HMGB1/TLR4 induces autophagy and promotes neuroinflammation after intracerebral hemorrhage

Abstract

Background and purpose: Intracerebral hemorrhage (ICH) causes autophagy as well as inflammation; the latter is known to involve the high-mobility group box 1 protein (HMGB1)/Toll-like receptor 4 (TLR4) axis. Here we investigated whether this axis may help mediate both the autophagy and inflammation associated with ICH.

Methods: ICH was induced by injecting autologous blood into Sprague-Dawley rats, followed in some cases by intracerebroventricular injection of short interfering RNA (siRNA) against HMGB1 or TLR4 at 6 h after ICH induction or by intraperitoneal injection of the autophagy inhibitor 3-methyladenine (3-MA) or autophagy activator rapamycin at 6, 24, and 48 h after ICH induction. Western blotting, immunohistochemistry or immunofluorescence was used to assess levels of HMGB1/TLR4 signaling pathway proteins as well as markers of autophagy (LC3B, Beclin1, Atg5) or inflammation (IL-1 beta, TNF-α). Numbers of apoptotic cells were determined using TUNEL staining. Changes in levels of these proteins were correlated with neurological deficits measured using the modified Neurological Severity Score.

Results: ICH caused HMGB1 to translocate from the nucleus into the cytoplasm, and it up-regulated expression of TLR4 and myeloid differentiation factor 88 (MyD88), and induced neurological deficits. Administering siRNA against HMGB1 or TLR4 reversed this up-regulation. Levels of markers of autophagy (LC3B, Beclin1, Atg5) or inflammation (IL-1 beta, TNF-α) were significantly higher 72 h after ICH than at baseline, as were the numbers of TUNEL-positive cells. Administering siRNA against HMGB1 or TLR4 markedly alleviated inflammation, and autophagy, apoptosis, and neurological deficits. Similarly, administering autophagy inhibitor 3-MA alleviated inflammation, apoptosis, and neurological deficits. Conversely, autophagy activator rapamycin exacerbated these effects of ICH.

Conclusions: During the acute phase of ICH, the HMGB1/TLR4/MyD88 axis acts via autophagy to promote inflammation.

Keywords: Autophagy; HMGB1; Inflammation; Intracerebral hemorrhage; TLR4.