Our results show that HG treatment led to a significant increase in the proportion of dead cells and ROS and a concurrent decrease in SIRT3 expression. HG treatment also disrupted mitochondrial dynamics and led to increased mitochondrial fission increased. The overexpression of SIRT3 reversed all of these effects, but this could be blocked by an OPA1 inhibitor. These findings support that HG may cause increased oxidative stress levels in diabetes and lead to endothelial cell apoptosis, which is consistent with previous research results14.
The SIRT family plays a crucial role in regulating cellular homeostasis and is involved in various cellular processes, including gene regulation, metabolism, aging, and the oxidative stress response15. Moreover, SIRTs show clear cellular localization; for example, SIRT3, -4, and − 5 are located in the mitochondria and participate in energy homeostasis, redox balance, mitochondrial unfolded protein response, biogenesis, dynamics, and mitophagy, making them important regulators of mitochondrial homeostasis16, 17. In previous studies, SIRT3, -4, and − 5 have been shown to protect endothelial cells through different pathways in cardiovascular disease18, 19, 20. However, there are few studies on the role of mitochondrial SIRTs in DR, and even fewer on those in RMEC. In this study, we examined the expression of mitochondrial SIRTs in RMEC under HG and found that the expressions of SIRT3 and SIRT5 decreased. This effect was enhanced with increased exposure to HG. Overall, SIRT3 was more affected by HG than SIRT5. These results suggest that SIRT3 is involved in endothelial cell injury caused by HG. To verify the role of SIRT3, SIRT3-overexpressed RMEC lines were constructed, and the overexpression of SIRT3 reversed apoptosis and reduced the ROS level, which had been increased by HG.
The pathological process of DR-related injury is based primarily on the excessive production of ROS21, which is produced mainly by mitochondria. Mitochondria are highly variable organelles that maintain homeostasis through multiple regulatory mechanisms, including mitochondrial biosynthesis, autophagy, and dynamics22. Our research found that the changes in OPA1 and Drp1, two important mitochondrial dynamics molecules, were abnormal under HG and reversed by SIRT3 overexpression. Mitochondrial dynamics are the dynamic balance that mitochondria maintain between fusion and fission. The morphological changes in mitochondria are closely related to their function. For further investigation, we labeled mitochondria with MitoTracker probes and used confocal microscopy to investigate the mitochondrial morphology in living cells. The mitochondrial morphologies were categorized into three morphotypes: tubular, mixed, and fragmented. Our results showed HG increased mitochondrial fragmentation, and SIRT3 overexpression reversed this pathological change and led to mitochondrial elongation.
Furthermore, our result showed that SIRT3 overexpression reversed the effect of HG on the mRNA and protein expressions of the mitochondrial fusion-related molecule OPA1. OPA1 is a fusion gene, first discovered in 2000, in dominant hereditary optic atrophy23 that mediates fusion of the inner mitochondrial membrane24.It was reported that SIRT3 could directly bind OPA1 and promote its deacetylation, thus increasing GTPase activity and fusion25. Yi et al. confirmed that SIRT3 can bind to OPA1 in melanocytes by co-immunoprecipitation and play a direct role in deacetylation modification to regulate mitochondrial dynamics26. However, in the LPS-mediated acute kidney injury model, SIRT3 overexpression indirectly promotes OPA1-mediated mitochondrial dynamics regulation through the deacetylation of the OPA1 upstream regulatory molecule i-AAA protease (YME1L1)27. In SIRT3 knockout mice, OPA1 shows excessive acetylation, abnormal mitochondrial cristae morphology, and severe heart damage, leading to a shortened lifespan28. Although the role of OPA1 has been proven in various pathological conditions, its involvement and role in DR has not been well reported. Herein, we showed that after inhibiting OPA1, the protective effect of SIRT3 on cell death, ROS decrease, and mitochondrial dynamics were reversed. This supported the protective effect of SIRT3 in RMEC being mediated through the OPA1 mitochondrial fusion pathway.
In conclusion, our research found that SIRT3 is involved in HG-induced endothelial impairment in DR through OPA1-mediated mitochondrial dynamics imbalance. Our investigation provides a theoretical basis for the potential prevention and treatment of DR.