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

doi:10.12300/j.issn.1674-5817.2025.077

Abstract

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

CLC Number:

Q95-33
R-332

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.

Figures/Tables 6

References 22

[1]	DI LORENZO F, DE CASTRO C, SILIPO A, et al. Lipopolysaccharide structures of Gram-negative populations in the gut microbiota and effects on host interactions[J]. FEMS Microbiol Rev, 2019, 43(3):257-272. DOI: 10.1093/femsre/fuz002 .
[2]	ULEVITCH R J, TOBIAS P S. Recognition of Gram-negative bacteria and endotoxin by the innate immune system[J]. Curr Opin Immunol, 1999, 11(1):19-22. DOI: 10.1016/S0952-7915(99)80004-1 .
[3]	GARCIA-VELLO P, DI LORENZO F, ZUCCHETTA D, et al. Lipopolysaccharide lipid A: a promising molecule for new immunity-based therapies and antibiotics[J]. Pharmacol Ther, 2022, 230:107970. DOI: 10.1016/j.pharmthera.2021.107970 .
[4]	SONG Z C, MENG L L, HE Z X, et al. LBP protects hepatocyte mitochondrial function via the PPAR-CYP4A2 signaling pathway in a rat sepsis model[J]. Shock, 2021, 56(6):1066-1079. DOI: 10.1097/SHK.0000000000001808 .
[5]	李思迪, 付彬, 郭中坤, 等. 利用CRISPR/Cas9技术构建脂多糖结合蛋白基因敲除小鼠[J]. 实验动物与比较医学, 2022, 42(4):294-300. DOI: 10.12300/j.issn.1674-5817.2022.002 .
	LI S D, FU B, GUO Z K, et al. Construction of lipopolysaccaride binding protein knockout mice using CRISPR/Cas9 technology[J]. Lab Anim Comp Med, 2022, 42(4):294-300. DOI: 10.12300/j.issn.1674-5817.2022.002 .
[6]	米传靓, 付彬, 李思迪, 等. Adra1a调节LPS诱导的Lbp^-/-小鼠原代肝细胞炎症反应[J]. 中国比较医学杂志, 2024, 34(5): 84-91. DOI: 10.3969/j.issn.1671-7856.2024.05.009 .
	MI C L, FU B, LI S D, et al. Adra1a regulates LPS-induced inflammation in primary hepatocytes of Lbp^-/- mice[J]. Chin J Comp Med, 2024, 34(5): 84-91. DOI: 10.3969/j.issn.1671-7856.2024.05.009 .
[7]	米传靓, 付彬, 李思迪, 等. Agtr1a调节LPS诱导的Lbp^-/- 小鼠原代肝细胞炎症[J]. 中国实验动物学报, 2023, 31(8): 1021-1027. DOI: 10.3969/j.issn.1005-4847.2023.08.007 .
	MI C L, FU B, LI S D, et al. Agtr1a regulates LPS-induced inflammation in primary hepatocytes of Lbp^-/- mice[J]. Acta Lab Anim Sci Sin, 2023, 31(8): 1021-1027. DOI: 10.3969/j.issn.1005-4847.2023.08.007 .
[8]	ZHOU F, CAI J, CHEN L,et al. ADRA2A contributes to airway inflammation and apoptosis in asthma through the ERK signaling in vitro and in vivo [J]. J Inflamm, 2025, 22(1): 55. DOI: 10.1186/s12950-025-00480-8 .
[9]	MIKSA M, DAS P, ZHOU M, et al. Pivotal role of the alpha(2A)-adrenoceptor in producing inflammation and organ injury in a rat model of sepsis[J]. PLoS One, 2009, 4(5):e5504. DOI: 10.1371/journal.pone.0005504 .
[10]	LIN Y, ZHU X, YAO W Z, et al. Yohimbine protects against endotoxin-induced acute lung injury by blockade of alpha 2A adrenergic receptor in rats[J]. Chin Med J, 2011, 124(7):1069-1074.DOI: 10.3760/cma.j.issn.0366-6999.2011.07.022 .
[11]	任文洁, 林哲绚. 胰蛋白酶和胶原酶灌注法分离提取小鼠原代肝细胞的比较[J]. 汕头大学医学院学报, 2022, 35(4):204-209. DOI: 10.13401/j.cnki.jsumc.2022.04.003 .
	REN W J, LIN Z X. Comparison of isolation and extraction of primary mouse hepatocytes by trypsin and collagenase perfusion[J]. J Shantou Univ Med Coll, 2022, 35(4):204-209. DOI: 10.13401/j.cnki.jsumc.2022.04.003 .
[12]	RYU J K, KIM S J, RAH S H, et al. Reconstruction of LPS transfer cascade reveals structural determinants within LBP, CD14, and TLR4-MD2 for efficient LPS recognition and transfer[J]. Immunity, 2017, 46(1):38-50. DOI: 10.1016/j.immuni. 2016.11.007 .
[13]	MANO S S, KANEHIRA K, TANIGUCHI A. Comparison of cellular uptake and inflammatory response via toll-like receptor 4 to lipopolysaccharide and titanium dioxide nanoparticles[J]. Int J Mol Sci, 2013, 14(7):13154-13170. DOI: 10.3390/ijms140713154 .
[14]	LI T X, BAI J J, DU Y C, et al. Thiamine pretreatment improves endotoxemia-related liver injury and cholestatic complications by regulating galactose metabolism and inhibiting macrophage activation[J]. Int Immunopharmacol, 2022, 108:108892. DOI: 10.1016/j.intimp.2022.108892 .
[15]	BRASS D M, SAVOV J D, WHITEHEAD G S, et al. LPS binding protein is important in the airway response to inhaled endotoxin[J]. J Allergy Clin Immunol, 2004, 114(3):586-592. DOI: 10.1016/j.jaci.2004.04.043 .
[16]	MINTER R M, BI X M, BEN-JOSEF G, et al. LPS-binding protein mediates LPS-induced liver injury and mortality in the setting of biliary obstruction[J]. Am J Physiol Gastrointest Liver Physiol, 2009, 296(1): G45-G54. DOI: 10.1152/ajpgi.00041.2008 .
[17]	LATTIN J, ZIDAR D A, SCHRODER K, et al. G-protein-coupled receptor expression, function, and signaling in macrophages[J]. J Leukoc Biol, 2007, 82(1):16-32. DOI: 10.1189/jlb.0107051 .
[18]	SHARMA N, SISTLA R, ANDUGULAPATI S B. Yohimbine ameliorates liver inflammation and fibrosis by regulating oxidative stress and Wnt/β-catenin pathway[J]. Phyto-medicine, 2024, 123:155182. DOI: 10.1016/j.phymed.2023. 155182 .
[19]	WANG J H, LIU Y J, GUO Y S, et al. Function and inhibition of P38 MAP kinase signaling: Targeting multiple inflammation diseases[J]. Biochem Pharmacol, 2024, 220:115973. DOI: 10. 1016/j.bcp.2023.115973 .
[20]	AHMED T, ZULFIQAR A, ARGUELLES S, et al. Map kinase signaling as therapeutic target for neurodegeneration[J]. Pharmacol Res, 2020, 160:105090. DOI:10.1016/j.phrs. 2020. 105090 .
[21]	REZATABAR S, KARIMIAN A, RAMESHKNIA V, et al. RAS/MAPK signaling functions in oxidative stress, DNA damage response and cancer progression[J]. J Cell Physiol, 2019, 234(9):14951-14965. DOI: 10.1002/jcp.28334 .
[22]	刘淑青. 肝细胞SOX9在肝再生中的作用和机制及肝细胞TGF-β信号通路对肝纤维化的影响和机制[D]. 上海: 中国人民解放军海军军医大学, 2023. DOI: 10.26998/d.cnki.gjuyu.2023.000324 .
	LIU S Q. The role and underlying mechanism of hepatocellular SOX9 in liver regeneration and hepatocellular TGF-β signaling pathway in liver fibrosis[D]. Shanghai: Naval Medical University, 2023. DOI: 10.26998/d.cnki.gjuyu.2023.000324 .

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

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