Administration of Tauroursodeoxycholic Acid Attenuates Early Brain Injury via Akt Pathway Activation - PubMed (original) (raw)
doi: 10.3389/fncel.2017.00193. eCollection 2017.
Gang Gu 1 2, Jianhao Wang 1 2, Yan Chai 1 2, Yueshan Fan 1 2, Mengchen Yang 1 2, Xin Xu 1 2, Weiwei Gao 1 2, Fei Li 1 2, Dongpei Yin 1 2, Shuai Zhou 1 2, Xin Chen 1 2, Jianning Zhang 1 2
Affiliations
- PMID: 28729823
- PMCID: PMC5498474
- DOI: 10.3389/fncel.2017.00193
Administration of Tauroursodeoxycholic Acid Attenuates Early Brain Injury via Akt Pathway Activation
Dongdong Sun et al. Front Cell Neurosci. 2017.
Abstract
Traumatic brain injury (TBI) is one of the leading causes of trauma-induced mortality and disability, and emerging studies have shown that endoplasmic reticulum (ER) stress plays an important role in the pathophysiology of TBI. Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, has been reported to act as an ER stress inhibitor and chemical chaperone and to have the potential to attenuate apoptosis and inflammation. To study the effects of TUDCA on brain injury, we subjected mice to TBI with a controlled cortical impact (CCI) device. Using western blotting, we first examined TBI-induced changes in the expression levels of GRP78, an ER stress marker, p-PERK, PERK, p-eIF2a, eIF2a, ATF4, p-Akt, Akt, Pten, Bax, Bcl-2, Caspase-12 and CHOP, as well as changes in the mRNA levels of Akt, GRP78, Caspase-12 and CHOP using RT-PCR. Neuronal cell death was assessed by a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, and CHOP expression in neuronal cells was detected by double-immunofluorescence staining. Neurological and motor deficits were assessed by modified neurological severity scores (mNSS) and beam balance and beam walking tests, and brain water content was also assessed. Our results indicated that ER stress peaked at 72 h after TBI and that TUDCA abolished ER stress and inhibited p-PERK, p-eIF2a, ATF4, Pten, Caspase-12 and CHOP expression levels. Moreover, our results show that TUDCA also improved neurological function and alleviated brain oedema. Additionally, TUDCA increased p-Akt expression and the Bcl-2/Bax ratio. However, the administration of the Akt inhibitor MK2206 or siRNA targeting of Akt abolished the beneficial effects of TUDCA. Taken together, our results indicate that TUDCA may attenuate early brain injury via Akt pathway activation.
Keywords: Akt signal pathway; apoptosis; endoplasmic reticulum stress (ER stress); tauroursodeoxycholic acid (TUDCA); traumatic brain injury (TBI).
Figures
Figure 1
The experimental protocol. Controlled cortical impact (CCI) animals were treated with tauroursodeoxycholic acid (TUDCA), saline, MK2206 or Akt siRNA. TUDCA, saline or MK2206 were treated for 3 days following traumatic brain injury (TBI), whereas Akt siRNA were treated for once. The time points at which we chose the samples from each group for western blotting, immunofluorescence, TUNEL staining and RT-PCR are also indicated.
Figure 2
TBI altered the expression of GRP78 and the ratio of p-Akt/Akt. Data described in (A,B) represented the time course of GRP78 and p-Akt/Akt following TBI. β-actin was used as the loading control. All the results are expressed as the mean ± SEM, and n = 5 for each group. *p < 0.05 vs. sham.
Figure 3
Coronal brain sections from C57/BL6 mice (Bregma −2.20) were double immune stained for CHOP protein (green) and NeuN (red). Levels of CHOP in neuronal cells were assessed immunofluorescence. Images show CHOP positive neuronal cells was considerably higher in the lesion area of the cerebral cortex 72 h following TBI. n = 5 for each group. Scale bar = 50 μm.
Figure 4
Effects of TUDCA on neurological function and brain oedema following TBI. Neurological function was evaluated with the modified neurological severity scores (mNSSs) (A) and beam walking latency (B) and beam balance tests (C). Brain oedema was evaluated by calculating brain water content (D). All data are expressed as the mean ± SEM, and n = 5 in each group. *p < 0.05 vs. the sham group, #p < 0.05 vs. the control group.
Figure 5
Effects of TUDCA on the PERK-ATF4-CHOP signaling pathway, the Bcl-2/Bax ratio and the expression level of Caspase-12 at 72 h after TBI. Representative results of p-PERK/PERK (A), p-eIF2α/ eIF2α (B), Bcl-2/Bax (C), ATF4 (D), Caspase-12 (E) and CHOP (F) alterations in the different groups. β-actin was used as the loading control. All the results are expressed as the mean ± SEM, and n = 5 for each group. *p < 0.05 vs. sham; #p < 0.05 vs. control.
Figure 6
Effects of TUDCA on the CHOP-positive neuronal cells at 72 h after TBI. Representative immunofluorescences images showing the colocalization of CHOP (green) with NeuN positive cells (red) in the lesion area of the cerebral cortex and the dentate gyrus of the ipsilateral hippocampus at 72 h after TBI (A). Quantitative data indicating that the expression of CHOP in neuronal cells was greater in TBI group than in TUDCA treated group both in the lesion area (B) and in the dentate gyrus (C). All the results are expressed as the mean ± SEM, and n = 5 for each group. *p < 0.05 vs. sham; #p < 0.05 vs. control. Scale bar = 50 μm.
Figure 7
Effects of TUDCA on the TUNEL-positive neuronal cells at 72 h after TBI. Representative microphotographs showing the colocalization of TUNEL (green)-positive cells with neuronal cells (red) in the lesion area of the cerebral cortex and the dentate gyrus of the ipsilateral hippocampus at 72 h after TBI (A). Quantitative data indicating that TUNEL-positive neuronal cells was greater in TBI group than in TUDCA treated group both in the lesion area (B) and in the dentate gyrus (C). All the results are expressed as the mean ± SEM, and n = 5 for each group. *p < 0.05 vs. sham; #p < 0.05 vs. control. Scale bar = 50 μm.
Figure 8
Effects of TUDCA on the expression of Pten and the ratio of p-Akt/Akt. Representative results of Pten (A), p-Akt/Akt (B) alterations in the different groups. β-actin was used as the loading control. All the results are expressed as the mean ± SEM, and n = 5 for each group. *p < 0.05 vs. sham; #p < 0.05 vs. control.
Figure 9
Effects of MK2206-mediated Akt inhibition on the protective effects of TUDCA at 72 h following TBI. Representative results of p-Akt/Akt (A), p-eIF2α/ eIF2α (B), Bcl-2/Bax (C), Pten (D), Caspase-12 (E) and CHOP (F) alterations in the different groups. β-actin was used as the loading control. All the results are expressed as the mean ± SEM, and n = 5 for each group. *p < 0.05 vs. sham; #p < 0.05 vs. control; &p < 0.05 vs. TUDCA.
Figure 10
Effects of MK2206-mediated Akt inhibition on the beneficial effects of TUDCA on oedema at 72 h after TBI. Brain water content was detected at 72 h after TBI. However, this protective effect of TUDCA was reversed by MK2206 administration. All the results are expressed as the mean ± SEM, and n = 5 for each group. *p < 0.05 vs. sham; #p < 0.05 vs. control; &p < 0.05 vs. TUDCA.
Figure 11
Detection of the mRNA levels of Akt, GRP78, Caspase-12 and CHOP by RT-PCR following Akt Silencing. Mice were transfected with Akt siRNA or control siRNA. Brains were removed for the detection of Akt (A), GRP78 (B), Caspase-12 (C) and CHOP (D) by RT- PCR 3 days after TBI. All the results are expressed as the mean ± SEM, and n = 5 for each group. *p < 0.05 vs. sham; #p < 0.05 vs. control; &p < 0.05 vs. TUDCA.
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