[Retracted] Therapeutical Significance of Serpina3n Subsequent Cerebral Ischemia via Cytotoxic Granzyme B Inactivation

Abstract

Ischemic stroke is a devastating CNS insult with few clinical cures. Poor understanding of underlying mechanistic network is the primary limitation to develop novel curative therapies. Extracellular accumulation of granzyme B subsequent ischemia promotes neurodegeneration. Inhibition of granzyme B can be one of the potent strategies to mitigate neuronal damage. In present study, we investigated the effect of murine Serpina3n and human (homolog) SERPINA3 against cerebral ischemia through granzyme B inactivation. Recombinant Serpina3n/SERPINA3 were expressed by transfected 293 T cells, and eluted proteins were examined for postischemic influence both in vitro and in vivo. During in vitro test, Serpina3n was found effective enough to inhibit granzyme B, while SERPINA3 was ineffectual to counter cytotoxic protease. Treatment of hypoxic culture with recombinant Serpina3n/SERPINA3 significantly increased cell viability in dosage-dependent manner, recorded maximum at the highest concentration (4 mM). Infarct volume analysis confirmed that 50 mg/kg dosage of exogenous Serpina3n was adequate to reduce disease severity, while SERPINA3 lacked behind in analeptic effect. Immunohistochemical test, western blot analysis, and protease activity assay’s results illustrated successful diffusion of applied protein to the ischemic lesion and reactivity with the target protease. Taken together, our findings demonstrate therapeutic potential of Serpina3n by interfering granzyme B-mediated neuronal death subsequent cerebral ischemia.

Potential role of extracellular granzyme B in wet age-related macular degeneration and fuchs endothelial corneal dystrophy

“…Serpina3n is a murine serine protease inhibitor homologous to the human α1-antichymotrypsin protein. Both Serpina3n and α1-antichymotrypsin can target and inactivate a multitude of proteases; however, unlike α1-antichymotrypsin, SerpinA3n is able to bind to and inhibit extracellular GrB ( Sipione et al, 2006 ; Aslam et al, 2022 ). In murine model of Aortic Aneurysms, systemic Serpina3n treatment was shown to reduce degradation of the proteoglycan decorin while increasing collagen density the aorta of mice, leading to a reduced frequency of aortic rupture and death ( Ang et al, 2011 ).…”

DIA-based technology explores hub pathways and biomarkers of neurological recovery in ischemic stroke after rehabilitation

“…Haptoglobin-related proteins bind to Hb and apolipoprotein-L, which not only link HPR to the cholesterol system, but the HPR/apo-L complex also has a specific trypanosomal lysis effect ( 85 ). SERPINA3 can attenuate neuronal injury by interfering with granzyme B-mediated neuronal death after cerebral ischemia ( 86 ). Presence of SERPINA3N/SERPINA3 aggregates in cortical oligodendrocytes in areas of brain injury ( 87 ).…”

Regulation of CNS pathology by Serpina3n/SERPINA3: The knowns and the puzzles

“…25 Because the resulting covalent serpinprotease complex is highly stable and resistant to heat, high salt and protein denaturants, detecting its presence by western blot provides a reliable surrogate indicative of Serpina3n/SERPINA3's protease inhibitory function. 26,27 In the alternative substrate pathway, the RCL cleavage is completed efficiently, and the target serine proteases are released, resulting in a cleaved, inactivated Serpina3n/SERPINA3 and an intact, active serine protease protein. The preference of the inhibitory vs substrate pathways is determined by how fast the serpinprotease interaction results in the cleaved conformation and the number of serpins required for inhibiting a single molecule of proteases (stoichiometry of inhibition, SI; greater SI correlates with less effective enzymatic inhibition).…”

“…Based on this premise, in vitro application of rSerpina3n seemed to slightly but significantly increase the viability of N2a neurons cultured in 'hypoxic medium' by inhibiting granzyme B activity. 27 Recent in vivo data have shown that Serpina3n upregulation in neurons mitigated neuronal injury and loss in ischaemic and traumatic brain injury. [53][54][55]61 These studies suggest that neuronal induction of Serpina3n may represent an endogenous adaptive mechanism to ischaemic and traumatic brain injury.…”