Anti-apoptotic activity of ETB receptor agonist, IRL-1620, protects neural cells in rats with cerebral ischemia

Seema Briyal, Amaresh K Ranjan, Mary G Hornick, Anupama K Puppala, Thanh Luu, Anil Gulati, Seema Briyal, Amaresh K Ranjan, Mary G Hornick, Anupama K Puppala, Thanh Luu, Anil Gulati

Abstract

Endothelin-B receptor agonist, IRL-1620, provides significant neuroprotection following cerebral ischemia in rats. Whether this neuroprotection is due to inhibition of apoptosis is unknown. IRL-1620-treated rats following permanent middle cerebral artery occlusion (MCAO) showed significant improvement in neurological and motor functions along with a decrease in infarct volume at 24 h (-81.3%) and day 7 (-73.0%) compared to vehicle group. Cerebral blood flow (CBF) significantly improved in IRL-1620-treated animals compared to vehicle by day 7 post MCAO. IRL-1620-treated rats showed an increase in phospho-Akt and decrease in Bad level 7 h post-occlusion compared to vehicle, while Akt and Bad expression was similar in cerebral hemispheres at 24 h post-MCAO. The phospho-Bad level was lower in vehicle- but not in IRL-1620-treated rats at 24 h. Anti-apoptotic Bcl-2 expression decreased, while pro-apoptotic Bax expression increased in vehicle-treated MCAO rats, these changes were attenuated (P < 0.01) by IRL-1620. Mitochondrial membrane-bound Bax intensity significantly decreased in IRL-1620 compared to vehicle-treated MCAO rats. IRL-1620 treatment reduced (P < 0.001) the number of TUNEL-positive cells compared to vehicle at 24 h and day 7 post MCAO. The results demonstrate that IRL-1620 is neuroprotective and attenuates neural damage following cerebral ischemia in rats by increasing CBF and reducing apoptosis.

Conflict of interest statement

Dr. Gulati is an employee of Pharmazz, Inc, he has issued and pending related patents. All other authors declare no competing interests.

Figures

Figure 1
Figure 1
Expression of total Akt and pAkt (A) and total Bad and pBad (B) protein levels with β-actin as a loading control. Lane 1- Sham [LH]; Lane 2 – Sham [RH]; Lane 3 –MCAO + Vehicle [LH]; Lane 4 –MCAO + Vehicle [RH]; Lane 5 – MCAO + IRL-1620 [LH]; Lane 5 – MCAO + IRL-1620 [RH]. LH = Left hemisphere; RH = Right hemisphere. *P < 0.01 compared to sham, #P < 0.05 compared LH; @P < 0.001 compared to MCAO + vehicle [RH]. Full-length blots are presented in supplementary file.
Figure 2
Figure 2
Expression of Bcl-2 (A) and Bax (B) protein levels with β-actin as a loading control. Lane 1 – sham [LH]; lane 2 – sham [RH]; lane 3 –MCAO + Vehicle [LH]; lane 4 –MCAO + vehicle [RH]; lane 5 –MCAO + IRL-1620 [LH]; lane 6 –MCAO + IRL-1620 [RH]. Values are expressed as mean ± S.E.M. *P #P < 0.05 compared LH; @P < 0.001 compare to MCAO + vehicle [RH]. Full-length blots are presented in supplementary file.
Figure 3
Figure 3
Bax translocation to mitochondria in cerebral ischemia-induced apoptosis. Bax was immuno-stained with anti-Bax, and the mitochondria were stained with MitoTracker (green). The merged image indicates colocalization of Bax on mitochondria. Values are expressed as mean ± S.E.M. *P @P < 0.001 compare to MCAO + vehicle.
Figure 4
Figure 4
TUNEL positive cells per 750 µm2 in the ischemic region were detected by TUNEL staining 24 h and day 7 after MCAO. Values are expressed as mean ± S.E.M. *p < 0.0001 compared to sham; @p < 0.001 compared to vehicle.
Figure 5
Figure 5
Effect of IRL-1620 on cerebral blood flow before, after and day 7 post MCAO in rat brains. Values are expressed as mean ± SEM. *P @P < 0.05 compared to MCAO + vehicle; $P < 0.0001 compared to IRL-1620 1 h post MCAO.
Figure 6
Figure 6
Effect of IRL-1620 on infarct volume in MCAO rats. 2 mm coronal sections of brains stained with TTC to visualize the infarct area 7 h, 24 h and day 7 post MCAO (red indicates normal tissue and white indicates infarct tissue). Values are expressed as mean ± SEM. *P @P < 0.05 compared to MCAO + vehicle.
Figure 7
Figure 7
Stimulation of ETB receptors by IRL-1620 can stimulate apoptotic signaling pathways which may be implicated in its neuroprotective effect.

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