Figure 6

Autophagy-induced TRAF2 degradation may be associated with the activation of RelB/p52 pathway in baicalin-treated TAMs. (a) Induced expression of IKKα in baicalin-treated TAMs is associated with autophagy induction. RNA interference against Atg5 was conducted as described and TAM was treated with 20 μM baicalin for 48 h. Expression of IKKα was detected by immunoblotting. It was shown that baicalin treatment resulted in increase of IKKα expression, which could be blocked by inhibition of autophagy by RNA interference against Atg5. (b) Silencing of IKKα expression attenuated RelB overexpression in baicalin-treated TAM. RNA interference against IKKα was conducted as described and TAM was treated with 20 μM baicalin for 48 h. It was shown that increased expression of RelB by baicalin was attenuated upon IKKα suppression. (c) Autophagy induction is independent to IKKα overexpression. (d) Suppression of IKKα by RNA interference induced re-skewing of baicalin-treated TAM to M2 phenotype. RNA interference was conducted as described and TAM was subjected to baicalin treatment. Suppression of IKKα expression significantly blocked change of CD86 and CD206 expression induced by baicalin. This was further evidenced by observation in cytokine expression profile (e) of baicalin-treated TAMs with or without RNA interference against IKKα. (f) RelA pathway may not be activated upon baicalin treatment. TAM was treated with baicalin in the presence of soluble TNF-α. Expression of RelA and IκBα was determined. (g) Baicalin treatment induced reduction of TRAF2 and TRAF3 expression. (h) Baicalin-suppressed TRAF2 expression may be blocked when autophagy was blocked by either Bafilomycin A1 or RNA interference against Atg5. (i) Baicalin induced TRAF2 degradation in M2-like macrophage but not in M1 phenotype. (j) Degradation of TRAF2 induced by baicalin may be dependent on autophagy-associated lysosomal pathway. TAM treated with or without baicalin was fixed and stained with TRAF2 antibody. Lysosome was stained with Lysotracker Green for 30 min. It was observed that TRAF2 was translocated to lysosome after treatment of baicalin in TAM (magnification: x60). (k) p62 cargo protein may dominate selective autophagic degradation of TRAF2 in baicalin-treated TAM. TAM was treated with or without baicalin in the presence of lysosome inhibitor leupeptin. Co-immunoprecipitation assay was conducted with TRAF2 antibody and association of p62 with TRAF2 was detected by immunoblotting. Recruitment of p62 to TRAF2 upon baicalin treatment was observed