Adra2a通过MAPK信号通路介导LPS诱导的Lbp-/- 小鼠肝细胞炎性反应

doi:10.12300/j.issn.1674-5817.2025.077

实验动物与比较医学 ›› 2026, Vol. 46 ›› Issue (2): 212-221.DOI: 10.12300/j.issn.1674-5817.2025.077

Adra2a通过MAPK信号通路介导LPS诱导的Lbp^-/- 小鼠肝细胞炎性反应

刘赛¹, 付彬¹, 李思迪¹^,², 陈志达¹, 张悦¹, 郭中坤¹, 王永安¹()(), 王可洲¹()()

^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.山东朋悦实验动物科技有限公司, 济南 250000

收稿日期:2025-05-22 修回日期:2025-09-11 出版日期:2026-04-25 发布日期:2026-04-18
通讯作者:
王可洲（1974—），男，博士，研究员，研究方向：药理毒理学研究。E-mail：wangkezhou_cn@163.com。ORCID：0000-0003-1919-

272X

王永安（1986—），男，博士，实验师，研究方向：生殖毒理学。E-mail：wya668@163.com。ORCID：0009-0006-5476-8877
作者简介:刘赛（2001—），男，硕士研究生，研究方向：免疫药理学。E-mail：1820739615@qq.com
基金资助:
山东省医学科学院医药卫生科技创新工程项目;济南市科学技术局“新高校20条”扶持项目(2021GXRC011);山东省生猪产业技术体系建设项目(SDAIT-08-17)

Adra2a Regulates LPS-Induced Inflammation in Hepatocytes of Lbp^-/- Mice via the MAPK Signaling Pathway

LIU Sai¹, FU Bin¹, LI Sidi¹^,², CHEN Zhida¹, ZHANG Yue¹, GUO Zhongkun¹, WANG Yongan¹()(), WANG Kezhou¹()()

^1.School of Laboratory Animal & Shandong Laboratory Animal Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Ji'nan 250117, China
^2.Shandong Pengyue Laboratory Animal Technology Co. , Ltd. , Ji'nan 250000, China

Received:2025-05-22 Revised:2025-09-11 Published:2026-04-25 Online:2026-04-18
Contact: WANG Kezhou (ORCID: 0000-0003-1919-272X), E-mail: wangkezhou_cn@163.com;
WANG Yongan (ORCID: 0009-0006-5476-8877), E-mail:wya668@163.com;

摘要/Abstract

摘要：

目的探究肾上腺素受体α2A（adrenoceptor alpha 2A，Adra2a）调节脂多糖（lipopolysaccharide，LPS）诱导脂多糖结合蛋白（lipopolysaccharide-binding protein，LBP）敲除小鼠（Lbp^-/- ）原代肝细胞炎症的机制。方法通过两步灌流法提取C57BL/6J小鼠和Lbp^-/- 小鼠（来自C57BL/6J小鼠）原代肝细胞，构建经LPS诱导的原代肝细胞体外炎症模型，同时通过腹腔注射LPS以构建炎症小鼠体内模型。将体外实验分为5组，即Control组、LPS组、BRL+LPS组、OE-NC+LPS组和OE-Adra2a+LPS组。其中，Control组为空白对照；LPS组加入LPS刺激原代肝细胞；BRL+LPS组加入BRL-44408 maleate预处理原代肝细胞后，再加入LPS；OE-NC+LPS组为加入空载病毒转染原代肝细胞后，再加入LPS；OE-Adra2a+LPS组为加入Adra2a过表达慢病毒转染原代肝细胞后，再加入LPS。同样，将体内实验分为5组，即Control'组、LPS'组、BRL+LPS'组、OE-NC+LPS'组、OE-Adra2a+LPS'组。其中，Control'组为空白对照；LPS'组为经腹腔注射LPS；BRL+LPS'组为经腹腔注射BRL-44408 maleate预处理小鼠后，再注射LPS；OE-NC+LPS'组为经腹腔注射空载病毒预处理小鼠后，再注射LPS；OE-Adra2a+LPS'组为经腹腔注射Adra2a过表达慢病毒预处理小鼠后，再注射LPS。通过CCK-8法检测经抑制和过表达Adra2a后的小鼠原代肝细胞存活率，采用实时荧光定量PCR法检测抑制和过表达Adra2a后肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素（interleukin，IL）-6和IL-1β这3种炎症因子基因表达量的变化，采用蛋白质印迹法检测经LPS刺激后Adra2a蛋白表达量以及细胞外调节蛋白激酶1/2（extracellular signal-regulated kinase 1/2，ERK1/2）、p38丝裂原激活的蛋白激酶（p38 mitogen-activated protein kinase，p38 MAPK）和c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）蛋白磷酸化的变化情况。结果体外实验中，在LPS刺激下，与Control组相比，C57BL/6J小鼠原代肝细胞中Adra2a蛋白表达量显著下降（P＜0.05），而Lbp^-/- 小鼠原代肝细胞中Adra2a蛋白表达量却显著升高（P＜0.001）；与LPS组相比，BRL+LPS组细胞的存活率显著升高（P＜0.01），TNF-α、IL-6和IL-1β基因转录水平显著降低（P＜0.01，P＜0.001，P＜0.001），ERK1/2、p38和JNK蛋白的磷酸化水平均显著降低（P＜0.01，P＜0.001，P＜0.001）；与OE-NC+LPS组相比，OE-Adra2a+LPS组细胞存活率显著下降（P＜0.001），TNF-α、IL-6和IL-1β基因转录水平显著升高（P＜0.001，P＜0.01，P＜0.001），ERK1/2、p38和JNK蛋白的磷酸化水平均显著升高（P＜0.001，P＜0.01，P＜0.001）。体内实验中，与LPS'组相比，BRL+LPS'组小鼠肝脏组织中ERK1/2、p38和JNK蛋白的磷酸化水平均显著降低（P＜0.001，P＜0.01，P＜0.01）；与OE-NC+LPS'组相比，OE-Adra2a+LPS'组小鼠肝脏ERK1/2、p38和JNK蛋白的磷酸化水平均显著升高（P＜0.01，P＜0.001，P＜0.01）。结论 LPS可引起Lbp^-/- 小鼠原代肝细胞Adra2a中蛋白表达量显著升高；Adra2a蛋白可以通过MAPK信号通路调节经LPS诱导的Lbp^-/- 小鼠原代肝细胞炎症水平。

关键词: 肾上腺素受体α2A, Lbp^-/- 小鼠, BRL-44408 maleate, 丝裂原激活的蛋白激酶信号通路

Abstract:

Objective To investigate the mechanism by which adrenoceptor alpha 2A (Adra2a) regulates lipopolysaccharide (LPS)-induced inflammation in primary hepatocytes from lipopolysaccharide-binding protein (LBP) knockout mice (Lbp^-/- ). Methods Primary hepatocytes from C57BL/6J and Lbp^-/- mice were isolated using a two-step perfusion method. An in vitro inflammatory model was established by LPS stimulation, and an in vivo inflammatory mouse model was established by intraperitoneal injection of LPS. The in vitro experiments were grouped as follows: Control group, LPS group, BRL+LPS group, OE-NC+LPS group, and OE-Adra2a+LPS group. The Control group served as the blank control. The LPS group involved stimulating primary hepatocytes with LPS. The BRL+LPS group involved pretreating primary hepatocytes with BRL-44408 maleate followed by LPS stimulation. The OE-NC+LPS group involved transfecting primary hepatocytes with an empty vector followed by LPS stimulation. The OE-Adra2a+LPS group involved transfecting primary hepatocytes with a lentivirus overexpressing Adra2a, followed by LPS stimulation. The in vivo experimental groups were divided into Control', LPS', BRL+LPS', OE-NC+LPS', and OE-Adra2a+LPS' groups. The Control' group served as the blank control. The LPS' group received intraperitoneal injection of LPS. The BRL+LPS' group received intraperitoneal injection of BRL-44408 maleate for pretreatment, followed by LPS injection. The OE-NC+LPS' group received intraperitoneal injection of empty vector for pretreatment, followed by LPS injection. The OE-Adra2a+LPS' group received intraperitoneal injection of a lentivirus overexpressing Adra2a for pretreatment, followed by LPS injection. Cell viability after Adra2a inhibition and overexpression was assessed via the Cell Counting Kit-8 (CCK-8) assay. RT-qPCR measured changes in gene expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) after Adra2a inhibition and overexpression. Western blotting was performed to detect Adra2a protein expression and phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase (JNK) following LPS stimulation. Results In vitro experiments revealed that LPS stimulation significantly decreased Adra2a protein expression in primary hepatocytes from C57BL/6J mice compared to the Control group (P<0.05), whereas it increased in primary hepatocytes from Lbp^-/- mice (P<0.001). Compared to the LPS group, the BRL+LPS group exhibited significantly increased cell viability (P<0.01), reduced TNF-α, IL-6, and IL-1β gene transcription levels (P<0.01, P<0.001, P<0.001), and decreased phosphorylation levels of MAPK signaling pathway-related proteins ERK1/2, p38, and JNK (P<0.01, P<0.001, P<0.001). Compared with the OE-NC+LPS group, the OE-Adra2a+LPS group showed significantly decreased cell viability (P<0.001), increased gene transcription levels of TNF-α, IL-6, and IL-1β genes (P<0.001, P<0.01, P<0.001), and elevated phosphorylation levels of MAPK signaling pathway-related proteins ERK1/2, p38, and JNK (P<0.001, P<0.01, P<0.001). In vivo experiments showed that, compared with the LPS' group, the BRL+LPS' group exhibited significantly reduced phosphorylation levels of MAPK signaling pathway-related proteins ERK1/2, p38, and JNK (P<0.001, P<0.01, P<0.01). In the OE-Adra2a+LPS' group, the phosphorylation levels of ERK1/2, p38, and JNK were significantly elevated compared to the OE-NC+LPS' group (P<0.01, P<0.001, P<0.01). Conclusion LPS stimulation can cause a significant increase in Adra2a protein expression in primary hepatocytes of Lbp^-/- mice. Adra2a protein can regulate the level of LPS-induced inflammation in primary hepatocytes of Lbp^-/- mice through the MAPK signaling pathway.

Key words: Adra2a, Lbp^-/- mice, BRL-44408 maleate, Mitogen-activated protein kinase signaling pathway

中图分类号:

Q95-33

刘赛,付彬,李思迪,等. Adra2a通过MAPK信号通路介导LPS诱导的Lbp^-/- 小鼠肝细胞炎性反应[J]. 实验动物与比较医学, 2026, 46(2): 212-221. DOI: 10.12300/j.issn.1674-5817.2025.077.

LIU Sai,FU Bin,LI Sidi,et al. Adra2a Regulates LPS-Induced Inflammation in Hepatocytes of Lbp^-/- Mice via the MAPK Signaling Pathway[J]. Laboratory Animal and Comparative Medicine, 2026, 46(2): 212-221. DOI: 10.12300/j.issn.1674-5817.2025.077.

图/表 6

表1 引物序列

Table 1 Primer sequences

基因 Gene	正向引物(5’-3’) Forward primer (5’-3’)	反向引物(5’-3’) Reverse primer (5’-3’)
Adra2a	GTGACACTGACGCTGGTTTG	CCAGTAACCCATAACCTCGTTG
β-actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT
TNF-α	CCCTCACACTCAGATCATCTTCT	GCTACGACGTGGGCTACAG
IL-6	TAGTCCTTCCTACCCCAATTTCC	TTGGTCCTTAGCCACTCCTTC
IL-1β	GCAACTGTTCCTGAACTCAACT	ATCTTTTGGGGTCCGTCAACT

注：C57BL/6J小鼠和Lbp^-/- 小鼠原代肝细胞中LBP蛋白的表达情况。LBP，脂多糖结合蛋白；β-actin，β-肌动蛋白。n=3。
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图1 原代肝细胞中LBP蛋白的表达情况
注：C57BL/6J小鼠和Lbp^-/- 小鼠原代肝细胞中LBP蛋白的表达情况。LBP，脂多糖结合蛋白；β-actin，β-肌动蛋白。n=3。

Figure 1 Expression of LBP protein in primary hepatocytes
Note： Expression of LBP protein in primary hepatocytes from C57BL/6J mice and Lbp^-/- mice. LBP， lipopolysaccharide-binding protein. n=3.

注：A~B，在LPS刺激12 h后，C57BL/6J和小鼠和Lbp^-/- 小鼠原代肝细胞中Adra2a蛋白的蛋白质印迹结果与定量分析；C，Lbp^-/- 小鼠原代肝细胞在LPS刺激、空载病毒转染及Adra2a基因过表达慢病毒转染后，细胞中Adra2a蛋白的蛋白质印迹结果与定量分析；D，Lbp^-/- 小鼠在经腹腔注射LPS、空载病毒及Adra2a基因过表达慢病毒后，肝脏组织内Adra2a蛋白的蛋白质印迹结果与定量分析。Control组，空白对照；LPS组，加入LPS刺激12 h；OE-NC+LPS组，加入空载病毒处理细胞24 h后再加入LPS刺激12 h；OE-Adra2a+LPS组，加入Adra2a基因过表达慢病毒处理细胞24 h后再加入LPS刺激12 h；Control’组为空白对照；LPS’组为经腹腔注射LPS刺激6 h，OE-NC+LPS’组为经腹腔注射空载病毒72 h后加入LPS刺激6 h；OE-Adra2a+LPS’组为经腹腔注射Adra2a基因过表达慢病毒72 h后加入LPS刺激6 h。LBP，脂多糖结合蛋白；β-actin，β-肌动蛋白；Adra2a，肾上腺素受体α2A；LPS，脂多糖。n=3；^*P<0.05，^**P<0.01，^***P<0.001，^****P<0.000 1。
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图2 LPS刺激和慢病毒处理后Adra2a蛋白表达的变化
注：A~B，在LPS刺激12 h后，C57BL/6J和小鼠和Lbp^-/- 小鼠原代肝细胞中Adra2a蛋白的蛋白质印迹结果与定量分析；C，Lbp^-/- 小鼠原代肝细胞在LPS刺激、空载病毒转染及Adra2a基因过表达慢病毒转染后，细胞中Adra2a蛋白的蛋白质印迹结果与定量分析；D，Lbp^-/- 小鼠在经腹腔注射LPS、空载病毒及Adra2a基因过表达慢病毒后，肝脏组织内Adra2a蛋白的蛋白质印迹结果与定量分析。Control组，空白对照；LPS组，加入LPS刺激12 h；OE-NC+LPS组，加入空载病毒处理细胞24 h后再加入LPS刺激12 h；OE-Adra2a+LPS组，加入Adra2a基因过表达慢病毒处理细胞24 h后再加入LPS刺激12 h；Control’组为空白对照；LPS’组为经腹腔注射LPS刺激6 h，OE-NC+LPS’组为经腹腔注射空载病毒72 h后加入LPS刺激6 h；OE-Adra2a+LPS’组为经腹腔注射Adra2a基因过表达慢病毒72 h后加入LPS刺激6 h。LBP，脂多糖结合蛋白；β-actin，β-肌动蛋白；Adra2a，肾上腺素受体α2A；LPS，脂多糖。n=3；^*P<0.05，^**P<0.01，^***P<0.001，^****P<0.000 1。

Figure 2 Changes in Adra2a protein expression after LPS and lentivirus treatment
Note： A–B， Western blotting and quantitative analysis of Adra2a protein in primary hepatocytes from C57BL/6J and Lbp^-/- mice after LPS stimulation for 12 h； C， Western blotting and quantitative analysis of Adra2a protein in primary hepatocytes from Lbp^-/- mice after LPS stimulation， empty vector transfection， or Adra2a-overexpressing lentivirus transfection； D， Western blotting and quantitative analysis of Adra2a protein in liver tissues of Lbp^-/- mice after intraperitoneal injection of LPS， empty vector， or Adra2a-overexpressing lentivirus. Control group， blank control； LPS group， stimulated with LPS for 12 h； OE-NC+LPS group， cells treated with empty vector for 24 h followed by LPS stimulation for 12 h； OE-Adra2a+LPS group， cells treated with Adra2a-overexpressing lentivirus for 24 h followed by LPS stimulation for 12 h； Control’ group， blank control； LPS’ group， intraperitoneal injection of LPS for 6 h； OE-NC+LPS’ group， intraperitoneal injection of empty vector for 72 h followed by LPS stimulation for 6 h； OE-Adra2a+LPS’ group， intraperitoneal injection of Adra2a-overexpressing lentivirus for 72 h followed by LPS stimulation for 6 h. LBP， lipopolysaccharide-binding protein； Adra2a， adrenoceptor alpha 2A； LPS， lipopolysaccharide. n=3； ^*P<0.05， ^**P<0.01， ^***P<0.001， ^****P<0.000 1.

注：A，Lbp^-/- 小鼠原代肝细胞在LPS刺激及加入BRL-44408 maleate预处理后细胞存活率的变化情况；B，Lbp^-/- 小鼠原代肝细胞在LPS刺激、空载病毒转染及Adra2a基因过表达慢病毒转染后细胞存活率的变化情况。Control组为空白对照；LPS组为加入LPS刺激12 h；BRL+LPS组为加入BRL-44408 maleate预处理细胞后再加入LPS刺激12 h；OE-NC+LPS组为加入空载病毒处理细胞24 h后加入LPS刺激12 h；OE-Adra2a+LPS组为加入Adra2a基因过表达慢病毒处理细胞24 h后加入LPS刺激12 h。Adra2a，肾上腺素受体α2A；LPS，脂多糖。n=6；^**P<0.01，^***P<0.001。
">

图3 抑制和过表达Adra2a对LPS刺激后Lbp^-/- 小鼠原代肝细胞存活率的影响
注：A，Lbp^-/- 小鼠原代肝细胞在LPS刺激及加入BRL-44408 maleate预处理后细胞存活率的变化情况；B，Lbp^-/- 小鼠原代肝细胞在LPS刺激、空载病毒转染及Adra2a基因过表达慢病毒转染后细胞存活率的变化情况。Control组为空白对照；LPS组为加入LPS刺激12 h；BRL+LPS组为加入BRL-44408 maleate预处理细胞后再加入LPS刺激12 h；OE-NC+LPS组为加入空载病毒处理细胞24 h后加入LPS刺激12 h；OE-Adra2a+LPS组为加入Adra2a基因过表达慢病毒处理细胞24 h后加入LPS刺激12 h。Adra2a，肾上腺素受体α2A；LPS，脂多糖。n=6；^**P<0.01，^***P<0.001。

Figure 3 Effects of Adra2a inhibition and overexpression on the survival rate of primary hepatocytes from Lbp^-/- mice following LPS stimulation
Note： A， changes in cell viability of primary hepatocytes from Lbp^-/- mice following LPS stimulation and pretreatment with BRL-44408 maleate； B， changes in cell viability of primary hepatocytes from Lbp^-/- mice following LPS stimulation， empty vector trans-fection， and Adra2a gene overexpression lentivirus trans-fection. Control group served as the blank control； LPS group received LPS stimulation for 12 h； BRL+LPS group received pretreatment with BRL-44408 maleate followed by LPS stimulation for 12 h； OE-NC+LPS group received empty vector treatment for 24 h followed by LPS stimulation for 12 h； OE-Adra2a+LPS group received Adra2a gene overexpression lentivirus treatment for 24 h followed by LPS stimulation for 12 h. Adra2a， adrenoceptor alpha 2A； LPS， lipopolysaccharide. n=6； ^**P<0.01， ^***P<0.001.

注：Lbp^-/- 小鼠原代肝细胞中TNF-α（A、D）、IL-6（B、E）和IL-1β（C、F）基因的mRNA水平变化情况。Control组为空白对照；LPS组为加入LPS刺激12 h；BRL+LPS组为加入BRL-44408 maleate预处理细胞后再加入LPS刺激12 h；OE-NC+LPS组为加入空载病毒处理细胞24 h后加入LPS刺激12 h；OE-Adra2a+LPS组为加入Adra2a基因过表达慢病毒处理细胞24 h后加入LPS刺激12 h。TNF -α，肿瘤坏死因子-α；IL-6，白细胞介素-6；IL-1β，白细胞介素-1β；Adra2a，肾上腺素受体α2A；LPS，脂多糖。n=3；^**P<0.01，^***P<0.001，^****P<0.000 1。
">

图4 抑制（A~C）和过表达（D~F）Adra2a对LPS刺激后Lbp^-/- 小鼠原代肝细胞ΤNF-ɑ、IL-6和IL-1β基因转录的影响

注：

Lbp^-/- 小鼠原代肝细胞中TNF-α（A、D）、IL-6（B、E）和IL-1β（C、F）基因的mRNA水平变化情况。Control组为空白对照；LPS组为加入LPS刺激12 h；BRL+LPS组为加入BRL-44408 maleate预处理细胞后再加入LPS刺激12 h；OE-NC+LPS组为加入空载病毒处理细胞24 h后加入LPS刺激12 h；OE-Adra2a+LPS组为加入Adra2a基因过表达慢病毒处理细胞24 h后加入LPS刺激12 h。TNF -α，肿瘤坏死因子-α；IL-6，白细胞介素-6；IL-1β，白细胞介素-1β；Adra2a，肾上腺素受体α2A；LPS，脂多糖。n=3；^**P<0.01，^***P<0.001，^****P<0.000 1。

Figure 4 Effects of Adra2a inhibition （A–C） and overexpression （D–F） on the gene transcription of TNF-ɑ， IL-6， and IL-1 β in primary hepatocytes from Lbp^-/- mice following LPS stimulation
Note： Changes in mRNA levels of the TNF-α （A， D）， IL-6 （B， E）， and IL-1β （C， F） genes in primary hepatocytes of Lbp^-/- mice. Control group served as the blank control； LPS group received LPS stimulation for 12 h； BRL+LPS group received LPS stimulation for 12 h after pretreatment with BRL-44408 maleate； OE-NC+LPS group received empty vector treatment for 24 h followed by LPS stimulation for 12 h； OE-Adra2a+LPS group received Adra2a gene over-expression lentivirus treatment for 24 h followed by LPS stimulation for 12 h. TNF-α， tumor necrosis factor-α； IL-6， interleukin-6； IL-1β， interleukin-1β； Adra2a， adrenoceptor alpha 2A； LPS， lipopolysaccharide. n=3； ^**P<0.01， ^***P<0.001， ^****P<0.000 1.

注：Lbp^-/- 小鼠原代肝细胞（A~B）和肝脏组织（C~D）中ERK1/2、p38、JNK及其磷酸化蛋白的蛋白质印迹结果与定量分析。Control组，空白对照组；LPS组，加入LPS刺激12 h；BRL+LPS组，加入BRL-44408 maleate预处理细胞后，再加入LPS刺激12 h；OE-NC+LPS组，加入空载病毒处理细胞24 h后，再加入LPS刺激12 h；OE-Adra2a+LPS组，加入Adra2a基因过表达慢病毒处理细胞24 h后，再加入LPS刺激12 h；Control’组，空白对照；LPS’组，加入LPS刺激12 h；BRL+LPS’组，腹腔注射BRL-44408 maleate预处理后，再加入LPS刺激6 h；OE-NC+LPS’组为经腹腔注射空载病毒处理细胞72 h后，再加入LPS刺激6 h；OE-Adra2a+LPS’组为经腹腔注射Adra2a基因过表达慢病毒72 h后，再加入LPS刺激6 h。ERK1/2，细胞外调节蛋白激酶1/2；p38，p38丝裂原活化蛋白激酶；JNK，c-Jun氨基末端激酶；Adra2a，肾上腺素受体α2A；LPS，脂多糖。n=3；^**P<0.01，^***P<0.001。
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图5 抑制和过表达Adra2a对Lbp^-/- 小鼠原代肝细胞和肝脏组织中ERK1/2、p38、JNK蛋白磷酸化的影响

注：

Lbp^-/- 小鼠原代肝细胞（A~B）和肝脏组织（C~D）中ERK1/2、p38、JNK及其磷酸化蛋白的蛋白质印迹结果与定量分析。Control组，空白对照组；LPS组，加入LPS刺激12 h；BRL+LPS组，加入BRL-44408 maleate预处理细胞后，再加入LPS刺激12 h；OE-NC+LPS组，加入空载病毒处理细胞24 h后，再加入LPS刺激12 h；OE-Adra2a+LPS组，加入Adra2a基因过表达慢病毒处理细胞24 h后，再加入LPS刺激12 h；Control’组，空白对照；LPS’组，加入LPS刺激12 h；BRL+LPS’组，腹腔注射BRL-44408 maleate预处理后，再加入LPS刺激6 h；OE-NC+LPS’组为经腹腔注射空载病毒处理细胞72 h后，再加入LPS刺激6 h；OE-Adra2a+LPS’组为经腹腔注射Adra2a基因过表达慢病毒72 h后，再加入LPS刺激6 h。ERK1/2，细胞外调节蛋白激酶1/2；p38，p38丝裂原活化蛋白激酶；JNK，c-Jun氨基末端激酶；Adra2a，肾上腺素受体α2A；LPS，脂多糖。n=3；^**P<0.01，^***P<0.001。

Figure 5 Effects of Adra2a inhibition and overexpression on ERK1/2， p38， and JNK protein phosphorylation inprimary hepatocytes and liver tissues of Lbp^-/- mice
Note： Western blotting and quantitative analysis of ERK1/2， p38， JNK and their phosphorylated proteins in primary hepatocytes from Lbp^-/- mice （A–B） and in the livers of Lbp^-/- mice （C–D）. Control group， blank control； LPS group， stimulated with LPS for 12 h； BRL+LPS group， cells pretreated with BRL-44408 maleate followed by LPS stimulation for 12 h； OE-NC+LPS group， cells treated with empty vector for 24 h followed by LPS stimulation for 12 h； OE-Adra2a+LPS group， cells treated with Adra2a-overexpressing lentivirus for 24 h followed by LPS stimulation for 12 h； Control’ group， blank control； LPS’ group， treated with LPS for 12 h； BRL+LPS’ group， mice pretreated with intraperitoneal injection of BRL-44408 maleate followed by LPS stimulation for 6 h； OE-NC+LPS’ group， mice treated with intraperitoneal injection of empty vector for 72 h followed by LPS stimulation for 6 h； OE-Adra2a+LPS’ group， mice treated with intraperitoneal injection of Adra2a-overexpressing lentivirus for 72 h followed by LPS stimulation for 6 h. ERK1/2， extracellular signal-regulated kinase 1/2； p38， p38 mitogen-activated protein kinase； JNK， c-Jun N-terminal kinase； Adra2a， adrenoceptor alpha 2A； LPS， lipopolysaccharide. n=3； ^**P<0.01，^***P<0.001.

图5 抑制和过表达Adra2a对Lbp^-/- 小鼠原代肝细胞和肝脏组织中ERK1/2、p38、JNK蛋白磷酸化的影响
注：Lbp^-/- 小鼠原代肝细胞（A~B）和肝脏组织（C~D）中ERK1/2、p38、JNK及其磷酸化蛋白的蛋白质印迹结果与定量分析。Control组，空白对照组；LPS组，加入LPS刺激12 h；BRL+LPS组，加入BRL-44408 maleate预处理细胞后，再加入LPS刺激12 h；OE-NC+LPS组，加入空载病毒处理细胞24 h后，再加入LPS刺激12 h；OE-Adra2a+LPS组，加入Adra2a基因过表达慢病毒处理细胞24 h后，再加入LPS刺激12 h；Control’组，空白对照；LPS’组，加入LPS刺激12 h；BRL+LPS’组，腹腔注射BRL-44408 maleate预处理后，再加入LPS刺激6 h；OE-NC+LPS’组为经腹腔注射空载病毒处理细胞72 h后，再加入LPS刺激6 h；OE-Adra2a+LPS’组为经腹腔注射Adra2a基因过表达慢病毒72 h后，再加入LPS刺激6 h。ERK1/2，细胞外调节蛋白激酶1/2；p38，p38丝裂原活化蛋白激酶；JNK，c-Jun氨基末端激酶；Adra2a，肾上腺素受体α2A；LPS，脂多糖。n=3；^**P<0.01，^***P<0.001。

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Adra2a通过MAPK信号通路介导LPS诱导的Lbp^-/- 小鼠肝细胞炎性反应

Adra2a Regulates LPS-Induced Inflammation in Hepatocytes of Lbp^-/- Mice via the MAPK Signaling Pathway

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