H3 Relaxin Alleviates Migration, Apoptosis and Pyroptosis Through P2X7R-Mediated Nucleotide Binding Oligomerization Domain-Like Receptor Protein 3 Inflammasome Activation in Retinopathy Induced by Hyperglycemia.

Kelaier Yang,Wenjian Lin,Xuefei Ma,Ming Hao,Xiaohui Zhang,Hongyu Kuang,Ziqi Ren,Ying Wang,Jiannan Liu,Lei Gao

doi:10.3389/fphar.2020.603689

Abstract

Introduction: P2X7R excitation-interrelated NLRP3 inflammasome activation induced by high glucose contributes to the pathogenesis of diabetic retinopathy (DR). Relaxin-3 is a bioactive peptide with a structure similar to insulin, which has been reported to be effective in diabetic cardiomyopathy models in vivo and in vitro. However, it is not known whether relaxin-3 has a beneficial impact on DR, and the underlying mechanisms of the effect are also remain unknown. Methods and Results: The retinas of male streptozotocin (STZ)-induced diabetic Sprague-Dawley (SD) rats were characterized. Human retinal microvascular endothelial cells (HRMECs) were used to evaluate the anti-inflammatory, antiapoptotic, antipyroptotic and anti-migration effects of H3 relaxin by transmission electron microscopy, wound-healing assay, transwell assay, flow cytometry, cytokine assays and western-blot analysis. After H3 relaxin treatment, changes of the ultrastructure and expression of NLRP3 inflammasome related proteins in the retinas of rats were compared with those in the diabetic group. In vitro, H3 relaxin played a beneficial role that decreased cell inflammation, apoptosis, pyroptosis and migration stimulated by advanced glycation end products (AGEs). Moreover, inhibition of P2X7R and NLRP3 inflammasome activation decreased NLRP3 inflammasome-mediated injury that similar to the effects of H3 relaxin. H3 relaxin suppressed the stimulation of apoptosis, pyroptosis and migration of HRMECs in response to AGEs mediated by P2X7R activation of the NLRP3 inflammasome. Conclusion: Our findings provide new insights into the mechanisms of the inhibitory effect of H3 relaxin on AGE-induced retinal injury, including migration, apoptosis and pyroptosis, mediated by P2X7R-dependent activation of the NLRP3 inflammasome in HRMECs.

Highlights

P2X7R excitation-interrelated Nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation induced by high glucose contributes to the pathogenesis of diabetic retinopathy (DR)
Treatment of Human retinal microvascular endothelial cells (hRMECs) with the combination of H3 relaxin and MCC950 reduced apoptosis and pyroptosis compared with those in hRMECs treated with advanced glycation end products (AGEs)-BSA, the reduction was incomplete (Figures 4A–E). These results indicated that H3 relaxin alleviated AGE-induced apoptosis and pyroptosis and the roles involved in these improvements might be partially mediated by the NLRP3 inflammasome
The results showed that hRMECs treated with MCC950 manifested a decrease in the levels of Vascular endothelial (VE)-cadherin, zonula occludens (ZO)-1, occludin, MMP2 and MMP9, accompanied by reduced expression levels of cleaved caspase1, IL-18 and IL-1β. hRMECs treated with H3 relaxin exhibited improvements in VE-cadherin, ZO-1, occludin, MMP2 and MMP9; unlike the MCC950 group, the H3 relaxin group showed improvements in all components of the NLRP3 inflammasome

Summary

Introduction

P2X7R excitation-interrelated NLRP3 inflammasome activation induced by high glucose contributes to the pathogenesis of diabetic retinopathy (DR). Relaxin-3 is a bioactive peptide with a structure similar to insulin, which has been reported to be effective in diabetic cardiomyopathy models in vivo and in vitro It is not known whether relaxin-3 has a beneficial impact on DR, and the underlying mechanisms of the effect are remain unknown. The NLRP3 inflammasome is the primary transducer that translates a decrease in cytoplasmic K+ induced by P2X7R receptor activation into pro-inflammatory signals (Franceschini et al, 2015). Treatment with P2X7R antagonists and data obtained from P2X7−/− mice demonstrates that suppression of P2X7R attenuates inflammatory processes, indicating that P2X7R plays the role of a trigger that decreases intracellular K+, and is an essential ATP-sensing receiver of NLRP3 inflammasome signaling (Karmakar et al, 2016). Inhibition of P2X7R expression indirectly reduces the activation of the NLRP3 inflammasome

Methods

Results

Discussion

Conclusion