南京医科大学第一附属医院麻醉科
江苏省自然科学基金青年项目
Department of Anesthesiology, The First Affiliated Hospital of Nanjing Medical University
Youth Program of Natural Science Foundation of Jiangsu Province
[摘 要] 目的：研究热休克蛋白A12A（heat shock protein A12A, HSPA12A）对内毒素血症肝损伤的影响及机制。方法：（1）采用脓毒症小鼠肝组织RNA测序的公共数据库，以生物信息学手段分析Hspa12a和多种载脂蛋白的mRNA表达变化；（2）采用6-8周龄HSPA12A基因敲除（HSPA12A knockout, Hspa12a-/-）鼠和野生型（wild type, WT）鼠，腹腔注射脂多糖（lipopolysaccharide, LPS）（5 mg/kg）诱导内毒素血症，以生理盐水（normal saline, NS）注射小鼠为对照组，分为NS-WT组、NS-Hspa12a-/-组、LPS-WT组和LPS-Hspa12a-/-组；LPS作用后6 h，收集肝组织，HE染色观察肝脏组织病理变化；免疫印迹和RT-PCR分析其中HSPA12A、APOA1、APOB、APOM表达水平；同时分离血清，测定肝功能标志物丙氨酸氨基转移酶（alanine aminotransferase, ALT）和天冬氨酸氨基转移酶（aspartate aminotransferase, AST）水平以及高密度脂蛋白胆固醇（high density lipoprotein cholesterol, HDL-C）和低密度脂蛋白胆固醇（low density lipoprotein cholesterol, LDL-C）水平；（3）WT鼠原代肝细胞过表达HSPA12A后，使用LPS（500 ng/ml）作用于肝细胞模拟内毒素血症肝损伤模型，6 h后检测肝细胞培养上清的ALT和AST水平。（4）根据患者是否发生脓毒症肝损伤分为脓毒症肝损伤组和对照组，比较两组患者ALT、AST、HDL-C和LDL-C的差异。 结果：（1）生物信息学分析显示，脓毒症导致小鼠肝脏Hspa12a、Apoa1、Apob和Apom的mRNA表达下降；（2）与NS-WT组相比，LPS-WT组肝组织出现明显损伤（P<0.001）、炎症病灶数量增多（P<0.01）、血清ALT（P<0.05）和AST（P<0.01）升高，同时肝组织中HSPA12A表达显著下降（P<0.05）；而与LPS-WT组相比，LPS-Hspa12a-/-组肝脏病理变化更严重（P<0.05）且血清ALT（P<0.01）和AST（P<0.05）水平升高，HDL-和LDL-C水平下降（P<0.01），肝组织载脂蛋白（APOA1、APOB、APOM）表达降低（P<0.05, P<0.01）（3）体外实验中LPS作用使肝细胞培养上清中的ALT和AST水平升高（P<0.001），而过表达HSPA12A能够减轻LPS作用引起的ALT和AST水平升高（P<0.01）。（4）临床数据显示，出现脓毒症肝损伤的患者相较于对照组血浆中的ALT和AST水平显著升高（P<0.001），HDL-C和LDL-C水平显著下降（P<0.001）。结论：内毒素血症导致肝脏HSPA12A表达下调，其介导了内毒素血症肝损伤的发生，过表达HSPA12A能够保护内毒素血症引起的肝损伤，该作用可能与维持肝脏载脂蛋白及脂蛋白稳态有关。
[Abstract] Objective: To explore the effects and mechanisms of heat shock protein A12A (HSPA12A) on liver injury during endotoxemia. Methods: (1) Bioinformatic analysis of mRNA expression changes in the expression of Hspa12a and multiple apolipoprotein using a public database of septic mouse liver tissue RNA sequencing. (2) Endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (LPS) (5 mg/kg) using 6-8-week-old HSPA12A knockout (Hspa12a-/-) mice and wild-type (WT) mice. Mice treated with normal saline (NS) served as controls. Expertiments were divided into four groups, NS-WT group, NS-Hspa12a-/- group, LPS-WT group and LPS-Hspa12a-/- group. Six hours after LPS, liver tissues were collected for examing tissue damage by HE and analyzing the expression levels of HSPA12A, APOA1, APOB, and APOM by immunoblotting and RT-PCR. At the same time, serum was separated for measuring levels of liver function markers (alanine aminotransferase, ALT; aspartate aminotransferase, AST) and lipoproteins( high density lipoprotein cholesterol, HDL-C; low density lipoprotein cholesterol, LDL-C). (3) Primary hepatocytes were treated with LPS (500 ng/ml) after HSPA12A overexpressed to emulate endotoxemia induced liver injury. Six hours after LPS, medium was collected for measuring levels of ALT and AST. (4) Patients were divided into sepsis induced liver injury group and control group according to whether the liver injury occurred. ALT, AST, HDL-C and LDL-C levels of patients were collected for analyse. Results: (1) Bioinformatics analysis showed decreases of Hspa12a, Apoa1, Apob and Apom mRNA levels in livers during sepsis. (2) LPS-WT mice displayed a decrease in HSPA12A (P<0.05) protein expression in livers along with the elevated serum ALT (P<0.05) and AST (P<0.01) activiaties compared with NS-WT mice. At the same time, liver tissues showed obvious histopathological injury (P<0.001) and the number of inflammatory foci was decreased (P<0.01). However, compare to LPS-WT mice, LPS- Hspa12a-/- group showed greater ALT (P<0.01) and AST (P<0.05) levels and lower HDL-C and LDL-C levels (P<0.01), along with more severe pathological damage of liver tissues (P<0.05), suggesting that HSPA12A deficiency exaggerated liver injury during endotoxemia. Hepatic apolipoproteins (APOA1, APOB, APOM) expression was reduced (P<0.05, P<0.01). (3) In vitro, ALT and AST levels in culture medium of hepatocytes were signaficantly increased after LPS treatment (P<0.001). However, overexpression of HSPA12A could alleviate the increases of ALT and AST levels (P<0.01). (4) Clinical results suggested that compared with control group, sepsis induced liver injury group shows signaficantly higher ALT and AST levels in serum (P<0.001). In contrast, HDL-C and LDL-C levels were signaficantly lower (P<0.001). Conclusions: Endotoxemia leads to downregulation of hepatic HSPA12A expression, ewhich mediates the development of endotoxemic liver injury. However, overexpression of HSPA12A can protect liver injury induced by endotoxemia. The action of HSPA12A may involve the regulation of hepatic apolipoprotein expression and serum lipoprotein cholesterol levels.
