Zinc (Zn²⁺) is essential for normal cellular functions and plays a signaling role in the brain (Qi et al., 2016; Mutlu and Baltaci, 2020; Alvarez et al., 2021). Zn²⁺ influx from the extracellular space and its mobilization from intracellular pools such as mitochondria, lysosomes, and cytosolic Zn²⁺ binding proteins lead to neuronal damage (Galasso and Dyck, 2007; Sensi et al., 2011). A growing number of basic and clinical experiments have suggested that cerebral ischemia/reperfusion (I/R) caused cellular injury resulting from activation of Zn²⁺ accumulation (Guo et al., 2019; Zhao et al., 2021; Manzanero et al., 2013). Removing Zn²⁺ with a specific Zn²⁺ chelator N,N,N’,N’-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) reduced Zn²⁺ accumulation in ischemic neurons, rescued them from cell death and improved functional outcomes (Qi et al., 2019). These findings implicate that Zn²⁺ acts as a critical mediator of neuronal death at high concentrations in the ischemic condition. In recent study, we showed that the overload of Zn²⁺ in mitochondria disrupted the function of mitochondria, resulting in reactive oxygen species generation and triggering cell death (Zhao et al., 2018; Qi et al., 2019). Reducing Zn²⁺ accumulation in mitochondria contributes to decreased cerebral ischemic injury by normobaric hyperoxia treatment (Dong et al., 2015). We also found that intracellular accumulated Zn²⁺ aggravates I/R injury through inducing endoplasmic reticulum stress (Zhao et al., 2022). However, the reason why Zn²⁺ produces severe brain damage remains to be elucidated.

Inflammation and immune responses have been proven to be crucial factors associated with stroke onset and progression (Lively et al., 2016; Li T. et al., 2022). In recent years, several cytokines have been proven especially promising as potential therapeutic targets for experimental ischemic stroke including the tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and the anti-inflammatory cytokine-10 (IL-10; Zhang et al., 2017; Lambertsen et al., 2019). TNF-α is a proinflammatory factor and works as an initiation factor of inflammatory response. The expression of TNF-α is rapidly upregulated after cerebral ischemia, leading to neurotoxicity (Vila et al., 2000; Shen et al., 2022). As a nuclear transcription factor, nuclear factor kappa B (NF-κB) constitutes the most basic NF-κB signaling pathway with IκB and IκB kinase complex, which is involved in the regulation of inflammation (Chen et al., 1998; Berti et al., 2002). In I/R injury, NF-κB is activated by a variety of stimuli including TNF-α (Jarosz et al., 2017; Kang et al., 2019). Production of TNF-α and activation of NF-κB have been documented to play critical roles in the process of cerebral disease (Hussain et al., 2016). Besides, the activation of NF-κB is also regulated by IL-6 (Jarosz et al., 2017; Kang et al., 2019). IL-6 is a pleiotropic cytokine involved in many central nervous system disorders including stroke. The expression of IL-6 is most prominently identified in neurons in the peri-ischemic regions (Kumari et al., 2016; Rasmussen et al., 2019; Ridwan et al., 2021). In contrast to IL-6, IL-10 is a pleiotropic anti-inflammatory cytokine, which binds to IL-10 receptors to reduce inflammation and limit apoptosis (Chen et al., 2013). Therapeutic administration of IL-10 has been shown to be neuroprotective in experimental stroke and to limit post-stroke inflammation (Chen et al., 2013; Garcia et al., 2017).

The immune system, especially the inflammation, is markedly susceptible to changes of Zn²⁺ levels (Haase and Rink, 2014). Zn²⁺ induces the synthesis of DNA, RNA, and proteins to meet the desired immune response (Baltaci et al., 2019). Recent reports have indicated that Zn²⁺ homeostasis affects neuroinflammation in the brain (Baltaci et al., 2022). Intracellular Zn²⁺ signals are required for the production of proinflammatory cytokines IL-6 and TNF-α, which are also directly induced by incubation with high extracellular Zn²⁺ concentrations (Haase and Rink, 2014; Olechnowicz et al., 2018). Also, in type 2 diabetes mellitus patients, impaired Zn²⁺ homeostasis leads to uncontrolled expression of immune mediators, such as IL-6 and NF-κB, which simultaneously exacerbate the immune response (Bonaventura et al., 2015; Olechnowicz et al., 2018). Zn²⁺ is necessary for the activation of NF-κB signaling pathway, whereas chelating Zn²⁺ with membrane permeable Zn²⁺ specific chelator TPEN completely blocked this pathway (Wang et al., 2015; Jarosz et al., 2017). However, in vitro, Zn²⁺ augments monocyte adhesion to endothelial cells, and its deficiency increases the production of proinflammatory cytokines (Jarosz et al., 2017). Therefore, the relationship between Zn²⁺ and inflammation is still under debate, especially in cerebral I/R. The detailed relationship between Zn²⁺ accumulation and inflammation in cerebral I/R needs to be further studied.

To investigate the interaction between Zn²⁺ accumulation and inflammation in the ischemic brain, a rat model of focal cerebral I/R was used in this study. We hypothesize that Zn²⁺ accumulation could exert neurotoxicity effect by promoting inflammation responses in ischemic penumbra of middle cerebral artery occlusion (MCAO) rats. These results provide a novel mechanism for the toxic effect of Zn²⁺ on cerebral I/R injury.

In the present study, we investigated whether the Zn²⁺ induces ischemic brain injury by regulating inflammation. We demonstrated that a drastic reperfusion time-dependent increase of proinflammatory cytokines TNF-α, IL-6, and NF-κB p65 nuclear translocation in MCAO rats at 6 and 24 h after reperfusion, while IκB-α, the inhibitory protein of NF-κB, as well as the anti-inflammatory cytokines IL-10 decreased in the ischemic brain tissue of MCAO rats, indicating that cerebral I/R injury triggers inflammation. Importantly, TNF-α-positive cells were colocalized with Zn²⁺ indicator, NG. Treatment with Zn²⁺ chelator TPEN leads to a significant reduction in TNF-α, IL-6, NF-κB p65 expression and an increase in IκB-α and IL-10 expression, indicating that cytosolic labile Zn²⁺ accumulation promotes inflammation following I/R injury. Our results also revealed that TNF-α, NF-κB p65, and IL-10 were colocalized with NeuN-positive cells respectively, and IL-6-positive cells were largely colocalized with TUNEL, indicating that inflammation occurs in neurons of cerebral ischemic rats, which contributes to neuronal death of the brain after cerebral I/R. Taken together, the present study demonstrated that excessive Zn²⁺ accumulation following cerebral ischemia could activate inflammatory mediators and inflammation-mediated neuronal apoptosis, which is a novel mechanism of cerebral ischemic injury.

The inflammatory response was characterized by neutrophil accumulation and proinflammatory factors release (Zhou et al., 2023). It has been reported that neutrophils begin to enter the cerebral cortex through leptomeninges after 6 h of MCAO (Kim et al., 2019), followed by neuronal apoptosis at 24 h after reperfusion (Zhao et al., 2018). Therefore, the expression changes of proinflammatory cytokines and anti-inflammatory cytokine in ischemic rats were studied at 6 and 24 h after reperfusion in this study. TNF-α is a key mediator in neuronal immunomodulatory system, and its abnormal expression plays an important role in various pathological processes (Lambertsen et al., 2012; Li B. et al., 2022). In the present study, very few TNF-α-positive cells were observed in the brain of the sham group, whereas TNF-α-positive cells was gradually upregulated in the ischemic penumbra of MCAO rats at 6 and 24 h after reperfusion, indicating that cerebral I/R resulted in increased expression of TNF-α. Under physiological conditions, Zn²⁺ has been demonstrated to decrease the generation of TNF-α (Prasad et al., 2011; Prasad, 2012; Eddie-Amadi et al., 2022). However, high concentrations of Zn²⁺ in septic shock have been reported to promote the expression of endotoxin-induced TNF-α produced by human monocytes (von Bulow et al., 2007). Our previous study showed that intracellular accumulated Zn²⁺ induced cerebral I/R injury, and chelating Zn²⁺ significantly reduced ischemia-induced cerebral infarct (Zhao et al., 2018). In the present study, we found that TNF-α colocalized with NG and TPEN treatment significantly suppressed the expression of TNF-α in the ischemic penumbra of MCAO rats at 24 h after reperfusion, implying that chelating Zn²⁺ reduces ischemic injury by decreasing the expression of TNF-α in ischemic tissue. Therefore, we speculated that excessive Zn²⁺ accumulation following cerebral I/R might lead to the upregulation of TNF-α, which in turn promote inflammation-mediated brain damage.

NF-κB is a dimer redox-sensitive transcription factor consisting of p50 and p65, and its transcriptional activity is silenced by IκB (Perkins, 2007). Under normal conditions, the NF-κB complex binds to IκB and is sequestered in the cytoplasm. Matsui et al. found that NF-κB is activated after I/R injury in animals (Matsui et al., 2005). As a sequence-specific DNA binding protein, NF-κB was previously reported to be Zn²⁺ dependent (Zabel et al., 1991). Recent studies further reported that Zn²⁺ regulates the activation of NF-κB by acting on TNF-α transcription (Haase et al., 2008). Following stimulation with TNF-α, IκB kinase is activated. The NF-κB dimer becomes active when activated IκB kinase phosphorylates IκB, which leads to ubiquitination and proteasomal degradation of IκB (Chen et al., 1998; Haase et al., 2008). Thus, in the present study, the decreased expression of IκB-α in MCAO rats at 6 and 24 h after reperfusion is possibly due to the increased expression of TNF-α. Chelating Zn²⁺ increased the IκB-α level in MCAO rats, suggesting that Zn²⁺ accumulation-induced increased TNF-α expression might lead to the degradation of IκB-α following cerebral I/R. With the degradation of IκB, NF-κB is released into the nucleus, leading to inflammatory response (Huang and Hung, 2013). In current study, we found that NF-κB p65-positive cells were colocalized with NeuN, NF-κB p65 mostly translocated to the neuronal nucleus in the ischemic penumbra of MCAO rats at 6 and 24 h after reperfusion, suggesting that NF-κB pathway is activated in neurons of ischemic rats. Moreover, nuclear NF-κB p65-positive cells were attenuated by TPEN treatment after reperfusion. Therefore, we speculated that excessive Zn²⁺ accumulation upregulates the expression of TNF-α, which in turn downregulates IκB, leading to the activation of NF-κB to promote neuronal apoptosis following I/R injury.

IL-6 is commonly regarded as a prominent mediator of the inflammatory response, which is involved in the elicitation of acute-phase inflammatory reactions/responses (Kumari et al., 2016; Rasmussen et al., 2019; Ridwan et al., 2021). During inflammatory response, IL-6 expression might be activated by TNF-α (Kumari et al., 2016; Hotter et al., 2019; Jenny et al., 2019; Lambertsen et al., 2019). Besides, it has been reported that Zn²⁺ levels affect IL-6 in DMBA-induced breast cancer in female rats (Gulbahce-Mutlu et al., 2021). In the present study, we found a reperfusion time-dependent increase in IL-6 expression in the ischemic penumbra of MCAO rats, which shows a similar trend with TNF-α. TPEN treatment decreased the expression of IL-6 in ischemic penumbra of MCAO rats at 6 and 24 h after reperfusion, indicating that inflammatory factor levels is associated with the reperfusion time in MCAO rats and the zinc-induced increased expression of TNF-α promotes the expression of IL-6, aggravating the inflammatory response of ischemic tissue. In addition, the results of this study showed that IL-6-positive cells were colocalized with TUNEL at 24 h after reperfusion, suggesting that Zn²⁺ accumulation exacerbates I/R injury through inflammatory response and inflammation-mediated neuronal apoptosis.

IL-10 inhibits the production of proinflammatory cytokines as shown by the development of hyper-inflammation and autoimmunity in IL-10-deficient mice (Frangogiannis et al., 2000; Yao et al., 2008; Jung et al., 2017). In animal models of cerebral ischemia, IL-10 significantly reduced infarct size and neurological deficits (Ooboshi et al., 2005; Jung et al., 2017). Our study found that IL-10-positive cells were largely colocalized with NeuN, and IL-10 expression decreased at 24 h after I/R compared with the sham group, indicating that along with the expression of proinflammatory factors increasing, the expression of anti-inflammatory factors decreased after cerebral ischemia. Besides, we found an obvious increase in IL-10 levels after TPEN treatment, suggesting that inhibition of Zn²⁺ accumulation has anti-inflammatory effect. Therefore, we speculated that high concentrations of Zn²⁺ might influence the expression of anti-inflammatory factors to promote inflammatory response, which in turn aggravates I/R neuronal injury. Although we demonstrated that ischemia-induced Zn²⁺ accumulation in MCAO rats could exacerbate cerebral I/R injury by promoting inflammation, further studies on the cellular and ionic mechanisms underlying the effects of Zn²⁺ on inflammatory factors should be investigated.

In summary, our results clearly demonstrated that cerebral I/R injury induced the upregulation of proinflammatory factors at 6 and 24 h post reperfusion, and downregulated anti-inflammatory factor at 24 h post reperfusion. More importantly, ischemia induced high concentration of Zn²⁺ caused inflammatory apoptosis in neurons, indicating that Zn²⁺ accumulation is an important inducement of inflammatory response and brain injury in ischemic stroke. Our study provided the evidence that Zn²⁺ accumulation following cerebral ischemia induced neuronal inflammation, and inflammation-mediated neuronal apoptosis, which provides an important mechanism of brain injury by Zn²⁺ following cerebral ischemia.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article.

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Xuanwu Hospital of Capital Medical University.

WL: investigation, methodology, visualization, writing—original draft. XY: data curation, visualization, methodology, and investigation. MD: data curation, resources, and investigation. WS: investigation, writing—review and editing. YH: investigation, resources. QA: investigation. ZQ: supervision, writing—review and editing. YZ: conceptualization, supervision, resources, methodology, writing—review and editing. All authors contributed to the article and approved the submitted version.

This study was supported by grants from the National Natural Science Foundation of China (No. 81971095, 82271308).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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