Gastrodin inhibits reactive astrocyte-mediated inflammation in hypoxic-ischemic brain damage through S100B/RAGE-Smad3 signaling

Abstract

Activated astrocytes and their associated inflammatory responses play critical roles in the pathogenesis of hypoxic-ischemic brain damage (HIBD). Gastrodin (GAS), an anti-inflammatory herbal agent, is known to suppress microglial activation. Here, we investigate whether it exerts a similar effect on activated astrocytes and whether it acts through S100B/RAGE-Smad3 signaling. The expression changes of S100B/RAGE-Smad3 signaling pathway-related proteins, inflammatory factors and A1/A2 astrocyte markers were detected by ELISA, western blot analysis, immunofluorescence and immunohistochemistry. The results show that GAS decreases the expression of sRAGE in the brain tissue and S100B in the serum and brain tissue of HIBD mice. However, it promotes the expression of sRAGE in the serum of HIBD mice. Moreover, GAS inhibits the expressions of RAGE, p-Smad3, TNF-α, and C3 (A1 astrocyte marker), and promotes the expressions of S100A10 (A2 astrocyte marker) and BDNF in HIBD model mice, as well as in oxygen glucose deprivation (OGD)-treated TNC-1 astrocytes. The immunofluorescence and immunohistochemical results of RAGE and p-Smad3, as well as the immunofluorescence results of C3 and S100A10, reveal the same trend. Interestingly, FPS-ZM1 (a specific inhibitor of RAGE) inhibits the expressions of p-Smad3, TNF-α, C3, and S100A10, but promotes that of BDNF compared with those in the OGD group. The combination of GAS and FPS-ZM1 further decreases the expression of C3. These results indicate that GAS can inhibit the activation of Smad3 through S100B/RAGE signaling and regulate the expression of A1/A2-type astrocytes.

1. Introduction

Hypoxic-ischemic brain damage (HIBD) is a leading cause of neonatal death in children. In addition, it has a high probability of causing significant long-term neurodisabilities, even with treatment [1]. HIBD triggers neuroinflammation induced by resident immune cells, including astrocytes and microglia, as well as that involving peripheral immune responses. Neuroinflammation is a major factor responsible for secondary brain tissue injury, which may even lead to neuronal death [2].

Emerging evidence has shown that astrocytes respond to injury, disease, or infection of the central nervous system (CNS) via morphological, molecular, and functional remodeling and turn into reactive astrocytes [3]. Reactive astrocytes play a dual role: they can promote and limit the inflammatory response triggered by HIBD [4]. On the one hand, they secrete tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which can induce neuronal death and other proinflammatory changes in nerve cells [5,6]. On the other hand, they can exhibit neuroprotective and anti-inflammatory effects by releasing brain-derived neurotrophic factor (BDNF) and IL-10 [7,8]. According to Liddelow et al. [9,10], there are at least two categories of astrocytes: A1 astrocytes (induced by neuroinflammation), which are neurotoxic reactive astrocytes marked by complement-3 (C3), and A2 astrocytes (induced by ischemia), which are neuroprotective reactive astrocytes marked by the S100A10 protein.

S100B is a member of a multigenic family of EF-hand calcium-binding proteins. Its primary expression in astrocytes and secretion into the extracellular space within the CNS have been extensively investigated [11,12]. Previous studies have indicated that nanomolar levels of S100B in the healthy brain can increase neuron survival and promote synapse formation [13]. However, when stimulated by brain injury, S100B can rapidly accumulate at micromolar concentrations and exert completely opposite effects. One example of the known deleterious effects of S100B is that it can induce neuronal apoptosis and hippocampus-dependent spatial learning impairment by interacting with the receptor for advanced glycation end products (RAGE) [14,15]. RAGE is a multiligand pattern recognition receptor that has been demonstrated to bind with several ligands, including advanced glycation end products (AGEs), S100B, high-mobility group box (HMGB1), and β amyloid (Aβ), and is associated with chronic inflammatory states in various diseases [16,17]. Additionally, RAGE can undergo alternative splicing to produce different isoforms at the mRNA level, such as secretory/soluble RAGE (sRAGE) and dominant negative RAGE (DNRAGE), with similar ligand-binding affinities to those of RAGE [18]. In addition, sRAGE can be derived by proteolytic cleavage of the extracellular domain of RAGE [19]. In addition, it can act as a decoy to intercept the interaction of ligands with the cell surface RAGE.