Expression and purification of recombinant glutaredoxin 1 and protection against oxidative stress injury during cerebral ischemia-reperfusion injury

Abstract

Glutaredoxin (Grx) is a small molecular protein widely found in both prokaryotes and eukaryotes, serving various biological functions, including participation in redox reactions and exerting anti-apoptotic effects[1]. To evaluate the protective effect of recombinant Grx1 against oxidative stress, we constructed the pET-30a (+)/Grx1 recombinant plasmid and performed soluble expression and purification of the recombinant Grx1. In vitro experiments, including ABTS and DPPH radical scavenging assays, showed that recombinant Grx1 has significant antioxidant activity. Reactive oxygen species detection revealed that the levels of reactive oxygen species in the Grx1 treatment group decreased by 33.01 % compared to the H₂O₂ group. Flow cytometry analyses indicated that the number of apoptotic cells in the Grx1 treatment group decreased by 23.51 % relative to the H₂O₂ group. Additionally, qRT-PCR analysis showed that Grx1 significantly reduced the expression levels of genes such as IL-1β, TNF-α, IL-6, and caspase-3 in PC12 cells. In vivo, recombinant Grx1 was utilized to treat cerebral ischemia-reperfusion injury (CIRI). Histological staining revealed that recombinant Grx1 significantly mitigated hippocampal tissue damage. Western blotting analysis demonstrated that Grx1 can reduce neuronal apoptosis following CIRI by decreasing Bax expression while increasing Bcl-2 expression. Furthermore, Grx1 was shown to modulate the HO-1/Nrf2 signaling pathway by elevating the expression of Nrf2 and HO-1. In summary, this study successfully overexpressed biologically active Grx1 in E. coli, and confirms that recombinant Grx1 exhibits remarkable antioxidant activity in both in vitro and in vivo experiments, effectively alleviating oxidative stress damage associated with ischemic stroke.