ObjectiveTo 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^-/-). MethodsPrimary 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. ResultsIn 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). ConclusionLPS 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.

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-1 β(P＜O.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.