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.