Objective To investigate the effects of penehyclidine hydrochloride (PHC) administered at different time points on neurological function and the blood-brain barrier (BBB) in rat model of severe intracerebral hemorrhage (ICH), and to preliminarily explore its potential mechanism of action based on the growth arrest-specific protein 6 (GAS6)/receptor tyrosine kinase (Axl) signaling pathway.
Methods ICH model rats were established via intracerebral injection of a collagenase type Ⅳ solution. The model rats were randomly divided into sham operation group (intracerebral injection of an equal volume of saline), model group (intracerebral injection of 0.4 μL of collagenase type Ⅳ solution), 24 h drug administration group(intracerebral injection of 0.4 μL of collagenase type Ⅳ solution combined with intraperitoneal injection of 2 mg/kg PHC 24 h after successful model establishment), 6 h drug administration group (intracerebral injection of 0.4 μL of collagenase type Ⅳ solution combined with intraperitoneal injection of 2 mg/kg PHC 6 h after successful model establishment), pre-drug administration group (intraperitoneal injection of 2 mg/kg PHC before modeling combined with intracerebral injection of 0.4 μL of collagenase type Ⅳ solution), and pathway inhibitor group (intraperitoneal injection of 2 mg/kg PHC before modeling combined with intracerebral injection of 0.4 μL of collagenase type Ⅳ solution and intraperitoneal injection of 75 mg/kg R428, a GAS6/Axl signaling pathway inhibitor, after successful model establishment). The degree of neurological impairment in rats was assessed after successful model establishment and at the end of treatment; brain tissue water content in rats was calculated; brain tissue damage and Evans blue (EB) content in rats were evaluated using hematoxylin-eosin (HE) and EB staining methods; western blot was used to detect the expression levels of Claudin-5, zonula occludens-1 (ZO-1), Occludin, matrix metalloproteinase-9 (MMP-9), and proteins related to the GAS6/Axl signaling pathway in brain tissue.
Results HE staining revealed that compared with the sham operation group, the model group exhibited irregular arrangement of brain tissue cells, a large number of necrotic cells, and significant infiltration of inflammatory cells; compared with the model group, the 24 h drug administration, 6 h drug administration, pre-drug administration, and pathway inhibitor groups showed more orderly brain tissue cells, reduced cell gaps, and decreased infiltration of inflammatory cells; compared with the 24 h drug administration group, the 6 h drug administration, pre-drug administration, and pathway inhibitor groups exhibited more intact brain tissue cell structures, reduced cell gaps, and decreased infiltration of inflammatory cells; compared with the pre-drug administration group, the 6 h drug administration and pathway inhibitor groups showed slight swelling of brain tissue cells and a small amount of inflammatory cell infiltration. Compared with the sham operation group, the model group had increased neurological function scores, brain tissue water content, EB content in brain tissue, and MMP-9 protein levels, along with decreased levels of ZO-1, Occludin, Claudin-5 proteins, and GAS6 and p-Axl protein expression; compared with the model group, the 24 h drug administration, 6 h drug administration, pre-drugadministration, and pathway inhibitor groups had decreased neurological function scores, brain tissue water content, EB content in brain tissue, and MMP-9 protein levels, along with increased levels of ZO-1, Occludin, Claudin-5 proteins, and GAS6 and p-Axl protein levels in brain tissue; compared with the 24 h drug administration group, the 6 h drug administration, pre-drug administration, and pathway inhibitor groups had decreased neurological function scores, brain tissue water content, EB content in brain tissue, and MMP-9 protein levels, along with increased levels of ZO-1, Occludin, Claudin-5 proteins, and GAS6 and p-Axl protein expression; compared with the pre-drug administration group, the 6 h drug administration and pathway inhibitor groups had increased neurological function scores, brain tissue water content, EB content in brain tissue, and MMP-9 protein levels, along with decreased levels of ZO-1, Occludin, Claudin-5 proteins, and GAS6 and p-Axl protein expression in brain tissue; the between-group differences mentioned above were statistically significant (P < 0.05).
Conclusion Early administration of PHC can improve neurological function and the BBB in ICH rats by reducing brain tissue damage and brain edema, and its mechanism may be related to the activation of the GAS6/Axl signaling pathway.
DownLoad: