Excessive ECM deposition and increased TM resistance to aqueous humor outflow (AHO) are common pathological traits that contribute to an elevated IOP in patients with POAG. To closely mimic the phenotypes of POAG, we used DEX-induced OHT mice, which manifested significant IOP elevation and AHO fibrosis, and DEX-treated HTMC in this study. We demonstrated that MET is a potential novel preventive and protective drug against TM damage and shed light on its underlying mechanism.
TM cells are sensitive to oxidative stress and typically undergo basic cellular metabolic disorders and excessive ECM deposition under mechanical stress induced by an abnormally fluctuating IOP. For protection against oxidative stress and the prevention of POAG, the prophylactic prescription of topical supplements with alleged antioxidant capacities is recommended. Ample evidence exists that MET has antioxidative and antifibrotic effects in several tissues, including the heart, lung, kidney, liver, skin, and ovarian[41]. Interestingly, early large population-based studies[35, 42] found that in patients with diabetes mellitus, those with the highest quartile of MET had a 25% reduced OAG risk relative to those without, and every 1 g increase was associated with a 0.16% reduction in OAG risk, indicating the preventive and protective effect of MET against OAG onset. Consequently, the role of MET in POAG pathogenesis has become a potential new target for preventive or therapeutic approaches and has become the focus of experimental studies[43].
Our previous study demonstrated the therapeutic potential of MET against DEX-induced oxidative damage in the TM, both in vivo and in vitro[34]. In this study, we confirmed the preventive effect of MET against DEX-induced TM damage and the onset of OHT using the following evidence: 1) Significantly lowered IOP in MET + DEX mice than in PBS + DEX mice, and 2) ECM signals (α-SMA, fibronectin) were blocked by MET in OHT mice or DEX-treated HTMC. These evidence suggested that MET contributes to the clearance of excessive ECM deposition in the AHO of OHT mice, which is closely linked to the increase in IOP induced by DEX.
Live cell staining and WB analysis showed increased lysosomes and mitochondria in HTMC after DEX exposure, whereas these physiological changes were partially rescued by pretreatment with MET. Increased organelle numbers emphasize enhanced metabolic dynamics and energy consumption, presumably leading to mitochondrial division and fusion imbalances, resulting in cell death[44]. Mitochondrial functions are shape-dependent and are regulated by a balance between fission and fusion[45]. Peng et al.[46] reported that contractions between these two organelles are critical for mitochondrial dynamics and cellular homeostasis.
TEM analysis showed many pathological characteristics of TM in PBS + DEX eyes, including rougher cell membrane edge, poor integrity of the cell membrane, blurred cytoplasm, and in specious mitochondria. However, these abnormal changes were attenuated in MET-preventive mice. These findings suggest that MET may protect the TM by preventing overactivation of lysosomes and mitochondria. Lin et al.[47] elucidated that low-dose (≤ 200µM) MET targeted PEN2, inhibiting proton pump v-ATPase of lysosomes and then down-regulating adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) signals. However, the MET doses in our study were higher (3–10 mM) than those in their study, and the correlations among MET dose, metabolic activity, and TM health remain unknown.
Autophagy, a cellular “house-keeping” mechanism, is a process that catalyzes the degradation of injured organelles and maintains cellular homeostasis, whereas excessive activation will further cause cell apoptosis[12]. In previous and this study, we observed that autophagy was induced in the TM tissues of OHT mice by DEX treatment, which is consistent with previous studies[48], and LC3B was even more increased in MET + DEX eyes. Moreover, autophagy-associated markers (Atg 5/12 and LC3-II/Ⅰ ratio) were activated together with a reduction in the autophagic substrate p62 in HTMC after MET treatment.
Mitophagy is a key process involved in the regulation of mitochondrial quality. DEX up-regulated mitophagy at an early stage and disrupted mitochondrial quality control later, which might represent a cellular adaptation to cellular damage[45]. In this study, we observed MET significantly activated mitophagy and promoted mitochondrial fusion in DEX-damaged HTMC, as evidenced by the significantly up-regulated PINK1 and MFN2, as well as the giant mitochondria observed by TEM scanning. This finding is consistent with recently published research on cardiomyocytes[49], indicating that the integration of these biogenesis pathways may contribute to improved cell health.
The mechanism diagram is shown in Fig. 10. We speculated that when TM is stimulated by DEX, cellular metabolism is enhanced. Under these physiological conditions, lysosomes and mitochondria are overactivated, an imbalance of fissures and fusion in mitochondria occurs, and more ROS are produced, along with mitophagy suppression while total macroautophagy activation. When cell metabolism is decompensated, mitochondria are reduced or even exhausted, autophagy may be inhibited, and excessive ROS and ECM accumulate. In contrast, MET pretreatment inhibits the respiratory chain in the mitochondria and blocks the ATPase pump in lysosomes, leaving the TM in a low energy consumption state. When these TM are exposed to DEX, fewer mitochondria and lysosomes are activated, and the fissures and fusion are closer to a balance in the mitochondria, together with less ROS and ECM production. Both mitophagy and the whole macroautophagy are activated to clear damaged organelles and degrade the ECM; thus, preventing IOP elevation.
This study had several limitations. First, the correlation between lysosomes and mitochondrial activity, mitophagy, macroautophagy, and TM fibrosis was not fully clarified. Second, the experimental period was too short to determine whether MET had a long-term protective effect against TM damage. Third, MET concentrations were not detected in the plasma and aqueous humor, neither did us test the blood glucose level in mice. Finally, the orientation of the tissues were inconsistent among groups. Additional studies are required to address this issue.
This study elucidated that MET protects TM against DEX-induced IOP elevation, probably by activating mitophagy and maintaining the fissure and fusion balance in the mitochondria. Our results suggest that MET is a potential therapeutic target for patients with POAG. However, the relationship between MET dose, lysosomes, mitochondrial metabolism, mitophagy, and autophagic activity requires further investigation.
In summary, this study elucidates the mechanisms of mitophagy activation by MET in the TM. Preemptive application of MET may be efficient in preventing IOP elevation in DEX-induced OHT in mice and may protect the TM by suppressing the overactivation of lysosomes and mitochondria.