Inhibitory effect of specnuezhenide on high glucose-induced human retinal microvascular endothelial cell injury and its mechanism

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.

Diabetic retinopathy; FOXO4 protein; Apoptosis; Oxidative stress; Inflammation; Specnuezhenide