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. 2019 Jul 18;9(1):10439.
doi: 10.1038/s41598-019-46203-x.

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

Seema Briyal  1 , Amaresh K Ranjan  1 , Mary G Hornick  1 , Anupama K Puppala  1 , Thanh Luu  2 , Anil Gulati  3   4
Affiliations

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

Seema Briyal et al. Sci Rep. .

Erratum in

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.

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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 < 0.01 compared to sham, #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 < 0.01 compared to sham, @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 < 0.001 compared to sham; @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 < 0.001 compared to sham; @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.
See this image and copyright information in PMC

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