3H-1,2-二硫醇-3-硫酮靶向GPX4调节铁死亡参与小鼠脓毒症所致的肠屏障损伤

3H-1,2-Dithiol-3-Thione Targeting GPX4 Me-diates Ferroptosis and Participates Sepsis-Induced Intestinal Barrier Injury in Mice

摘要: 目的：通过观察3H-1,2-二硫醇-3-硫酮(D3T)预处理对脓毒症小鼠的肠道屏障损伤的影响，初步探讨铁死亡参与脓毒症所致的肠屏障损伤调节的可能机制，为临床脓毒症诊治提供新思路。方法：采用盲肠结扎穿刺术(CLP)建立小鼠脓毒症模型，3H-1,2-二硫醇-3-硫酮(3H-1,2-dithiole-3-thione, D3T)间接诱导GPX4高表达。32只雄性C57BL/6J小鼠随机分为假手术组(Sham)、假手术给药组(Sham + D3T)、脓毒症组(CLP)、脓毒症给药组(D3T)，每组各8只小鼠。给药组在实验前30分钟，腹腔注射D3T悬液(二甲基亚砜(DMSO)溶解，50 mg/kg)，假手术组及脓毒症组同时腹腔注射等剂量的DMSO+PBS混合液，术后20小时观察小鼠生存情况，内眦静脉取血测定TNF-α、IL-6表达以评估小鼠炎症水平；部分小肠组织行苏木精－伊红(HE)染色观察小肠组织的损伤情况，Westernblotting检测肠道紧密连接蛋白ZO-1、Occludin及铁死亡相关蛋白GPX4、ACSL4及SLC7A11/xCT表达水平。结果：在72小时生存率观察实验中，CLP组小鼠生存率(33%)较Sham组相比明显降低(P < 0.01)，而D3T预处理改善了脓毒症小鼠的生存情况(68%, P < 0.01)；造模后20小时，CLP组小鼠脓毒症中毒症状明显，腹腔感染严重，小肠病理损伤明显加重，血清TNF-α及IL-6水平异常升高(P < 0.01)，而D3T预处理改善了脓毒症中毒症状，腹腔感染及小肠病理损伤，血清炎症因子水平下降(P < 0.05)；通过蛋白印迹法，我们发现脓毒症小鼠小肠组织肠道紧密连接蛋白ZO-1和Occludin、铁死亡相关蛋白GPX4和SLC7A11/xCT蛋白水平明显下降(P < 0.01)，ACSL4蛋白水平异常升高(P < 0.01)，而D3T预处理一定程度上调了小肠ZO-1、Occludin、GPX4及SLC7A11/xCT的表达(P < 0.01)，抑制了ACSL4的表达(P < 0.01)。结论：3H-1,2-二硫醇-3-硫酮发挥其抗氧化特性，靶向GPX4抑制铁死亡，改善小鼠脓毒症所致的肠屏障损伤。

Abstract: Objective: The research was designed to investigate the effect of pretreatment with 3H-1,2-dithiole- 3-thione (D3T) on intestinal barrier injury in septic mice and explore the possible mechanism of ferroptosis in the regulation of intestinal barrier injury induced by sepsis, which may provide new strategy for clinical diagnosis and treatment of sepsis. Methods: Sepsis model was induced by cecal ligation and puncture (CLP) in mice. The expression of GPX4 was up-regulated by using 3H-1,2- di-thiole-3-thione (D3T). Thirty-two male C57BL/6J mice were randomly divided into four groups, in-cluding sham operation group (Sham), sham operation treatment group (Sham + D3T), sepsis group (CLP) and sepsis treatment group (D3T), with 8 mice in each group. 30 minutes before the experi-ment, the mice in the treatment group were injected with D3T suspension (dissolved in dimethyl sulfoxide (DMSO), 50 mg/kg) by intraperitoneal injection, while the mice in the sham operation group and sepsis group were injected with DMSO + PBS at the same dose by intraperitoneal injec-tion. After 20 hours, we observe the survival condition in each group and detect the contents of se-rum TNF-α and IL-6 to evaluate the level of inflammation in mice of each group. Partial small intes-tine was collected to evaluate the intestinal injury by using hematoxylinand eosin (HE) staining. The remaining small intestine was stored for detecting theexpression of intestinal tight junction pro-teins ZO-1, Occludin and ferroptosis relatedproteins GPX-4, ACSL-4 and SLC7A11/xCT by using Western blotting. Results: In the 72-hours survival observation experiment, the survival rate of CLP group (33%) was significantly lower than that of Sham group (P < 0.01), while D3T pretreatment improved the survival of septic mice (68%, P < 0.01). 20 hours after the establishment of the model, the CLP group shows obvious septic symptoms, including serious abdominal infection, aggravated pathological intestinal injury, and the higher levels of serum TNF-α and IL-6. However, D3T pre-treatment improved the symptoms of sepsis, alleviated abdominal infection and pathological injury of small intestine, and decreased the level of serum inflammatory factors. By Western blotting, we found that the levels of intestinal tight junction proteins ZO-1 and Occludin, ferroptosis related proteins GPX4 and SLC7A11/xCT were significantly decreased, while the level of ACSL4 protein was abnormally increased in septic mice. D3T pretreatment up-regulated the expression of ZO-1, Oc-cludin, GPX4 and SLC7A11/xCT and inhibited the expression of ACSL4 in small intestine of septic mice. Conclusion: 3H-1,2-dithiole-3-thione exerts its antioxidant properties by targeting GPX4 to in-hibit ferroptosis and improve intestinal barrier injury induced by sepsis in mice.