Effect of pregabalin administration upon reperfusion in a rat model of hyperglycemic stroke: Mechanistic insights associated with high-mobility group box 1

Abstract

Hyperglycemia, which reduces the efficacy of treatments and worsens clinical outcomes, is common in stroke. Ability of pregabalin to reduce neuroexcitotoxicity may provide protection against stroke, even under hyperglycemia. We investigated its protective effect against hyperglycemic stroke and its possible molecular mechanisms. Male Wistar rats administered dextrose to cause hyperglycemia, underwent middle cerebral artery occlusion for 1 h and subsequent reperfusion. Rats were treated with an intraperitoneal injection of 30 mg/kg pregabalin or an equal amount of normal saline at the onset of reperfusion (n = 16 per group). At 24 h after reperfusion, neurological deficit, infarct volume, and apoptotic cell count were assessed. Western blot analysis was performed to determine protein expression of high-mobility group box 1 (HMGB1), toll-like receptor-4 (TLR-4), phosphorylated nuclear factor-kappa B (p-NF-κB), interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), phosphorylated inducible and endothelial nitric oxide synthase (p-iNOS, p-eNOS), Bcl-2, Bax, Cytochrome C, and caspase-3 in the brain. Pregabalin-treated rats showed significantly improved neurological function (31% decrease in score), reduced infarct size (by 33%), fewer apoptotic cells (by 63%), and lower expression levels of HMGB1, TLR4, p-NF-κB, IL-1β, and TNF- α, compared with control rats. Decreased p-iNOS and increased p-eNOS expressions were also observed. Expression of Bax, Cytochrome C, and cleaved caspase-3/caspase3 was significantly downregulated, while Bcl-2 expression was increased by pregabalin treatment. Pregabalin administration upon reperfusion decreased neuronal death and improved neurological function in hyperglycemic stroke rats. Cogent mechanisms would include attenuation of HMGB1/TLR-4-mediated inflammation and favorable modulation of the NOS.

Fig 1. Determination of the dose range for pregabalin treatment.

Rats were treated with the indicated dose (mg/kg) of pregabalin (PGB). Representative pictures of TTC-stained brain sections from rat in the different group (a); Infarct volumes expressed as a percentage of area at risk (b); Neurological deficit scores (c). Values are mean ± SD from 8 independent rats. *P <0.05. Scale bar: 1 cm.

Fig 2. Effect of pregabalin treatment upon reperfusion on apoptosis in the brain of hyperglycemic stroke rat.

Pregabalin reduced the number of TUNEL-positive cells compared with the control group. Quantitative representation of TUNEL-positive cells was determined by random counting of four fields per section. Values are mean ± SD from 8 independent rats. *P <0.05. Scale bar: 50 μm.

Fig 3. Pregabalin treatment attenuated expressions of HMGB1/TLR-4/p-NF-κB/IL-1β and TNF-α in the brain of hyperglycemic stroke rat.

The protein levels of HMGB1, TLR-4, P-NF-kB, NF-kB, IL-1β and TNF-ɑ were detected in the ipsilateral hemisphere of rat brain in each group using western blot analysis. Right panels were quantified signal by scanning densitometry. Values are mean ± SD from 5 independent rats. *P <0.05.

Fig 4. Pregabalin treatment favorably modulated expressions of NOS in the brain of hyperglycemic stroke rat.

The western blot analysis performed. The protein levels of P-iNOS and iNOS were detected in the ipsilateral hemisphere of rat brain in each group. Right panel was quantified signal by scanning densitometry. Values are mean ± SD from 5 independent rats. *P <0.05.

Fig 5. Pregabalin treatment favorably modulated expressions of apoptosis-related molecules in the brain of hyperglycemic stroke rat.

The protein levels of Bcl-2, Bax, Cytochrome C (a), Cleaved caspase-3 and Caspase-3 (b) were detected in the ipsilateral hemisphere of rat brain in each group using western blot analysis. Right panels were quantified signal by scanning densitometry. Values are mean ± SD from 5 independent rats. *P <0.05.