Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals

Abstract

Background: This study aimed to investigate the protective effect of metformin on the trabecular meshwork (TM) and explore its molecular mechanisms in vivo and in vitro. Methods: Ocular hypertension (OHT) mouse models were induced with dexamethasone (DEX) and further treated with metformin to determine its IOP lowering effect. Cultured human TM cells (HTMC) were pre-stimulated with tert-butyl hydroperoxide (tBHP) to induce oxidative damage and then supplemented with metformin for another 24 h. The expression of fibrotic markers and integrin/ROCK signals, including α-SMA, TGF-β, fibronectin, F-actin, integrin beta 1, Rho-associated kinase (ROCK)1/2, AMP-activated protein kinase (AMPK), myosin light chain 1 (MLC 1), and F-actin were determined by western blotting (WB) and immunofluorescence (IF). Reactive oxygen species (ROS) content was analysed using flow cytometry (FCM). Results: Administration of metformin reduced the elevated IOP and alleviated the fibrotic activity of aqueous humour outflow in OHT models. Additionally, metformin rearranged the disordered cytoskeleton in the TM both in vivo and in vitro. Furthermore, metformin significantly inhibited ROS production and activated integrin/ROCK signalling induced by tBHP in HTMC. Conclusion: Metformin reduced the elevated IOP in steroid-induced OHT mouse models and exerted its protective effects against oxidative injury by regulating cytoskeleton remodelling through the integrin/ROCK pathway. This study provides new insights into metformin use and preclinical evidence for the potential treatment of primary open-angle glaucoma. Funding: This work was supported by the Key R&D Program of Zhejiang (2022C03112), Leading Scientific and Technological Innovation Talents in Zhejiang Province (2021R52012), National Key R&D Program of China (2020YFC2008200), Zhejiang Provincial National Science Foundation of China (LQ18H120010), Key Innovation and Guidance Program of the Eye Hospital, School of Ophthalmology & Optometry,Wenzhou Medical University (YNZD 2201903), and the Wenzhou Municipal Technological Innovation Program of High-level Talents (No. 604090352/577).

Metformin in Glaucoma Treatment

“…In addition, 54 preclinical studies that explored pathophysiological pathways and cytokines involved in the proposed possible mechanisms were retrieved in full text after a manual search of the 10 initially identified studies. 38 Metformin effect in glaucoma risk factors…”

“…34 Furthermore, a recent study has demonstrated metformin's effect on IOP reduction and cytoskeleton rearrangement in an animal mice model, caused by excessive reactive oxygen species (ROS) and TGF-β, through the activation of integrin/Rho-associated kinase Rho-Associated Protein Kinase signaling. 38…”

“…2,[87][88][89][90] ECM remodeling resulting from this pathway further stiffens and compresses the connective tissue microarchitecture, leading to damage to RGCs. 2,38 These fibrotic changes, in addition to the disruption of cytoskeletal integrity and apoptosis by ROS in TM, increase resistance to aqueous outflow and IOP, setting in motion a vicious cycle. 2,38,91 Another key contributing factor to ECM remodeling is aging, as older tissues contain fibroblasts that are resistant to apoptosis, have decreased levels of cadherin and catenincytoplasmic proteins to maintain epithelial integrity, and experience progressive collagen glycation, which drives tissue stiffness and rigidity.…”

“…2,38 These fibrotic changes, in addition to the disruption of cytoskeletal integrity and apoptosis by ROS in TM, increase resistance to aqueous outflow and IOP, setting in motion a vicious cycle. 2,38,91 Another key contributing factor to ECM remodeling is aging, as older tissues contain fibroblasts that are resistant to apoptosis, have decreased levels of cadherin and catenincytoplasmic proteins to maintain epithelial integrity, and experience progressive collagen glycation, which drives tissue stiffness and rigidity. 2,91,92 Metformin has been shown to exert its antifibrotic effects through both the direct inhibition of TGF-β and the modulation of various signaling pathways in vivo mouse and cultivated cells.…”

“…2,91,92 Metformin has been shown to exert its antifibrotic effects through both the direct inhibition of TGF-β and the modulation of various signaling pathways in vivo mouse and cultivated cells. 2,38,93 Other observed effects of metformin that may play a role in protecting RGCs include the inhibition of apoptosis through the upregulation of antiapoptotic genes and the downregulation of proapoptotic genes in response to environmental stressors, the maintenance of intercellular junctions, regulation of fatty acid synthesis and glycolysis processes, cellular protein trafficking, mitophagy, and autophagy. 19,48,61,81,[94][95][96][97]…”