Involvement of Circadian Clock Gene BMAL1 in Doxorubicin-Induced Inflammation in Vascular Smooth Muscle Cells

The molecular clock component Brain and Muscle Arnt-Like protein-1 (BMAL-1) affects various biologic processes, including cell survival, in numerous cell types. We previously demonstrated that BMAL1 positively regulates cell proliferation in Vascular Smooth Muscle Cells (VSMCs). However, its role in VSMC inflammation remains unelucidated. Because doxorubicin causes phlebitis associated with vascular inflammation, the present study used cultured VSMCs to investigate whether BMAL1 affected doxorubicin-induced vascular inflammation. Doxorubicin treatment led to Increased Interleukin (IL)-6 mRNA expression with an increase in BMAL1 expression in VSMCs. BMAL1 knockdown significantly increased IL-6 mRNA and further enhanced doxorubicin-induced IL-6 mRNA expression in VSMCs. BMAL1 knockdown also significantly decreased cell viability and affected the expression of other clock genes, including Per1 and Clock. Furthermore, the levels of nuclear factor erythroid 2-related factor 2, which has anti-inflammatory effects, increased in VSMCs with BMAL1 knockdown. Finally, BMAL1 knockdown increased NADPH oxidase 4 mRNA, p38α mRNA, and p38β mRNA levels, leading to increased total p38 Mitogen-Activated Protein Kinase (MAPK) and phosphorylated p38 MAPK. IL-6 mRNA induction caused by BMAL1 knockdown was significantly inhibited in VSMCs following pretreatment with SB203580, a p38 MAPK inhibitor. Our findings demonstrated that decreased BMAL1 expression caused VSMC inflammation via p38 MAPK activation. Moreover, doxorubicin-induced inflammation in VSMCs was further enhanced when BMAL1 expression levels were low. Thus, BMAL1 may be a novel therapeutic target to treat inflammatory disease, including doxorubicin-induced phlebitis.