Myeloid Differentiation Factor 2 Inhibition Protects Against Doxorubicin‐induced Microglial Activation and Senescence

Abstract

AbstractBackgroundDoxorubicin, an anti‐tumor drug, generates neuroinflammation that leads to microglial senescence and chemo‐brain (1, 2). Toll‐like receptor 4 (TLR4) signaling pathway is mainly expressed in microglia and it plays a key role in developing doxorubicin‐induced neuroinflammation, microglial activation and chemo‐brain (3). Myeloid differentiation factor 2 (MD‐2) is known as an essential protein in activation of TLR4 signaling pathway (1). However, the effect of blocking TLR4 signaling via targeting MD‐2 on doxorubicin‐induced microglial activation and senescence has never been investigated.MethodThe human microglial clone 3 cell line (HMC3) was treated with three different doses of MD‐2 inhibitor (L6H21: 5μM, 10μM, 20μM) for 30 minutes, then with doxorubicin hydrochloride (DOX) (50nM) for 24 hours. After 24 hours treatment with L6H21 and DOX, the microglial cells were assessed for the TLR4 signaling, microglial activation, cell death and cellular senescence pathways.ResultDOX treatment activated TLR4 signaling pathway as well as microglia, as evidenced by significantly increased protein expressions of TLR4, MD‐2, tumor necrosis factor alpha and ionized calcium‐binding adapter molecule 1. In addition, DOX treatment induced microglial cell death and senescence, as shown by significantly increased protein expressions of Bcl‐2 associated X‐protein to B‐cell lymphoma 2 ratio and p16Ink4a. Treatment with L6H21 (5μM) attenuated not only TLR4 signaling along with microglial activation, but also apoptosis and cellular senescence in DOX‐treated microglia. L6H21 (10μM and 20μM) protected against TLR4 signaling along with microglial activation, apoptosis as well as cellular senescence in DOX‐treated microglia.ConclusionThese findings imply that MD‐2 inhibition could be the potential therapeutic strategy for attenuating DOX‐induced microglial activation and senescence.