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. 2018 Dec;96(12):1900-1913.
doi: 10.1002/jnr.24279. Epub 2018 Jul 19.

Combined treatment with GSNO and CAPE accelerates functional recovery via additive antioxidant activities in a mouse model of TBI

Mushfiquddin Khan  1 , Anandakumar Shunmugavel  1 , Tajinder S Dhammu  1 , Hamza Khan  2 , Inderjit Singh  1   3 , Avtar K Singh  3   4
Affiliations

Combined treatment with GSNO and CAPE accelerates functional recovery via additive antioxidant activities in a mouse model of TBI

Mushfiquddin Khan et al. J Neurosci Res. 2018 Dec.

Abstract

Traumatic brain injury (TBI) is the major cause of physical disability and emotional vulnerability. Treatment of TBI is lacking due to its multimechanistic etiology, including derailed mitochondrial and cellular energy metabolism. Previous studies from our laboratory show that an endogenous nitric oxide (NO) metabolite S-nitrosoglutathione (GSNO) provides neuroprotection and improves neurobehavioral function via anti-inflammatory and anti-neurodegenerative mechanisms. To accelerate the rate and enhance the degree of recovery, we investigated combining GSNO with caffeic acid phenethyl ester (CAPE), a potent antioxidant compound, using a male mouse model of TBI, controlled cortical impact in mice. The combination therapy accelerated improvement of cognitive and depressive-like behavior compared with GSNO or CAPE monotherapy. Separately, both GSNO and CAPE improved mitochondrial integrity/function and decreased oxidative damage; however, the combination therapy had greater effects on Drp1 and MnSOD. Additionally, while CAPE alone activated AMPK, this activation was heightened in combination with GSNO. CAPE treatment of normal animals also significantly increased the expression levels of pAMPK, pACC (activation of AMPK substrate ACC), and pLKB1 (activation of upstream to AMPK kinase LKB1), indicating that CAPE activates AMPK via LKB1. These results show that while GSNO and CAPE provide neuroprotection and improve functional recovery separately, the combination treatment invokes greater recovery by significantly improving mitochondrial functions and activating the AMPK enzyme. Both GSNO and CAPE are in human consumption without any known adverse effects; therefore, a combination therapy-based multimechanistic approach is worthy of investigation in human TBI.

Keywords: RRIDs; S-nitrosoglutathione; antioxidant activity; caffeic acid phenethyl ester; combination therapy; functional recovery; mitochondria quality; traumatic brain injury.

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Conflict of interest statement

Conflict of interests

The author(s) declare that they have no conflict of interests.

Figures

Figure 1.
Figure 1.
Effect of GSNO, CAPE, and combination GSNO+CAPE for 7 days on neurobehavioral functions. Treatment of TBI animals with GSNO, CAPE, and GSNO+CAPE improved motor functions (Beam walk; A and foot fault: B), cognitive (NOR, C) and depressive- like behavior (FST; D). Sham animals showed deficits neither in motor nor in cognition and depressive-like behavior. The combination therapy of GSNO+CAPE did not show additional improvement in motor function. However, both cognition and depressive-like behavior were significantly accelerated and greatly improved in the combination group compared with GSNO- or CAPE-treated group. Data are presented as mean+SD (n=8). ***p<0.001 vs. Sham, +++p<0.001, ++p<0.01, +p<0.05 vs. TBI, #p<0.05 vs. GSNO, $p<0.05 and $ $p<0.01 vs. CAPE.
Figure 2.
Figure 2.
Effect of GSNO, CAPE, and combination GSNO+CAPE for 7 days on neuronal survival/degeneration and tissue histology. Photomicrographs of DAB staining of immunoreactivity and % of area of NeuN (A, Ai), neuronal viability staining, viable neuron count (Nissl; B, Bi), and brain tissue H&E staining (C) showing enhanced inflammatory infiltration in the traumatic penumbra region of TBI group. Data are presented as mean+SD (n=4). ***p<0.001 vs. Sham, +++p<0.001 and ++p<0.01 vs. TBI, #p<0.05 and ##p<0.01 vs. GSNO, $p<0.05 vs. CAPE.
Figure 3.
Figure 3.
Effect of GSNO, CAPE, and combination GSNO+CAPE for 7 days on expression of Drp1, Fis1, and OPa1. The expression of Drp1, Fis1, and OPa1 was measured in the traumatic penumbra region using western analysis (A) and its densitometry (B). TBI had an increased expression of Drp1 and Fis1 and a decreased expression of OPa1. The levels of both Drp1 and Fis1 were decreased in both GSNO and CAPE groups compared with TBI group. Treatment with GSNO+CAPE further decreased the levels of Drp1. All treatment groups also had increased expression of Opa1. Data are presented as mean ± SD (n=4). ***p<0.001 vs. Sham, +++p<0.001 vs. TBI, ###p<0.001 vs. GSNO, $ $p<0.01 vs. CAPE.
Figure 4.
Figure 4.
Effect of GSNO, CAPE, and combination GSNO+CAPE for 7 days on the expression of PGC1α, HO-1, and MnSOD. The expression of PGC1α, HO-1, and MnSOD was measured in the traumatic penumbra region using western analysis (A) and its densitometry (B). TBI brain had reduced expression of PGC1α, HO-1 and increased expression of MnSOD. All treated groups had increased expression of PGC1α, HO-1, and MnSOD compared with TBI group. However, GSNO+CAPE group had increased expression of HO-1 and MnSOD compared with GSNO or CAPE alone. Data are presented as mean ± SD (n=4). ***p<0.001 vs. Sham, +++p<0.001, ++p<0.01, +p<0.05 vs. TBI, ##p<0.01, #p<0.05 vs. GSNO, $p<0.05 vs. CAPE.
Figure 5.
Figure 5.
Effect of GSNO, CAPE, and combination GSNO+CAPE for 7 days on activation and expression of AMPK. Activation of AMPK (measured as increased levels of AMPK phosphorylation) and expression of total AMPK (AMPKα) were measured in traumatic penumbra region using western analysis (A) and its densitometry (B). While CAPE treatment increased AMPK activation levels, GSNO treatment had no significant effects on AMPK activation. Treatment with GSNO+CAPE had synergistic effects. Data are presented as mean ± SD (n=4). ***p<0.001 vs. Sham, +++p<0.001 vs. TBI, ###p<0.001 vs. GSNO, $p<0.05 vs. CAPE.
Figure 6.
Figure 6.
Effect of CAPE on activation and expression of AMPK, ACC, and LKB1 in wild type mouse brain. Normal control mice were treated iv with either 5mg/kg CAPE in 50% DMSO or 50% DMSO alone. The animals were sacrificed 2 h after the injection and the expressions of pAMPK and AMPK with densitometry (A, A1); pACC and ACC with densitometry (B, B1); and pLKB1 and LKB1 with densitometry (C, C1) in cortical area were determined using western blot analysis. Data are presented as mean ± SD (n=4). ***p<0.001, **p<0.01 vs. DMSO.
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