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摘要：
目的 评价受体相互作用蛋白激酶3-混合谱系激酶结构域样蛋白（receptor interacting protein kinase 3- mixed-lineage kinase domain-like, RIPK3-MLKL）通路激活介导的坏死性凋亡在小鼠脓毒症相关性肺损伤中的作用，坏死性磺酰胺治疗是否通过抑制MLKL激活缓解肺损伤。
方法 本研究采用两阶段实验设计，第一阶段采用盲肠结扎穿孔术（cecal ligation and puncture, CLP）复制小鼠脓毒症模型并观察不同时间点的肺损伤程度及坏死性凋亡信号通路激活情况。即采用单臂实验设计，取24只6-8周雄性C57BL/6J小鼠行CLP，分别于术后0h、6h、12h和24h对小鼠进行取样，每个时间点n=6，观察小鼠肺部组织病理学、肺水肿情况、动脉血气分析及坏死性凋亡相关蛋白（RIPK3，MLKL）表达情况，通过第一阶段实验明确RIPK3-MLKL通路激活导致的坏死性凋亡是否参与了脓毒症肺损伤。第二阶段实验，验证MLKL的特异性抑制剂坏死性磺胺（Necrosulfonamide, NSA）是否可阻断脓毒症小鼠坏死性凋亡，逆转肺损伤。采用随机对照干预性实验研究设计，即另选24只小鼠采用随机数字表法分为4组：假手术组（Sham组，n=6）、NSA+假手术组（NSA组，n=6）、CLP手术组（CLP组，n=6）和NSA+CLP手术组（NSA+CLP组，n=6）。在造模后24h观察肺部组织病理学、肺水肿情况、动脉血气分析及坏死性凋亡相关蛋白表达情况。
结果 第一阶段实验发现CLP术后脓毒症相关性肺损伤程度随着时间延长逐渐加重，CLP后24h肺湿/干重比（wet to dry ratio, W/D）上升（6.05±0.70 vs 4.33±0.22, P0.01），动脉血氧分压降低（72.05±2.47 vs 88.65±2.31, P0.01），二氧化碳分压升高（50.02±2.70 vs 40.53±2.59, P0.01）。坏死性凋亡相关蛋白p-RIPK3及p-MLKL表达明显升高（P0.01）。第二阶段实验中应用NSA后，与CLP组相比，NSA+CLP组W/D值下降（4.73±0.24 vs 6.07±1.10, P0.01），肺水肿程度减轻，p-RIPK3及p-MLKL表达明显下调（P0.01），动脉血气氧分压升高（79.83±2.59 vs 71.93±3.50, P0.01），但二氧化碳分压差异无统计学意义（46.70±2.67 vs 41.80±2.49, P=0.18）。
结论 RIPK3-MLKL通路激活引起的坏死性凋亡参与了小鼠脓毒症相关肺损伤的过程，NSA通过抑制MLKL激活，抑制坏死性凋亡，缓解小鼠脓毒症相关性肺损伤。
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Abstract: Objective To evaluate the role of inhibiting receptor interacting protein kinase 3- mixed-lineage kinase domain-like (RIPK3-MLKL) pathway activation in mice sepsis-related lung injury and to determine whether necrosulfonamide (NSA) can alleviate lung injury by inhibiting this pathway.
Methods A two-stage experiment is designed. In the first stage, the sepsis model developed by cecal ligation and puncture (CLP) was used for observing the severity of lung injury and necroptosis pathway activation. Twenty-four C57BL/6J mice aged 6-8 weeks underwent CLP. Samples were taken at 0h, 6h, 12h, and 24h after surgery, with 6 mice randomly at each time point. Histopathological changes, pulmonary edema, arterial blood gas analysis, and necroptosis-related protein (RIPK3, MLKL) expression were measured. RIPK3-MLKL pathway activation was involved in the development of sepsis-related lung injury. In the second stage, a randomized controlled intervention study was conducted to verify whether necrosulfonamide (NSA), the specific inhibitor of MLKL, can alleviate lung injury by inhibiting necroptosis. According to random number table method, another twenty-four mice were divided into four groups: sham operation group (Sham group, n=6), NSA + sham operation group (NSA group, n=6), operation group (CLP group, n=6) and NSA + operation group (NSA+CLP group, n=6). Histopathological changes, pulmonary edema, arterial blood gas analysis, and necroptosis-related protein expression were observed 24h after modeling.
Results The first-stage experiment found that the degree of sepsis-related lung injury gradually worsened within 24 hours after CLP. Compared with 0h after CLP, the lung wet to dry ratio (W/D) at 24h after CLP was significantly higher (6.05±0.70 vs 4.33±0.22, P0.01), the arterial blood gas analysis showed a decrease in oxygen partial pressure (2.05±2.47 vs 88.65±2.31, P0.05), and an increase in carbon dioxide partial pressure (50.02±2.70 vs 40.53±2.59, P0.01). The expression of necroptosis-related proteins p-RIPK3 and p-MLKL was up-regulated (P0.05). In the second-stage experiment, with the application of inhibitor necrosulfonamide (NSA), the NSA+CLP group showed a decrease in W/D (4.73±0.24 vs 6.07±1.10, P0.01), alleviation of pulmonary edema, down-regulation in p-RIPK3 expression and p-MLKL expression and an increase in arterial oxygen partial pressure (79.83±2.59 vs 71.93±3.50, P0.01) compared with the CLP group. The partial pressure of carbon dioxide was decreased in NSA+CLP group, but there was no statistical significance (46.70±2.67 vs 41.80±2.49, P=0.18).
Conclusion RIPK3-MLKL pathway activation-mediated necroptosis is associated with lung injury in sepsis mice. NSA alleviates sepsis-related lung injury in mice by inhibiting MLKL pathway activation.
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