3.5. Gal-3 activates TLR4/ PI3K/AKT signaling axis in vitro
A previous study showed that Gal-3 increased both RNA and protein levels
for TLR4 in WD. To further investigated the effect and mechanism of
Gal-3 on the TLR4 signaling pathway, we treated the HT22 cells with
Gal-3-shRNA vector, lipopolysaccharide(LPS) (1μg/ml), a TLR4 activator
and TAK-242 (10μg/ml), a TLR4 inhibitor. We firstly found that OGD/R
injury caused the high expression of TLR4 protein, but reduced the
phosphorylation of PI3K(p-PI3K) and AKT(p-AKT) as compared with the
control group in vitro (P <0.01, Fig 5 A). Transfection
with the Gal-3 shRNA vector significantly decreased TLR4 protein
expression and increased the expression level of p-PI3K and p-AKT after
OGD/R as compared to OGD/R group in HT22 cells (P <0.01,
Fig 5 B). Then, based on cells transfected with the Gal-3 KO vector, as
the results of Fig. 5C showed, compared with OGD/R group and
OGD/R+sh-Gal-3 group, the cell viability and the expression of
Bcl-2/Bax, p-PI3K and p-AKT(P <0.01, Fig 5 C, D) were
reduced, but the release of LDH, the level of Gal-3, TLR4, cleaved of
Caspase3, as well as the ratio of cell apoptosis
(P <0.01, Fig5 C, E), were enhanced in HT22 cells
treated with LPS. In contrast, HT-22 cells treated with the TAK-242 had
significantly increased cell viability, the level of Bcl-2/Bax, p-AKT
and p-PI3K (P <0.01, Fig 5C, D), and decrease LDH
release, Gal-3, TLR4 protein expression, as well as cleaved Caspase3 and
TUNEL positive cells (P <0.01, Fig 5C, E, F) as compared
with the OGD/R group or damaged cells transfected with vector only.
Meanwhile, an opposite expression trend and a significant increase or
decrease in the peak value of cleaved Caspase1, IL-1β, IL-6 and TNF-α
(P <0.01, Fig 5 C, G-I) were observed in
OGD/R+sh-Gal-3+LPS group and OGD/R+sh-Gal-3+ TAK-242 group. These
results demonstrated that Gal-3 interaction with TLR4 and activating
TLR4/PI3K/AKT signaling axis aggravated OGD/R-induced inflammatory
damage and apoptosis in vitro.
3.6. D-allose
regulated TLR4/ PI3K/AKT pathway
through Gal-3 to protect
HT22 cells from OGD/R-induced injury
To examine whether Gal-3 and TLR4/ PI3K/AKT signaling axis get involved
in the mechanism of the D-allose in HT22 cells, the cell cytotoxicity,
inflammatory response and apoptosis were determined by Western blot and
immunoblot analysis and so on. Following transfection with sh-Gal-3 or
sh-NC, HT22 cells were treated with D-allose. As the result in Fig 6A-B
showed, cell viability and the LDH release of HT22 cells transfected
with sh-Gal-3 was higher than that of HT22 cells transfected with sh-NC
(P <0.01), while expression of inflammatory factors,
IL-1β, IL-6 and TNF-α (P <0.01, Fig 6 C-E) of HT22 cells
transfected with sh-Gal-3 were lower than that of HT22 cells transfected
with sh-NC. Moreover, compared with OGD/R+D-allose+sh-NC, we found that
the expression of p-PI3K and p-AKT and Bcl-2/Bax were higher, expression
of Gal-3, TLR4 and cleaved Caspase3, cleaved Caspase1 as well as TUNEL
positive cells were lower in OGD/R+D-allose+sh-Gal-3 group
(P <0.01, Fig 6 F-G). Together, these results indicated
that D-allose could inhibit TLR4/PI3K/AKT-induced cell cytotoxicity,
inflammatory damage and apoptotic cell death by induction of Gal-3.