100 Hz EA，持续30 min；HIOP + 2/100 Hz EA: RIR损伤+ 2/100 Hz EA在新明和BL1处持续30分钟；HIOP + 4/20 Hz EA: RIR损伤+ 4/20 Hz EA在新明和BL1处持续30 min。其次，采用苏木精和伊红（HE）染色评价视网膜形态。20只大鼠随机分为4组（n = 5/组）：对照组：不治疗；HIOP：急性眼压升高所致RIR损伤；HIOP + SHAM EA: RIR损伤+新明、BL1假EA 30 min（浅针插入，无电刺激）；HIOP + 2 Hz EA: RIR损伤+ 2 Hz EA在新明和BL1处持续30 min。第三，采用异硫氰酸荧光素(FITC)-葡聚糖泄漏法评估OBRB的通透性。15只大鼠随机分为5组（n = 3/组）：对照组：不治疗；HIOP：急性眼压升高所致RIR损伤；HIOP + SHAM EA: RIR损伤+新明、BL1假EA 30 min（浅针插入，无电刺激）；HIOP + 2 Hz EA: RIR损伤+ 2 Hz EA在新明和BL1处持续30 min；Nal + HIOP + 2hz EA：在RIR损伤诱导前30分钟，在玻璃体内注射δ-阿片受体拮抗剂纳曲多（10µl, 100 nM），然后在新明和BL1进行2hz EA治疗30分钟。体外研究了脑啡肽对ARPE‐19细胞氧-葡萄糖剥夺/再灌注（OGD/R）损伤的影响。采用细胞计数试剂盒-8 （CCK-8）法测定细胞活力，用Molecular Devices仪记录细胞形态变化。Annexin V-FITC流式细胞术检测细胞凋亡。western blotting （WB）检测总蛋白和膜蛋白中δ阿片受体（DOR）的表达。免疫荧光（IF）染色和WB检测ZO-1和Claudin-19。对于基于细胞的检测，n表示生物独立复制的数量。结果2 Hz EA处理可使脑啡肽（蛋氨酸-脑啡肽和亮氨酸-脑啡肽）水平升高(P <；0.01)，恢复增加的视网膜厚度(P <；0.05)和减轻rgc损失(P <；0.05)后rir损伤。2 Hz EA可改善外视网膜fitc -葡聚糖渗漏(P <；0.05)，纳曲多可可逆逆转(P <；0.05)，一种DOR拮抗剂。30µM脑啡肽增强ARPE-19细胞活力(P <；0.001, P <；0.0001)，抑制细胞凋亡(P <；0.0001)。脑啡肽升高总蛋白DOR水平(P <；0.05)和膜蛋白组分(P <；0.001, P <；0.0001)， ZO-1升高(P <；0.001, P <；0.01)和Claudin-19 (P <；0.0001, P <；0.001) OGD/R后的水平，被纳曲多抵消。结论2hz EA可通过脑啡肽激活DOR抑制RIR损伤中OBRB的分解。

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

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.

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来源期刊

World Journal of Acupuncture-Moxibustion INTEGRATIVE & COMPLEMENTARY MEDICINE-

CiteScore

1.30

自引率

28.60%

发文量

1089

审稿时长

50 days

期刊介绍： The focus of the journal includes, but is not confined to, clinical research, summaries of clinical experiences, experimental research and clinical reports on needling techniques, moxibustion techniques, acupuncture analgesia and acupuncture anesthesia.