Cerebral I/R injury is a complicated neurodegenerative disease that leads to serious outcomes and affects individuals’ lives (Wu et al. 2022). The aim of the current was to elucidate the protective effect of Sert against cerebral I/R injury in rats. Oxidative stress is a mainstay pathophysiological mechanism of cerebral injury, which occurs due to the over-liberation of reactive oxygen species (ROS), resulting in an imbalance of oxidant-antioxidant equilibrium in favour of oxidation, and consequently, mitochondrial dysfunction occurs. Mitochondrial dysfunction itself acts as a ROS generator, resulting in a vicious cycle of ROS generation (Wu et al. 2020; Mei et al. 2022).

Consistent with previous studies (Zhang and Cui 2022; Yang et al. 2022), our results showed increased oxidative stress in the cerebral I/R group as manifested by elevated MDA, total nitrate/ nitrite contents and decreased GSH level.

Moreover, inflammation is another deleterious pathophysiological event involved in cerebral deterioration. It is documented that up-regulation of neuroinflammatory mediators in cerebral I/R causes persistent damage to the brain, which plays a crucial role in the injury of neurons occurring in the acute phase of ischemic stroke (Zhao et al. 2020; Nie et al. 2022). This pathway leads to the activation of glial cells through multiple molecular pathways as activated NF-ĸB p65. Glial cells preserve the integrity of the brain tissue and protect it from injury resulting from a dangerous insult. It was stated that microglial cells, representing about 15% of brain cells, are in a resident state until activated by noxious factors as pathogen or neurodegenerative disease. Microglial cells act as a first defence line for CNS microenvironment, which is activated minutes to a few hours following ischemia. In the same context, activation of microglial cells is detected by measurement of Iba-1 expression (Hernández et al. 2021). Additionally, it was found that astrocytes account for 50% of the brain volume. The main function of astrocytes is to maintain the efficient processing of brain tissue by balancing ion-water content, downregulating excitatory neurotransmitters and getting rid of wastes. Astrocyte activation occurs during cerebral I/R, leading to astrogliosis that is detected by GFAP. (Hernández et al. 2021; Jurcau and Simion 2022).

In our study, cerebral I/R resulted in enhanced NF-ĸB p65, GFAP, and Iba-1 protein expression, along with enhanced IL-1, TNF-α levels and decreased IL-10 content. Earlier studies showed that in the cerebral I/R model, oxidative stress enhances transcription factors such as NF-ĸB, which is distributed in the neurons, microglial cells, and astrocytes. This leads to the activation of astrocytes and microglial cells with the subsequent overproduction of inflammatory mediators and a decrease in the production of anti-inflammatory mediators (Zhou et al. 2018; Jurcau and Simion 2022).

The microglial cells activation to classic (M1) or alternative (M2) phenotypes is considered as a macrophage response which occurs due to ischemia. M1 phenotype acts as an indicator of the inflammatory phase by releasing inflammatory cytokines such as IL-1, TNF-α. Many upstream regulatory cascades lead to M1 activation, such as the ERK pathway, activated NF-Kβ p65 and IL-1. Conversely, the M2 phenotype indicates the anti-inflammatory phase, leading to enhanced release of anti-inflammatory cytokines as IL-10 (Zhao et al. 2017).

In the same context, the current data showed a remarkable increase in the expression of ERK, NF-Kβ p65 and CD 86 (M1 marker of glial cells) of the cerebral I/R group. Previous results showed that microglial/microphage polarisation to the proinflammatory M1 phenotype in the cerebral I/R model was attributed to oxidative stress, upregulation of NF-KB and ERK (Gaire et al. 2019; Yang et al. 2019; Jurcau and Simion 2022).

Ischemic stroke can be converted to haemorrhagic stroke due to multiple factors such as ROS and inflammation (Shao et al. 2021; Spronk et al. 2021). During haemorrhagic stroke, lysis of red blood cells occurs. Released haemoglobin (Hb), which participates in the aggravation of oxidative stress, is bound to haptoglobin, forming a complex that binds to CD 163 (a receptor found on neuron cells) and is engulfed by macrophages. Consequently, the upregulation of CD 163 is an indisputable indication of the occurrence of haemorrhagic stroke (Garton et al. 2017). Moreover, a previous study revealed that the expression of CD 163 in neurons is increased after intracranial haemorrhage (ICH) to quarantine the hemolytic product of red blood cells (Hb) and decrease the arising inflammatory cascade as well as generation of ROS. It was found that CD163 expression is increased in both hematoma and perihematomal regions within 6 h after ICH (Garton et al. 2017; Liu et al. 2017).

In our study, Ischemic stroke was transformed into haemorrhagic stroke in the ischemic group, as evidenced by enhanced CD 163 expression in neuron cells. It is noteworthy that haemorrhagic stroke plays a crucial role in maintaining microglial cells in the proinflammatory M1 phase through NF-ĸB-p65 upregulation and ERK phosphorylation (Bi et al. 2021).

In the present study, it was found that levels of MMP-2,9 elevated in I/R group as compared to the sham group. Conversion of ischemic stroke to haemorrhagic stroke was evidenced by measuring matrix metalloproteinase enzymes (MMPs), which have a fundamental role in disrupting BBB via degrading tight junction proteins, resulting in the release of RBCs (Spronk et al. 2021). Previous studies documented that MMP-2 and MMP-9 are essential in BBB disruption during ischemic stroke. It was found that MMP-2 is the initiator of BBB derangement following cerebral ischemia, while MMP-9 is the principal contributor to BBB dysregulation in the delayed phase after ischemic stroke (Lakhan et al. 2013). Moreover, it was demonstrated that upregulated levels of MMPs in the cerebral ischemia group resulted from the generation of reactive oxygen and nitrogen species following reperfusion, leading to haemorrhagic transformation (HT) (Hong et al. 2021). In addition, the cerebral I/R group significantly enhanced HO-2 activity compared to the sham group.

As mentioned before, after leakage, RBCs undergo lysis with the subsequent release of Hb molecules, which are engulfed by the neuronal cells via binding to the neuronal receptor CD163. In the presence of HO-2, an enzyme that is mainly expressed in the neurons, Hb molecules are degraded, leading to the release of intracellular iron, which gives rise to reactive oxygen species (Garton et al. 2017; Liu et al. 2017; Ma et al. 2016). In the same context, as an undisputable confirmation of induction of HT, the Prussian blue stain revealed a significant upregulation of ferric ion deposits in brain tissue as a result of HT in I/R compared to the sham group.

Autophagy plays a severe role in injury caused to neurons due to cerebral I/R. Autophagy is mainly initiated by the liberation of ROS that leads to the downregulation of phosphorylation of PI3K with the consequent inhibition of the downstream mTOR, an essential autophagy regulatory protein. Inhibition of mTOR increases the relative expression of autophagic mediators as LC3 II/I and Beclin-1 (Yan et al. 2019; Yang et al 2019; Zhao et al. 2022). LC3 is present on the autophagosome membrane and is engaged in forming an intact autophagosome. In addition, Beclin-1 is critically involved in lysosome fusion and autophagy initiation (Wei et al. 2021). Moreover, many studies noted that autophagic upregulation is accompanied by apoptotic enhancement (Li et al. 2015; Chen et al. 2022). In our study, PI3K, mTOR, Beclin-1 and LC3 II/I expression were elevated in the cerebral I/R group, along with increased levels of apoptotic makers, caspase-3, Bax, decrease of Bcl-2 content, as well as downregulation of intact neurons number in both cortex and hippocampal CA1 region.

Pre-treatment with Sert exhibited a protective effect against the cerebral I/R model, as shown by decreased MDA, total nitrate/nitrite levels and enhanced GSH content as relevant to the cerebral I/R group. Conforming with our results, a previous study showed that Sert decreased oxidative stress markers in heart failure patients (Michalakeas et al. 2011).

Moreover, Sert down-regulated NF-ĸB-p65, p-ERK, GFAP, Iba-1 expression as well as IL-1, TNF-α contents and increased IL-10 content as relevant to the cerebral I/R group. These outcomes were confirmed by previous studies, which reported that Sert prevented the activation of microglial cells by suppressing NF-ĸB expression (Sitges et al. 2014; Lu et al. 2019).

In addition, pre-treatment with Sert shifted activated microglial cells from the M1 phenotype to the anti-inflammatory M2 phenotype, as detected by increased CD163 and decreased CD 86 expression in comparison with the cerebral I/R group. It was previously documented that M2 polarisation is attributed to many factors, such as decreased activation of ERK, mitigated liberation of ROS and suppressed NF-ĸB-p65 expression (Su et al. 2015; Yang et al. 2019). In the same context, our study showed that Sert inhibits haemorrhagic transformation as detected by suppressed expression of CD163 in neurons, MMP-2,9 levels, ferric ion deposits and activity of HO-2 as compared to the cerebral I/R group.

Finally, compared to the cerebral I/R group, Sert enhanced PI3K and mTOR expression, leading to inhibition of autophagy as indicated by decreased Beclin-1 and LC3II/I expression. Moreover, in consistency with a previous study (Wann et al. 2009), apoptosis was inhibited as demonstrated by the decline of apoptotic markers, Bax, caspase-3, enhancement of anti-apoptotic marker, Bcl-2, and restoration of the number of intact neuron cells. It is worth mentioning that all the aforementioned results were further confirmed by histological examination.