Scutellarein alleviates the dysfunction of inner blood-retinal-barrier initiated by hyperglycemia-stimulated microglia cells

Abstract

Aim: To investigate the alleviation of scutellarein (SN) against inner blood-retinal-barrier (iBRB) dysfunction in microglia cells stimulated by hyperglycemia and to elucidate the engaged mechanism.

Methods: Microglia BV2 cells were stimulated by using 25 mmol/L D-glucose. The same concentration of mannitol (25 mmol/L) was applied as an isotonic contrast. Real-time PCR, Western-blot assay and immunofluorescence staining assay was performed. The dysfunction of iBRB in vitro was detected by using transendothelial electrical resistance (TEER) assay. Additionally, the leakage of fluorescein isothiocyanate (FITC)-conjugated dextran (70 kDa) was detected.

Results: SN abrogated microglia BV2 cells activation and reduced the phosphorylated activation of extracellular signal-regulated protein kinase (ERK)1/2. SN also decreased the transcriptional activation of nuclear factor κB (NFκB) and the elevated expression of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1β in BV2 cells treated with D-glucose (25 mmol/L). SN attenuated iBRB dysfunction in human retinal endothelial cells (HRECs) or choroid-retinal endothelial RF/6A cells when those cells were treated with TNFα, IL-1β or IL-6, or co-cultured with microglia cells stimulated by D-glucose. Moreover, SN restored the decreased protein expression of tight junctions (TJs) in TNFα-treated HRECs and RF/6A cells.

Conclusion: SN not only alleviate iBRB dysfunction via directly inhibiting retinal endothelial injury caused by TNFα, IL-1β or IL-6, but also reduce the release of TNFα, IL-1β and IL-6 from microglia cells by abrogating hyperglycemia-mediated the activation of microglia cells.

Keywords: blood-retinal-barrier; inflammation; scutellarein; tight junctions; tumor necrosis factor α.

Figure 1. SN reduced microglia cells activation in vitro

A: The chemical structure of SN; B: Representative immunofluorescence image (n=3); C: Iba1 expression (n=4) ^aP<0.05 versus control; ^dP<0.05 versus D-glucose.

Figure 2. SN abrogated the D-glucose-initiated NFκB and ERK1/2 activation in BV2 cells

A: The expression of phosphorylated IKK and NFκBp65 is detected; B: The quantitative analysis of phosphorylated IKK (n=3); C: The quantitative analysis of nuclear and cytosolic NFκBp65 (n=4); D: Elevated ERK1/2 phosphorylation caused by D-glucose (25 mmol/L) was reduced by SN; E: The quantitative analysis of ERK1/2 phosphorylation (n=4) ^aP<0.05, ^bP<0.01 versus control; ^dP<0.05, ^eP<0.01, ^fP<0.001 versus D-glucose.

Figure 3. SN inhibited the D-glucose-induced up-regulated expression of TNFα, IL-6 and IL-1β in vitro

A-C: Cellular mRNA level of IL-1β (n=4), IL-6 (n=3), and TNFα (n=4); D: Content of TNFα in the supernatants from D-glucose (25 mmol/L)-treated BV2 cells (n=3) ^aP<0.05, ^bP<0.01 versus control; ^dP<0.05, ^eP<0.01 versus D-glucose.

Figure 4. SN rescued the D-glucose-stimulated BV2 cells or TNFα-induced iBRB injury in RF/6A cells

A, B: Upper-chamber seeded with RF/6A cells and lower-chamber seeded with BV2 cells in 24-well plates composed a co-cultured environment, and 6h before the D-glucose (25 mmol/L) stimulation on BV2 cells, SN (20, 50 µmol/L) was added into the bottom of transwells for pre-treatment. TEER (n=3) and FITC-dextran leakage (n=4) were determined. C, D: TNFα (20 ng/mL) was added into the plates under the chambers, and RF/6A cells were cultured in the upper-chamber with the pretreatment with or without SN (20, 50 mmol/L) for 6h. TEER (n=3) and FITC-dextran leakage (n=4) were detected. E: The same treatment as C&D was performed and the cell samples were collected for detecting the content of claudin-1 (n=4), claudin-5 (n=4), claudin-19 (n=3) and occludin (n=4) ^aP<0.05, ^bP<0.01, ^cP<0.001 versus control; ^dP<0.05, ^eP<0.01, ^fP<0.001 versus D-glucose-treated BV2 cells or TNFα.

Figure 5. SN rescued the D-glucose-stimulated BV2 cells or TNFα-initiated iBRB injury in HRECs

A, B: Upper-chamber seeded with HREC cells and lower-chamber seeded with BV2 cells in 24-well plates composed a co-cultured environment, and 6h before the D-glucose (25 mmol/L) stimulation on BV2 cells, SN (20, 50 µmol/L) was added into the bottom of transwells for pre-treatment. TEER (A) (n=3) and FITC-dextran leakage (B) (n=4) were determined. C, D: TNFα (20 ng/mL) was added into the plates under the chambers, and HRECs were cultured in the upper-chamber with the pretreatment with or without SN (20, 50 mmol/L) for 6h. TEER (n=3; C) and FITC-dextran leakage (n=4; D) were detected. E: The same treatment as C&D was performed and the cell samples were collected for detecting the content of claudin-1 (n=3), claudin-5 (n=4), claudin-19 (n=3) and occludin (n=3) ^aP<0.05, ^bP<0.01, ^cP<0.001 versus control; ^dP<0.05, ^eP<0.01, ^fP<0.001 versus D-glucose-treated BV2 cells or TNFα.

Figure 6. SN rescued the IL-1β or IL-6-mediated iBRB injury in HRECs

A, B: IL-1β (20 ng/mL) was added into the plates under the chambers, and HRECs were cultured in the upper-chamber with the pretreatment with or without SN (20, 50 mmol/L) for 6h. A: TEER was detected (n=4). B: FITC-dextran leakage was detected (n=3). C, D: IL-6 (20 ng/mL) was added into the plates under the chambers, and HRECs were cultured in the upper-chamber with the pretreatment with or without SN (20, 50 mmol/L) for 6h. C: TEER was detected (n=4). D: FITC-dextran leakage was detected (n=3) ^aP<0.05, ^cP<0.001 versus control; ^dP<0.05, ^eP<0.01, versus IL-1β or IL-6.