PSTK基因/GPx-1/过氧化氢信号通路异常是百草枯所致肺损伤的重要机制

Paraquat (PQ) poisoning caused high mortality in China . Its main cause of death is oxidative stress-induced lung injury. Previously, we assayed gene spectrum of lung tissue of PQ-induced lung injury, and identified 35 core genes related to PQ poisoning. Among them, phosphoseryl-tRNA kinase (PSTK) is the only gene that is related to selenium proteins, and we also observed that knockout of PSTK gene in both C-elegans and mice model exhibited shorter survival time than wide type when challenged by PQ. The blood PSTK level in healthy volunteers was much lower than that in patients with PQ poisoning. In addition, Glutathione peroxidase-1 (GPx-1) is an intracellular antioxidant enzyme that enzymatically reduces hydrogen peroxide to water to limit its harmful effects. Knockout of GPX-1 in both Caenorhabditis elegans and in C57BL/6 mice shortens their survival time. PSTK influences the function of selenoproteins through the selenocysteine insertion sequence. Thus, we hypothesized that the aberrant PSTK/GPx-1/H2O2 signaling plays a key role in PQ-induced acute lung injury (ALI). To confirm this, we initially investigate the physiological role of PSTK, and function of PSTK/GPx-1/H2O2 signaling in pathological mechanism of PQ induced ALI. Our studies will not only deepen the understanding of mechanism of PQ-induced ALI, but also provide a promising treatment of using both PSTK intervention and selenoprotein.

百草枯(PQ)中毒死亡率高，其直接死因为急性肺损伤(ALI)。前期我们检测了PQ中毒肺组织基因表达谱，确定了35个关键调控基因，其中PSTK是唯一调控硒蛋白的基因；PSTK基因和蛋白表达与PQ中毒小鼠肺损伤程度成负相关；PQ中毒PSTK基因敲除线虫和PSTK基因敲除小鼠生存时间均显著缩短；百草枯中毒患者血液PSTK水平较正常人显著降低 。由于硒蛋白GPx-1主要通过还原过氧化氢（H2O2）为水而起抗氧化作用，且PQ中毒的GPx-1基因敲除小鼠生存时间显著缩短而过表达则显著延长；PSTK通过调控硒代半胱氨酸而调控GPx-1功能，故认为PSTK/GPx-1/H2O2信号通路异常是PQ致肺损伤的重要机制。为此，拟采用基因工程小鼠通过阐明肺PSTK基因的生理作用和PSTK/GPx-1/H2O2通路在PQ所致肺损伤中的作用机理，为研究基于干预PSTK基因和采用硒蛋白联合治疗PQ中毒新方案提供证据。

百草枯中毒死亡率高，主要是百草枯诱导的氧化应激器官损伤。但是百草枯如何诱导氧化应激的机理不清。为研究PSTK基因在百草枯所致肺损伤中的作用机制，我们首先构建了PSTK全基因敲除小鼠模型，发现其胚胎致死。由于PSTK基因主要在肝脏细胞表达，故采用CRISPR/Cas9技术进行肝细胞PSTK基因条件性敲除小鼠的构建。PSTK是硒蛋白调控的核心基因，其下游有SPS1和SPS2两条途径，但是这两条通路的生理与病理意义不清楚？那条通路与百草枯中毒肺损伤有关亦不清楚? 故我们分别构建了SPS1和SPS2的基因敲除小鼠模型。采用肝细胞PSTK基因条件性敲除小鼠百草枯中毒模型验证发现:肝细胞PSTK基因异常表达是PQ所致急性肺损伤加重的重要机理。从而确定肝细胞PSTK基因是肺组织生理和病理调控的关键基因。最后采用单细胞基因测序技术研究了PSTK基因加重肺损伤的肺细胞表达谱特征及其与GPx-1的关系。另外我们评估了序贯性血液灌流连续性血液净化技术（CVVH）治疗急性百草枯中毒患者的有效性和安全性，为最终克服急性百草枯患者救治的难题奠定了基础。