Subarachnoid hemorrhage (SAH) remains a life-threatening disease with poor prognosis. How to improve the clinical outcome after SAH is still a tough challenge. Multiple pathophysiological processes occurring after SAH, including early brain injury (EBI), cerebral vasospasm, and delayed cerebral ischaemia (1). Till now, mounting evidence has demonstrated that the neurological outcome after SAH is seriously influenced by early brain injury (EBI) (2–4). Multiple cellular mechanisms are involved in the EBI pathophysiology after SAH. Among them, activation of inflammatory response and oxidative damage contribute greatly to the development of EBI (5–7). Thus, a potential therapy for improving the prognosis after SAH is intervening neuroinflammation and oxidative stress.

Transforming growth factor-β-activated kinase 1 (TAK1), a member of the mitogen‐activated protein kinase (MAPK) family, has a wide range of biological functions (8–10). It has demonstrated that TAK1 activation could strongly elicit pro-inflammatory cytokines release and drive microglial toward a proinflammatory phenotype (11–14). In addition, TAK1 mediates reactive oxygen species (ROS) generation to aggravate oxidative damage in different diseases models (15, 16). In central nervous system (CNS), TAK1 is mainly expressed in neurons and could regulate a variety of intracellular signaling pathways, including nuclear factor‐κB (NF‐κB), AMP-activated protein kinase (AMPK), and nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome (17–19). Since these molecular mechanisms contribute to EBI pathophysiology, targeting TAK1 might be an effective treatment for SAH.

Takinib, a novel and specific TAK1 inhibitor, has been recently evaluated in a variety of disease models (20–22). Previous studies have demonstrated that takinib could inhibit inflammation and apoptosis by suppression of TAK1-mediated signaling pathway (20, 21). However, whether takinib could protect against EBI after SAH and the potential molecular mechanisms remain unclear. Hence, this study aimed to investigate the possible role of takinib in EBI after SAH and its underlying mechanisms.

All the experimental procedures were approved by the Animal Ethics Review Committee of Beijing Friendship Hospital and carried out in accordance with the National Institutes of Health guidelines. A total of 190 rats (weighing 250 to 300 g) were used in our study. Among them, 154 rats underwent SAH insults and 8 rats were excluded due to mild SAH grading score (less than 8) and intracerebral hematoma. All rats were numbered consecutively and randomization was conducted by using the website Randomization (http://www.randomization.com).

In this study, we verified the beneficial effects of takinib on EBI after SAH. Our data indicated that takinib could ameliorate SAH-induced inflammatory response by inhibiting M1-microglial phenotype polarization and promoting microglial polarization to M2 phenotype. In addition, takinib reduced ROS generation and suppressed oxidative damage after SAH. Concomitant with the reduced neuroinflammation and oxidative stress, takinib improved functional outcome and neuronal survival after SAH. Mechanistically, takinib inhibited TAK1 activation and the subsequent ROS-NLRP3 inflammasome signaling pathway following SAH. In contrast, NLRP3 activation by nigericin abated the neuroprotective effects of takinib against EBI after SAH ( Figure 7 ).

microglial polarization plays a critical role in the pathogenesis of neuroinflammation after SAH (29–31). Activated microglial exhibit different phenotypes under different stimulants and have distinct functions. M1-polarized microglial could increase proinflammatory cytokines release and aggravate ROS production. In contrast, M2-polarized microglial produce anti-inflammatory cytokines to keep the immune balance (29, 32). It has demonstrated that inhibition of M1 microglial polarization and induction microglial polarization into M2 could alleviate neuroinflammation and improve neurological outcomes after SAH (29). In addition, oxidative stress also participates in the development of EBI after SAH. Numerous studies have reported that ROS overproduction could induce neuroinflammation to exacerbate brain damage after SAH (1, 24, 33). Therefore, inhibition of M1-like microglial polarization and oxidative damage might be a successful strategy to reduce EBI after SAH.

Multiple studies have proved that TAK1 inhibition is a promising therapeutic application for various CNS diseases including traumatic brain injury (TBI), ischemic stroke, and SAH (34–36). For example, Shen et al. demonstrated that microglial-selective deletion of Tak1 inhibited IL-18 production and ameliorated ischemic stroke injury in prolonged obesity (37). Shi et al. reported that pharmacological inhibition of TAK1 with 5Z-7-oxozeaenol provided long-lasting improvement of stroke outcomes (38). In SAH area, TAK1 inhibition also attenuated EBI by reducing neuroinflammation and neuronal death (34). These indicated that TAK1 might be a promising target for treating SAH. However, the effects of TAK1 inhibition on microglial polarization and oxidative damage after SAH remain unclear.

It has been reported that 5Z-7-oxozeaenol is a potent selective inhibitor of TAK1 and vascular endothelial growth factor receptor 2 (39). Different with 5Z-7-oxozeaenol, takinib is a novel and highly selective TAK1 inhibitor. Recent studies have showed that takinib could effectively inhibit TAK1 activation-mediated inflammatory and apoptotic signaling cascades in different research fields (20, 21). However, to date, no study investigated the potential role of takinib on EBI after SAH. In our study, we first evaluated the effects of takinib on TAK1 expression after SAH. In accordance with previous studies, our data revealed that p-TAK1 (Thr 187) was significantly increased in neurons after SAH. In contrast, takinib treatment significantly decreased p-TAK1 expression. We then evaluated the possible influence of takinib on microglial polarization. Intriguingly, it showed that SAH insults significantly induced M1 microglial polarization, which could be inhibited by takinib treatment. Moreover, TAK1 inhibition by takinib increased M2 phenotype microglial. These suggested that TAK1 inhibition by takinib might have the potential to inhibit M1 microglial polarization and promote the microglial phenotype toward M2.

ROS overproduction plays an important role in the development of EBI after SAH. It can disrupt cellular functions by damaging nucleic acids, proteins, and lipids (1). Moreover, ROS production can aggravate neuroinflammation by activation of NLRP3 inflammasome signaling (25, 40). NLRP3 inflammasome has been demonstrated to participate in microglial polarization after SAH. Previous studies have demonstrated that inhibition of ROS/NLRP3 inflammasome signaling could decrease microglial M1 polarization and promote microglial polarization to M2 phenotype (29, 41). Our experiments revealed that TAK1 inhibition by takinib regulated microglial M1-M2 phenotype transition after SAH. However, the possible mechanisms remain elusive. Mounting evidence has showed that TAK1 activation could induce the aggravation of oxidative stress by promoting ROS production (16, 42). Inhibition of TAK1 is able to attenuate ROS overproduction and might be a potential therapeutic target for oxidative stress-related injuries. Interestingly, TAK1 has been verified as a key regulator of NLRP3 inflammasome activation (11, 34, 43). In our experiments, our data showed that takinib successfully inhibited the ROS overproduction and the subsequent activation of NLRP3 inflammasome signaling after SAH. Moreover, nigericin, a NLRP3 activator, abated the protective effects of takinib against EBI after SAH, validating the interaction between TAK1 and NLRP3 inflammasome. Concomitant with the reduced oxidative damage and neuroinflammation, takinib treatment significantly reduced neuronal apoptosis and improved functional behavior after SAH. Together with our experimental results, we provided the evidence that TAK1-ROS-NLRP3 inflammasome axis involved in the development of EBI after SAH. By intervening with TAK1-ROS-NLRP3 inflammasome axis, takinib modulated microglial polarization and inhibited oxidative damage.

It should be noted that NLRP3 inflammasome could modulate microglial polarization in a variety of disorders, including intracerebral hemorrhage, depression, ischemic stroke, white matter injury, as well as Alzheimer’s disease (44–46). These suggested that targeting NLRP3 inflammasome might be a feasible method to relieve neuroinflammation. In SAH area, Xu et al. reported that TAK1 inhibition by siRNA could reduce NLRP3 inflammasome-mediated neuronal pyroptosis (34). However, the previous studies did not investigate whether TAK1 inhibition affected microglial polarization. In the present study, we demonstrated that TAK1 could affect microglial polarization by modulating NLRP3 inflammasome. Inhibition TAK1 by takinib suppressed NLRP3 inflammasome and decreased M1 microglial polarization. However, how TAK1 regulates NLRP3 inflammasome is not fully investigated. In addition to ROS, K⁺ efflux, endosomal rupture, and mitochondrial dysfunction could trigger NLRP3 inflammasome activation (47). Hindi et al. indicated that TAK1 plays a critical role in regulating skeletal muscle mass and oxidative metabolism. TAK1 activation could induce an accumulation of dysfunctional mitochondria as well as oxidative damage in skeletal muscle (19). We suspected that TAK1 might also affect mitochondrial dysfunction to trigger NLRP3 inflammation. But further studies are still needed to decipher this question and whether other molecular targets are involved in this modulation.

Our study has several limitations. Firstly, the long-term effects of TAK1 inhibition in the delayed phase of SAH remains unclear. Meanwhile, no studies have performed to investigate the influence of TAK1 on cerebral vasospasm and delayed ischemic neurologic deficits after SAH. A recent study by Shen et al. indicated that prolonged high-fat diet (for 32 weeks) feeding-induced obesity has a significant cerebrovascular dysfunction, which is closely associated with microglial TAK1 activation. They showed that microglial TAK1 activation significantly aggravated basilar artery abnormalities. Both pharmacological suppression and genetic microglial-selective TAK1 deletion relieved basilar artery dysfunction and improved the outcome of ischemic stroke (37). On this background, we suspected that TAK1 inhibition might ameliorate cerebral vasospasm and relieve delayed ischemic neurologic deficits in the late phase of SAH. Secondly, the intracerebroventricular administration limits the clinical utility of takinib. Other administration routes instead of ventricle injections should be conducted to validate its clinical translatability. Thirdly, our data indicated that takinib could protect against M1 microglial polarization and oxidative damage after SAH by modulation of TAK1-ROS-NLRP3 inflammasome axis. However, whether these effects are mediated by NF‐κB, AMPK, and p38 should be further determined (48, 49). Lastly, in addition to activate NLRP3, nigericin might modulate other signaling targets to aggravate neuroinflammation. Thus, more experiments are needed to solve these questions.

In conclusion, we provided the first evidence that takinib could modulate microglial polarization and inhibit oxidative damage after SAH primarily by targeting the TAK1-ROS-NLRP3 inflammasome axis. These findings implied that takinib might be a potential new therapeutic candidate for the treatment of SAH.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The animal study was approved by the Animal Ethics Review Committee of Beijing Friendship Hospital. The study was conducted in accordance with the local legislation and institutional requirements.

WW: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing. CP: Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing – original draft. JZ: Investigation, Methodology, Validation, Writing – review & editing. LP: Methodology, Writing – review & editing. XZ: Methodology, Supervision, Writing – review & editing. LS: Conceptualization, Methodology, Project administration, Writing – original draft, Writing – review & editing. HZ: Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Writing – review & editing.