Siramesine attenuates early brain injury through TMEM97/NPC1 pathway after experiment subarachnoid hemorrhage in rats

Introduction: Subarachnoid hemorrhage (SAH) is a devastating event, and early brain injury (EBI) has been confirmed as the primary determinant of poor outcomes in SAH. The pathophysiologic process by which SAH leads to EBI is complicated. We thought that Transmembrane protein 97 (TMEM97) protein may be involved in the regulation of mitochondrial dynamics. For this reason, in this study we try to verify the role of TMEM97 after SAH.

Methods: 187 rats were subjected to an endovascular perforation model of SAH. Siramesine was administered intraperitoneally 1 h after SAH. SM-21 and siRNA for NPC1 were administered by intraperitoneally or intracerebroventricular (i.c.v) injection at 1 h and 48 h before SAH induction. Western blot and immunofluorescence were performed to determine the expression and location of TMEM97. To investigate the effects of TMEM97, the mortality, SAH grade, neurological tests, Fluoro-Jade C, Nissal staining, DHE staining, and Western blot experiments were performed. Finally, SM-21 and NPC1 siRNA were used to further clarify the effect of Siramesine on EBI and mitochondrial fission of SAH through TMEM97/NPC1 signaling pathway.

Results: The results showed that the expression of TMEM97 decreased after SAH induction and reached to the lowest level at 24 h when compared to sham group. Double immunofluorescence staining showed that TMEM97 was mainly expressed in neurons, and the number of TMEM97-positive neurons was decreased in SAH (24h) group when compared with that in Sham group. We found that TMEM97 activation with Siramesine significantly improved neurobehavioral scores and reduced oxidative stress and neuronal apoptosis in SAH+Siramesine group compared with SAH+vehicle group. Moreover, Siramesine treatment significantly promoted the expression TMEM97, NPC1, DJ-1 and Bcl-2, and downregulated expression of Drp1, Roma-1 and Bax. The protective effects of Siramesine were reversed by i.p. injection of SM-21 and NPC1 siRNA in SAH+Siramesine+SM-21 and SAH+Siramesine+NPC1 siRNA groups.

Conclusion : TMEM97/NPC1 signaling pathway was involving in the regulating the process of EBI. The activation of TMEM97/NPC1 signaling pathway can reduce neuronal oxidative stress and apoptosis after SAH in rats.