摘要
目的探究Adra1a调节LPS诱导的LBP敲除小鼠(Lbp^(-/-))原代肝细胞炎症反应。方法利用二步灌流法提取WT型、Lbp^(-/-)型小鼠原代肝细胞,构建由LPS诱发的原代肝细胞原发炎症模型;采用加入抑制剂哌唑嗪、转染siRNA来下调LBP敲除小鼠原代肝细胞Adra1a的表达;抑制剂法将原代肝细胞分为3组分别是对照组A、LPS组A、抑制剂哌唑嗪组,转染siRNA主要是对原代肝细胞进行分组,包括对照组B、LPS组B、si-NC组、si-Adra1a组;将WT型小鼠的原代肝细胞分为两组分别为对照组(空白对照)、LPS组(LPS刺激12 h)。本研究以WT型、Lbp^(-/-)型小鼠原代肝细胞为研究对象利用Western blot方法验证Adra1a在LPS刺激下的变化情况,采用CCK-8、qRT-PCR、Western blot等实验方法验证哌唑嗪及si-Adra1a对Lbp^(-/-)小鼠的原代肝细胞的炎症及存活率的改善情况。结果在LPS刺激下Lbp^(-/-)小鼠的原代肝细胞Adra1a蛋白表达显著升高(P<0.01),而野生型没有显著变化;抑制剂哌唑嗪组及干扰组的细胞存活率显著升高(P<0.01,P<0.05);抑制剂哌唑嗪组及si-Adra1a组的TNF-α、IL-1β炎症因子表达情况显著降低(P<0.01),与细胞损伤及炎症相关的蛋白p-p38、p-ERK、p-JNK的表达量也显著降低(P<0.01)。结论LPS刺激Lbp^(-/-)小鼠原代肝细胞后Adra1a表达上调、炎症信号因子上调,使用哌唑嗪与si-Adra1a特异性降低Adra1a表达后使LPS相关的Lbp^(-/-)小鼠原代肝细胞炎症因子明显下降,可验证敲除LBP导致Adra1a在LPS诱导的炎症调节中参与反应。
Objective To explore the role of Adra1a in regulating the LPS-induced inflammation response in primary hepatocytes of lipopolysaccharide-binding protein knockout(Lbp^(-/-))mice.Methods Primary hepatocytes were extracted from WT and Lbp^(-/-)mice using a two-step perfusion method,and an inflammation model was established using LPS induction.Expression of Adra1a in primary hepatocytes of Lbp^(-/-)mice was suppressed by administering the inhibitor prazosin and transfection with si-Adra1a.The cells were divided into three groups under inhibitor conditions:control group A,LPS group A,and prazosin group.For siRNA transfection,cells were also divided into groups:control group B,LPS group B,si-NC group,and si-Adra1a group.WT primary hepatocytes were divided into two groups:control group(blank)and LPS group(12 h stimulation).Changes in the Adra1a response to LPS stimulation were verified by Western blot.Other method ologies,such as CCK-8,qRT-PCR,and Western blot assays,were used to confirm improvements in cell inflammation and the survival rate by prazosin and si-Adra1a.Results Significant elevation in Adra1a protein expression in Lbp^(-/-)primary hepatocytes was observed post-LPS stimulation(P<0.01),whereas no notable change was found in the wildtype.A remarkable increase in the cell survival rate was noted in prazosin and si-Adra1a groups(P<0.01,P<0.05).Furthermore,prazosin and si-Adra1a groups exhibited significantly reduced expression of proinflammatory factors TNF-αand IL-1β(P<0.01),p-p38,p-ERK,and p-JNK(P<0.01),which are associated with cell damage and inflammation.Conclusions Following LPS stimulation,upregulation of Adra1a and proinflammatory cytokine expression was observed in Lbp^(-/-)primary hepatocytes.Specific downregulation of Adra1a expression using prazosin and si-Adra1a significantly decreased LPS-induced proinflammatory cytokines in Lbp^(-/-)primary hepatocytes.Adra1a is implicated in the regulation of the LPS-induced inflammation response in primary hepatocytes of Lbp^(-/-)mice.
作者
米传靓
付彬
李思迪
陈志达
郭中坤
王可洲
MI Chuanliang;FU Bin;LI Sidi;CHEN Zhida;GUO Zhongkun;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)
出处
《中国比较医学杂志》
CAS
北大核心
2024年第5期84-91,共8页
Chinese Journal of Comparative Medicine
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)。
