HMGB1/TLR4 induces autophagy and promotes neuroinflammation after intracerebral hemorrhage

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