Dihydroartemisinin up-regulates VE-cadherin expression in human renal glomerular endothelial cells

Abstract

The antimalarial agent dihydroartemisinin (DHA) has been shown to be anti-inflammatory. In this study, we found that DHA increased the expression of the junctional protein vascular endothelial (VE)-cadherin in human renal glomerular endothelial cells. In addition, DHA inhibited TGF-β RI-Smad2/3 signalling and its downstream effectors SNAIL and SLUG, which repress VE-cadherin gene transcription. Correspondingly, DHA decreased the binding of SNAIL and SLUG to the VE-cadherin promoter. Together, our results suggest an effect of DHA in regulating glomerular permeability by elevation of VE-cadherin expression.

Keywords: TGF-β signalling; VE-cadherin; dihydroartemisinin; glomerular endothelial cells.

Figure 1

DHA up‐regulates the expression of VE‐cadherin and inhibits TGF‐β signalling in HRGECs. (A) Relative VE‐cadherin mRNA expression in HRGECs treated with vehicle or DHA (n = 4; **P < 0.01). (B) Immunoblots of VE‐cadherin protein from HRGECs treated with vehicle or DHA. GAPDH was used as loading control. (C) Representative images of VE‐cadherin immunostaining on HRGECs treated with vehicle or DHA. Magnification: 200X. (D) Immunoblots of TGF‐β RI protein from HRGECs treated with vehicle or DHA. (E) Immunoblots of phospho‐Smad2 and total Smad2 from HRGECs treated with vehicle or DHA (F) Immunoblots of phospho‐Smad3 and total Smad3 from HRGECs treated with vehicle or DHA. GAPDH was used as loading control.

Figure 2

DHA inhibits the expression of SNAIL/SLUG in HRGECs and suppresses SNAIL/SLUG binding affinity to the human VE‐cadherin promoter. HRGECs were treated with 25 μM DHA for 24 hrs before measurements. (A, C) Relative mRNA expression of SNAIL (A) and SLUG (C) measured by qRT‐PCR in HRGECs treated with vehicle or DHA (n = 4; **P < 0.01). (B, D) Immunoblots of SNAIL (B) and SLUG (D) protein from HRGECs treated with DHA. GAPDH was used as loading control. (E, G) Representative images of PCR products from the DNA fragments pulled down by SNAIL (E) and SLUG (G) antibodies. Primers were designed to detect the ‐240 E‐box on the human VE‐cadherin promoter. (F, H) Binding ratios relative to the total input of chromatin used for ChIP with the SNAIL (F) and SLUG (H) antibodies (n = 4; **P < 0.01).