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.