Agtr1a调节LPS诱导的Lbp^(-/-)小鼠原代肝细胞炎症  

Agtr1a regulates LPS-induced inflammation in primary hepatocytes of Lbp^(-/-)mice

作　　者：米传靓 付彬 李思迪 陈志达 郭中坤 张振宇 王可洲 MI Chuanliang;FU Bin;LI Sidi;CHEN Zhida;GUO Zhongkun;ZHANG Zhenyu;WANG Kezhou(School of Laboratory Animal&Shandong Laboratory Animal Center,Shandong First Medical University&Shandong Academy of Medical Sciences,Jinan 250117,China.;Jinan Pengyue Experimental Animal Breeding Co.,Ltd,Jinan 250000)

机构地区：[1]山东第一医科大学(山东省医学科学院)实验动物学院(省实验动物中心),济南250117 [2]济南朋悦实验动物繁育有限公司,济南250000

出　　处：《中国实验动物学报》2023年第8期1021-1027,共7页Acta Laboratorium Animalis Scientia Sinica

基　　金：山东省医学科学院医药卫生科技创新工程,济南市科技局“高校20条”(2021GXRC011);山东省医药卫生科技发展计划(2019WS177);山东省生猪产业技术体系(SDAIT-08-17)。

摘　　要：目的基于LBP敲除小鼠(Lbp^(-/-))原代肝细胞探讨LBP缺失后Agtr1a调节LPS诱导炎症的作用。方法通过两步灌流法提取WT组、Lbp^(-/-)组小鼠原代肝细胞,构建LPS诱导的原代肝细胞炎症模型;分别采取加入抑制剂氯沙坦、转染siRNA两种方法抑制Lbp^(-/-)小鼠原代肝细胞Agtr1a表达;抑制剂法将细胞分为对照组A(空白对照)、LPS组A(LPS刺激)、抑制剂氯沙坦组(氯沙坦干预3 h后加入LPS刺激),转染siRNA法将细胞分为对照组B(空白对照)、LPS组B(LPS刺激)、阴性对照组(si-NC干扰12 h后加入LPS刺激)、干扰组(si-agtr1a干扰12 h后加入LPS刺激);WT组小鼠原代肝细胞则分为对照组(空白对照)、LPS组(LPS刺激)。本研究利用Western Blot验证Lbp^(-/-)小鼠原代肝细胞中LBP蛋白敲除情况,从WT组、Lbp^(-/-)组小鼠原代肝细胞的Western Blot方面验证Agtr1a在LPS刺激下的变化情况,使用CCK8、qPCR、Western Blot等方法探讨抑制剂氯沙坦及si-agtr1a对Lbp^(-/-)小鼠原代肝细胞炎症的抑制情况。结果Lbp^(-/-)小鼠原代肝细胞中LBP蛋白已敲除完全;与WT组相比,在LPS诱导下Lbp^(-/-)小鼠原代肝细胞Agtr1a表达显著升高(P<0.001);抑制剂组细胞存活率显著升高(P<0.01);抑制剂组以及干扰组的炎症因子表达显著降低(P<0.01),同时与炎症相关蛋白p-ERK的表达也显著降低(P<0.01)。结论LPS刺激Lbp^(-/-)小鼠原代肝细胞后Agtr1a表达上调能够代偿脂多糖结合蛋白(LBP)的作用来促进炎症的发生,抑制Agtr1a表达能显著降低LPS诱导的Lbp^(-/-)小鼠原代肝细胞炎症反应。Objective To investigate the role of Agtr1a in regulating LPS-induced inflammation after LBP deletion in primary hepatocytes of LBP knockout(Lbp^(-/-))mice.Methods Primary hepatocytes of WT and Lbp^(-/-)groups were isolated by a two-step perfusion method to establish an inflammation model induced by LPS.Agtr1a expression in Lbp^(-/-)mouse primary hepatocytes was inhibited by losartan and siRNA.Cells were divided into a control group A(blank control),LPS group A(LPS stimulation),and losartan group(LPS stimulation was applied after 3 h of losartan treatment).Cells were divided into a control group B(blank control),LPS group B(LPS stimulation),negative control group(LPS stimulation was applied after 12 h of si-NC transfection),and interference group(LPS was applied after 12 h of si-agtr1a transfection).Primary hepatocytes in the WT group were divided into a control group(blank control)and LPS group(LPS stimulated).Western Blot was used to confirm knockout of LBP protein in Lbp^(-/-)mouse primary hepatocytes.Changes in Agtr1a expression in primary hepatocytes of WT and Lbp^(-/-)mice under LPS stimulation were verified by Western Blot.CCK8 assays,qPCR,and Western Blot were used to investigate the inhibitory effects of losartan and si-agtr1a on inflammation in primary hepatocytes of Lbp^(-/-)mice.Results LBP protein was completely knocked out in primary liver cells of Lbp^(-/-)mice.Compared with the wildtype group,Agtr1a expression in primary hepatocytes of Lbp^(-/-)mice was significantly increased under LPS induction(P<0.001).The cell survival rate of the inhibitor group was significantly increased(P<0.01).Expression of proinflammatory factors in inhibitor and interference groups was significantly decreased(P<0.01).Expression of inflammation-related protein p-ERK was also significantly decreased(P<0.01).Conclusions Upregulation of Agtr1a expression in primary hepatocytes of Lbp^(-/-)mice after LPS stimulation compensates for the effect of lipopolysaccharide-binding protein to promote inflammation.Inhibition of Agtr1a

关 键 词：血管紧张素受体1a Lbp^(-/-)小鼠 原代肝细胞 抑制剂 干扰RNA 细胞外信号调节激酶/磷酸化细胞外信号调节激酶 

分 类 号：Q95-33[生物学—动物学]