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. 2024 Mar 20;44(3):563-570.
doi: 10.12122/j.issn.1673-4254.2024.03.18.

[PI3K/Akt/Erk signaling pathway mediates neuroprotection of CaMKⅡγ and CaMKⅡδ against ischemic reperfusion injury in mice]

[Article in Chinese]
H Liu  1   2 , Z Lin  1   3 , J Ye  1   3
Affiliations

[PI3K/Akt/Erk signaling pathway mediates neuroprotection of CaMKⅡγ and CaMKⅡδ against ischemic reperfusion injury in mice]

[Article in Chinese]
H Liu et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To observe neuroprotective effects of Ca2+/calmodulin-dependent kinase Ⅱ (CaMK Ⅱ)γ and CaMkII δ against acute neuronal ischemic reperfusion injury in mice and explore the underlying mechanism.

Methods: Primary cultures of brain neurons isolated from fetal mice (gestational age of 18 days) were transfected with two specific siRNAs (si-CAMK2G and si-CAMK2D) or a control sequence (si-NT). After the transfection, the cells were exposed to oxygen-glucose deprivation/reperfusion (OGD/R) conditions for 1 h followed by routine culture. The expressions of phosphatidylinositol-3-kinase/extracellular signal-regulated kinase (PI3K/Akt/Erk) signaling pathway components in the neurons were detected using immunoblotting. The expressions of the PI3K/Akt/Erk signaling pathway proteins were also detected in the brain tissues of mice receiving middle cerebral artery occlusion (MCAO) or sham operation.

Results: The neuronal cells transfected with siCAMK2G showed significantly lower survival rates than those with si-NT transfection at 12, 24, 48, and 72 h after OGD/R (P < 0.01), and si-CAMK2G transfection inhibited OGD/R-induced upregulation of CaMKⅡγ expression. Compared to si-NT, transfection with si-CAMK2G and si-CAMK2D both significantly inhibited the expressions of PI3K/Akt/Erk signaling pathway components (P < 0.01). In the mouse models of MCAO, the expressions of CaMKⅡδ and CaMKⅡγ were significantly increased in the brain, where activation of the PI3K/Akt/Erk signaling pathway was detected. The expression levels of CaMKⅡδ, CaMKⅡγ, Erk, phosphorylated Erk, Akt, and phosphorylated Akt were all significantly higher in MCAO mice than in the sham-operated mice at 24, 48, 72, and 96 h after reperfusion (P < 0.05).

Conclusion: The neuroprotective effects of CaMKⅡδ and CaMKⅡγ against acute neuronal ischemic reperfusion injury are mediated probably by the PI3K/Akt/Erk pathway.

目的: 观察钙/钙调蛋白依赖性激酶Ⅱ（CaMKⅡ）的同工型CaMKⅡγ和CaMKⅡδ对鼠神经元细胞缺血缺氧再灌注损伤的影响，并探究其作用机制。

方法: 分离胚胎期第18天胎鼠大脑用于提取原代神经元，在5% CO2、37 ℃条件下培养，分为常规培养的对照组、空白对照组（si-NT）、CaMKⅡγ敲除组（si-CAMK2G）和CaMKⅡδ敲除组（si-CAMK2D）。研究组神经元转染处理后，更换为无糖培养基，并将其置于缺氧环境中模拟氧糖剥夺（OGD/R）条件，持续1 h，随后复原标准培养环境。通过对神经元裂解物行免疫蛋白印迹检测磷脂酰肌醇-3-激酶/细胞外信号调节激酶（PI3K/Akt/Erk）信号通路构件的表达量，并建立小鼠大脑中动脉闭塞（MCAO）模型，通过对比假手术组（Sham）（n=25）和MCAO组（n=25）PI3K/Akt/Erk信号通路表达来进行验证。

结果: si-CAMK2G组的胎鼠神经元细胞在OGD/R 12、24、48、72 h的生存率明显低于si-NT组（P < 0.01或0.001），并可逆转OGD/R介导的胎鼠神经元细胞CaMKⅡγ表达上调。si-CAMK2G组和si-CAMK2D组与si-NT组相比，PI3K/Akt/Erk信号通路表达受到明显抑制（P < 0.01）。在MCAO模型中，MCAO组小鼠脑CaMKⅡδ和CaMKⅡγ的表达显著增加并激活了PI3K /Akt/Erk信号通路，CaMKⅡδ和CaMKⅡγ，Erk、磷酸化Erk、Akt和磷酸化Akt在MCAO造模成功再灌注后24、48、72和96 h的表达显著高于Sham组再灌注24 h（P < 0.05，0.01或0.0001）。

结论: CaMKⅡγ和CaMKⅡδ在神经元细胞发生缺血缺氧损伤时的神经保护作用可能是通过PI3K/Akt/Erk信号通路介导的。

Keywords: Ca2+/calmodulin-dependent kinase Ⅱ; cerebral ischemia/reperfusion injury; extracellular signal-regulated kinase; neuroprotection; phosphatidylinositol-3-kinase; signaling pathway.

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Figures

图 1
图 1
OGD/R后0~72 h钙黄绿素AM/碘化丙啶染色的原代神经元图像 Representative images of calcein-AM/propidium iodide (PI) staining of the primary neurons from 0 h to 72 h following oxygen-glucos deprivation/reperfusion (OGD/R) (Original magnification: × 200). The red channel shows PI staining for cell death, and the green shows calcein-AM staining of live cells.
图 2
图 2
敲减CAMK2G可增加OGD/R诱导的原代神经元细胞死亡 Knockdown of CAMK2G exacerbates OGD/R-induced neuronal cell death. Cell survival was assessed by calcein-AM/PI staining. The total PI-positive or calcein-AM-positive cells were counted from 10 random fields in each image. The neuron survival rate was calculated as the ratio of calcein-AM-positive cell number over the total cell number by Image J. **P < 0.01, ***P < 0.001.
图 3
图 3
OGD/R对si-NT、si-CAMK2D、si-CAMK2G转染后的大鼠原代神经元细胞中PI3K/Ark/Erk通路组件的影响 Knockdown of CAMK2D or CAMK2G inhibits upregulation of CaMKⅡδ or CaMKⅡγ, respectively, and suppresses the expressions of Erk, p-Erk, Akt and pS473-Akt induced by OGD/R. GAPDH was blotted as the loading control. Representative images from 3 independent experiments are shown.
图 4
图 4
各灰度值表达量化图 Quantification of the expression levels of CaMKⅡδ, CaMKⅡγ, Akt, phosphorylate-Akt, Erk and phosphorylate-Erk in the neurons with or without CAMK2G and CAMK2D knockdown by densitometry analysis. Percent control (y-axis) represents the expression of the target genes to that of the controls under normoxic condition (100%). Con: Control. p-Erk: Phosphorylate-Erk. Data are Mean±SD from 4 independent experiments. **P < 0.01 vs control.
图 5
图 5
MCAO诱导小鼠脑梗死 Cerebral infarction in mice induced by MCAO observed at 24, 48, 72, and 96 h after reperfusion.
图 6
图 6
通过Western blotting分析两组梗死灶周围脑组织的CaMKⅡγ和CaMKⅡδ的表达 Analysis of CaMKⅡγ and CaMKⅡδ expressions in the penumbra of sham-operated and MCAO mice at 24, 48, 72 and 96 h after reperfusion by Western blotting.
图 7
图 7
通过灰度值分析将CaMKⅡδ和CaMKⅡγ的表达量化 Quantification of the levels of CaMKⅡδ and CaMKⅡγ by densitometry analysis. The value in sham-operated group at 24 h served as the control (100%). Data are Mean±SD from 4 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 vs sham 24 h.
图 8
图 8
Sham组和MCAO组织梗死灶周围组织中PI3K/Akt /Erk信号通路的Western blot分析 PI3K /Akt /Erk signaling pathway in the penumbra of sham-operated and MCAO mice at 24, 48, 72 and 96 h after reperfusion by Western blotting. Representative images from 4 independent experiments are shown.
图 9
图 9
各灰度值表达量化表 Quantification of the levels of Akt, phosphorylate-Akt, Erk and phosphorylate-Erk in the penumbra of sham-operated and MCAO mice by densitometry analysis. Data are Mean±SD from 4 independent experiments. *P < 0.05; **P < 0.01 vs sham 24 h.
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