Effects of probucol on high glucose-induced specificity protein 1/Keap1/Nrf2/glutamate-cysteine ligase catalytic in the cultured human müller cells

Abstract

Objective To observe the expression of probucol on high glucose-induced specificity protein 1(SP1), kelchlike ECH associated protein1 (Keap1), NF-E2-related factor 2 (Nrf2) and glutamate-cysteine ligase catalytic (GCLC) in the cultured human muller cells and preliminary study the antioxidation of the probucol on muller cells. Methods Primary cultured human muller cells were randomly divided into four groups: normoglycaemia group (5.5 mmol/L glucose), normoglycaemia with probucol group (5.5 mmol/L glucose+100 μmol/L probucol), hyperglycemia group (25.0 mmol/L glucose), hyperglycemia with probucol group (25.0 mmol/L glucose + 100 μmol/L probucol). Immunofluorescence staining was used to assess distribution of SP1, Keap1, Nrf2, GCLC in human Muller cells. SP1, Keap1, Nrf2 and GCLC messenger RNA (mRNA) expression was evaluated by quantitative real-time RT-PCR (qRT-PCR). Independent sample t test was used to compare the data between the two groups. Results All muller cells expressed glutamine synthetase(>95%), which confirmed the cultured cells in vitro were the purification of generations of muller cells. The expressions of SP1, Keap1, Nrf2, and GCLC protein were positive in human muller cells. qRT-PCR indicated that SP1 (t=28.30, P<0.000), Keap1 (t=5.369, P=0.006), and Nrf2 (t=10.59, P=0.001) mRNA in the hyperglycemia group increased obviously compared with the normoglycaemia group; GCLC (t=4.633, P=0.010) mRNA in the hyperglycemia group decreased significantly compared with the normoglycaemia group. However, SP1 (t=12.60, P=0.000) and Keap1 (t=4.076, P=0.015) in the hyperglycemia with probucol group decreased significantly compared with the hyperglycemia group; Nrf2 (t=12.90, P=0.000) and GCLC (t=15.96, P<0.000) mRNA in the hyperglycemia with probucol group increased obviously compared with with the hyperglycemia group. Conclusion Probucol plays an antioxidant role by inhibiting the expression of SP1, Keap1 and up-regulating the expression of Nrf2, GCLC in muller cells induced by high glucose. Key words: Probucol/therapeutic use; Sp1 transcription factor; NF-E2-related factor 2; Glutamate-cysteine ligase; Muller cell; Kelchlike ECH assoiated protein1