Effect and mechanism of doxorubicin on integrity of blood-brain barrier in young mice based on AP-1/miR-155 signaling pathway

HU Nan; YIN Yiqing; ZHANG Yu; WANG Jiefu

doi:10.3969/j.issn.1002-266X.2025.11.009

Abstract：Objective To observe the effect of doxorubicin(DOX)on the integrity of the blood-brain barrier(BBB)in young mice and to explore its possible mechanism.Methods In vivo experiment:96 young mice were randomly divid-ed into the control group 1(C1 group),DOX group 1(D1 group),DOX+microRNA-155(miR-155)antagonist group 1(D+L1 group),DOX+activator protein 1(AP-1)inhibitor group 1(D+S1 group),miR-155 antagonist group 1(L1 group),and AP-1 inhibitor group 1(S1 group),with 16 rats in each group.The D1 group received intraperitoneal(i.p.)injection of DOX(4 mg/kg).The D+L1 group received i.p.injection of a locked nucleic acid(LNA)-modified miR-155 antagonist(LNA-antimiR-155)(5 mg/kg),followed by i.p.injection of DOX(4 mg/kg)1 h later.The D+S1 group re-ceived i.p.injection of the AP-1 inhibitor SP600125(10 mg/kg),followed by i.p.injection of DOX(4 mg/kg)1 h lat-er.The L1 group received i.p.injection of LNA-antimiR-155(5 mg/kg).The S1 group received i.p.injection of SP600125(10 mg/kg).All interventions were administered once a week for 5 weeks.BBB integrity was assessed using Evans Blue(EB)staining.The expression of tight junction proteins Occludin and Claudin-5 in hippocampal tissues was detected by Western blotting.In vitro experiment:The young mouse microvascular endothelial cell line bEnd.3 was divid-ed into groups corresponding to the in vivo experiment:C2,D2,D+L2,D+S2,L2,and S2 groups.The C2 group was rou-tinely cultured without drug intervention.The D2 group was treated with DOX(1 µmol/L)for 48 h.The D+L2 group was pretreated with LNA-antimiR-155(50 nmol/L)for 24 h,followed by intervention with DOX(1 µmol/L)for 48 h.The D+S2 group was pretreated with the AP-1 inhibitor SR11302(50 µmol/L)for 24 h,followed by intervention with DOX(1 µmol/L)for 48 h.The L2 group was treated with LNA-antimiR-155(50 nmol/L)alone for 24 h.The S2 group was treated with SR11302(50 µmol/L)alone for 24 h.Annexin A1 expression in the cells was detected by immunofluores-cence staining.MiR-155 expression was detected by real-time quantitative polymerase chain reaction(RT-qPCR).AP-1 activity was assessed by detecting c-Jun protein expression through Western blotting.Results In vivo experiment re-sults:Compared with the C1 group,the EB content in the hippocampal tissues of the D1 group was higher(P<0.05).Compared with the D1 group,the EB content in the hippocampal tissues of the D+L1,D+S1,L1,and S1 groups was lower(all P<0.05).Compared with the C1 group,the relative protein expression levels of Occludin and Claudin-5 in the brain tissues of the D1 group were lower(both P<0.05).Compared with the D1 group,the relative protein expression levels of Occludin and Claudin-5 in the brain tissues of the D+L1,D+S1,L1,and S1 groups were higher(all P<0.05).In vitro ex-periment results:Compared with the C2 group,the expression of Annexin A1 in the brain microvascular endothelial cells of the D2,D+L2,and D+S2 groups was lower(all P<0.05).Compared with the D2 group,the expression of Annexin A1 in the brain microvascular endothelial cells of the D+S2,D+L2,L2,and S2 groups was higher(all P<0.05).Compared with the C2 group,the relative expression of miR-155 in the microvascular endothelial cells of the D2 group was higher(P<0.05).Compared with the D2 group,the relative expression of miR-155 in the microvascular endothelial cells of the D+L2,D+S2,L2,and S2 groups was lower(all P<0.05).Compared with the C2 group,the relative expression of c-Jun pro-tein in the brain microvascular endothelial cells of the D2 and D+L2 groups was higher(both P<0.05),while that in the D+S2 and S2 groups was lower(P<0.05).Compared with the D2 group,the relative expression of c-Jun protein in the brain microvascular endothelial cells of the D+S2,L2,and S2 groups was lower(all P<0.05).Conclusion DOX may inhibit the expression of Annexin A1 and the synthesis of tight junction proteins by regulating the expression of AP-1 and miR-155,thereby disrupting BBB integrity of young mice.