Expression of microRNA-124 and cortical proteins in ischemic penumbra of rats pretreated with “Tongdu Tiaoshen” electroacupuncture

doi:10.12307/2023.596

Abstract

Abstract: BACKGROUND: “Tongdu Tiaoshen” electroacupuncture pretreatment is an emerging physiotherapy combining traditional acupuncture with modern electrical stimulation, which has been reported to have significant therapeutic effects on ischemic stroke, but its specific mechanism is still unclear.
OBJECTIVE: To investigate the effects of “Tongdu Tiaoshen” electroacupuncture pretreatment on cell apoptosis, the relative expression of microRNA-124 (miR-124) in the ischemic penumbra and Notch-1, p-JNK and cysteinyl-aspartate specific protease-3 (caspase-3) proteins in the cortex of rats and their possible mechanisms of action.
METHODS: Seventy-five male Sprague-Dawley rats were randomly divided into five groups (n=15 per group): model group, sham-operated group, electroacupuncture pretreatment group, miR-124 antagomir pretreatment group and electroacupuncture+miR-124 agomir pretreatment group. In the electroacupuncture pretreatment group, 20-minute electroacupuncture at Fengchi, Baihui, and Dazhui was performed once a day, for 7 continuous days. In the miR-124 antagomir pretreatment group, miR-124 antagomir was injected into the lateral ventricle of rats on day 7. In the electroacupuncture+miR-124 agomir pretreatment group, miR-124 agomir was injected based on electroacupuncture pretreatment. Model and sham-operated groups were not treated. After the intervention, animal models were created. In the sham-operated group, the rats were only fixed on the operating board and the vessels were blunt-separated. In the other groups, the right cerebral ischemia-reperfusion injury model was established by the middle cerebral artery occlusion method and the Zea Longa suture method. Twenty-four hours after the modeling, the modified neurological severity scoring was performed to determine the damage. TUNEL staining method was then used to measure cell apoptosis in the rat cerebral cortex. Ultrastructure of the rat cerebral cortex was observed under electron microscopy.
RESULTS AND CONCLUSION: Compared with the sham-operated group, the modified neurological severity scores were significantly elevated in the other groups (all P < 0.01). Compared with the model group, the miR-124 antagomir pretreatment group and electroacupuncture pretreatment group had significantly reduced modified neurological severity scores (both P < 0.05). Compared with the sham-operated group, the number of apoptotic cells was elevated in the model group (P < 0.01). Compared with the model group, the number of apoptotic cells was reduced in the pretreatment groups (all P < 0.01). The model group had more severe defects in cell structure and contents. The electroacupuncture pretreatment group had mild defects in cell structure and contents. Cells in the miR-124 antagomir pretreatment group were more intact. There was damage to cell structure and contents in the electroacupuncture+miR-124 agomir pretreatment group. The relative expressions of caspase-3, Notch-1 and p-JNK proteins in the cortex of rats were significantly lower in the miR-124 antagomir pretreatment and electroacupuncture pretreatment group compared with the model group (all P < 0.01). The relative expression of miR-124-3p and Notch-1 mRNA in the cortex of the pretreated rats was significantly reduced compared with that of the model group (all P < 0.01). The relative expression of caspase-3 mRNA and p-JNK mRNA in the cortex of rats was significantly reduced in the miR-124 antagomir pretreatment and electroacupuncture pretreatment groups compared with the model group (all P < 0.01). The relative expression of p-JNK mRNA in the cortex of rats in the electroacupuncture+miR-124 agomir pretreatment group was also reduced compared with the model group (P < 0.05). To conclude, “Tongdu Tiaoshen” electroacupuncture pretreatment can inhibit the apoptosis of neuronal cells after cerebral ischemia-reperfusion injury by interfering with the expression level of miR-124 in the brain tissue of rats, thereby reducing neurological deficits after cerebral ischemia-reperfusion injury. The mechanism of this action may be related to the positive regulation mechanism of miR-124 on the Notch pathway.

Key words: cerebral ischemia-reperfusion injury, apoptosis, microRNA-124, Tongdu Tiaoshen, Notch signaling pathway

CLC Number:

Zhang Junyu, Zhang Lida, Hu Yanqi, Jin Zikai, Luo Fuci, Wu Xiaoqing, Tong Tingting, Song Xiaoge, Han Wei. Expression of microRNA-124 and cortical proteins in ischemic penumbra of rats pretreated with “Tongdu Tiaoshen” electroacupuncture[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(26): 4139-4146.

Figures/Tables 5

2.5 各组大鼠皮质脑区各蛋白相对表达量的比较与假手术组比较，模型组大鼠皮质脑区caspase-3蛋白相对表达量显著升高(P < 0.01)；与模型组比较，miR-124抑制剂预处理组和电针预处理组大鼠皮质脑区caspase-3蛋白相对表达量显著降低(均P < 0.01)，电针+miR-124激动剂预处理组大鼠皮质脑区caspase-3蛋白相对表达量的降低无统计学意义(P > 0.05)；且miR-124抑制剂预处理组大鼠皮质脑区caspase-3蛋白相对表达量低于电针预处理组(P < 0.05)和电针+miR-124激动剂预处理组(P < 0.01)，电针预处理组大鼠皮质脑区caspase-3蛋白相对表达量亦低于电针+miR-124激动剂预处理组(P < 0.01)。与假手术组比较，模型组大鼠皮质脑区Notch-1蛋白相对表达量显著升高(P < 0.01)；与模型组比较，miR-124抑制剂预处理组和电针预处理组大鼠皮质脑区Notch-1蛋白相对表达量显著降低(均P < 0.01)，电针+miR-124激动剂预处理组大鼠皮质脑区Notch-1蛋白相对表达量的降低无统计学意义(P > 0.05)；并且miR-124抑制剂预处理组大鼠皮质脑区Notch-1蛋白相对表达量低于电针预处理组(P < 0.05)和电针+ miR-124激动剂预处理组(P < 0.01)，电针预处理组大鼠皮质脑区Notch-1蛋白相对表达量亦低于电针+miR-124激动剂预处理组(P < 0.01)。与假手术组比较，模型组大鼠皮质脑区p-JNK蛋白相对表达量升高无统计学意义(P > 0.05)；与模型组比较，miR-124抑制剂预处理组和电针预处理组大鼠皮质脑区p-JNK蛋白相对表达量明显降低(均P < 0.01)，电针+miR-124激动剂预处理组大鼠皮质脑区p-JNK蛋白相对表达量的降低无统计学意义(P > 0.05)；且miR-124抑制剂预处理组大鼠皮质脑区p-JNK蛋白相对表达量低于电针预处理组和电针+miR-124激动剂预处理组(均P < 0.01)，电针预处理组大鼠皮质脑区p-JNK蛋白相对表达量亦低于电针+miR-124激动剂预处理组(P < 0.01)。见图4。"

2.6 各组大鼠皮质脑区各mRNA相对表达量的比较与假手术组比较，模型组大鼠皮质脑区miR-124-3p相对表达量明显升高(P < 0.01)；与模型组比较，miR-124抑制剂预处理组、电针预处理组和电针+miR-124激动剂预处理组大鼠皮质脑区miR-124-3p相对表达量均明显降低(均P < 0.01)；其中miR-124抑制剂预处理组大鼠皮质脑区miR-124-3p相对表达量明显低于电针预处理组和电针+miR-124激动剂预处理组(均P < 0.01)，且电针预处理组大鼠皮质脑区miR-124-3p相对表达量显著低于电针+miR-124激动剂预处理组(P < 0.01)。与假手术组比较，模型组大鼠皮质脑区caspase-3 mRNA相对表达量明显升高(P < 0.01)；与模型组比较，miR-124抑制剂预处理组和电针预处理组大鼠皮质脑区caspase-3 mRNA相对表达量明显降低(均P < 0.01)，而电针+miR-124激动剂预处理组大鼠皮质脑区caspase-3 mRNA相对表达量的降低无统计学意义(P > 0.05)；此外，miR-124抑制剂预处理组大鼠皮质脑区caspase-3 mRNA相对表达量显著低于电针预处理组和电针+miR-124激动剂预处理组(均P < 0.01)，而电针预处理组和电针+miR-124激动剂预处理组间差异无显著性意义 (P > 0.05)。与假手术组比较，模型组.大鼠皮质脑区Notch-1 mRNA相对表达量明显升高(P < 0.01)；与模型组比较，miR-124抑制剂预处理组、电针预处理组和电针+miR-124激动剂预处理组大鼠皮质脑区Notch-1 mRNA相对表达量均明显降低(均P < 0.01)；且miR-124抑制剂预处理组大鼠皮质脑区Notch-1 mRNA相对表达量显著低于电针预处理组和电针+miR-124激动剂预处理组(均P < 0.01)，电针预处理组大鼠皮质脑区Notch-1 mRNA相对表达量显著低于电针+miR-124激动剂预处理组(P < 0.01)。与假手术组比较，模型组大鼠皮质脑区p-JNK mRNA相对表达量明显升高(P < 0.01)；与模型组比较，miR-124抑制剂预处理组、电针预处理组和电针+miR-124激动剂预处理组大鼠皮质脑区p-JNK mRNA相对表达量均降低(P < 0.01，P < 0.01和P < 0.05)；且miR-124抑制剂预处理组大鼠皮质脑区p-JNK mRNA相对表达量显著低于电针预处理组和电针+miR-124激动剂预处理组(均P < 0.01)，但电针预处理组和电针+miR-124激动剂预处理组间差异无显著性意义(P > 0.05)。见图5。"

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