Meisoindigo, but not its core chemical structure indirubin, inhibits zebrafish interstitial leukocyte chemotactic migration

Figures & data

Figure 1. Leukocyte migration in response to acute injury is visualized in zebrafish embryo model (Tg:zlyz-EGFP). (a).Tg:zlyz-EGPF embryos (at 3dpf) were subjected with tail transection for generating zebrafish leukocyte migration model. The red line means the incision of tail transection, and the square region means the highly inflamed region. (b–c). Leukocytes that migrated to the highly inflamed regions [white boxes in (b)] in Tg:zlyz-EGFP embryos (3 dpf, n denotes the number of zebrafish embryos) with tail transection were quantitatively analyzed at different time points(c). (d). Schematic diagram of zebrafish leukocyte recruitment and resolution in acute inflammation.

Figure 2. Identification of meisoindigo that inhibits zebrafish leukocyte recruitment to the injury site. (a) Chemical screening strategy. (b) Chemical structure of meisoindigo. (c,d) Tg:zlyz-EGFP embryos (3 dpf) without tail transection were treated with vehicle(n = 9) or meisoindigo(M, 100 μM, n = 11) for 6 h. Leukocytes around the whole trunk of zebrafish embryo [white boxes in (c)] were quantitiatively analyzed. (e,f) Tg:zlyz-EGFP embryos (3 dpf) with tail transection were treated with vehicle (n = 13), 25 μM (n = 13), 50 μM (n = 18) or 75 μM (n = 14) meisoindigo, respectively. Leukocytes that migrated to the highly inflamed regions [white boxes in (e)] at 6 hptt were quantitatively analyzed (f). The p values were annotated as obtained from a two-tailed t test.

Table 1. List of the compounds in the chemical screening.

Compound	Concentration	Biological effectof inhibiting leukocytechemotatic migration
Arsenic trioxide	25 μM	No effect
Meisoindigo	25 μM	Significantly effective
Indirubin	25 μM	No effect
Tanshinone II A	25 μM	No effect

Figure 3. Meisoindigo has no effect on zebrafish leukocyte reverse migration from the injury site. (a) Treatment schedule. Tg:zlyz-EGFP embryos (3dpf) were subjected with tail transection and administrated with vehicle (n = 14) or meisoindigo (M, 100 μM, n = 11) from 6 hptt to 12 hptt. (b, c) Leukocytes migrated to the highly inflamed regions [white boxes in (b)] at 12hptt were quantitatively analyzed (c). The p value was annotated as obtained from a two-tailed t test.

Figure 4. Indirubin, the core chemical structure of meisoindigo, did not inhibit leukocyte chemotactic migration, and meisoindigo does not regulate pAKT/AKT or pERK/ERK level. (a) Chemical structure of indirubin (I) and meisoindigo (M). Meisoindigo is a synthetic derivative of indirubin with a methyl substitution at N element. (b,c) Tg:zlyz-EGFP embryos (3dpf) with tail transection were treated with vehicle (n = 15), indirubin (I, 100 μM, n = 14) or meisoindigo (M, 100 μM, n = 11), respectively. Leukocytes that migrated to the highly inflamed regions [white boxes in (b)] at 6 hptt were quantitatively analyzed (c). The p values were annotated as obtained from a two-tailed t test. (d) Westernblot analysis showing the effect of meisoindigo on the selected signaling pathway components. 20 zebrafish embryos (3dpf, n = 20) with tail transection were treated with vehicle, meisoindigo or indirubin at the indicated concentration from 0 hppt to 6 hptt, respectively. Then, the lysate of whole zebrafish embryos was subjected to westernblot analysis for detecting pAKT/AKT or pERK/ERK level.