Figure 5

Comparison of the protective activity of monoclonal antibodies and CIGB-258 against carboxymethyllysine (CML, final 500 ng) toxicity in zebrafish embryos. (A) Survivability of zebrafish embryos during 24 h post-injection in the presence of Infliximab (43 ng), Etanercept (15 ng), Tocilizumab (44 ng), or CIGB-258 (final 1 ng). *, p < 0.05; **, p < 0.01. (B) Stereo image of the zebrafish embryos at 5 h and 24 h post-injection. The red arrowheads indicate defective development and death of embryos in the CML alone group (photograph b). The blue arrowhead indicates the slowest developmental speed in eye pigmentation and tail elongation in CML alone group at 24 h post-injection (photograph c). Fluorescence image of dihydroethidium (DHE, Ex = 585 nm, Em = 615 nm) stained and acridine orange (AO, Ex = 505 nm, Em = 535 nm) stained embryo at 5 h post-injection. The extent of ROS production and apoptosis was visualized by DHE and AO staining, respectively. The white arrow indicates the CML-injected site to show that ROS production and cellular apoptosis occurred simultaneously by acute inflammation. Photo a, PBS alone; photo b, CML + PBS; photo c, CML + Infliximab; photo d, CML + Etanercept; photo e, CML + Tocilizumab; photo f, CML + CIGB-258 (C) Quantification of the fluorescence from DHE-stained and AO-stained embryo images using Image J software version 1.53r (http://rsb.info.nih.gov/ij/ accessed on 17 January 2023). AU, arbitrary unit; NS, not significant.