Chen Yuan-Li, Xu Guo, Liang Xiao, Wei Juan, Luo Jing, Chen Guan-Nan, Yan Xiao-Di, Wen Xue-Ping, Zhong Ming, Lv Xin
Department of Anesthesiology, Shanghai Pulmonary Hospital, Tongji University School of MedicineShanghai, P. R. China; Shanghai Key Lab of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of MedicineShanghai, P. R. China; Jiangsu Province Key Laboratory of Anesthesiology, Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical UniversityXuzhou, P. R. China.
Department of General Surgery, Huai'an First People's Hospital, Nanjing Medical University Huai'an, P. R. China.
Am J Transl Res. 2016 Dec 15;8(12):5685-5695. eCollection 2016.
Pyroptosis is a programmed cell death associated with caspase-1 and accompanied by the secretion of a large number of pro-inflammatory cytokines. In the acute stage of sepsis, the release of several pro-inflammatory cytokines aggravates hepatic cell death, and acute liver injury is aggravated with the progress of the disease, resulting in acute liver failure with a very high mortality rate. The present study investigated the effect of inhibiting hepatic cell pyroptosis on the septic acute liver injury. Septic acute liver injury mice model was established by cecal ligation and puncture (CLP model). The liver tissues were assessed for inflammatory infiltration by HE, serum concentrations of ALT, AST, IL-1β, and IL-18 were examined by ELISA, hepatic cell pyroptosis was determined by flow cytometry, and expressions of caspase-1 and NLRP3 were assessed by Western blot. CLP-induced acute liver injury was distinct at 24 h post-operation, with the highest hepatic cell pyroptosis rate. The pyroptosis rate and liver injury indexes were positively correlated. Western blot showed that the expressions of pyroptosis-related proteins, caspase-1, and NLRP3, were increased. Normal mouse hepatic cells were cultured in vitro and LPS+ATP introduced to establish the cell model of septic acute liver injury. The expressions of caspase-1, NLRP3, IL-1β, and IL-18 in LPS+ATP group were significantly higher than the control group by Western blot and ELISA. The inhibitors of NLRP3 (Glyburide) and caspase-1 (AC-YVAD-CMK) alone or in combination were used to pre-treat the hepatic cells, which revealed that the pyroptosis rate was decreased and the cell damage alleviated. The in vivo assay in rats showed that post inhibitor treatment, the 10-days survival was significantly improved and the liver damage reduced. Therefore, inhibiting the hepatic cell pyroptosis could alleviate CLP-induced acute liver injury, providing a novel treatment target for septic acute liver injury.
细胞焦亡是一种与半胱天冬酶 -1 相关的程序性细胞死亡，并伴有大量促炎细胞因子的分泌。在脓毒症急性期，几种促炎细胞因子的释放会加重肝细胞死亡，且随着疾病进展急性肝损伤会加重，进而导致急性肝衰竭，死亡率极高。本研究探讨抑制肝细胞焦亡对脓毒症急性肝损伤的影响。通过盲肠结扎和穿刺建立脓毒症急性肝损伤小鼠模型（CLP 模型）。用苏木精 - 伊红染色（HE）评估肝组织的炎症浸润情况，用酶联免疫吸附测定法（ELISA）检测血清中谷丙转氨酶（ALT）、谷草转氨酶（AST）、白细胞介素 -1β（IL -1β）和白细胞介素 -18（IL -18）的浓度，用流式细胞术测定肝细胞焦亡情况，并用蛋白质免疫印迹法评估半胱天冬酶 -1 和 NOD 样受体蛋白 3（NLRP3）的表达。CLP 诱导的急性肝损伤在术后 24 小时明显，此时肝细胞焦亡率最高。焦亡率与肝损伤指标呈正相关。蛋白质免疫印迹法显示，细胞焦亡相关蛋白、半胱天冬酶 -1 和 NLRP3 的表达增加。体外培养正常小鼠肝细胞，引入脂多糖（LPS）+三磷酸腺苷（ATP）建立脓毒症急性肝损伤细胞模型。蛋白质免疫印迹法和 ELISA 检测显示，LPS + ATP 组中半胱天冬酶 -1、NLRP3、IL -1β 和 IL -18 的表达明显高于对照组。单独或联合使用 NLRP3 抑制剂（格列本脲）和半胱天冬酶 -1 抑制剂（AC - YVAD - CMK）预处理肝细胞，结果显示焦亡率降低，细胞损伤减轻。大鼠体内实验表明，抑制剂处理后，10 天生存率显著提高，肝损伤减轻。因此，抑制肝细胞焦亡可减轻 CLP 诱导的急性肝损伤，为脓毒症急性肝损伤提供了新的治疗靶点。
