RAGE silencing deters CML-AGE induced inflammation and TLR4 expression in endothelial cells

The Nε-(carboxymethyl)lysine (CML), the predominant advanced glycation end products (AGEs) in diabetes and its RAGE induced cytokine release has been well explored. But the CML mediated multiple AGEs receptor expression is still not understood and the role played by RAGE silencing in modulating CML generated pro-inflammatory cytokines in micro and macrovascular endothelial cells is yet to be studied. HUVEC and HREC cells were exposed with CML for 24 h. RAGE, AGER1, AGER2, Gal-3, TLR4, TLR2, CD36, FEEL-1, FEEL-2, and chemokine HMGB1 were quantified by either qPCR/western blotting. The receptor's expression was also determined in control vs diabetic retina. Expression of pro-inflammatory genes, ROS, and mitochondrial membrane potential change were assessed using ELISA, DCFDA, and JC-1 method respectively. RAGE expression was silenced either by Si-RAGE or neutralising antibody with anti-RAGE and expression of other AGE receptors, adaptors, and signalling pathway were studied compared with Si-Control. CML activated RAGE, TLR4, HMGB1(p