Journal of Traditional Chinese Medical Sciences (Oct 2023)
Guilu Erxian glue mitigates oxidative damage in mouse GC-1 spermatogonial cells by inhibiting autophagy via the Keap1/Nrf2 pathway
Abstract
Objective: To explore the effects and underlying mechanisms of Guilu Erxian glue (GLEXG) on oxidative damage in a mouse GC-1 spermatogonial (MGS) cell model. Methods: A cellular model for oxidative damage was created using MGS cells exposed to hydrogen peroxide (H2O2). Cell viability was assessed using the cell counting kit-8 assay, while reactive oxygen species (ROS) and malondialdehyde (MDA) levels were measured via flow cytometry and enzyme-linked immunosorbent assay, respectively. Western blotting and immunofluorescence techniques were employed to quantify the relative expression levels of sequestosome-1 (p62), nuclear factor erythroid 2-related factor 2 (Nrf2), microtubule-associated protein light chain 3β (LC3B), and Kelch-like ECH-associated protein 1 (Keap1). Quantitative real-time PCR was used to evaluate Keap1 mRNA expression. Transmission electron microscopy (TEM) was conducted to observe structural changes in autophagy-related vesicles. Results: The cellular model of oxidative damage induced by H2O2 showed reduced cell viability along with elevated levels of ROS and MDA. Treatment with 10% GLEXG-enriched serum significantly enhanced cell viability (P = .0002) while decreasing ROS and MDA levels (P = .0105 and P = .0033, respectively). In rapamycin-treated MGS cells, GLEXG treatment substantially upregulated the relative protein expression of p-mTOR, Nrf2, and p62 (all P < .01), and downregulated the expression of Keap1 and the LC3B-II/LC3B-I ratio (P = .002 and P = .0043, respectively). It also lowered ROS and MDA levels. TEM analysis revealed that GLEXG treatment considerably reduced the number of abnormally enlarged autolysosomes in rapamycin-treated MGS cells. In Keap1-siRNA-transfected MGS cells, the siRNA-Keap1-2311 knockdown site demonstrated higher efficiency. Furthermore, GLEXG treatment in these Keap1-siRNA-transfected cells notably upregulated the relative protein expression of Nrf2 and p62, decreased Keap1 expression and the LC3B-II/LC3B-I ratio, and reduced ROS and MDA levels. Conclusion: GLEXG effectively mitigated oxidative damage in the MGS cell model by inhibiting autophagy through the Keap1/Nrf2 pathway.
