Intracerebral hemorrhage (ICH) is a non-traumatic intraparenchymal hemorrhage causing high mortality and poor outcomes. The incidence of ICH increases with age, with reported one- and five-year mortalities of 46% and 29%, respectively. Moreover, patients with ICH demonstrate less functional independence, with only 12–39% of survivors living independently (Poon et al., 2014, van Asch et al., 2010). Despite the advancement in understanding the cause and pathophysiology of ICH, its treatment remains challenging for neurologists (Hostettler et al., 2019). Therefore, identifying effective drugs for treating ICH is essential.

Studies have shown that ICH pathology can be divided into two types: primary brain injury (PBI) and secondary brain injury (SBI) (Wu et al., 2020; Xue and Yong, 2020). PBI could be due to the compression and space occupation of an ICH hematoma, while SBI is mainly caused by toxic damage to the brain tissue by the components of a hematoma and its metabolites after ICH. Neuroinflammation is one of the most important causes of neuronal death (Magid-Bernstein et al., 2022), the immune cells activated after ICH release inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, aggravating brain injury (Tschoe et al., 2020). As a damage-associated molecular pattern, high mobility group protein 1 (HMGB1) is released to induce inflammation by dead cells or activated immune cells after cell injury or death (Andersson et al., 2018). The binding of HMGB1 to toll-like receptor-4 (TLR4) aggravates neuroinflammation (Lei et al., 2022), and inhibition of TLR4 is reported to attenuate the ICH-induced neuroinflammation (Lai et al., 2019). Moreover, one of the components of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor family, NF-κB p65, is upregulated over time ranging from minutes to days and is associated with the pathological death of nerve cells surrounding the hematoma (Hickenbottom et al., 1999; Zhao et al., 2007). The role of HMGB1 during the early stages of ICH-mediated neuroinflammation has been identified and could be a potential therapeutic target in treating ICH (Tian et al., 2017).

Annao Pingchong decoction (ANPCD), a TCM prescription developed from ZGXFD, has better heat-clearing effects than ZGXFD. Zhengan Xifeng Decoction (ZGXFD), designed by a famous doctor named Zhang Xichun, is a well-known Traditional Chinese Medicine (TCM) prescription recorded in Records of Traditional Chinese and Western Medicine in Combination. ZGXFD is empirically used to treat stroke, Parkinson's disease, and hypertension in TCM clinical practice, which belongs to “Yin Deficiency and Wind Movement” (Cui, 2014; Huang et al., 2019). A Meta-analysis also showed that ZGXFD is effective against hemorrhagic stroke (Liang et al., 2015).

ANPCD comprises Longgu (Fossilia Ossia Mastodi), Muli (Ostrea gigas Thunberg), Chuan NiuXi (Cyathula officinalis K. C. Kuan), BaiJiLi (Tribulus terrestris L.), and GouTeng (Uncaria rhynchophylla (Miq.) Miq. ex Havil.), ZeXie (Alisma plantago-aquatica L.), BaiShao (Paeonia lactiflora Pall.), Zhizi (Gardenia jasminoides J. Ellis), HuangQin (Scutellaria baicalensis Georgi), DaHuang (Rheum palmatum L.), and Gancao (Glycyrrhiza uralensis Fisch.). The daily dose of ANPCD for adults is shown in Table 1. ANPCD has achieved excellent therapeutic effects in treating ICH by alleviating brain edema and improving neurological impairments (Long et al., 2018; Zhou et al., 2003a, Zhou et al., 2003b). The therapeutic principle of TCM involves settling the liver, extinguishing wind, calming chong qi, down-bearing counterflow, and quieting blood and the brain.

A network pharmacology study of 1679 stroke-related TCM formulas has identified Licorice, Paeoniae Radix Alba, Scutellariae Radix, and Rhei Radix et Rhubarb among the top 30 commonly used herbs, and are components of ANPCD (Ren et al., 2019). One hundred fifty-five traditional Chinese medicines used to treat acute cerebral hemorrhage were screened from the China National Knowledge Infrastructure (CNKI) database, which consists of nine herbs of ANPCD with a frequency of use >10 (Liao et al., 2022). Researchers have identified certain ANPCD compounds, such as geniposide, genipin, baicalin, wogonoside, and Paeoniflorin, with anti-inflammatory properties, playing a therapeutic role in central nervous system diseases (Liang et al., 2017; Liao et al., 2021; Lv et al., 2018; Zhang and Wei, 2020). Glycyrrhizic acid, extracted from Licorice root, has been extensively studied owing to its robust anti-inflammatory and immune-regulatory functions (Richard, 2021). It is also an HMGB1 inhibitor and inhibits neuroinflammation to attenuate hypoxic-ischemic brain damage (K. Zhu et al., 2022). In previous studies, ANPCD effectively inhibited ferroptosis and decreased matrix metalloprotease (MMP)-2/9 and aquaporin (AQP)-4/9 expression (Guo et al., 2020; Zhou et al., 2011, 2011, 2013, 2011). However, the therapeutic mechanisms underlying ANPCD remain unclear. Therefore, in this study, we aimed to identify the role of the HMGB1/TLR4/NF-κB p65 pathway in exerting anti-inflammatory and anti-apoptotic effects of ANPCD during the early stages of ICH-induced brain injury.