Introduction
Significant advances have been made to lessen the immense public health problem of stroke. Endeavors in prevention have brought down stroke incidence and mortality, and the establishment of exceptional intensive care units has ameliorated the functional consequence of stroke victims [
1,
2]. However, limited approaches have been made in developing therapies to decrease the detrimental effects of cerebral ischemia. It has been demonstrated that the brain has a marvelous capacity for self-preservation, elucidated by the protective responses induced by ischemia and preconditioning [
2]. Understanding the mechanisms underlying these protective actions will help to minimize ischemic brain injury.
Minocycline, a tetracycline antibiotic, manifests anti-inflammatory, anti-apoptotic, and neuroprotective effects independent of its broad-spectrum antimicrobial activity [
3-
7]. Its superior penetration to the brain tissue, good clinical safety, and prolonged therapeutic window makes it an ideal candidate for application in the treatment of stroke [
8]. Although the observed neuroprotection of minocycline in ischemic stroke reflects some properties that mimic selected features of endogenous neuroprotection, it is still unclear whether minocycline acts solely by engaging endogenous neuroprotective mechanisms [
2].
Monocyte chemotactic protein-induced protein 1 (MCPIP1) was discovered as an inducible protein expressed in human peripheral blood monocytes treated with monocyte chemotactic protein 1 (MCP-1) [
9]. Our previous reports demonstrated that MCPIP1 was a negative regulator of macrophage activation and that MCPIP1 inhibited the production of proinflammatory cytokines, such as TNFα, IL-1β, IL-6, and MCP-1, by inhibiting the activity of JNK and NF-κB proinflammatory signal pathways [
10,
11]. Our further studies indicated that MCPIP1 was also inducibly expressed in macrophages, endothelial cells, and microglia with LPS stimulation [
12-
14], and MCPIP1 participates in LPS preconditioning/electroacupuncture-pretreatment-induced ischemic brain tolerance [
14,
15]. The present study examined whether MCPIP1 is involved in minocycline-treatment-induced neuroprotection against cerebral ischemia/reperfusion injury in neuronal cultures and
in vivo using MCPIP1-deficient mice.
Discussion
Many studies have shown that doxycycline and minocycline play anti-inflammatory and neuroprotective effects in ischemic brain injury. This action is completely independent and distinct from their antimicrobial action [
3,
8,
19-
22]. It has been well known that ischemic stroke includes secondary inflammation that plays a significant part in the devastating consequences of ischemic insult [
23-
27]. Although numerous studies have shown that minocycline can provide significant neuroprotection against ischemic brain injury and inhibit inflammatory responses such as microglial activation and proinflammatory cytokine generation [
3,
27-
29], the molecular mechanisms that contribute to the alleviation of brain inflammation after ischemia by minocycline are not well understood. The present study is the first to reveal the role of MCPIP1 in minocycline-treatment-induced cerebral ischemic protection. MCPIP1 was first identified as a protein induced in human peripheral blood monocyte by the chemokine MCP-1 [
9]. MCPIP1 has been demonstrated to have the ability to suppress inflammation [
10,
11,
30]. MCPIP1 negatively affects inflammatory gene expression and NF-κB activation in response to LPS [
10,
11]. In our previous studies, MCPIP1 was also found to be inducibly expressed in macrophages, macroglia, and endothelial cells with LPS stimulation [
12-
14]. It was also shown that MCPIP1 participates in LPS and electroacupuncture preconditioning-induced ischemic brain tolerance [
14]. The recent study [
31] showed that MCPIP1 can be translationally controlled by some other proinflammatory cytokines such as IL-17. All of these investigations indicated that MCPIP1 may act as an inducible endogenous negative feedback regulator of inflammation and could play a highly beneficial role in human inflammation-related diseases including stroke. In the present study, we found that MCPIP1 was significantly induced in mouse brain by minocycline treatment, which suggested that MCPIP1 may be involved in minocycline-induced neuroprotection after ischemic stroke. In addition, we observed that brain infarct volumes, brain edema, and neurological functions after stroke were significantly alleviated by minocycline treatment in wild type mice, whereas in MCPIP1-deficient mice, minocycline treatment failed to improve these outcomes of ischemia, which indicated that MCPIP1 may participate in minocycline-induced neuroprotection in ischemic stroke. Many studies have demonstrated that minocycline is an ideal therapeutic agent for stroke. Our results show that administration of minocycline before or after stroke afforded protection against stroke damage. Of course, the post-treatment paradigm of minocycline indicates more clinical significance in its potential therapeutic application for stroke in the future. The novelty of the present study is that MCPIP1 mediates minocycline-induced neuroprotection against cerebral ischemia/reperfusion injury whether the drug is ministered before or after stroke. In both cases, minocycline induced MCPIP1 in the brain and afforded protection against ischemic brain damage.
It has been well established that proinflammatory gene expression leads to stroke damage [
25,
32]. During brain ischemia, proinflammatory cytokines such as TNF-α, IL-1β, IL-6, and chemokines such as CINC and MCP-1 are produced by a variety of activated cell types, including endothelial cells, microglia, astrocytes, and neurons [
32]. Blocking the production of these proinflammatory cytokines would be an important strategy to protect against ischemia brain injury. In this study, we observed that TNFα, IL-1β, IL-6, and MCP-1 expressions were significantly reduced at 24 h after MCAO by minocycline treatment. In MCPIP1-deficient mice, minocycline treatment failed to suppress the production of such cytokine. Furthermore, we found that expression of MCPIP1 protein was upregulated by minocycline treatment mainly in neurons and microglia, which are the main source of proinflammatory cytokines during ischemia. These results implied that MCPIP1 induction by minocycline may play a beneficial role by inhibiting the generation of proinflammatory cytokines in neurons and microglia during brain ischemia. Activation of NF-κB-signaling pathways leads to inflammatory cytokine production [
11]. In our previous study, we found that MCPIP1 can also act as a deubiquitinase to negatively regulate NF-κB signaling by targeting TNF receptor-associated factors (TRAFs) [
11,
33]. In the present study, we found that phosphorylation of p-65 was significantly reduced at 24 h after MCAO in minocycline-treated wild type mice compared to that of the control. In MCPIP1-deficient mice, there was no significant difference on p-65 phosphorylation level between the minocycline-treated and control group without minocycline treatment. Our study suggested that enhanced activation of NF-κB-signaling pathway in MCPIP1-deficient mice leads to increased proinflammatory cytokine production after brain ischemia with minocycline treatment and that MCPIP1 is involved in minocycline-treatment-induced inhibition of the NF-κB-signaling pathway after ischemic stroke. In the present study, minocycline treatment showed significant neuroprotection in mice subjected to focal brain ischemia by MCAO, which is consistent with other reports. However, we found there was significant loss of minocycline-treatment-induced ischemic brain tolerance in MCPIP1-deficient mice after ischemic stroke, which indicated that MCPIP1 may mediate the beneficial role in minocycline-treatment-induced neuroprotection after focal brain ischemia.
It is possible that the absence of MCPIP1 in the mice creates systemic inflammation that minocycline treatment cannot overcome. However, our
in vitro studies in mixed neuron-glia cells and primary cortical neurons indicated that minocycline-pretreatment-induced neuroprotection is, at least partly, via MCPIP1, as absence of MCPIP1 in mixed neuron-glia cells and knockdown of MCPIP1 in primary cortical neurons with OGD treatment resulted in a loss of minocycline-induced protection, which could largely exclude the potential affects of systemic consequence resulting from
in vivo studies of general knockout mice. Considering the other experimental results in this study, including that MCPIP1 can be significantly induced by minocycline treatment in the brain, and that MCPIP1 has been identified as an important inducible anti-inflammatory regulator in stroke pathophysiology [
11], it would appear more likely that MCPIP1 actually participates in the minocycline-treatment-induced neuroprotection. Mice with brain-specific MCPIP1 deficiency might provide a suitable model to explore the role of MCPIP1 in ischemic-stroke-induced brain damage without the complication caused by the general knockout of MCPIP1. Based on our
in vivo and
in vitro studies, we may conclude that MCPIP1 induction is involved in minocycline-induced neuroprotection from ischemic brain injury.
All these findings indicate that the endogenous inducible MCPIP1 protein may serve as an important common shared mediator which performs negative feedback to reduce the inflammatory responses caused by a wide range of hazardous stresses.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JL designed the experiments, performed all experiments, analyzed the data, generated the figures, and wrote the manuscript. ZJ did all experiments. JW did parts of the animal surgery and performed the experiments. PEK provided advice in the design of the study and in interpreting of the data and revising the manuscript. All authors have read and approved the manuscript.