MCP-induced protein 1 mediates the minocycline-induced neuroprotection against cerebral ischemia/reperfusion injury in vitro and in vivo

Abstract

Background: Minocycline, a broad-spectrum tetracycline antibiotic, has shown anti-inflammatory and neuroprotective effects in ischemic brain injury. The present study seeks to determine whether monocyte chemotactic protein-induced protein 1 (MCPIP1), a recently identified modulator of inflammatory reactions, is involved in the cerebral neuroprotection conferred by minocycline treatment in the animal model of focal cerebral ischemia and to elucidate the mechanisms of minocycline-induced ischemic brain tolerance.

Methods: Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 2 h in male C57BL/6 mice and MCPIP1 knockout mice followed by 24- or 48-h reperfusion. Twelve hours before ischemia or 2 h after MCAO, mice were injected intraperitoneally with 90 mg/kg of minocycline hydrochloride. Thereafter, the animals were injected twice a day, at a dose of 90 mg/kg after ischemia until sacrificed. Transcription and expression of MCPIP1 gene was monitored by quantitative real-time PCR (qRT-PCR), Western blot, and immunohistochemistry. The neurobehavioral scores, infarction volumes, and proinflammatory cytokines in brain and NF-κB signaling were evaluated after ischemia/reperfusion.

Results: MCPIP1 protein and mRNA levels significantly increased in mouse brain undergoing minocycline pretreatment. Minocycline treatment significantly attenuated the infarct volume, neurological deficits, and upregulation of proinflammatory cytokines in the brain of wild type mice after MCAO. MCPIP1-deficient mice failed to evoke minocycline-treatment-induced tolerance compared with that of the control MCPIP1-deficient group without minocycline treatment. Similarly, in vitro data showed that minocycline significantly induced the expression of MCPIP1 in primary neuron-glial cells, cortical neurons, and reduced oxygen glucose deprivation (OGD)-induced cell death. The absence of MCPIP1 blocked minocycline-induced protection on neuron-glial cells and cortical neurons treated with OGD.

Conclusions: Our in vitro and in vivo studies demonstrate that MCPIP1 is an important mediator of minocycline-induced protection from brain ischemia.

Figures

Figure 1

Minocycline-treatment-induced MCPIP1 in the brain. (A) MCPIP1 mRNA expression in mouse brain by minocycline treatment as measured by qRT-PCR. Values represent mean ± SD, *P < 0.05, #P < 0.01, §P < 0.001 versus control. (B) MCPIP1 protein levels in mouse brain by minocycline treatment as measured by Western blot. Results are representative of three independent experiments. *P < 0.05 versus control. Values represent mean ± SD. MCPIP1, monocyte chemotactic protein-induced protein 1; qRT-PCR, quantitative real-time PCR.

Figure 2

MCPIP1 is expressed in neurons and microglia. Co-localization of MCPIP1 expression (A, D, G) of NSE (B), CD11b (E), and GFAP (H) at 24 h after minocycline treatment. Yellow fluorescence indicates co-localization of MCPIP1/NSE (C) and MCPIP1/CD11b (F). MCPIP1/GFAP (I) showed no yellow-fluorescent structures. n = 5 mice per group. GFAP, glial fibrillary acidic protein; MCPIP1, monocyte chemotactic protein-induced protein 1; NSE, neuron-specific enolase.

Figure 3

Reduction in infarct size and improvement of neurological function by minocycline treatment in the wild type, but not in MCPIP1-deficient mice. The brain infarct size was assessed 48 h after MCAO. (A) Infarct images obtained by TTC staining at 48 h after MCAO. The normal tissue was stained deep red and the infarct was stained milky. (B) Brain infarcts were quantified as percentage area of ischemic hemisphere. The infarct size of minocycline-pretreated wild type mice was significantly reduced compared to that of the control. There was no significant difference in brain infarct size between the minocycline-pretreated and control in MCPIP1-deficient mice. (C) Brain water content as a measure of brain edema of the ischemic hemisphere. The brain edema was significantly reduced at 48 h after MCAO in minocycline-pretreated wild type mice compared to that of the control group. In the MCPIP1-deficient mice, there was no significant difference in brain water content between minocycline-pretreated and control group without minocycline treatment. (D) Neurological function assessment was performed 24 h after MCAO. The neurological scores of minocycline-pretreated wild type mice were significantly improved compared to that of the control. In MCPIP1-deficient mice, there was no significant difference in neurological deficits between minocycline-pretreated and control group. Values represent mean ± SD, *P < 0.05, n = 10 mice per group. MCAO, middle cerebral artery occlusion; MCPIP1, monocyte chemotactic protein-induced protein 1; TTC, 2,3,5-triphenyltetrazolium chloride.

Figure 4

Reduction in infarct size and improvement of neurological function by 2-h post-treatment of minocycline on stroke in the wild type, but not in MCPIP1-deficient mice. (A) The results showed that the infarct size of minocycline-treated wild type mice was significantly reduced compared to that of the control (40.3% ± 6.1% versus 23.3% ± 4.7%, respectively; values represent mean ± SD, *P < 0.05, n = 10 mice per group). MCPIP1-deficient mice failed to evoke minocycline treatment-induced neuroprotection compared with that of control MCPIP1 knockout group without minocycline treatment (55.1% ± 4.9% versus 52.8% ± 5.3%). There was no significant difference in brain infarct size between minocycline-treated and control in MCPIP1 knockout mice. (B) The neurological functions of mice were determined, and the results showed that the neurological scores of minocycline-treated wild type mice were significantly improved compared to that of the control. In MCPIP1-deficient mice, there was no significant difference in neurological deficits between minocycline-treated and control group without minocycline treatment. MCPIP1, monocyte chemotactic protein-induced protein 1.

Figure 5

Reduction in inflammatory cytokine expression in ischemic brain by minocycline treatment in the wild type, but not in MCPIP1-deficient mice. The results showed that the expression levels of TNFα, IL-1β, IL-6, and MCP-1 were significantly elevated in shams in MCPIP1-deficient mice than that of wild type and that the expression levels of TNFα, IL-1β, IL-6, and MCP-1 were significantly reduced at 24 h after MCAO in minocycline-pretreated wild type mice compared to that of the control. In the MCPIP1-deficient mice, there was no significant difference in proinflammatory cytokine expression between minocycline-pretreated and control group without minocycline treatment. The MCPIP1 mRNA level in mouse brain was significantly induced by minocycline treatment compared to controls in wild type mice. Significant increase of MCPIP1 transcript level was detected at 24 h after minocycline treatment after ischemic stroke (P < 0.01). Values represent mean ± SD, n = 6 mice per group. MCPIP1, monocyte chemotactic protein-induced protein 1; MCP-1, monocyte chemotactic protein 1.

Figure 6

Minocycline neuroprotection to OGD-induced neuronal damage via MCPIP1 in neuron-glia cells from MCPIP1−/−mice. (A, B) Cell viability assays by MTT and cell death rate by trypan blue inclusion revealed that treatment of OGD on the mixed neuron-glia cells resulted in cell death and pretreatment with minocycline can increase the resistance to OGD-induced neuronal damage. Conversely, there was no significant difference on OGD-induced neuronal damage in neuron-glia cells from MCPIP1−/− mice with the treatment of minocycline compared with that of the control group without minocycline treatment. (C, D) Pretreatment of minocycline decreased OGD-induced expression of TNFα and IL-1β in the mixed neuron-glia cells. There was no significant reduction of OGD-induced TNFα and IL-1β expression in mixed neuron-glia cells from MCPIP1−/− mice with the treatment of minocycline compared with that of the control group without minocycline treatment. MCPIP1, monocyte chemotactic protein-induced protein 1; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; OGD, oxygen glucose deprivation.

Figure 7

Minocycline neuroprotection to OGD-induced neuronal damage via MCPIP1 in primary cortical neurons from wild type mice. (A) A representative Western blot shows the induced expression of MCPIP1 by minocycline and the knockdown of MCPIP1 by siMCPIP1 in primary cortical neurons from C57BL/6 wild type mice. OGD induced MCPIP1 mildly, and minocycline induced MCPIP1 to a much higher level. siMCPIP1 knocked down MCPIP1 very efficiently. (B) MTT cell viability analysis shows that pretreatment with minocycline increased the resistance to OGD-induced neuronal damage and that knockdown of MCPIP1 by siMCPIP1 reduced the protective effects of minocycline on primary cortical neurons subjected to OGD. (C) Knockdown of MCPIP1 significantly increased the expression of TNFα induced by OGD in primary cortical neurons pretreated with minocycline compared with that of control group of siControl. MCPIP1, monocyte chemotactic protein-induced protein 1; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; OGD, oxygen glucose deprivation; siControl, negative control silencing RNA; siMCPIP1, MCPIP1 silencing RNA.

Figure 8

Inhibition of NF-κB activation in the ischemic brain by minocycline treatment in the wild type, but not in MCPIP1-deficient mice. A representative Western blot shows protein levels of p-65 phosphorylation. The phosphorylation of p-65 was significantly reduced at 24 h after MCAO in minocycline-pretreated wild type mice compared to that of the control. In MCPIP1-deficient mice, there was no significant difference in p-65 phosphorylation level between the minocycline-pretreated and control group without minocycline treatment. Densitometric analysis was used to quantify phospho-p-65 protein levels versus total p-65 in three independent Western blots, and the data are expressed as the normalized folds with respect to sham. Values represent mean ± SD. MCAO, middle cerebral artery occlusion; MCPIP1, monocyte chemotactic protein-induced protein 1.