Tetrahydrocurcumin provides neuroprotection in rats after traumatic brain injury: autophagy and the PI3K/AKT pathways as a potential mechanism  Yongyue.

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Tetrahydrocurcumin provides neuroprotection in rats after traumatic brain injury: autophagy and the PI3K/AKT pathways as a potential mechanism  Yongyue Gao, MD, Jie Li, MD, PhD, Lingyun Wu, MD, PhD, Chenhui Zhou, MD, PhD, Qiang Wang, MD, PhD, Xiang Li, MD, Mengliang Zhou, MD, PhD, Handong Wang, MD, PhD  Journal of Surgical Research  Volume 206, Issue 1, Pages 67-76 (November 2016) DOI: 10.1016/j.jss.2016.07.014 Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 1 Administration of tetrahydrocurcumin provided neuroprotection after TBI. (A) Beam-walking score was tested in 24 h and 72 h after TBI. Both 25 and 50 mg/kg tetrahydrocurcumin treatment induced marked improvements in neurobehavioral function at 24 h and 72 h. The higher dose induced a better effect. The data are presented as the mean ± SD (n = 6, per group). ∗∗∗P < 0.001, ###P < 0.001 versus Sham group; ∗P < 0.05, ##P < 0.01 versus TBI + vehicle (V) group; @P < 0.05, $P < 0.05 versus TBI + tetrahydrocurcumin (25 mg/kg). (B) The effect of tetrahydrocurcumin on brain edema. The TBI and TBI + V groups exhibited significantly increased brain water contents, whereas 25 mg/kg tetrahydrocurcumin treatment significantly reduced the brain water content, and 50 mg/kg treatment induced an even greater difference. The data are presented as the mean ± SD (n = 6, per group). ∗∗∗P < 0.001 versus Sham group; ##P < 0.01 versus TBI + V; @P < 0.05 versus TBI + tetrahydrocurcumin (25 mg/kg). Journal of Surgical Research 2016 206, 67-76DOI: (10.1016/j.jss.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 2 The effect of tetrahydrocurcumin on neuronal damage at 24 h after TBI. (A) Representative images showing Nissl staining of damaged neurons (arrows) in the rat cortex. Magnification 40×. (B) The rate of neuronal survival in the four groups. Treatment with 50 mg/kg tetrahydrocurcumin significantly increased the survival of neurons following TBI. The data are presented as the mean ± SD (n = 6, per group). ∗∗∗P < 0.001 versus Sham group; #P < 0.05 versus TBI + V. (Color version of figure is available online.) Journal of Surgical Research 2016 206, 67-76DOI: (10.1016/j.jss.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 3 The effect of tetrahydrocurcumin on apoptosis at 24 h after TBI. (A) Representative images of TUNEL-positive cells (arrows) surrounding the injury site in the four groups. Magnification 40×. (B) The number of the TUNEL-positive cell in the TBI and TBI + vehicle (V) groups significantly increased, and tetrahydrocurcumin reversed this increase. (C) The protein levels of cleaved caspase-3 were measured by Western blot. (D) The TBI and TBI + V groups exhibited significantly increased expression levels of cleaved caspase-3. Tetrahydrocurcumin treatment decreased the apoptosis induced by TBI at 24 h. The data are presented as the mean ± SD (n = 6, per group). ∗∗∗P < 0.001 versus Sham group; ##P < 0.01 versus TBI; ###P < 0.001 versus TBI + V. (Color version of figure is available online.) Journal of Surgical Research 2016 206, 67-76DOI: (10.1016/j.jss.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 4 The effect of tetrahydrocurcumin on autophagy after TBI. Representative images of immunofluorescence for LC3 surrounding the injured cortex for the four groups. Magnification 40×. LC3 punctate dots were observed in the cytoplasm by immunofluorescent staining of LC3 (red). Neuron cells and nuclei are labeled with NeuN (green) and DAPI (blue), respectively. (Color version of figure is available online.) Journal of Surgical Research 2016 206, 67-76DOI: (10.1016/j.jss.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 5 The effect of tetrahydrocurcumin on autophagy at the molecular level following TBI. (A) The expression of autophagy-associated protein was measured by Western blot in the four groups. (B) The expressions of LC3 and Beclin-1 were slightly increased, whereas p62 decreased after TBI. Tetrahydrocurcumin treatment significantly increased the expressions of LC3 and Beclin-1 and decreased the expression of p62. The data are presented as the mean ± SD (n = 6, per group). ∗∗∗P < 0.001 versus Sham group; ∗∗P < 0.01 versus Sham group; ##P < 0.01 versus TBI + V. Journal of Surgical Research 2016 206, 67-76DOI: (10.1016/j.jss.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 6 The effect of autophagy on apoptosis after TBI. (A) Administration of tetrahydrocurcumin with 3-MA after TBI; the expressions of LC3 and cleaved caspase-3 were measured by Western blot. (B) The protein levels of LC3-II and cleaved caspase-3 were increased after TBI. Tetrahydrocurcumin treatment increased the expression of LC3-II, whereas it decreased the expression of cleaved caspase-3; however, 3-MA reversed the effect of tetrahydrocurcumin on LC3 and cleaved caspase-3 levels. The data are presented as the mean ± SD (n = 6, per group). ∗∗∗P < 0.001 versus Sham group; ##P < 0.01 versus TBI + V; @@P < 0.01 versus TBI + tetrahydrocurcumin group; #P < 0.05 versus TBI group. Journal of Surgical Research 2016 206, 67-76DOI: (10.1016/j.jss.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 7 The effect of tetrahydrocurcumin on the PI3K/AKT signaling pathway after TBI. (A) Treatment with tetrahydrocurcumin after TBI; the expressions of p-AKT and AKT were measured by Western blot. The protein level of p-AKT exhibited no significant change after TBI, whereas tetrahydrocurcumin remarkably increased the expression of p-AKT. The data are presented as the mean ± SD (n = 6, per group). ##P < 0.01 versus TBI + vehicle (V) group. (B) The effect of the PI3K/AKT signaling pathway on apoptosis when treated with tetrahydrocurcumin after TBI. The expression level of protein was measured via Western blot. Tetrahydrocurcumin increased the expression the p-AKT, whereas the level of cleaved caspase-3 decreased; however, the PI3K inhibitor LY294002 decreased the expression of p-AKT and abrogated the effect of tetrahydrocurcumin-induced antiapoptosis. The data are presented the mean ± SD (n = 6, per group). ∗∗∗P < 0.001 versus Sham group; @@@P < 0.001 versus TBI + tetrahydrocurcumin group; ##P < 0.01 versus TBI group; #P < 0.05 versus TBI group; @P < 0.05 versus TBI + tetrahydrocurcumin group. Journal of Surgical Research 2016 206, 67-76DOI: (10.1016/j.jss.2016.07.014) Copyright © 2016 Elsevier Inc. Terms and Conditions