OBJECTIVES:To investigate the neuroprotective effects of formononetin (FMN) against hypoxic-ischemic brain damage (HIBD) in neonatal mice and the underlying mechanism. METHODS:Twenty-four neonatal C57BL/6J mice were randomly divided (n=6) into sham-operated group, HIBD model group, HIBD+FMN-L (50 mg/kg) group, and HIBD+FMN-H (100 mg/kg) group. Mouse models of HIBD were established by left common carotid artery ligation followed by hypoxia (92% N₂, 8% O₂) for 40 min. FMN at the two doses was administered by intraperitoneal injection, and 3 days later, brain tissues from the cortical ischemic penumbra were collected for assessing expressions of ferroptosis-related proteins (P53, SAT1, and ACSL4) using Western blotting and immunofluorescence staining and for detecting the levels of Fe²⁺, superoxide, malondialdehyde (MDA), and glutathione (GSH). In cultured HT22 neurons with oxygen-glucose deprivation (OGD), the effects of 100 μmol/L FMN, 10 μmol/L Nutlin-3 (a P53 agonist), or their combination on expressions of ferroptosis proteins, intracellular Fe²⁺, reactive oxygen species (ROS), lipid peroxidation, GSH, mitochondrial membrane potential, and cell viability were evaluated. RESULTS:In the neonatal mouse models of HIBD, FMN treatment significantly suppressed the protein expression of P53, SAT1, and ACSL4, reduced Fe²⁺, ROS, and MDA levels and increased GSH content in the cortical ischemic penumbra. In HT22 neurons with OGD, FMN obviously alleviated OGD-induced ferroptosis as shown by lowered expressions of the key ferroptosis proteins, reduced Fe²⁺ accumulation and lipid peroxidation, and significant increases of GSH levels, mitochondrial membrane potential, and cell viability. Mechanistic experiments showed that activation of P53 signaling by Nutlin-3 markedly reversed the protective effects of FMN. CONCLUSIONS:FMN produces neuro-protective effects against HIBD in neonatal mice by mitigating neuronal ferroptosis, primarily through downregulation of the P53/SAT1/ACSL4 signaling pathway.

In hypoxic-ischemic brain damage (HIBD), the programmed cell death known as ferroptosis is significantly activated. Microglial cells demonstrate a high level of sensitivity to iron accumulation. Understanding how to regulate the dual role of microglia and transforming the microglial ferroptosis to a moderate and controllable process has considerable implications for the targeted treatment in HIBD. This paper serves as an overview of microglia-mediated ferroptosis in HIBD as a disease model. We discuss various aspects centered around microglia, including pathophysiological mechanisms, polarization and functions of microglia, molecular mechanisms of ferroptosis, signaling pathways, and therapeutic strategies. The review aims to provide a reference for studies of ferroptosis in microglia.

BACKGROUND:Ischemic stroke (IS) remains a major contributor to global morbidity and mortality, largely due to limited therapies and unclear pathogenesis. Gastrodin (GAS), a bioactive ingredient from traditional Chinese medicine, has shown potential in mitigating apoptosis, inflammation, and pyroptosis. Recent research highlights ferroptosis as a crucial factor in IS development. PURPOSE:In this study, we aimed to examine the effect and mechanism of GAS on ferroptosis in IS. METHODS:Mendelian randomization (MR) was applied to evaluate the effect of GAS on IS risk. The neuroprotective and anti-ferroptotic effects of GAS were assessed through TTC staining, neurological deficit scoring, and detection of ferroptosis-related biomarkers. GAS target was predicted via network pharmacology and molecular docking, and validated using surface plasmon resonance and cellular thermal shift assay. Phosphorylation, nuclear translocation, and N⁶-methyladenosine methylation were analyzed using Western blotting, immunofluorescence, and MeRIP-qPCR. RESULTS:MR analyses identified a negative association between GAS and IS. GAS treatments attenuated cerebral infarct volume, improved neurological function, and alleviated ferroptosis both in vivo and in vitro. Mechanistically, GAS binds to PI3K, activating the PI3K/Akt signaling pathway through phosphorylation. Phosphorylated Akt induces serine/threonine phosphorylation and nuclear translocation of Alkbh5, which reduces the m6A methylation levels of glutamate-cysteine ligase modifier subunit (Gclm), resulting in increased Gclm expression and enhanced GSH synthesis, and ultimately contributing to the inhibition of ferroptosis in IS. CONCLUSION:This study is the first to demonstrate that GAS mitigates IS-induced ferroptosis via the PI3K/Akt-Alkbh5-Gclm axis, bridging epigenetic regulation and iron metabolism.

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.

Hypoxic-ischemic brain damage (HIBD) is a significant cause of neonatal death and neurological dysfunction. Following this injury, activated microglia can lead to a series of inflammatory responses. Gastrodin (GAS), a polyphenol extracted from the Chinese herbal medicine Gastrodia elata Blume, has demonstrated antioxidant and anti-inflammatory effects. This study investigated the neuroprotective impact of GAS in HIBD mice model and in BV2 cells subjected to oxygen-glucose deprivation (OGD) treatment. Expression of various members of the Ccr2/Akt/Gsk-3β, including Ccl2, Ccr2, Akt, p-Akt, Gsk-3β, p-Gsk-3β and inflammatory factors TNF-α and IL-1β in activated microglia was assessed by Western blotting, immunofluorescence, and qRT-PCR in HIBD in postnatal mice, and in OGD-induced BV2 microglia in vitro with or without GAS treatment. The present results showed that GAS effectively reduces the expression of Ccl2 and Ccr2, increases the phosphorylation levels of Akt and Gsk-3β, and decreases the expression of the TNF-α and IL-1β. Additionally, we have shown that inhibition of Ccr2 by RS102895 increased the expression of p-Akt and p-Gsk-3β, and attenuate production of proinflammatory mediators in activated microglia. Of note, the expression of p-Akt, p-Gsk-3β, TNF-α and IL-1β remained unchanged after the combination of gastrodin and RS102895. Taken together, we conclude that GAS can play a protective role in reducing the neuroinflammatory response after HIBD. It is suggested that this is mainly through up-regulating the Akt/Gsk-3β signaling pathway via the Ccr2 receptor in the present experimental paradigm.

ABSTRACT Objective Emerging evidence highlights the involvement of long non‐coding RNAs (lncRNAs) and ferroptosis in the pathogenesis of traumatic brain injury (TBI). However, the regulatory role of lncRNAs in TBI‐induced ferroptosis remains poorly understood. This study aims to investigate the role of a specific lncRNA, noncoding transcript of chemokine (C‐C motif) ligand 4 ( Ccl4 ) overlapping ( Ntoco ), in the regulation of ferroptosis following TBI and explore its potential as a therapeutic target. Methods The expression levels of Ntoco following controlled cortical injury (CCI) in mice were measured using real‐time PCR. Behavioral tests post‐injury were assessed using the rotarod test and Morris water maze, and lesion volume was evaluated using micro‐MRI. Ntoco binding proteins were identified using RNA pull‐down and RNA immunoprecipitation. RNA sequencing was employed to identify Ntoco ‐related pathways. Western blotting and co‐immunoprecipitation were used to measure protein levels and ubiquitination processes. Results Ntoco upregulation was observed in CCI mice. Ntoco knockdown inhibited neuron ferroptosis, reduced lesion volume, and improved spatial memory following TBI. Ntoco overexpression promoted ferroptosis in neurons. Mechanistically, Ntoco facilitated K48‐linked ubiquitination and degradation of proteins by binding to Hnrnpab, suppressing the NF‐κB/Lcn2 signaling pathway. This included reduced phosphorylation of IkBα, increased phosphorylation of IKKα/β, nuclear translocation of the NF‐κB p65 subunit, and elevated Lcn2 expression. Conclusion Our findings suggest that Ntoco plays a crucial role in TBI‐induced ferroptosis by modulating the NF‐κB/Lcn2 signaling pathway. Targeting Ntoco may provide a promising therapeutic strategy to mitigate ferroptosis and improve outcomes following TBI.

Gastrodin, an anti-inflammatory herbal agent, is known to suppress microglia activation. Here, we investigated whether it would exert a similar effect in reactive astrocytes and whether it might act through the renin-angiotensin system (RAS) and sirtuin 3 (SIRT3). Angiotensinogen (ATO), angiotensin-converting enzyme (ACE), angiotensin II type 1 (AT1) and type 2 (AT2) receptor and SIRT3 expression was detected in TNC-1 astrocytes treated with BV-2 microglia conditioned medium (CM) with or without gastrodin and lipopolysaccharide (LPS) pre-treatment by RT-PCR, immunofluorescence and western blotting analysis. Expression of C3 (A1 astrocyte marker), S100A10 (A2 astrocyte marker), proinflammatory cytokines and neurotrophic factors was then evaluated. The results showed a significant increase of ATO, ACE, AT1, SIRT3, C3, proinflammatory cytokines and neurotrophic factors expression in TNC-1 astrocytes incubated in CM + LPS when compared with cells incubated in the CM, but AT2 and S100A10 expression was reduced. TNC-1 astrocytes responded vigorously to BV-2 CM treated with gastrodin + LPS as compared with the control. This was evident by the decreased expression of the abovementioned protein markers, except for AT2 and S100A10. Interestingly, SIRT3, IGF-1 and BDNF expression was enhanced, suggesting that gastrodin inhibited the expression of RAS and proinflammatory mediators but promoted the expression of neurotrophic factors. And gastrodin regulated the phenotypic changes of astrocytes through AT1. Additionally, azilsartan (a specific inhibitor of AT1) inhibited the expression of C3 and S100A10, which remained unaffected in gastrodin and azilsartan combination treatment. These findings provide evidence that gastrodin may have a therapeutic effect via regulating RAS-SIRT3.

Objective Formononetin (FOR), a traditional Chinese medicine, has been widely used for nerve protection and nerve function rehabilitation after cerebral stroke. However, the role of FOR in autophagic lysosome function in cerebral ischemia reperfusion damage has not been investigated. This study aimed to explore whether the therapeutic benefits of FOR were influenced by the regulation of autophagy flux. Methods Male Sprague-Dawley rats were separated into sham, model, and MCAO+FOR (30 mg/kg) groups after undergoing middle cerebral artery occlusion (MCAO) and ischemia-reperfusion (I/R). Then, the brain tissues in the ischemic penumbra were obtained to detect the proteins in autophagic/lysosomal pathway with antibodies of Beclin-1, LC3, SQSTM1/P62, Ubiquitin, LAMP-2, Cathepsin B (CTSB) and Cathepsin D (CTSD) by Western blot and immunofluorescence, respectively. Meanwhile, the therapeutic effectiveness was evaluated by measuring infarct volume, neurological impairments, and neuronal necrosis. Results The findings of this study demonstrate that FOR treatment exhibits a dual effect by enhancing the autophagic activities of Beclin-1 and LC3 in neurons, while simultaneously improving the autophagic clearance function, as evidenced by reinforced lysosomal activities of LAMP-2, CTSB, and CTSD, as well as reduced autophagic accumulation of Ubiquitin and P62 in the MCAO+FOR group compared to the MCAO group. Additionally, 7 d of FOR treatment dramatically reduced neurological deficits, infarct volume, and neuronal death caused by cerebral ischemia. Conclusion These findings suggest that the neuroprotective mechanism of FOR therapy in accelerating recovery from ischemic stroke may involve the increase of autophagy flux in the penumbra.

Activated microglia and their mediated inflammatory responses play an important role in the pathogenesis of hypoxic-ischemic brain damage (HIBD). Therefore, regulating microglia activation is considered a potential therapeutic strategy. The neuroprotective effects of gastrodin were evaluated in HIBD model mice, and in oxygen glucose deprivation (OGD)-treated and lipopolysaccharide (LPS)activated BV-2 microglia cells. The potential molecular mechanism was investigated using western blotting, immunofluorescence labeling, quantitative realtime reverse transcriptase polymerase chain reaction, and flow cytometry. Herein, we found that PI3K/AKT signaling can regulate Sirt3 in activated microglia, but not reciprocally. And gastrodin exerts anti-inflammatory and antiapoptotic effects through the PI3K/AKT-Sirt3 signaling pathway. In addition, gastrodin could promote FOXO3a phosphorylation, and inhibit ROS production in LPSactivated BV-2 microglia. Moreover, the level P-FOXO3a decreased significantly in Sirt3-siRNA group. However, there was no significant change after gastrodin and siRNA combination treatment. Notably, gastrodin might also affect the production of ROS in activated microglia by regulating the level of P-FOXO3a via Sirt3. Together, this study highlighted the neuroprotective role of PI3K/AKT-Sirt3 axis in HIBD, and the anti-inflammatory, anti-apoptotic, and anti-oxidative stress effects of gastrodin on HIBD.

目的:研究天麻素(GAS)对氧糖剥夺(OGD)激活的BV2细胞Notch通路表达的影响.方法:BV2细胞分成4组,分别为对照组(control)、氧糖剥夺组(OGD)、OGD联合0.17 mmol/L GAS处理组(OGD+GAS-L),和OGD联合0.34 mmol/L GAS处理组(OGD+GAS-H).免疫荧光双标染色和Western Blot检测Notch通路相关蛋白Notch-1、NICD、RBP-JK和Hes-1的表达变化.结果:与control组相比,OGD组中的Notch-1、NICD、RBP-JK和Hes-1表达明显增高(P＜0.05);GAS干预后上述蛋白表达均明显降低(P＜0.05);而不同浓度的GAS治疗组之间无明显差异(P＞0.05).结论:GAS能抑制OGD诱导的BV2细胞中Notch通路相关分子的表达.

Notch-1 and renin angiotensin system (RAS) are involved in microglia activation. It has been reported that gastrodin inhibited inflammatory responses mediated by activated microglia. This study explored the possible interaction between this two pathways, and to determine whether gastrodin would exert its effects on both of them. Expression of RAS, Notch-1 signaling and proinflammatory mediators in lipopolysaccharide (LPS) activated BV-2 microglia subjected to various treatments was determined by Western blot and immunofluorescence. The protein expression of RAS, Notch-1 pathway and TNF-α and IL-1β was significantly increased in activated microglia. Exogenous Ang II markedly enhanced the expression of these biomarkers. Meanwhile, Azilsartan [a specific inhibitor of AT1 (AT1I)] inhibited the expression of Notch-1 pathway and proinflammatory cytokines. When Notch-1 signaling was inhibited with DAPT, ACE and AT1 expression remained unaffected, indicating that RAS can regulate the Notch-1 pathway in activated microglia but not reciprocally. Additionally, we showed here that gastrodin inhibited the RAS, Notch-1 pathway and inflammatory response. Remarkably, gastrodin did not exert any effect on expression of Notch-1 signaling when RAS was blocked by AT1I, suggesting that gastrodin acts on the RAS directly, not through the Notch-1 pathway. Furthermore, TNF-α and IL-1β expression was significantly increased in activated microglia treated with exogenous Ang II; the expression, however, was suppressed by gastrodin. Of note, expression of proinflammatory cytokines was further decreased in gastrodin and AT1I combination treatment. The results suggest that gastrodin acts via the RAS which regulates the Notch-1 signaling and inflammation in LPS-induced microglia.

目的:研究天麻素(GAS)对新生大鼠缺血缺氧性脑损伤后脑内激活的小胶质细胞表达NADPH氧化酶2(NOX2)的影响.方法:将39只3 d龄新生SD大鼠随机分为假手术组(sham)、缺血缺氧模型组(HIBD)、以及HIBD与GAS联合处理组(HIBD+GAS).利用左侧颈总动脉结扎结合缺氧箱(8％O2,92％N2)缺氧2 h制备HIBD模型.采用Western Blot和免疫荧光双标染色检测大鼠左侧胼胝体区NOX2的表达.结果:与sham组相比,HIBD组大鼠胼胝体区NOX2表达明显增高(P＜0.05),并且主要表达在活化的小胶质细胞;GAS处理后HIBD大鼠胼胝体区小胶质细胞活化减弱,同时NOX2表达降低(P＜0.05).结论:GAS可能通过降低新生大鼠脑内小胶质细胞NOX2的表达,发挥其神经保护作用.