Abstract
Background: Ischemic stroke (IS) occurs when a blood vessel
supplying the brain becomes obstructed, resulting in cerebral ischemia.
This type of stroke accounts for approximately 87% of all strokes.
Globally, IS leads to high mortality and poor prognosis, which is
associated with neuroinflammation, neuronal apoptosis, etc. D-allose is
a bio-substrated glucose that is widely expressed in a series of plants.
Our previous study showed that D-allose exerts neuroprotective effects
against acute cerebral ischemic/reperfusion (I/R) injury by reducing
neuroinflammation. Here, we aim to clarify the beneficial effects and
underlying mechanism of D-allose in suppressing IS-induced
neuroinflammation damage, cytotoxicity and neuro-specific apoptosis and
neurological deficits in vitro and in vivo.
Methods: For in vivo study, the I/R model was induced by middle
cerebral artery occlusion and reperfusion (MCAO/R) in C57BL/6J mice and
D-allose was given intraperitoneal injection within 5min after
reperfusion. In vitro, mouse hippocampal neuronal cells (HT22) with
oxygen–glucose deprivation and reperfusion (OGD/R) were established as
the cell model of IS. The neurological score evaluation, some cytokines,
cytotoxicity and apoptosis in the brain and cell lines were measured.
Moreover, Gal-3 short hairpin RNA, lentivirus and adeno-associated virus
were employed to modulate Gal-3 expression in neuronal both in vitro and
in vivo to reveal the molecular mechanism.
Results: D-allose alleviated cytotoxicity, including cell
viability, LDH release and apoptosis in HT22 cells after OGD/R, which
also relieved brain injury, such as lesion volume, brain edema, neuronal
apoptosis, as well as neurological functional deficits in a mouse model
of I/R. Meanwhile, D-allose decreased inflammatory releases, such as
IL-1β, IL-6 and TNF-α. Furthermore, the expression of Gal-3 was
augmented after I/R in wild-type mice and HT22 cells, which further
bound to TLR4 confirmed by three-dimension structure prediction and
Co-IP. Then, silencing the Gal-3 gene with its shRNA inhibition
decreased the activation of TLR4 signaling and alleviated IS-induced
neuroinflammation, apoptosis and brain injury. Importantly, loss of
Gal-3 enhanced the D-allose’s protection against I/R-induced HT22 cell
injury, inflammation insults and apoptosis, whereas activation of TLR4
by selective agonist LPS increased the degree of injury of neuron, as
well as abolished the protective effects of D-allose.
Conclusions: In summary, D-allose plays a crucial role in
inhibiting inflammation after IS by suppressing Gal-3/TLR4/PI3K/AKT
signaling pathway in vitro and in vivo.