Anti-apoptosis and anti-inflammation effects and mechanism of berberine on high-concentration glucose induced rat retinal Müller cells

Yiping JIN; Haohao ZHU; Yujie LIAO; Xiaoyan YU

ABSTRACT

Objective:

To investigate the effects of berberine on Sprague Dawley (SD) rat retinal Müller cells cultured by high concentration glucose.

Methods:

The cultured SD rat retinal Müller cells were divided into normal-glucose group, high-glucose group, high-glucose+ 10 μmol/L berberine group and high-glucose+ 25 μmol/L berberine group, and the cells were cultured in 5 mmol/L glucose, 25 mmol/L glucose, 25 mmol/L glucose+ 10 μmol/L berberine, and 25 mmol/L glucose+ 25 μmol/L berberine, respectively.After 72 hours cultured, cell apoptosis rate was detected by flow cytometry; the expressions levels of tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) in the culture supernatant were detected by enzyme linked immunosorbent assay (ELISA); the L-glutamate-L-aspartate transporter (GLAST) and the related protein expression levels were detected by real-time fluorescence quantitative PCR; the expressions levels of GLAST, protein phosphatase magnesium-dependent 1A (PPM1A), nuclear factor-κB (NF-κB) and cleaved caspase-3 in the cytoplasm, and the expression level of NF-κB protein in the nucleus were detected by Western blot.

Results:

The cell apoptosis rate was (1.37±0.21)%, (17.67±1.17)%, (10.60±0.17)% and (5.57±0.35)% in the normal-glucose group, high-glucose group, high-glucose+ 10 μmol/L berberine group and high-glucose+ 25 μmol/L berberine group, respectively, and the overall comparative difference was statistically significant ( F=375.97, P<0.01). The cell apoptosis rates in the high-glucose group was increased in comparison with those in the normal-glucose group ( P<0.01). The cell apoptosis rates in the high-glucose+ 10 μmol/L berberine group and high-glucose+ 25 μmol/L berberine group were significantly reduced in comparison with that in the high-glucose group (both at P<0.01). And the cell apoptosis rate in the high-glucose+ 25 μmol/L berberine group was lower than that in the high-glucose+ 10 μmol/L berberine group ( P<0.01). ELISA results revealed that the overall comparative differences of the concentrations of TNF-α, IL-8 and COX-2 among the four groups were statistically significant ( F=28.36, 35.88, 41.59; all at P<0.01). The concentrations of TNF-α, IL-8 and COX-2 in the high-glucose group were significantly higher than those in the normal-glucose group ( P<0.01). Compared with the high-glucose group, the concentrations of TNF-α and COX-2 were significantly decreased in the high-glucose+ 10 μmol/L berberine group and high-glucose+ 25 μmol/L berberine group (both at P<0.05). The concentrations of TNF-α and IL-8 in the high-glucose+ 25 μmol/L berberine group were lower than those in the high-glucose+ 10 μmol/L berberine group (both at P<0.05). Real-time fluorescence quantitative PCR showed that the relative expression levels of GLAST mRNA in Müller cells among the four groups were statistically significant ( F=268.60, P<0.01). Compared with the normal-glucose group, the relative expression level of GLAST mRNA was significantly decreased in the high-glucose group ( P<0.01). In the high-glucose+ 10 μmol/L berberine group and high-glucose+ 25 μmol/L berberine group, the relative expression level of GLAST mRNA was significantly elevated compared with the high-glucose group (both at P<0.05). Western blot analysis results showed that the overall comparative differences of the GLAST, PPM1A, cleaved caspase-3, NF-κB in cytoplasm and NF-κB in nucleus among the four groups were statistically significant ( F=135.20, 156.98, 80.96, 128.07, 47.36; all at P<0.01). The relative expression levels of GLAST, PPM1A and NF-κB protein in cytoplasm in the high-glucose group were significantly lower than those in the normal-glucose group, high-glucose+ 10 μmol/L berberine group and high-glucose+ 25 μmol/L berberine group (all at P<0.05). The relative expression levels of cleaved caspase-3 and NF-κB protein in nucleus in the high-glucose group were significantly higher than those in the normal-glucose group, high-glucose+ 10 μmol/L berberine group and high-glucose+ 25 μmol/L berberine group (all at P<0.05).

Conclusions:

High-concentration glucose can induce cell apoptosis and inflammatory response of SD rats retinal Müller cells in vitro.However, berberine can inhibit cell apoptosis and inflammatory response induced by high-concentration glucose via suppressing NF-κB translocation and transcription activity, and thereby inhibiting the expression of inflammatory cytokines.