Diabetic retinopathy (DR) is a serious and relatively under-recognized complication of diabetes. Müller glial cells extend throughout the retina and play vital roles in maintaining retinal homeostasis. Previous studies have demonstrated that TGR5, a member of the bile acid-activated GPCR family, could ameliorate DR. However, the role of TGR5 in regulating Müller cell function and the underlying mechanism remains to be ascertained. To address this, high glucose (HG)-treated human Müller cells and streptozotocin-treated Sprague-Dawley rats were used in the study. The IP3R1-GRP75-VDAC1 axis and mitochondrial function were assessed after TGR5 ablation or agonism. Cytosolic mitochondrial DNA (mtDNA)-mediated cGAS-STING activation was performed. The key markers of retinal vascular leakage, apoptosis, and inflammation were examined. We found that mitochondrial Ca²⁺ overload and mitochondrial dysfunction were alleviated by TGR5 agonist. Mechanically, TGR5 blocked the IP3R1-GRP75-VDAC1 axis mediated Ca²⁺ efflux from the endoplasmic reticulum into mitochondria under diabetic condition. Mitochondrial Ca²⁺ overload led to the opening of the mitochondrial permeability transition pore and the release of mitochondrial DNA (mtDNA) into the cytosol. Cytoplasmic mtDNA bound to cGAS and upregulated 2’3’ cyclic GMP-AMP. Consequently, STING-mediated inflammatory responses were activated. TGR5 agonist prevented retinal injury, whereas knockdown of TGR5 exacerbated retinal damage in DR rats, which was rescued by the STING inhibitor. Based on the above results, we propose that TGR5 might be a novel therapeutic target for the treatment of DR.

Cellular senescence is a hallmark of aging and has been linked to age-related diseases. Age-related macular degeneration (AMD), the most common aging-related retinal disease, is prospectively associated with retinal pigment epithelial (RPE) senescence. However, the mechanism of RPE cell senescence remains unknown. In this study, tert-butyl hydroperoxide (TBH)-induced ARPE-19 cells and D-galactose-treated C57 mice were used to examine the cause of elevated iron in RPE cell senescence. Ferric ammonium citrate (FAC)-treated ARPE-19 cells and C57 mice were used to elucidated the mechanism of iron overload-induced RPE cell senescence. Molecular biology techniques for the assessment of iron metabolism, cellular senescence, autophagy, and mitochondrial function in vivo and in vitro. We found that iron level was increased during the senescence process. Ferritin, a major iron storage protein, is negatively correlated with intracellular iron levels and cell senescence. NCOA4, a cargo receptor for ferritinophagy, mediates degradation of ferritin and contributes to iron accumulation. Besides, we found that iron overload leads to mitochondrial dysfunction. As a result, mitochondrial DNA (mtDNA) is released from damaged mitochondria to cytoplasm. Cytoplasm mtDNA activates the cGAS-STING pathway and promotes inflammatory senescence-associated secretory phenotype (SASP) and cell senescence. Meanwhile, iron chelator Deferoxamine (DFO) significantly rescues RPE senescence and retinopathy induced by FAC or D-gal in mice. Taken together, these findings imply that iron derived from NCOA4-mediated ferritinophagy causes cellular senescence via the cGAS-STING pathway. Inhibiting iron accumulation may represent a promising therapeutic approach for age-related diseases such as AMD.

Endoplasmic reticulum (ER) and mitochondrial dysfunction play fundamental roles in the pathogenesis of diabetic retinopathy (DR). However, the interrelationship between the ER and mitochondria are poorly understood in DR. Here, we established high glucose (HG) or advanced glycosylation end products (AGE)-induced human retinal vascular endothelial cell (RMEC) models in vitro, as well as a streptozotocin (STZ)-induced DR rat model in vivo. Our data demonstrated that there was increased ER–mitochondria coupling in the RMECs, which was accompanied by elevated mitochondrial calcium ions (Ca²⁺) and mitochondrial dysfunction under HG or AGE incubation. Mechanistically, ER–mitochondria coupling was increased through activation of the IP3R1–GRP75–VDAC1 axis, which transferred Ca²⁺ from the ER to the mitochondria. Elevated mitochondrial Ca²⁺ led to an increase in mitochondrial ROS and a decline in mitochondrial membrane potential. These events resulted in the elevation of mitochondrial permeability and induced the release of cytochrome c from the mitochondria into the cytoplasm, which further activated caspase-3 and promoted apoptosis. The above phenomenon was also observed in tunicamycin (TUN, ER stress inducer)-treated cells. Meanwhile, BAPTA-AM (calcium chelator) rescued mitochondrial dysfunction and apoptosis in DR, which further confirmed of our suspicions. In addition, 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, was shown to reverse retinal dysfunction in STZ-induced DR rats in vivo. Taken together, our findings demonstrated that DR fueled the formation of ER–mitochondria coupling via the IP3R1–GRP75–VDAC1 axis and accelerated Ca²⁺-dependent cell apoptosis. Our results demonstrated that inhibition of ER–mitochondrial coupling, including inhibition of GRP75 or Ca²⁺ overload, may be a potential therapeutic target in DR.

Background: Diabetic retinopathy (DR) is one of the most important microvascular diseases of diabetes. Our previous research demonstrated that bile acid G-protein-coupled membrane receptor (TGR5), a novel cell membrane receptor of bile acid, ameliorates the vascular endothelial cell dysfunction in DR. However, the precise mechanism leading to this alteration remains unknown. Thus, the mechanism of TGR5 in the progress of DR should be urgently explored.Methods: In this study, we established high glucose (HG)-induced human retinal vascular endothelial cells (RMECs) and streptozotocin-induced DR rat in vitro and in vivo. The expression of TGR5 was interfered through the specific agonist or siRNA to study the effect of TGR5 on the function of endothelial cell in vitro. Western blot, immunofluorescence and fluorescent probes were used to explore how TGR5 regulated mitochondrial homeostasis and related molecular mechanism. The adeno-associated virus serotype 8-shTGR5 (AAV8-shTGR5) was performed to evaluate retinal dysfunction in vivo and further confirm the role of TGR5 in DR by HE staining, TUNEL staining, PAS staining and Evans Blue dye.Results: We found that TGR5 activation alleviated HG-induced endothelial cell apoptosis by improving mitochondrial homeostasis. Additionally, TGR5 signaling reduced mitochondrial fission by suppressing the Ca²⁺-PKCδ/Drp1 signaling and enhanced mitophagy through the upregulation of the PINK1/Parkin signaling pathway. Furthermore, our result indicated that Drp1 inhibited mitophagy by facilitating the hexokinase (HK) 2 separation from the mitochondria and HK2-PINK1/Parkin signaling. In vivo, intraretinal microvascular abnormalities, including retinal vascular leakage, acellular capillaries and apoptosis, were poor in AAV8-shTGR5-treated group under DR, but this effect was reversed by pretreatment with the mitochondrial fission inhibitor Mdivi-1 or autophagy agonist Rapamycin.Conclusion: Overall, our findings indicated that TGR5 inhibited mitochondrial fission and enhanced mitophagy in RMECs by regulating the PKCδ/Drp1-HK2 signaling pathway. These results revealed the molecular mechanisms underlying the protective effects of TGR5 and suggested that activation of TGR5 might be a potential therapeutic strategy for DR.