Abstract

Organophosphates (OPs), widely used as insecticides, exhibit severe acute toxicity and pose a significant threat to public health. Current standard therapy, which mainly relies on atropine and oxime reactivators, alleviates symptoms but fails to effectively eliminate circulating toxin or excess acetylcholine (ACh), thus representing a non-causal intervention. This study proposes a novel, efficient synergistic dual-enzyme strategy for the treatment of acute OP poisoning in a Sprague-Dawley rat model. Intravenous co-administration of organophosphorus hydrolase (OPH) from Pseudomonas diminuta , site-specifically PEGylated at its N-terminus, together with recombinant human butyrylcholinesterase (BuChE), effectively mitigated cholinergic crisis and accelerated systemic clearance of residual OPs. The hydrolysis rate of ethyl paraoxon by PEG-OPH is 90.9-fold higher than its binding rate to BuChE. This kinetic advantage enables rapid degradation of free OPs in the circulation during the early intoxication phase, thereby dominating initial toxin clearance and preventing excessive BuChE consumption. Concurrently, the administered BuChE hydrolyzes accumulated ACh due to acetylcholinesterase (AChE) inhibition, helping restore the balance of cholinergic neurotransmission. Compared with BuChE monotherapy, the synergistic regimen reduced the required BuChE dose by up to 83%, while achieving full restoration of respiratory parameters to baseline levels within 30 min post-treatment. Moreover, 24-h open-field testing revealed significantly enhanced locomotor activity in the combination group, as evidenced by a markedly greater total distance traveled compared with either PEG-OPH or BuChE monotherapy. Critically, this dual-enzyme strategy conferred complete protection against lethality, yielding a 100% 7-d survival rate. This work pioneers a therapeutic strategy using exogenous enzymes