Hesperetin treatment attenuates glycation of lens proteins and advanced‑glycation end products generation

Abstract

Advanced glycation end products (AGEs) in lens proteins increase with aging, thus inducing cataracts and/or presbyopia. Hesperetin (Hst), which is an abundant plant flavanone largely derived from citrus species, and its derivatives attenuate cataracts and presbyopia in vivo and in vitro; however, no reports have described its effects on AGE formation in lens proteins. The present study demonstrated that AGEs in lens proteins increase with age in mice. Additionally, it showed that Hst can prevent AGEs and N(ε)‑carboxymethyl‑lysine generation and modification of lens proteins using in vitro in human lens epithelial cell lines and ex vivo in mouse lens organ cultures. Furthermore, treatment with Hst prevented lens hardening and decreased chaperone activity in lens proteins. These results suggested that Hst and its derivatives are good candidates for the prevention of presbyopia and cataracts.

Keywords: advanced glycation end product; hesperetin; lens protein; natural flavonoid; presbyopia.

Figure 1.

AGE modifications of lens proteins and lens elasticity changes with aging. (A) AGE and CML modifications of lens proteins of 10, 25, and 75-week-old mice were detected using an immunoblot analysis. (B) Lens elasticity was measured using SoftMeasure (Horiuchi Electronics Co., Ltd.) and (C) indentations of the lens were assessed under 0.05 N of force. The experiments used three to five independent samples per group. Data are presented as the mean ± standard error of the mean. *P<0.05 vs. the control group (n=6-8 mice/group). AGE, advanced glycation end product; CML, N(ε)-carboxymethyl-lysine.

Figure 2.

Effect of Hst treatment on AGE modifications of lens epithelial cell lines. (A) The structural formula of Hst. (B) Immunoblot analysis of AGE and CML modifications of lens proteins stimulated with high-glucose medium, MGO and CML administration with Hst. Hst treatment prevented AGE and CML modifications of lens proteins in a dose-dependent manner. The experiments used three independent samples per group. Hst, hesperetin; AGE, advanced glycation end product; CML, N(ε)-carboxymethyl-lysine; MGO, methylglyoxal; HG, High glucose stimulation (31 mM glucose).

Figure 3.

Effect of Hst treatment on AGE and CML formations in lens epithelial cell lines. The immortalized human lens epithelial cells were incubated using high-glucose medium with or without Hst for 5 days. After incubation, lens proteins were fixed with 4% paraformaldehyde and immunostained with (A) AGE antibody and (B) CML antibody. The nuclei are labeled with DAPI (blue). Hst, hesperetin; AGE, advanced glycation end product; CML, N(ε)-carboxymethyl-lysine.

Figure 4.

Lens elasticity and lens chaperone activity ex vivo. Lenses were incubated in M199 medium for up to 2 days. Lens elasticity and chaperone activity in the lens were measured. (A) Lenses incubated with high-glucose solution with or without Hst were mounted, and measurements of the pressure on and indentations of the lenses were performed. (B) Strain when lenses were pushed with 0.05 N of force is shown (bar graph). (C) The chaperone activity of water-soluble lens fractions was measured by light scattering with ALDH at 360 nm following the administration of high-glucose solution and/or Hst. Increased light scatting at 360 nm indicates ALDH aggregation, and inhibition of light scattering is dependent on chaperone activity. (D) Relative chaperone activity of lens proteins. The relative chaperone activity of cultured lens proteins with Hst was calculated using light scattering with ALDH for 60 min after the addition of 1,10-phenanthroline. The change in light scattering with ALDH without water-soluble lens proteins was defined as 100%. Bars represent the mean value. Error bars represent the standard error in (B) and (D). *P<0.05). Hst, hesperetin; NG, normal glucose stimulation (5.5 mM glucose); HG, high glucose stimulation (31 mM glucose); ALDH, aldehyde dehydrogenase.

Figure 5.

Effect of Hst treatment on AGE and CML formations in organ-cultured lenses with or without high-glucose stimulation. At two days following stimulation, lenses were sectioned and stained using AGE or CML antibody. (A) The effect of AGE formation on organ-cultured cells. (B) Effect of CML formation on organ-cultured cells. AGE and CML immunolabeling are presented in green. Membranes and nuclei are labeled with wheat germ agglutinin (WGA; red) and 4′,6′-diamidino-2-phenylindole (DAPI; blue), respectively. Hst, hesperetin; AGE, advanced glycation end product; CML, N(ε)-carboxymethyl-lysine; HG, high glucose stimulation (31 mM glucose).