Inhibiting expression of HSP60 and TLR4 attenuates paraquat-induced microglial inflammation

Abstract

Accumulating evidences suggest that heat shock protein 60 (HSP60) and toll-like receptor 4 (TLR4) are involved in triggering inflammatory response in microglia. Paraquat (PQ) evokes microglial inflammation by up-regulating expression of HSP60-TLR4-myeloid differentiation factor 88 (Myd88)-nuclear factor-kappa B (NF-κB) in vitro. The aim of this study is to investigate the potential modulatory roles of HSP60 and TLR4 in PQ-induced inflammation. Before treated with PQ, microglia BV2 cells were pretreated using siRNA to knockdown HSP60 or with specific inhibitor to inhibit TLR4 expression. Expression of TLR4 and MyD88, and nuclear translocation of NF-κB subunit p65 were studied with immunoblotting and immunofluorescence, respectively. Expression of pro-inflammatory factors was assessed with quantitative real-time PCR. Knockdown of HSP60 or inhibition of TLR4 significantly reduced the expression of TLR4 and MyD88 and decreased the accumulation of NF-κB p65 in the nucleus. Gene expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) were also significantly decreased in response to PQ. These results suggest that HSP60 and TLR4 can modulate intracellular signaling of PQ-induced inflammation. Inhibiting HSP60 or TLR4 reduces significantly the intensity of inflammation in PQ-activated microglia.