Discussion
Inflammation response of neuroimmune cells and its induced apoptotic
cell death of neurons are two main pathophysiological mechanisms of
secondary brain injury (SBI) after IS45,46. Previous
studies had showed that many drugs, which are extracted from plants,
targeting inflammatory injury and apoptotic cell death are useful
therapy for IS-induced SBI47. Recently, as a safe
“rare sugar” for consumption by mammals, D-allose has been developed a
potential drug for cancer and different tissues
ischemia/reperfusion(I/R) injury4,48-51. However, the
relationships between D-allose, inflammation and apoptosis have not been
elucidated. Taken together, this current study showed the following
novel findings: Firstly, D-allose administration after IS was beneficial
to reduce cell cytotoxicity, inflammation and neuronal apoptosis, as
well as improve neurological function in vitro and in vivo. Secondly,
Gal-3 was directly combined with TLR4 and activated TLR4/PI3K/AKT
signaling pathway in OGD/R and MCAO/R models. Thirdly, D-allose
regulates TLR4/PI3K/AKT signaling to attenuate
neuroinflammation and apoptosis
by inhibiting Gal-3 after IS in HT22 cells and mice.
IS often causes inflammatory injury, neuronal death and neurological
deficiency. Growing evidence indicated that D-allose exerts distinct
cytoprotective effects in different organ I/R injuries. However, the
mechanism underlying the protective effect of D-allose in I/R injury in
vitro and in vitro is still largely unclear. Previous studies indicated
that D-allose reduces the production of to regulate oxidative stress in
many diseases7,35,52,53. Furthermore, in our previous
study, we concluded that D-allose protects brain microvascular
endothelial cells from hypoxic/reoxygenated injury by inhibiting the UPR
pathway and attenuating eIF2αphosphorylation and endoplasmic reticulum
stress54. Moreover, similar to the previous reports
about the anti-inflammation of D-allose through inhibiting LPS-induced
increases in TNF-α, tissue cytokine-induced neutrophil chemoattractant-1
and myeloperoxidase concentrations, as well as the subsequent
neutrophil-mediated renal, cerebral and skin slap injury. Interestingly,
there are contrary results on the effect of D-allose on programmed cell
apoptosis in a variety of cells that have been reported. On the one
hand, D-allose induced tumor cell apoptosis in lung cancer and so
on55,56. On the other, D-allose decreased apoptotic
cell death in I/R-induced injury in different organs and tissues.
Meanwhile, some further studies demonstrate that D-allose exerts its
anti-inflammatory and anti-apoptotic effects to protect the
liver57, kidney4,
retina58 and skin flap51 from I/R
injury. Our previous studies also provided pieces of evidence that
D-allose may have therapeutic potential against cerebral I/R injury
through attenuating BBB disruption, inflammatory damage and apoptosis
via PPARγ-dependent regulation of NF-κB35.
Consistent with the previous reports, in the present study, we found
that intraperitoneally administered D-allose markedly reduced neuron
cytotoxicity, including the release of LDH concentration and cell
viability in HT22 cells, diminished infarct volume ratio, brain edema,
and improved neurological dysfunction in mouse after I/R injury.
Additionally, we found that D-allose treatment significantly reduced
cerebral I/R-induced neuronal apoptosis probably by inhibiting
inflammatory response. Together, our findings suggested that D-allose
had protective effects against apoptotic cell death via activating its
anti-inflammation effects after I/R injury.
The next that should be noted is the mechanism of the protective effect
of D-allose in vitro and in vivo after IS. Gal-3 belongs to the type
three β-galectin binding lectin family and plays an important
disease-exacerbating role in autoimmune/inflammatory, I/R injury and
malignant diseases. Like D-allose up-, or down-regulate apoptosis in
different diseases, the function of Gal-3 in apoptotic cell death is
also controversial. Some reports had showed that Gal-3 contributes to
the regulation of ischemic stroke by inhibiting neuronal apoptotic cell
death in rats, but other studies had indicated that Gal-3 tends to
modulation of glioma via enhancing tumor cell apoptosis, and increased
levels of Gal-3 are found in some CNS
diseases24,59-61. Meanwhile, whether Gal-3 is a
proinflammatory factor or anti-inflammation protein remains unclear so
far. In this study, compared with the I/R group in brains and HT22
cells, D-allose treatment significantly decreased the expression level
of Gal-3, together with apoptotic markers, Bax and cleaved-Caspase3, as
well as inflammatory mediators, such as cleaved Caspase1, IL-1β, IL-6
and TNF-α, suggesting reduced expression of Gal-3 induced by D-allose
might mediate the neuroprotective effects we observed in this study. To
confirm this, Gal-3 knockdown experiment was further performed. As the
results found, both in Gal-3 down-regulate cerebral and HT22 cells, the
anti-inflammation and anti-apoptosis effects of D-allose were enhanced.
These results suggested that increased Gal-3 expression induced
inflammatory insults and lots of neuronal apoptosis in vitro and in vivo
after I/R injury, and the protective effects of D-allose following IS
might be via the Gal-3 pathway.
Furthermore, one of the members of the TLRs family, TLR4, mediates
several cellular processes, such as cell apoptosis, oxidative stress,
infection-induced inflammation and sterile inflammation by endogenous
molecules27,62. Recent shreds of evidence had found
that serves as a ligand of TLR4, Gal-3 activated TLR4/NF-κB signaling to
promote cell proliferation and migration in lung adenocarcinoma and
reduced myocardial injury and improve cardiac dysfunction via inhibiting
apoptosis63. And the other studies also revealed the
TLR4-mediated pro-inflammatory and apoptotic effects of
Gal-320. Additionally, PI3K/AKT signaling is an
important axis regulated by ROS production and the product of
metabolism, which participates in the regulation of oxidative stress,
inflammatory response, and apoptosis in I/R injury64.
TLR4 reportedly is an upstream regulator of the PI3K/AKT axis and
upregulates p-PI3K and p-AKT levels resulting in the activation of
TLR4/PI3K/AKT signaling, which promotes the proliferation and migration
in the development of cardiac hypertrophy65. Recent
research efforts have revealed Gal-3 is involved in the modulation of
angiogenesis and apoptosis via inhibiting AKT signaling to enhance
neuro-vascular protection and functional recovery after
IS24. In this study, we first found Gal-3 directly
combined with TLR4, and loss of Gal-3 reduced the rate of apoptosis and
apoptotic proteins, Bax, cleaved Caspase3 and cleaved Caspase1, as well
as pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α, which through
inhibiting TLR4/PI3K/AKT signaling pathway in OGD/R and MCAO/R model.
Therefore, as the results showed, compared I/R injury+Gal-3 KO in vitro
and in vivo, the anti-inflammatory damage and anti-apoptotic cell death
effects of D-allose were both suppressed in Gal-3 KO HT22 cells and mice
cerebral which treatment with TLR4 agonist LPS. To our best knowledge,
it is the first report to confirm that D-allose treatment inhibits
TLR4/PI3K/AKT signaling pathway to significantly reduced cerebral
I/R-induced neuroinflammation and neuronal apoptosis probably via
attenuating Gal-3 in vitro and in vivo.
There are some limitations in our study. In terms of research objects,
although HT22 cells have been widely used as an IS model in vitro for
neuronal function, diverse results about the effects of D-allose in IS
may be observed in primary neurons. In an aspect of the mechanism study,
the exact mechanism of Gal-3 activating TLR4/PI3K/AKT signaling was not
identified. Therefore, we need to do more studies in other neuronal
cells to mimic IS model in vitro would obtain the identification of
D-allose neuroprotection in I/R injury and sheds light on an accurate
anti-inflammatory and anti-apoptotic mechanism.
In conclusion, our findings in mouse brains and HT22 cells suggest that
D-allose attenuates post-I/R brain damage, neuronal cytotoxicity and
apoptosis via reducing inflammation. This protective effect appears to
be largely due to the regulated Gal-3/TLR4 complex inhibiting
TLR4/PI3K/AKT signaling axis in SBI of IS (Fig. 8 Created with
BioRender.com.). Therefore, D-allose may serve as a promising candidate
for the treatment of IS patients.
Authors’ contributions: YL, LZ and DG designed the project and
reviewed the manuscript; YL, JC, YF performed the experiments; YL, MZ,
MG and XL analyzed the data; YL, MZ, YZ and JB interpreted the data; YL,
LZ and DG drafted and edited the manuscript. All authors read and
approved the final.
Funding: This work was supported by the National Natural
Science Foundation of China (No.81971227).