3.3. Increased expression of Gal-3 in ischemic/reperfusion(I/R)
injury-induced inflammation and neuronal apoptosis in vivo and in vitro
As a very important inflammatory factor, Gal-3 participates in the
process of inflammation, oxidative stress, cell apoptosis and pyroptosis
in the brain. Compared with the Sham group, the mRNA and protein
expression levels of Gal-3 increased steadily and reached the peak at
48h after MCAO/R (P < 0.01, Fig 3 A-B), as well as
enhanced at 2h, 4h, 8h and 12h, peaking at 8h following with OGD/R
(P < 0.01, Fig 3 D-E). Therefore, the 48 h after MCAO/R
and the 8h after ODG/R were used to determine the expression and effect
in the following experiments. Meanwhile, similar results were observed
by immunohistochemical staining in vitro and in vivo, compared with the
Sham group, Gal-3 located on cytoplasm and nuclei of neuron and HT22
cells was observed, as well as Gal-3 positive-cells increased at 48h
after MCAO/R and 8h after OGD/R (P <0.01, Fig 3 C、F).
The above results suggested that Gal-3 is expressed in neurons, except
for neuroimmune cells, and maybe plays a crucial effect in
inflammation-induced neuron injury and death.
To further evaluate the role of Gal-3 in OGD/R-induced HT-22 cell
injury, lentivirus-based Gal-3-shRNA vectors were used to knockout (KO)
the expression of Gal-3(P <0.01, Fig 3 G). The results
of Fig 3 showed that, compared with the control group, Gal-3-shRNA
significantly decreased Gal-3 mRNA and protein levels by 50% and 70%
in HT22 cells (P <0.05, Fig 3 H-I). Then, we evaluated
the neuroprotection of Gal-3 deficiency against OGD/R-induced HT-22 cell
injury. As shown in Fig3 J-P, increased the levels of cell viability,
Bcl-2/Bax, and decreased the release of LDH, cleaved Caspase 3 levels
and TUNEL-positive neurons, as well as the level of cleaved Caspase1,
IL-1β, IL-6 and TNF-α were observed in OGD/R+sh-Gal-3 group as compared
to the control group(P <0.01), suggesting Gal-3 might be
a key mediator of OGD/R-induced HT-22 cell cytotoxicity and apoptosis.