Abstract：
Neuroinflammation is one of the important mechanisms of trimethyltin chloride (TMT) central neurotoxicity. Artemisinin (ARS) is a well-known antimalarial drug that also has significant anti-inflammatory effects. Prokineticin 2 (PK2) is a small molecule secreted protein that is widely expressed in the nervous system and plays a key role in the development of neuroinflammation. However, it remains unclear whether ARS can ameliorate neuroinflammation caused by TMT and whether PK2/PKRs signaling pathway plays a part in it. In this research, male Balb/c mice were administered TMT (2.8 mg/kg, i.p.) followed by immunohistochemistry to assess the expression of PK2, PKR1, and PKR2 proteins in the hippocampus. Network pharmacology was used to predict the intersection targets of ARS, central nervous system(CNS) injury and TMT. The neurobehavior of mice was evaluated by behavioral scores. Histopathological damage of the hippocampus was evaluated by HE, Nissl and Electron microscopy. Western blotting was used to identify the expression of synapse-related proteins (PSD95, SYN1, Synaptophysin), PK system-related proteins (PK2, PKR1, PKR2), and inflammation-related proteins (TNF-α, NF-κB p65). Immunohistochemistry showed that TMT resulted in elevated PK2 and PKR2 protein expression in the CA2 and CA3 regions of the hippocampus in mice, while PKR1 protein was not significantly altered. Network pharmacology showed that PK2 could interact with the intersectional targets of ARS, CNS injury, and TMT. ARS remarkably attenuated TMT-induced seizures and hippocampal histological damage. Further studies demonstrated that ARS treatment attenuated TMT-induced hippocampal ultrastructural damage, possibly by increasing the number of rough endoplasmic reticulum and mitochondria as well as upregulating the levels of synapse-associated proteins (PSD95, SYN1, Synaptophysin). Western blotting results revealed that ARS downregulated TMT-induced TNF-α and NF-κB p65 protein levels. In addition, ARS also decreased TMT-induced protein expression of PK2 and PKR2 in the mouse hippocampus, but had no significant effect on PKR1 protein expression. Our results suggested that ARS ameliorated TMT-induced abnormal neural behavior and hippocampal injury, which may be achieved by regulating PK2/PKRs inflammatory pathway and ameliorating synaptic injury. Therefore, we suggest that PK2/PKRs pathway may be involved in TMT neurotoxicity and ARS may be a promising drug that can relieve TMT neurotoxicity.
摘要：
神经炎症是三甲基氯化锡(TMT)中枢神经毒性的重要机制之一。青蒿素（ARS）是一种众所周知的抗疟药，也具有显着的抗炎作用。前动力蛋白2（PK2）是一种在神经系统中广泛表达的小分子分泌蛋白，在神经炎症的发生发展中起关键作用。然而,目前尚不清楚ARS是否能改善TMT引起的神经炎症，以及PK2/PKRs信号通路是否参与其中。在这项研究中,雄性Balb/c小鼠给予TMT(2.8mg/kg,i.p.），然后进行免疫组织化学以评估海马中PK2，PKR1和PKR2蛋白的表达。网络药理学用于预测ARS的交叉目标，中枢神经系统（CNS）损伤和TMT。通过行为评分评价小鼠的神经行为。通过HE评估海马的组织病理学损伤，Nissl和电子显微镜。蛋白质印迹用于鉴定突触相关蛋白（PSD95，SYN1，突触素）的表达，PK系统相关蛋白（PK2，PKR1，PKR2），和炎症相关蛋白(TNF-α，NF-κBp65)。免疫组化显示TMT导致小鼠海马CA2和CA3区PK2和PKR2蛋白表达升高,而PKR1蛋白无明显改变。网络药理学表明PK2可以与ARS的交叉靶标相互作用，中枢神经系统损伤,和TMT。ARS可显着减轻TMT引起的癫痫发作和海马组织学损伤。进一步的研究表明，ARS治疗可以减轻TMT诱导的海马超微结构损伤，可能通过增加粗面内质网和线粒体的数量以及上调突触相关蛋白（PSD95，SYN1，突触素）的水平。Western印迹结果显示ARS下调TMT诱导的TNF-α和NF-κBp65蛋白水平。此外,ARS还降低了TMT诱导的小鼠海马中PK2和PKR2的蛋白表达，但对PKR1蛋白表达无显著影响。我们的结果表明ARS改善了TMT引起的异常神经行为和海马损伤，这可能是通过调节PK2/PKRs炎症通路和改善突触损伤来实现的。因此,我们认为PK2/PKRs通路可能参与了TMT的神经毒性,ARS可能是一种有希望的减轻TMT神经毒性的药物.
