Gas6 attenuates lipopolysaccharide-induced TNF-[alpha] expression and apoptosis in H9C2 cells through NF-[kappa]B and MAPK inhibition via the Axl/PI3K/Akt pathway

Therapeutic agents used to treat sepsis-induced cardiac dysfunction are designed to suppress tumor necrosis factor (TNF)-[alpha] release and inhibit cell apoptosis. Exogenous administration of growth arrest-specific 6 (Gas6) exerts several biological and pharmacological effects; however, the role of Gas6 in sepsis-induced myocardial dysfunction remains unclear. In this study, H9C2 cardiomyocytes were stimulated with LPS (10 [micro]g/ml) to mimic septic cardiac dysfunction and Gas6 (100 ng/ml) was applied exogenously. Subsequently, mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-[kappa]B activation, TNF-[alpha] expression, and apoptosis in the presence or absence of TP-0903 (15 nM) and Wortmannin (3 nM) were evaluated. The morphological alterations of H9C2 cells were visualized by phase-contrast microscopy. Cell viability was determined using the Cell Counting kit 8 assay and lactate dehydrogenase release, and TNF-[alpha] release was analyzed by ELISA analysis. Cell apoptosis was analyzed by flow cytometry and TUNEL assay. Nuclear morphological alterations were detected by Hoechst staining and caspase-3 activity was measured using biochemical methods. The expression levels of Bax and Bcl-2, and the phosphorylation and expression levels of Axl, Akt, I[kappa]B-[alpha], p65, c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 were determined by western blotting. Furthermore, immunofluorescence analysis was performed to visualize translocation of NF-[kappa]B p65. The results demonstrated that Gas6 suppressed TNF-[alpha] release and inhibited cell apoptosis, and attenuated nuclear factor (NF)-[kappa]B and mitogen-activated protein kinase (MAPK) activation via the Axl/PI3K/Akt pathway. Furthermore, the cardioprotective properties of Gas6 on the suppression of LPS-induced TNF-[alpha] release and apoptosis were abolished by treatment with TP-0903 (an Axl inhibitor) and Wortmannin (a PI3K inhibitor). Pretreatment with TP-0903 and Wortmannin abrogated the effects of Gas6 on phosphorylated-I[kappa]B-[alpha], I[kappa]B-[alpha], NF-[kappa]B, ERK1/2, JNK and p38 MAPK. These findings suggested that activation of Axl/PI3K/Akt signaling by Gas6 may inhibit LPS-induced TNF-[alpha] expression and apoptosis, as well as MAPK and NF-[kappa]B activation.