ABSTRACT
Objective:To investigate the effect of specnuezhenide on high glucose-induced human retinal microvascular endothelial cells (hRMECs) injury and its mechanism.Methods:The hRMECs were divided into a normal control group cultured in a culture medium containing 5.5 mmol/L glucose, a hypertonic group cultured in a culture medium containing 5.5 mmol/L glucose + 24.5 mmol/L mannitol, a high glucose group cultured in a culture medium containing 30 mmol/L glucose, as well as high glucose+ low-, medium-, and high-dose specnuezhenide groups cultured in culture media containing 30 mmol/L glucose + 25, 50, 100 μmol/L specnuezhenide for 24 hours, respectively.In addition, hRMECs were divided into a high glucose+ small interfering RNA-negative control (si-NC) group cultured in a culture medium containing 30 mmol/L glucose, a high glucose+ si-forkhead box O4 (FOXO4) group cultured in a culture medium containing 30 mmol/L glucose, a high glucose+ specnuezhenide+ pcDNA group cultured in a culture medium containing 100 μmol/L specnuezhenide + 30 mmol/L glucose, and a high glucose+ specnuezhenide+ pcDNA-FOXO4 group cultured in a culture medium containing 100 μmol/L specnuezhenide+ 30 mmol/L glucose for 24 hours after transfection by corresponding reagents.Cell apoptosis was detected by flow cytometry.The malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity in cells were detected by the thiobarbituric acid method and xanthine oxidase method, respectively.The concentrations of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in the cell culture supernatant were detected by enzyme linked immunosorbent assay.The relative expression level of FOXO4 protein in cells was determined by Western blot.Results:The apoptosis rates of normal control group, hypertonic group, high glucose group, high glucose+ low-, medium- and high-dose specnuezhenide groups were (7.32±0.72)%, (7.44±0.70)%, (23.96±1.32)%, (19.84±1.09)%, (14.13±0.85)% and (9.84±0.70)%, respectively.There were significant differences in cell apoptosis rate, MDA concentration, SOD activity, the concentration of IL-1β, the concentration of TNF-α, and the relative expression level of FOXO4 protein among the six groups ( F=498.545, 1 186.693, 516.629, 654.247, 638.238, 472.655; all at P<0.001). Compared with high glucose group, the apoptosis rate, MDA concentration, IL-1β and TNF-α concentration, FOXO4 protein expression level were significantly decreased in high glucose+ low-, medium- and high-dose specnuezhenide groups, and SOD activity was significantly increased in a dose-dependent manner.Compared with high glucose+ si-NC group, the expression level of FOXO4 protein, cell apoptosis rate, MDA concentration, IL-1β and TNF-α mass concentrations were decreased in high glucose + si-FOXO4 group, while the SOD activity was increased.Compared with high glucose+ specnuezhenide+ pcDNA group, the apoptosis rate, MDA concentration, IL-1β and TNF-α concentrations, FOXO4 protein expression level of hRMECs in high glucose+ specnuezhenide+ pcDNA-FOXO4 group were significantly increased, and SOD activity was significantly decreased (all at P<0.05). Conclusions:Specnuezhenide can protect hRMECs from high glucose-induced apoptosis, oxidative stress and inflammatory response by down-regulating FOXO4.
ABSTRACT
RATIONALE: Inflammation in post-myocardial infarction (MI) is necessary for myocyte repair and wound healing. Unfortunately, it is also a key component of subsequent heart failure pathology. Transcription factor forkhead box O4 (FoxO4) regulates a variety of biological processes, including inflammation. However, its role in MI remains unknown. OBJECTIVE: To test the hypothesis that FoxO4 promotes early post-MI inflammation via endothelial arginase 1 (Arg1). METHODS AND RESULTS: We induced MI in wild-type and FoxO4(-/-) mice. FoxO4(-/-) mice had a significantly higher post-MI survival, better cardiac function, and reduced infarct size. FoxO4(-/-) hearts had significantly fewer neutrophils, reduced expression of cytokines, and competitive nitric oxide synthase inhibitor Arg1. We generated conditional FoxO4 knockout mice with FoxO4 deleted in cardiac mycoytes or endothelial cells. FoxO4 endothelial cell-specific knockout mice showed significant post-MI improvement of cardiac function and reduction of neutrophil accumulation and cytokine expression, whereas FoxO4 cardiac mycoyte-specific knockout mice had no significant difference in cardiac function and post-MI inflammation from those of control littermates. FoxO4 binds the Foxo-binding site in the Arg1 promoter and activates Arg1 transcription. FoxO4 knockdown in human aortic endothelial cells upregulated nitric oxide on ischemia and suppressed monocyte adhesion that can be reversed by ectopic-expression of Arg1. Furthermore, chemical inhibition of Arg1 in wild-type mice had similar cardioprotection and reduced inflammation after MI as FoxO4 inactivation and administration of nitric oxide synthase inhibitor to FoxO4 KO mice reversed the beneficial effects of FoxO4 deletion on post-MI cardiac function. CONCLUSIONS: FoxO4 activates Arg1 transcription in endothelial cells in response to MI, leading to downregulation of nitric oxide and upregulation of neutrophil infiltration to the infarct area.
