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.