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

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

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.

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.

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.

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.

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.

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