Oral administration of resveratrol or lactic acid bacterium improves lens elasticity

A decrease in the elasticity of the ocular lens during aging is associated with loss of the accommodative ability of the eye, leading to presbyopia. Although near vision impairment is a social issue affecting the length of healthy life expectancy and productivity of elderly people, an effective treatment to improve near vision has not yet become available. Here we examined the effect of Enterococcus faecium WB2000, Lactobacillus pentosus TJ515, and resveratrol on lens elasticity in rats, where the stiffness of the ocular lens increases exponentially during the aging process. A combination of WB2000 and resveratrol improved lens elasticity not only in the long term but also with just short-term treatment. In addition, TJ515 decreased stiffness in the eye lens with long-term treatment. Therefore, the oral administration of WB2000 and resveratrol or TJ515 may be a potential approach for managing the progression of near vision impairment.


Scientific Reports
| (2021) 11:2174 | https://doi.org/10.1038/s41598-021-81748-w www.nature.com/scientificreports/ In the present study, we measured the stiffness of the rat lens to confirm that the elasticity of the lens is decreased during aging. Further, we evaluated the effects of the application of resveratrol and two lactic acid bacteria, WB2000 and TJ515, on the stiffness of the rat lens. Both the short-and long-term administration of resveratrol and WB2000 in combination mitigated the increase in stiffness of the rat lens typically experienced during aging, whereas the administration of TJ515 alone decreased the lens stiffness with long-but not shortterm administration. These results indicate that the oral supplementation of an antioxidative diet could be a potential candidate to ameliorate the near vision impairment and presbyopia.

Results
Measurement of the lens elasticity of young and aged rats. To compare the elasticity of the lens during aging, crystalline lenses were extracted from young (8-week-old), middle-aged (24-week-old and 43-week-old) and old-aged (73-week-old) male rats and their stiffness was measured using a force-displacement measuring instrument. Previously, a coverslip lens-squeezing method and a height gauge relying on an electronic balance method were used to measure the distance of strain when pushed with a specific force 17,31 . In this study, we quantified the stiffness of the lens at 0.05 Newtons (N) with the instrument (Fig. 1a-c). The group of aged rats showed decreased strain (%, the changes of lens anteroposterior diameter) relative to that in the group of young rats, indicating that the stiffness of the lens was increased in the former ( Fig. 1d; Table 1). Changes in the elasticity of lens during aging are conserved across species ranging from mice to humans 3,5,31-33 .
The long-term effects of resveratrol and lactic acid bacteria on the elasticity of the rat lens. To investigate the effects of resveratrol and lactic acid bacteria on rat lens elasticity, we treated male rats with resveratrol and the lactic acid bacterium WB2000 in combination or with another lactic acid bacterium, TJ515, The lenses extracted from young (8-week-old, n = 8), middle-aged (24-week-old and 43-week-old) and old-aged (73-week-old, n = 5) male rats were assessed. The percentages of strain for the four age groups when lenses were pushed with 0.05 newtons (N) of force are shown in the bar graph. Data are presented as mean ± SEM. The asterisk indicates a statistically significant difference compared to 8-week-old (*P < 0.05, one-way analysis of variance followed by Tukey's post-hoc test). Table 1. The elasticity of young, middle and aged rat lenses was measured with a force-displacement measuring instrument. As shown in Fig. 1d, the lenses extracted from young (8-week-old, n = 8), middle (24-week-old, n = 5 and 43-week-old, n = 4) and aged (73-week-old, n = 5) male rats were assessed. The percentages of strain for the four age groups when lenses were pushed with 0.05 newtons (N) of force are shown. Data are presented as mean ± SEM. The asterisk indicates a statistically significant difference compared to 8-week-old (*P < 0.05, one-way analysis of variance followed by Tukey's post-hoc test). www.nature.com/scientificreports/ alone by daily oral administration from 12 to 52 weeks of age. After 40 weeks of treatment, ocular lenses were extracted, then assessed for stiffness when pushed with 0.05 N of force (Fig. 2a). The combination of resveratrol and WB2000 yielded results of increased strain as compared with those of control vehicle-treated rat lenses ( Fig. 2a; Table 2). Additionally, treatment with TJ515 alone for 40 weeks also increased the strain relative to that recorded in control vehicle-treated rats ( Fig. 2a; Table 2). These results indicate that the elasticity of the rat lens is affected by the long-term oral administration of the combination of resveratrol and WB2000 or TJ515 alone.
The short-term effects of resveratrol and lactic acid bacteria on the elasticity of the rat lens. Considering the improvements in rat lens elasticity gained with long-term treatment with TJ515 alone or the combination of resveratrol and WB2000, we next examined whether these supplements ameliorate the stiffness of the rat lens with only short-term treatment. At this stage of the research, male rats were treated with TJ515 or the combination of resveratrol and WB2000 by daily oral administration for 4 weeks instead of 40 weeks. The distance of strain in the group of rats treated with the combination of resveratrol and WB2000 was increased, indicating that the lens elasticity was improved by oral supplementation with resveratrol and WB2000 in a short period of time ( Fig. 2b; Table 2). Meanwhile, rats treated with TJ515 alone for four weeks showed decreased lens strain as compared with that measured in control vehicle-treated rats ( Fig. 2c; Table 2). These  www.nature.com/scientificreports/ results together indicate that the combination of resveratrol and WB2000 possesses the potential to improve rat lens elasticity both in the short and long term, while TJ515 only functions as a long-term treatment to decrease the degree of lens stiffness in rats. To gain an understanding of the effects of resveratrol and lactic acid bacteria in the context of near vision and presbyopia, a clinical study assessing resveratrol, WB2000, and TJ515 is necessary in human.

Discussion
Here, we showed that the stiffness of a crystalline lens in rats is increased with age and that daily oral administration of resveratrol and lactic acid bacteria increased the lens elasticity. In Helmholtz theory, short-range accommodation is associated with the degree of lens elasticity. Previously, we showed that lens elasticity was decreased in a smoking model in rats and pirenoxine eye drops, which have been used for the management of cataract, could improve the lens elasticity in rats 7 . Furthermore, the progression of near vision impairment was prevented by pirenoxine treatment in a clinical study 7 . Pirenoxine is known to maintain transparency in the crystalline lens by reducing the number of calcium ions, which have a crucial role in the aggregation of lens crystallin 34,35 . The homeostasis of lens proteins is associated with modifications including oxidation, deamidation, glycation, and the formation of disulfide bonds 36 . Indeed, in previous research, advanced glycation end-products mediated crosslinking and disulfide exchange among the proteins in the crystalline lens that are increased during aging 11,37 . Resveratrol, contained in grapes and skin of peanuts, is used already as a dietary supplement and is known to have a beneficial effect on cancer, type 2 diabetes, and cardiovascular disease, owing to the activation of SIRT1 and its protective antioxidant properties [38][39][40] . In addition, mixed dietary supplementation containing lactoferrin, fish oil, and Enterococcus faecium WB2000 decreased the amount of ROS production from the lacrimal gland in rats 41 . The oxidation of cysteine thiol by ROS is known to lead to the formation of protein disulfide bonds, which is a cause of protein aggregation 42,43 . The antioxidant response facilitated through redox-regulated proteins and the reduced form of glutathione possesses an ability to prevent protein disulfide bonding 44 . Therefore, the combination of resveratrol and WB2000 may serve as an antioxidant and could improve the elasticity of the crystalline lens. Although resveratrol has the antimicrobial activity against lactic acid bacteria, adding lactic acid bacteria concurrently may support the function of gut microbiota 45 . Separately, we observed that treatment with Lactobacillus pentosus TJ515 isolated from fermented food in Thailand increased the elasticity of the crystalline lens in rats, so dietary supplementation with TJ515 could also have a potential implication for the near vision impairment. Some types of lactic acid bacteria, Lactobacilus species are implicated in modulating immune response on autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease [46][47][48][49][50][51] . Since aging accelerates constitutive low-grade inflammation and higher prevalence of autoimmunity even in the eye, the dietary supplement of TJ515 could also be a potential implication for the near vision 52,53 .
It is reported that symptoms of asthenopia including irritating and tired eyes, blurred vision, and a dry eye sensation, accompany the pre-and early stages of presbyopia 54 . Therefore, the treatment of presbyopia may ameliorate those symptoms of asthenopia. The pre-and early stages of presbyopia, those can be detected by the measurement of the DCNFVA (distance-corrected near functional visual acuity) begin at about 30 years old 54,55 . It is interesting that our short-term experiment exhibited ameliorated effects on the lens elasticity of younger rats (Fig. 2b,c). The profits of any investigation to treat the near vision impairment and presbyopia will not only improve health life expectancy but also work productivity, which will serve as checkpoints in efforts to meet sustainable development goals coordinated by the World Health Organization 56 .

Measurement of rat lens elasticity with a force-displacement measuring instrument with flat indentation.
The elasticity of rat lenses was measured by using a force-displacement measuring instrument with flat indentation (YAWASA, Tec Gihan Co., Ltd., Tokyo, Japan). Briefly, lenses isolated from rats were immediately put on a metallic flat stage and the 12 mm-diameter flat indenter was positioned above the lens. Then the indenter gradually applied a load on the lens along the anteroposterior axis of the lens from above until 5.0 N (N) with the speed of 0.1 mm/s. The force and indentation displacement were recorded every 180 -1 s to generate the force-displacement curve. We calculated the mean and SEM of the strain (%) under 0.05 N and assessed the difference statistically.

Animals.
We consulted the ARRIVE guideline (https ://arriv eguid eline s.org/) to ensure proper reporting of animal experiments. The Keio University Institutional Animal Care and Use Committee approved all animal experiments (approval number: 17074), which were performed in Keio University School of Medicine, according to the Institutional Guidelines on Animal Experimentation at Keio University.
Male Wistar rats were purchased from Oriental Yeast (Tokyo, Japan). For the comparison of lens elasticity between young and aged rats, eight 8-week-old male rats, five 24-week-old male rats, four 43-week-old male rats and five 73-week-old male rats were prepared and each groups' lenses were extracted to measure the elasticity using the YAWASA device.
For the evaluation of the effects of long-term treatment, 12-week-old male rats were divided into six groups and orally administrated the vehicle (n = 6), TJ515 (0.0070 mg daily) (n = 6), or a mixture of resveratrol (0.088 mg daily) and WB2000 (0.042 mg daily) (n = 6) for 40 weeks. After treatment, rat lenses were extracted to measure the elasticity using the YAWASA device. www.nature.com/scientificreports/ For the evaluation of the effects of short-term treatment with the mixture of resveratrol and WB2000, 5-weekold male rats were divided into two groups and orally administrated the vehicle (n = 5) or a mixture of resveratrol (0.44 mg daily) and WB2000 (0.21 mg daily) (n = 6) for 4 weeks.
Finally, for the evaluation of the effects of short-term treatment with TJ515, 5-week-old male rats were divided into two groups and orally administrated either the vehicle (n = 12) or TJ515 (0.0070 mg daily) (n = 6) for 3 weeks. After treatment, rat lenses were extracted to measure the elasticity using the YAWASA device.
Statistical analysis. Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Data were expressed as mean ± standard error of the mean (SEM). The analysis of experimental data was performed using the two tailed Student's t-test or one-way analysis of variance, followed by Tukey's post-hoc test. A P value < 0.05 was considered to be statistically significant.