Protective Mechanism Of Phosphatidylinositol3-kinase Pathway In Gastrodin Inhibits Lipopolysaccharide-induced Inflammatory Responses In H9C2Cardiomyocytes

Objective:The study was to investigate the role and precise molecular mechanisms for explaining how gastrodin suppresses the inflammatory response in the lipopolysaccharide (LPS) induced H9c2cardiomyocytes.Methods:Rat H9c2cardiomyocytes were cultured and treated differently in vitro and randomly divided into following groups, including the blank control group, the LPS-stimulated (1μg/ml,30min to12h) group, gastrodin (5,10and20μM) was added1h before LPS treatment group, pretreatment with1μM wortmannin for1h then added gastrodin and LPS treatment group and corresponding positive control group. The cytotoxicity of gastrodin and/or the PI3-K inhibitor wortmannin were evaluated in the presence or absence of LPS by MTT assay.The variation of protein and mRNA expression levels of inducible nitric oxide synthase(iNOS), cyclooxygenase-2(COX-2), tumor necrosis factor(TNF-a)and interleukin-6(IL-6) were analyzed by RT-PCR assay and double immunofluorescence labeling, respectively. Phosphorylation levels of phosphatidylinositol3-OH kinase cellular Akt/protein kinaseB (PI3K-Akt), inhibitor κB (IκB) and mitogen-activated protein kinases (MAPKs) cascades including extracellular signal-regulated kinase1/2(ERK1/2), c-Jun N-terminal kinases (JNK) and p38mitogen-activated protein kinases(p38MAPK) were measured by western blot.The nuclear protein level of nuclear factor-κB(NF-κB) was assayed by electrophoretic mobility shift assay (EMSA).Results:Gastrodin treatment strongly suppressed nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) family activation and upregulation of the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2(COX-2), tumor necrosis factor-a(TNF-a), and interleukin-6(IL-6) in LPS-stimulated H9c2cardiomyocytes. Gastrodin obviously upregulated the phosphatidylinositol3-kinase (PI3-K)/Akt signaling in a dose-dependent manner. Wortmannin, a specific PI3-K inhibitor, blocked the inhibitory effects of gastrodin on LPS-stimulated H9c2cardiomyocytes. PI3-K/Akt inhibition partially abolished the inhibitory effects of gastrodin on the phosphorylation of inhibitor κB-a(IKB-a), extracellular signal-regulated kinase1/2(ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38mitogen-activated protein kinase (p38MAPK), and activity of NF-κB.Conclusions:This report demonstrates that gastrodin and/or LPS and/or wortmannin do not affect the viability of H9c2cardiomyocytes; gastrodin significantly suppresses the release of proinflammatory mediators and cytokines by activation of PI3-K/Akt signaling in LPS-stimulated H9c2cardiomyocytes; gastrodin effectively attenuates LPS-induced acute inflammatory responses in H9c2cardiomyocytes by activating the PI3-K/Akt signaling pathway to inhibit the phosphorylation of IκB-α, and activation of NF-κB.