The aim of this study is to investigate the effect of extracellular vesicles isolated from T regulatory cells (rEXS) on the inner blood-retinal barrier (IBRB).

BV2 microglial cells were divided into five groups: control (no LPS or rEXS), LPS-stimulated (100 ng/ml LPS), and three rEXS-treated groups (10 μg/ml, 20 μg/ml, and 50 μg/ml rEXS+ 100 ng/ml LPS). A co-culture system of BV2 cells and human umbilical vein endothelial cell (HUVEC) was used to simulate retinal vessels, with groups divided as follows: HUVEC control (no BV2), co-culture (BV2+ HUVEC), co-culture stimulated (BV2+ HUVEC+ 100 ng/ml LPS), and co-culture exosome (BV2+ HUVEC+ 50 μg/ml rEXS). ELISA was used to measure interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-10 levels, while Western blot analyzed zona occludens (ZO)-1 and occludin expression. Data were expressed as ±s and compared by one-way ANOVA and LSD post-hoc test.

Nanoparticle tracking analysis showed that rEXS had a characteristic size range of 50 to 155 nm, with a peak at 106 nm and 155 nm. The rEXS concentration was 4.74×10¹⁰particles/ml. Specific markers were detected in rEXS. Transmission electron microscopy confirmed rEXS particles were approximately 100 nm in diameter. The TNF-αconcentrations in the control group, LPS-stimulated group, 10 μg/ml rEXS groups, 20 μg/ml rEXS groups, 50 μg/ml rEXS groups were (1035.96±32.36)pg/ml, (2965.25±4.08)pg/ml, (2960.41±35.65)pg/ml, (2863.32±30.55)pg/ml, (2586.32±33.25)pg/ml; IL-1βwere (2.60±0.05)pg/ml, (17.18±0.13)pg/ml, (10.98±0.06)pg/ml, (6.39±0.04)pg/ml, (4.05±0.06)pg/ml; IL-10 were (28.24±2.66)pg/ml, (24.55±0.77)pg/ml, (33.96±3.58)pg/ml, (34.29±4.32)pg/ml, and (48.32±1.55)pg/ml, respectively. There were significant differences between them (F=23.01, 1.96, 29.53; P<0.05). In the co-culture system, compared with relative control group, the ZO-1 expression levels in the co-culture group, co-culture stimulated group, co-culture exosome group were (1.28±0.010), (0.8±0.02), and (1.23±0.01); occludin were (1.37±0.04), (0.81±0.02), (1.18±0.03), respectively. There were significant differences between them (F=808.3, 222.2; P<0.05).

The results of the present study revealed that rEXS may have a protective effect on the IBRB by inhibiting the release of TNF-α and IL-1β cytokines, promoting IL-10-mediated anti-inflammatory effects, and alleviating LPS-induced tight junction disruption.