Takinib inhibits microglial M1 polarization and oxidative damage after subarachnoid hemorrhage by targeting TAK1-dependent NLRP3 inflammasome signaling pathway

Abstract

Transforming growth factor-β-activated kinase 1 (TAK1) positively regulates oxidative stress and inflammation in different diseases. Takinib, a novel and specific TAK1 inhibitor, has beneficial effects in a variety of disorders. However, the effects of takinib on early brain injury (EBI) after subarachnoid hemorrhage (SAH) and the underlying molecular mechanisms remain unknown. Our study showed that takinib administration significantly inhibited phosphorylated TAK1 expression after SAH. In addition, takinib suppressed M1 microglial polarization and promoted M2 microglial polarization. Furthermore, blockade of TAK1 by takinib reduced neuroinflammation, oxidative damage, brain edema, and neuronal apoptosis, and improved neurological behavior after SAH. Mechanistically, we revealed that TAK1 inhibition by takinib mitigated reactive oxygen species (ROS) production and ROS-mediated nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome activation. In contrast, NLRP3 activation by nigericin abated the neuroprotective effects of takinib against EBI after SAH. In general, our study demonstrated that takinib could protect against EBI by targeting TAK1-ROS-NLRP3 inflammasome signaling. Inhibition of TAK1 might be a promising option in the management of SAH.

Keywords: NLRP3; TAK1; early brain injury; subarachnoid hemorrhage; takinib.

Figure 1

Dose-response effects of takinib on TAK1 activation after SAH. (A) Representative western blot bands and quantitative analyses of p-TAK1 (B) and TAK1 (C) after SAH (n = 6 each group). (D) Representative photomicrographs and quantification (E) of p-TAK1 immunofluorescence staining in the basal cortex at 24 h after SAH (n = 6 each group). One-way ANOVA with Tukey, bars represent the mean ± SD. ^* P < 0.05.

Figure 2

Effects of takinib on TAK1-NLRP3 inflammasome activation after SAH. (A) Representative western blot bands and quantitative analyses of p-TAK1 (B), TAK1 (C), NLRP3 (D), caspase1 (E), cleaved caspase1 (F), IL-1β (G), and IL-18 (H) after SAH (n = 6 each group). One-way ANOVA with Tukey, bars represent the mean ± SD. ^* P < 0.05.

Figure 3

Effects of takinib on microglial phenotypic transformation after SAH. (A) Double immunofluorescence staining for CD16/32 in microglial in the basal cortex after SAH. (B, C) Quantification of CD16/32 immunofluorescence staining in the basal cortex at 24 h after SAH (n = 6 each group). (D) Double immunofluorescence staining for CD206 in microglial in the basal cortex after SAH. (E, F) Quantification of CD206 immunofluorescence staining in the basal cortex at 24 h after SAH (n = 6 each group). One-way ANOVA with Tukey, bars represent the mean ± SD. ^* P < 0.05.

Figure 4

Effects of takinib on ROS and oxidative damage after SAH. Western blot assay (A) and quantification (B) for expression of 3-nitrotyrosine in different groups (n = 6 each group). (C) Quantification of MDA level in brain tissue at 24 h post-SAH (n = 6 each group). (D) Representative photomicrographs and quantification (E) of 8-OhdG immunofluorescence staining in the basal cortex after SAH (n = 6 each group). One-way ANOVA with Tukey, bars represent the mean ± SD. ^* P < 0.05.

Figure 5

Effects of takinib on neurological behavior, brain water content, and neuronal survival after SAH. Quantification of (A) neurological deficits scores, (B) rotarod performance, and (C) brain water content in different groups after SAH (n = 10-12 each group). (D) Representative photomicrographs and quantification (E) of TUNEL staining in the basal cortex after SAH (n = 6 each group). One-way ANOVA with Tukey, bars represent the mean ± SD. ^* P < 0.05.

Figure 6

Effects of takinib on histopathological change and neurological behavior at day 3 after SAH. Quantification of (A) neurological deficits scores and (B) rotarod performance in different groups after SAH (n = 6 - 7 each group). (C) Representative photomicrographs and quantification (D) of survival neurons in the basal cortex after SAH (n = 6 each group). One-way ANOVA with Tukey, bars represent the mean ± SD. ^* P < 0.05.

Figure 7

Schematic illustrating the possible mechanisms of takinib action after SAH. As illustrated, SAH significantly increases the expression level of p-TAK1(Thr187) indicating that TAK1 is activated after SAH. TAK1 activation triggers reactive oxygen species (ROS) generation. In response to ROS accumulation after SAH, NLRP3 recruits the adaptor apoptosis-related speck-like protein (ASC) and pro-caspase-1 to form a large multiprotein complex. NLRP3 inflammasome activation promotes microglial phenotype toward M1 and inhibits M2 microglial polarization after SAH, subsequently aggravating neuroinflammation and brain damage. Takinib, a novel and highly selective TAK1 inhibitor, could suppress TAK1 activation and TAK1-mediaed ROS-NLRP3 inflammasome signaling after SAH. In contrast, NLRP3 inflammasome activator nigericin reverses the beneficial effects of takinib against SAH, eventually aggravating SAH-induced brain damage.