Investigation of synergistic mechanism and identification of interaction site of aldose reductase with the combination of gigantol and syringic acid for prevention of diabetic cataract

Abstract

Background: Gigantol and syringic acid (SA) have been shown to synergistically prevent formation of diabetic cataract (DC). However, the exact mechanism of this effect is unknown. Here, we investigate the effect of these compounds on the activity of aldose reductase (AR) and cataract formation.

Methods: We examined the synergistic anti-cataract efficacy of gigantol and SA in the high glucose- and streptozotocin -induced DC rat model; synergism was evaluated using Jin's formula. We investigated possible mechanisms of action by measuring AR expression and activity and levels of sorbitol using enzyme kinetics, Western blot, and RT-PCR. Finally, we examined binding interaction between AR and both compounds using a combination of site-directed mutagenesis, recombinant expression of wild-type and mutant proteins, and enzyme kinetics.

Results: Combination treatment of gigantol and SA synergistically protected both HLECs(human lens epithelial cells) grown in vitro and DC formation in STZ-induced rats in vivo. Synergism was attributed to inhibition of AR activity, downregulation of AR expression via impaired transcription, and decreased sorbitol levels. Enzyme kinetics studies showed that the activity of an AR Asn160Ala mutant protein was significantly decreased compared to wild-type AR, confirming that Asn160 is a key residue for interaction between AR and both compounds.

Conclusion: Combined administration of gigantol and SA synergize to enhance anti-cataract efficacy. The synergistic effect is mainly attributed to disruption of the polyol pathway and inhibition of AR activity.

Keywords: Aldose reductase; Diabetic cataract; Gigantol; Site-directed mutagenesis; Synergism; Syringic acid.

Fig. 1

Structure and protein sequence of plasmid pET-28a-AR and its mutants. a plasmid map of pET-28a and pET-28a-AR. pET-28a was digested with HindIII and XhoI and ligated with a 951-bp fragment from either wild-type or mutant AR. b Protein sequences of wild-type and mutant AR

Fig. 2

Cataract progression in different groups as observed by slit lamp microscope. The results are expressed as mean ± SD (n = 15). Compared with the model group (Group II), turbidity in the pirenoxine group (GroupVI) and all 3 treatment groups (Group III, IV, V) was significantly reduced, ^* P < 0.01, vs all other groups. Optimal effects were observed in the combination (Group V); ^** P < 0.05, vs group III, IV

Fig. 3

Inhibitory effect of gigantol and SA eye drops on STZ-induced diabetic cataracts in Wistar rats. Representative photographs of lens from each group at the end of 60 days eye drop treatment. a normal control (group I); b diabetic cataracts untreated (group II); f diabetic cataracts treated with pirenoxine sodium eye drops each time (group VI); c, d, and e diabetic cataracts treated with SA, gigantol, or the combination of gigantol and SA, respectively (group III, IV and V)

Fig. 4

AR expression in the lens after treatment with gigantol, syringic acid, and the combination. a Representative image of Western blot, lanes 1 to 5: Group I to Group V. β-actin was used as the internal control. b AR expression levels after normalization for β-actin. The values are mean ± SD (n = 20), *p < 0.01, vs Group I, III, IV, V; **p < 0.05, vs Group III, IV

Fig. 5

AR mRNA expression after treatment with gigantol, syringic acid, and the combination. The expression of AR mRNA is normalized using the housekeeper gene (β-actin), which is evenly expressed in rat lens cells. Compared with the normal control group, mRNA levels of the AR gene increased 4.85-fold in Group II and 1.23-fold in Group V; *p < 0.01 vs Group I, Group III, Group IV and Group V. **p < 0.05 vs Group III and IV

Fig. 6

Sorbitol levels in the lens after treatment with gigantol, syringic acid, and the combination. The sorbitol level in lens in DC rats is significantly higher than levels in control rats (p < 0.01). Gigantol, syringic acid, and the combination reduced STZ-induced lenticular sorbitol accumulation. Sorbitol levels in lenses treated with the combination are lower than in the gigantol or syringic acid groups alone (p < 0.05)

Fig. 7

Expression of wild-type and mutant AR (sodium dodecyl sulfate polyacrylamide gel electrophoresis). BL21 (DE3)-pET28b-AR and the mutant were induced by 0.5 mM IPTG at 18 °C and then purified. Sampling volume was 8 μg per well. 2 proteins were successfully purified. AR protein molecular mass was 34.8 kDa, and was 38 kDa after being fused to His. 1: Marker; 2: blank control; 3: wild-type AR; 4: AR N160A. The arrow indicates the target protein

Fig. 8

Wild-type AR (wt-AR) and the mutant AR (Q160A) activity in the presence of different concentrations of gigantol and syringic acid. Data are expressed as mean ± SD (n = 3)