MicroR-146 protects against rat ischemia-reperfusion injury by targeting NF-κB-mediated PI3K/AKT/mTOR signaling pathway

MicroR-146 (miR-146) plays crucial roles in attenuating nerve injury during cerebral ischemia-reperfusion (I/R) injury. The purpose of this study was to investigate the neuroprotective effect of miR-146 against cerebral I/R injury. Rat model of cerebral I/R injury was established using 4-vessel occlusion and reperfusion. In this study, miR-146 expression was significantly decreased in neurons in cerebral I/R injury rat compared to sham group. In addition, miR-146+/+ significantly decreased inflammatory cytokines, oxidative stress, neuron cell apoptosis and the infarct size in cerebral I/R injury rats (p<0.05). Transfection of miR-146 reduced apoptosis, autophagy and autophagy-related proteins LC-3, Beclin-1 and increased p62 in the neuron cells compared to control group. Furthermore, overexpression of miR-146 was indicated to directly targeting NF-κB and activating PI3K/AKT/mTOR expression in neuron cells. In conclusion, these findings suggested that miR-146 could protect against rat ischemia-reperfusion injury by targeting NF-κB-mediated PI3K/AKT/mTOR signaling pathway, which offered a potential molecular for the treatment of cerebral I/R injury.

Keywords:
miR-146; cerebral I/R injury; apoptosis; PI3K/AKT/mTOR

Authorship

Leyuan WANG

The Cerebrovascular Intervention Department, The People's Hospital of Changle County, Changle County, Shandong Province, ChinaThe People's Hospital of Changle CountyChinaChangle County, Shandong Province, ChinaThe Cerebrovascular Intervention Department, The People's Hospital of Changle County, Changle County, Shandong Province, China

Guofang LIU

The Neurosurgery Department, The People's Hospital of Laoling City, Laoling City, Shandong Province, ChinaThe People's Hospital of Laoling CityChinaLaoling City, Shandong Province, ChinaThe Neurosurgery Department, The People's Hospital of Laoling City, Laoling City, Shandong Province, China

Zetao SHAO

Qianqian ZHANG

Lili YIN

Enbo XU

Biao LI

Xiangxing CUI

Hongtao TENG ^**Corresponding author:hongtao_teng@hotmail.com

The Neurosurgery Department, The Fourth People's Hospital of Jinan City, Jinan City, Shandong Province, ChinaThe Fourth People's Hospital of Jinan CityChinaJinan City, Shandong Province, ChinaThe Neurosurgery Department, The Fourth People's Hospital of Jinan City, Jinan City, Shandong Province, China

https://orcid.org/0000-0001-8550-3456

*Corresponding author:hongtao_teng@hotmail.com

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (6)

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Figure 1
miR-146+/+ decreased brain damage in cerebral I/R injury rat. (A) Expression of miR-146 in hippocampal CA1 pyramidal layer in cerebral I/R, miR-146+/+ and sham group; (B) Neurological score of cerebral I/R, miR-146+/+ and sham group; (C) Apoptosis in hippocampal CA1 pyramidal layer in cerebral I/R, miR-146+/+ and sham group; (D) Infarct size of hippocampal CA1 pyramidal layer in cerebral I/R, miR-146+/+ and sham group. **P<0.01 compared with miR-NC group. nsP>0.05 compared with miR-NC group.

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Figure 2
miR-146+/+ down-regulated inflammation and oxidative stress in cerebral I/R injury rat. (A) The levels of serum IL-1β, IL-6 and TNF-α in cerebral I/R, miR-146+/+ and sham group; (B-D) The contents of MDA (B), the activity of SOD (C) and ROS (D) in serum in cerebral I/R, miR-146+/+ and sham group. **P<0.01 compared with miR-NC group. nsP>0.05 compared with miR-NC group.

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Figure 3
miR-146 reduced apoptosis and autophagy in neuronal cells. (A) Effects of miR-146 transfection on apoptosis of neuronal cells compared to control group; (B) Transfection of miR-146 reduced autophagy in neuronal cell basing LC3 fluorescence; (C, D) Transfection of miR-146 decreased autophagy-related geneand proteinexpression of LC-3, Beclin-1 and increased p62 in neuronal cells. **P<0.01 compared with miR-NC group. nsP>0.05 compared with miR-NC group.

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Figure 4
miR-146 directly modulated NF-κB expression in neuronal cell. (A) The relative luciferase activity of co-transfection with miR-146 mimics carrying the 3’UTR of NF-κB compared with the negative control; (B) Transfection of miR-146 decreased NF-κB and pNF-κB expression in neuronal cells. **P<0.01 compared with miR-NC group. nsP>0.05 compared with miR-NC group.

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Figure 5
miR-146 activated PI3K/Akt/mTOR signaling pathway by targeting NF-κB gene in neuronal cells. (A) Effects of miR-146 transfection on PI3K, p-PI3K, Akt, p-Akt, mTOR and p-mTOR expression in neuronal cells; (B) Effects of inhibitor (NF-κBIR) on PI3K, Akt and mTOR expression in neuronal cells compared to control. **P<0.01 compared with miR-NC group. nsP>0.05 compared with miR-NC group.

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Figure 6
Effects of NF-κB overexpression on miR-146-decreased apoptosis of neuronal cells. Capital letters compare means between cycles in the same treatment.

Figure 1 miR-146+/+ decreased brain damage in cerebral I/R injury rat. (A) Expression of miR-146 in hippocampal CA1 pyramidal layer in cerebral I/R, miR-146+/+ and sham group; (B) Neurological score of cerebral I/R, miR-146+/+ and sham group; (C) Apoptosis in hippocampal CA1 pyramidal layer in cerebral I/R, miR-146+/+ and sham group; (D) Infarct size of hippocampal CA1 pyramidal layer in cerebral I/R, miR-146+/+ and sham group. **P<0.01 compared with miR-NC group. nsP>0.05 compared with miR-NC group.

Figure 2 miR-146+/+ down-regulated inflammation and oxidative stress in cerebral I/R injury rat. (A) The levels of serum IL-1β, IL-6 and TNF-α in cerebral I/R, miR-146+/+ and sham group; (B-D) The contents of MDA (B), the activity of SOD (C) and ROS (D) in serum in cerebral I/R, miR-146+/+ and sham group. **P<0.01 compared with miR-NC group. nsP>0.05 compared with miR-NC group.

Figure 3 miR-146 reduced apoptosis and autophagy in neuronal cells. (A) Effects of miR-146 transfection on apoptosis of neuronal cells compared to control group; (B) Transfection of miR-146 reduced autophagy in neuronal cell basing LC3 fluorescence; (C, D) Transfection of miR-146 decreased autophagy-related geneand proteinexpression of LC-3, Beclin-1 and increased p62 in neuronal cells. **P<0.01 compared with miR-NC group. nsP>0.05 compared with miR-NC group.

Figure 4 miR-146 directly modulated NF-κB expression in neuronal cell. (A) The relative luciferase activity of co-transfection with miR-146 mimics carrying the 3’UTR of NF-κB compared with the negative control; (B) Transfection of miR-146 decreased NF-κB and pNF-κB expression in neuronal cells. **P<0.01 compared with miR-NC group. nsP>0.05 compared with miR-NC group.

Figure 5 miR-146 activated PI3K/Akt/mTOR signaling pathway by targeting NF-κB gene in neuronal cells. (A) Effects of miR-146 transfection on PI3K, p-PI3K, Akt, p-Akt, mTOR and p-mTOR expression in neuronal cells; (B) Effects of inhibitor (NF-κBIR) on PI3K, Akt and mTOR expression in neuronal cells compared to control. **P<0.01 compared with miR-NC group. nsP>0.05 compared with miR-NC group.

Figure 6 Effects of NF-κB overexpression on miR-146-decreased apoptosis of neuronal cells. Capital letters compare means between cycles in the same treatment.

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MicroR-146 protects against rat ischemia-reperfusion injury by targeting NF-κB-mediated PI3K/AKT/mTOR signaling pathway

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[1] *Corresponding author:hongtao_teng@hotmail.com