Neuroprotective mechanism of taurine due to up-regulating calpastatin and down-regulating calpain and caspase-3 during focal cerebral ischemia

Abstract

Aims: Taurine as an endogenous substance possesses a number of cytoprotective properties. In the study, we have evaluated the neuroprotective effect of taurine and investigated whether taurine exerted neuroprotection through affecting calpain/calpastatin or caspase-3 actions during focal cerebral ischemia, since calpain and caspase-3 play central roles in ischemic neuronal death.

Methods: Male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo), and 22 h of reperfusion. Taurine was administrated intravenously 1 h after MCAo. The dose-responses of taurine to MCAo were determined. Next, the effects of taurine on the activities of calpain, calpastatin and caspase-3, the levels of calpastatin, microtubule-associated protein-2 (MAP-2) and alphaII-spectrin, and the apoptotic cell death in penumbra were evaluated.

Results: Taurine reduced neurological deficits and decreased the infarct volume 24 h after MCAo in a dose-dependent manner. Treatment with 50 mg/kg of taurine significantly increased the calpastatin protein levels and activities, and markedly reduced the m-calpain and caspase-3 activities in penumbra 24 h after MCAo, however, it had no significant effect on mu-calpain activity. Moreover, taurine significantly increased the MAP-2 and alphaII-spectrin protein levels, and markedly reduced the ischemia-induced TUNEL staining positive score within penumbra 24 h after MCAo.

Conclusions: Our data demonstrate the dose-dependent neuroprotection of taurine against transient focal cerebral ischemia, and suggest that one of protective mechanisms of taurine against ischemia may be blocking the m-calpain and caspase-3-mediated apoptotic cell death pathways.

Fig. 1

Dose–response of taurine to focal cerebral ischemia. The rats received 2-h ischemia and then reperfused. Vehicle or various dose of taurine was injected intravenously at 1 h after ischemia. (A) Dose–response of taurine to neurological deficits (mean ± SEM, n = 15, *P < 0.05 and **P < 0.01 versus vehicle). (B) The infarct zone was displayed by TTC staining in vehicle- or various dose of taurine-treated rats. (C) The bar graph reflected the infarct volumes from TTC staining in various groups (mean ± SEM, n = 14, *P < 0.01 versus vehicle)

Fig. 2

Effects of taurine on calpastatin protein levels and activities in cytosolic fractions of penumbras 24 h after MCAo in rats. The rats received 2-h ischemia and then reperfused. Taurine or saline was administrated intravenously 1 h after ischemia. (A) Western blot analysis using anti-calpastatin antibody. (B) The bar graph reflected the densitometric data from the experiment of calpastatin Western blot analysis (mean ± SEM, n = 8, *P < 0.05 versus vehicle). (C) The bar graph reflected the calpastatin activities through determining the inhibition of purified m-calpain by samples (mean ± SEM, n = 12, *P < 0.01 versus vehicle)

Fig. 3

Effects of taurine on μ- and m-calpain activities in cytosolic fractions of penumbras 24 h after MCAo in rats. The rats received 2-h ischemia and then reperfused. Taurine or saline was administrated intravenously 1 h after ischemia. (A) Casein zymography of μ- and m-calpain. (B) The bar graph reflected the densitometric data from the casein zymography (mean ± SEM, n = 12, μ-calpain: *P < 0.05 versus sham, m-calpain: *P < 0.01 versus sham,^# P < 0.05 versus vehicle)

Fig. 4

Effects of taurine on caspase-3 activities in cytosolic fractions of penumbras 24 h after MCAo. The rats received 2-h ischemia and then reperfused. Taurine or saline was administrated intravenously 1 h after ischemia. Fluorescent arbitrary units were converted into picomoles of AFC released per hour and milligrams of protein (mean ± SEM, n = 8. *P < 0.05 versus sham,^# P < 0.05 versus vehicle)

Fig. 5

Effects of taurine on MAP-2 and αII-spectrin levels in cytosolic fractions of penumbras 24 h after MCAo in rats. The rats received 2-h ischemia and then reperfused. Taurine or saline was administrated intravenously 1 h after ischemia. (A) Western blot analysis using anti-MAP-2 antibody. (B) Western blot analysis using anti-αII-spectrin antibody. (C) The bar graph reflected the densitometric data from the experiment of MAP-2 Western blot (mean ± SEM, n = 8, *P < 0.05 versus sham,^# P < 0.01 versus vehicle). (D) The bar graph reflected the densitometric data from the experiment of αII-spectrin Western blot (mean ± SEM, n = 8, *P < 0.01 versus sham,^# P < 0.01 versus vehicle)

Fig. 6

The representative feature of TUNEL-positive cells in the frontoparietal cortex motor area (cortical penumbra, B–D) and medial part of striatum (striatal penumbra, E–G) 24 h after MCAo in rats (Original magnification 400×). The rats received 2-h ischemia and then reperfused. Taurine or saline was administrated intravenously 1 h after ischemia. (A) Schematic representation of the distribution of neuronal damage in rat brain after transient focal ischemia delineated by TUNEL. Two areas subjected to analysis of TUNEL are illustrated: FPCM, frontoparietal cortex motor area (cortical penumbra); MS, medial part of striatum (striatal penumbra). (B and E) Sham-operated rats. (C and F) Vehicle-treated rats. (D and G) Taurine-treated rats. (H) The bar graph reflected the TUNEL positive staining score in each group (mean ± SEM, n = 9, *P < 0.05 versus vehicle)

Fig. 7

Overview of the neuroprotection of taurine against focal cerebral ischemia through blocking the calpain- and caspase-3-mediated apoptotic cell death pathway. “➝” indicates positive actions. “⇨” indicates inhibitory actions