热休克蛋白A12A对内毒素血症肝损伤的作用及机制研究  

Study on the role and mechanism of heat shock protein A12A in hepatic injury induced by endotoxemia

作　　者：王诏鹤 孔秋月 丁正年 WANG Zhaohe;KONG Qiuyue;DING Zhengnian(Department of Anesthesiology and Perioperative Medicine,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]南京医科大学第一附属医院麻醉与围术期医学科,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2024年第5期615-625,共11页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省自然科学基金(BK20201087)。

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6 h后,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;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)根据是否发生脓毒症肝损伤将患者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,脓毒症小鼠肝脏Hspa12a、Apoa1、Apob和Apom的m RNA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-C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�Objective:To explore the role and mechanism of heat shock protein A12A(HSPA12A)in hepatic injury induced by endotoxemia.Methods:The mRNA expression changes of Hspal2a and multiple apolipoproteins were analyzed by bioinformatics using a public database of RNA sequencing results from septic mice liver tissue.②Endotoxemiawasinducedby intraperitoneal injection of lipopolysaccharide(LPS,5 mg/kg)using 6-8-week-old Hspal2a knockout(Hspal2a-)mice and wild-type(WT)mice.Mice treated with normal saline(NS)served as controls.Animals were divided into four groups,NS-WT group,NS-Hspal2a'group,LPS-WT group,and LPS-Hspal2a'group.Six hours after LPS treatment,liver tissues were collected to evaluate the tissue damage by HE and analyze the expression levels of HSPA12A,ApoA1,ApoB,and ApoM by immunoblotting and RT-PCR.Serum was separated for measuring the 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).Primary hepatocytes overexpressed Hspal2a were treated with LPS(500 ng/mL)to emulate endotoxemia induced liver injury.Six hours after LPS treatment,culture medium was collected for measuring levels of ALT and AST.@Patients were divided into the sepsis induced liver injury group and the control group according to whether the septic liver injury occurred.ALT,AST,HDL-C and LDL-C levels were collected and compared between the two groups.Results:Bioinformatic analysis showed that the levels of Hspal2a,Apoal,Apob and Apom mRNA were decreased in livers of septic mice.②Compared withNS-WT mice,LPS-WT micedisplayed obvioushistopathological injury inliver tissues(P<0.001)and the number of inflammatory foci was increased(P<0.01)along with the elevated serum ALT(P<0.05)and AST(P<0.01)activiaties.At the same time,the expression of HSPA12A protein in liver was decreased(P<0.05).However,compared with LPS-WT mice,LPS-Hspal2a'-mice showed more severe pathological damage of liver tissues(P<0.05),along wi

关 键 词：热休克蛋白A12A 内毒素血症 肝损伤 载脂蛋白 

分 类 号：R631.1[医药卫生—外科学]