Untreated cataract is the leading cause of blindness worldwide, which is characterized by opacification of the crystalline lens [23]. Epidemiological studies have shown that age is the dominant risk for cataracts, known as ARC, and the only treatment is surgical removal [24]. Previous studies have shown that senescence of LECs is a main cause of ARC [25]. Until now, there are no effective therapeutic agents to inhibit the cloudiness of lens and attenuate the senescence of LECs without undesirable side effects. Exploring the molecular biological basis of the senescence of lens epithelial cells, and the mechanism of protecting normal LECs against senescence are the focus of present study. Our results showed that inactivation of AMPK and impairment of autophagy were associated with senescence of LECs and ARC. Furthermore, we found that MET might protect LECs against senescence and inhibit the cloudiness of lens via activation of AMPK and improvement of autophagy.
Senescence is a fundamental biological process accompanied by a general decline in tissue function. Indeed, as the lifespan increases, age-related dysfunction, such as ARC, has become a growing public health issue [26]. Senescence of LECs has been documented involved in the occurrence and the development of ARC [5]. Our results showed that the LECs in the naturally aged mice (Aged) were flat, arranged unevenly and the density of LECs decreased markedly (Fig. 1b). Additionally, the expressions of aging-associated gene (P21, P53) were evidently up-regulated in the LECs in the naturally aged mice (Aged) (Fig. 1c, d, e). These results above implied that the senescence of LECs was associated with the occurrence and the development of ARC. Thus, an accurate understanding of the molecular mechanisms of LECs senescence may be required to provide a rationale for new treatment strategies.
MET, a biguanide, is the most widely-prescribed oral hypoglycemic medication for type 2 diabetes worldwide. Emerging evidence indicates that MET has beneficial effects on health beyond those associated with amelioration in glycemia [27]. It has been confirmed that the mechanisms of MET in targeting fundamental pathways in biological aging, including activation of AMPK, augmenting autophagy, inhibiting inflammation, exerting antioxidative effects [28]. In humans, MET has been in clinical use for over 60 years, has a high safety profile and is uniquely positioned to intervene several crucial pathways responsible for aging and age-related diseases. However, the effects of MET on ARC and the regulatory mechanism of those effects have never been reported. Consequently, chronic low-dose MET administration was innovatively performed on ARC in naturally aged mice. Our results demonstrated that chronic low-dose MET administration was able to serve transparency lens (Fig. 2a) and attenuate the senescence of LECs in vivo (Fig. 2b, c).
It is widely acknowledged that MET activates AMPK pathway, which is the potential mechanism of MET to exert anti-aging effect [29]. AMPK, cellular energy sensor, plays a crucial role in regulating cellular energy balance [30]. Several studies have illustrated that AMPK serves as an integrator and mediator of several pathways and processes linking energetics to longevity. In our study, we found that the inactivation of AMPK was the feature of senescent LECs in naturally aged mice (Fig. 3). Later, MET was shown to stimulate AMPK pathway to prevent the senescence of LECs (Fig. 4). As an established AMPK activator, MET’s role in aging prevention is generally attributed to its effects on modulating downstream signaling pathways, such as restoration of autophagy, activation of Sirt1 and Foxo1, as well as the suppression of mTOR [31, 32]. Our study provided a new line of evidence stressing the importance of AMPK function activated by MET on preventing the occurrence of ARC.
As mentioned above, AMPK thus communicates with numerous pathways and proteins to exert the anti- senescence effect, including augmentation of autophagy. Autophagy, emerging as a core process for longevity assurance, has attracted widespread interest as a potential therapeutic target for age-associated diseases[21]. Autophagy is a fluid, multi-step complex biological process. The complete process of autophagy is called autophagic flux, which is widely used to reflect the level of autophagy. As an indicator of autophagy, LC3-II is tightly bound to autophagosomal membrane, which is degraded by lysosomal enzymes. Likewise, P62, autophagy-specific substrate, usually is degraded in autophagolysosome and its expression indirectly reflects the level of autophagy. In present study, Our findings implied that the level of autophagy was distinctly declined in senescent LECs (Fig. 5), while MET reversed of age-related impairment of autophagy (Fig. 6). With regard to the anti-aging molecular mechanism of MET stems from the primary function of AMPK in cells because it generally enhances autophagy via AMPK activation [33]. On the other hand, MET is shown to augment autophagy directly, thereby contributing to prevention of senescence [34]. However, more detailed studies are absolutely needed to elucidate the mechanism(s) that MET augments autophagy via independent or dependent AMPK pathway.