Therapeutic Effect of Gypenosides on Antioxidant Stress Injury in Orbital Fibroblasts of Graves' Orbitopathy

Abstract

Purpose: To examine the impact of gypenosides (Gyps) on oxidative stress damage of orbital fibroblasts (OFs) from Graves' ophthalmopathy (GO) patients.

Methods: The relationship between Gyps and GO oxidative stress was understood by bioinformatics analysis. Orbital connective tissues of GO and non-GO patients were obtained for primary OF culture. The proliferation level of OFs was measured by Cell Counting Kit-8 method, and the appropriate intervention concentration of Gyps and H₂O₂ was obtained. The expression of apoptosis-related protein mRNA was analyzed by RT-qPCR technique. ROS and SOD test suites were employed to detect the oxidative stress level in OFs. Flow cytometry apoptosis detection, TUNEL detection, and lactate dehydrogenase detection were used to analyze the level of apoptosis. Western blotting detection was utilized to examine the regulatory pathway of oxidative stress, apoptosis, and autophagy-related proteins. The changes of cell morphology, autophagosome, and autophagy lysosome were observed by transmission electron microscope.

Results: The suitable intervention concentration of Gyps is 100 μg/mL, and the suitable intervention concentration of high concentration H₂O₂ is 350 μM. In comparison with the blank control group, the H₂O₂ intervention group enhanced the expression of apoptosis-related mRNA, the expression of ROS and SOD, the apoptosis rate, the expression of autophagy activation-related protein and Nrf2/ERK/HO-1 protein, and the number of autophagosomes and autophagy lysosomes. Compared with H₂O₂ intervention group, the expression of apoptosis-related mRNA decreased, ROS expression decreased, SOD expression increased, apoptosis rate decreased, autophagy activation-related protein expression decreased, Nrf2/ERK/HO-1 protein expression increased, and the quantity of autophagosomes and autophagy lysosomes decreased in H₂O₂ + Gyps intervention group.

Conclusion: Gyps can decrease the oxidative stress level of OFs generated by H₂O₂, reduce cell autophagy, and reduce apoptosis. Gyps may regulate the oxidative stress response of OFs in GO patients via the Nrf2/ERK/HO-1 signaling pathway.