Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in relatively healthy individuals worldwide, and it leads to a severe decline in patients' quality of life. At present, there is no effective treatment for TBI, some patients through hematoma drainage or even craniotomy to remove hematoma surgery, which the effect is not ideal (Hu et al., 2022a). >70% of patients survive with comorbidities, including varying degrees of language, sensory, motor, and other higher neurological deficits, and some patients even survive in a long-term vegetative state. Although significant progress has been made in the study of the mechanism of TBI, it is unclear whether brain injury can significantly improve the prognosis of TBI after active and effective clinical treatment of TBI (Lynde et al., 2022; Hu et al., 2022b). The pathophysiological mechanism of TBI is very complex, involving a series of events, such as neuroinflammation; oxidative stress; cell apoptosis, which leads to increased cerebral edema and permeability of blood-brain barrier (BBB). These factors largely determine the prognosis and outcome of patients with TBI, and therefore, extensive studies on the pathogenesis and protective effects of TBI are urgently needed. (Neil et al., 2023; Wang et al., 2018).

Necroptosis is implicated in numerous pathological and physiological processes, such as embryonic tissue development, immune responses, tumor growth，inflammatory injury. (He et al., 2009; Wang et al., 2014a; Sun et al., 2011). Dysregulation of necroptosis has been involved in a variety of diseases, including neurodegenerative disorders, inflammatory diseases and cancer, (He and Wang, 2018; Weinlich et al., 2017). As a result, targeting necroptosis has emerged as an underlying therapeutic strategy for these diseases. Several small molecules have been developed to inhibit RIPK1 and/or RIPK3 and are currently in clinical trials for various indications (Cho et al., 2009; Wang et al., 2014b). Necroptosis is interacted by RIPK1 and RIPK3 (Wang et al., 2012). RIPK1 interacts with RIPK3 in TNF-induced necroptosis, through their RIP homotypic interaction motif (RHIM) domain, they lead to the phosphorylation of RIPK3 and activation (Liu et al., 2017). One of the key features of necroptosis is its dependence on RIPK3, a downstream effector of necroptosis, contains a C-terminal RHIM domain and an N-terminal serine/threonine kinase domain. The RHIM domain of RIPK3 is responsible for binding to the RIPK1 and other proteins to form a signaling complex that triggers necroptosis (Zhou et al., 2019). The RIPK3 kinase activity is essential for MLKL activation and phosphorylation. (He et al., 2011). Phosphorylated MLKL is translocated to the membrane, where it oligomerizes and forms polymers, leading to membrane rupture and cell death.

Further evidence shows that RIPK3 can be activated by ZBP1 and other PHIM-containing protein for necroptosis. ZBP1 is a cytoplasmic DNA sensor that senses viral or bacterial RNA and DNA, which induces antiviral immune responses through activation of RIPK3-mediated necroptosis (Lawlor et al., 2015). TRIF is an adaptor protein involved in RIG-I-like receptor (RLR) and Toll-like receptor (TLR) signaling pathways, can activate RIPK3-mediated necroptosis in response to viral infections (Mifflin et al., 2020). These observations highlight the importance of RHIM-containing proteins in the regulation of necroptosis and their role in innate immune responses. In addition to its role in inducing necroptosis, RIPK3 can also participate in other signaling pathways that are independent of MLKL-mediated necroptosis (Park et al., 2018). For example, RIPK3 has been shown to regulate inflammatory responses by activating nuclear factor-κB (NF-κB) and producing inflammatory factor such as IL-1β and IL-6. RIPK3 can also induce apoptosis through activation of caspase-8 in the absence of MLKL or in the presence of caspase-8 inhibitors (Zhang et al., 2019). These observations suggest that RIPK3 can have diverse functions and that its role in cell death and survival is highly context dependent. Several studies have shown that RIPK3 is a critical mediator of necroptosis, and inhibition of RIPK3 can prevent necroptosis (Mompeán et al., 2018). Thus, targeting RIPK3 may be a potential therapeutic option for treating necroptosis-related diseases.

Given the essential role of RIPK3 in necrosis, its kinase activity has been extensively studied, and number of small molecule compounds that act on the kinase have been studied. For the classical GSK ‘872, GSK’ 843, GSK ‘840 studies, and also for the exploration of Braf, Bcr-Abl, VEGRF with RIPK3 as a potential drug target. (Hart et al., 2019; Xia et al., 2020). Due to the uncertainty of these compounds, RIPK3 inhibitors are currently not well used in clinical practice to treat tumors or suppress inflammatory reactions such as sepsis. Therefore, we are trying to find and study compounds that can inhibit RIPK3 with less toxic side effects and high efficacy. Here, we screened databases of FDA-approved small molecule compounds that are under investigation for possible anti-tumor compound. Our ultimate goal is to screen for novel compounds that inhibit RIPK3 kinase activity. We screened thousands of compounds and identified four compounds including Foretinib (GSK1363089,1D6), Poziotinib(HM781-36B,15F6), Dasatinib Monohydrate(15F9), Pexmetinib(ARRY-614,15A10), that could inhibit the activity of RIPK3 kinase. These four compounds (NBCs) exhibited potent inhibition of necroptosis induced cellular effects, by multiple programmed necrotizing stimuli in humans, mice, and rat cells. These compounds have good in vivo pharmacokinetic parameters and in vitro safety. Pretreatment the four compounds significantly ameliorated TNF induced systemic Inflammatory response syndrome (SIRS) mouse model. Further studies of the four compounds are needed to determine the precise role of RIPK3 and other RHIM-containing proteins in the regulation of necroptosis and immune responses.

After TBI, the neurogenesis of the injured peripheral neurons and the peri-hippocampal region was significantly increase. On the other hand, neuroinflammation contributed significantly to brain injury, and necroptosis is involved in neuroinflammatory processes after TBI. (Zille et al., 2017). But in fact, the exact and specific role of necroptosis on TBI is still unclear and requires further study. For the study, we hypothesized that neuronal injury is induced by the pathologic activation of RIPK3 after TBI, selecting specific inhibition of RIPK3 after TBI may reduce or delay the progression of neuronal injury. We aimed to explore the activity of necroptosis in a mouse model of TBI and further discuss the effects of necroptosis in neuronal injury and neuroprotection after TBI and its potential mechanisms. Dysregulation of necroptosis has been implicated in various pathological conditions, highlighting the importance of understanding the molecular mechanisms of the four compounds that regulate neuroinflammation after TBI (Aronowski and Zhao, 2011; Chen et al., 2015).