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.

Objective To investigate the effect and mechanisms of factor fibroblast growth factor inducible 14(Fn14)in the high glucose induced-cardiomyocyte hypertrophy. Method To observe the expression of collagenⅠ, connective tissue growth factor ( CTGF ) , transforming growth factor-β1 ( TGF-β1 ) , and Fn14 in high glucose induced-cardiomyocyte hypertrophy. Fn14 expressions was down-regulated by siRNA interference technique, and then the expressions of collagen Ⅰ, CTGF, and TGF-β1 were observed, and the mechanism was also explored. Results The expression of collagen I, CTGF and TGF-β1 was significantly up-regulated after high glucose induced-cardiomyocyte hypertrophy for 72 h. At the same time, the expression of Fn14 was increased after 72 h-treatment, and reached the peak at concentration of 30 mmol/L high glucose. High glucose could not up-regulated the expression of collagenⅠ, CTGF, and TGF-β1 after siFn14 interference, while the same result was observed in the expression of p-JNK. Conclusion The expressions of collagenⅠ, CTGF, TGF-β1, and Fn14 in cardiomyocyte of neonatal rats were induced by high glucose. While Fn14 expression was inhibited, the expressions of collagenⅠ, CTGF, and TGF-β1 were down-regulated, which seems to be involved with p-JNK signaling pathway.

Objective Routine perioperative intravenous antimicrobial agents,was administered as surgical prophylaxis.However,whether balanced ultrafiltration during extracorporeal circulation can remove antimicrobial agent remains unclear.The concentrations of antimicrobial agent in plasma and ultrafiltrate samples were measured in this pseudo-extracorporeal circulation model.Methods Extracorporeal circulation consisted of cardiotomy reservoir (Ningbo Fly Medical Healthcare CO.,LTD.Ningbo,China),D902 Lilliput 2 membrane oxygenator (Sorin Group Asia Pte Ltd,Beijing,China) and Capiox (R) AF02 pediatric arterial line filter (Terumo Corporation,Beijing,China).HEMOCONCENTRATOR BC 20 plus (MAQUET Cardiopulmonary AG,Hirrlingen,Germany) was placed between arterial purge line and oxygenator venous reservoir.Fresh donor human whole blood was added into the circuit and mixed with Ringer's solution to obtain a final hematocrit of 24％-28 ％.After 30 minutes of extracorporeal circulation,zero-balanced ultrafiltration was initiated and arterial line pressure was maintained at approximately 100 mm Hg(1 mm Hg =0.133 kPa) with Hoffman clamp.The rate of ultrafiltration (12 ml/min) was controlled by ultrafiltrate outlet pressure.Identical volume of plasmaslyte A was dripped into the circuit to maintain stable hematocrit during 45 minutes of experiment.Plasma and ultrafiltrate samples were drawn every 5 minutes and concentrations of antimicrobial agent (including Cefmetasole and cefotiam) were measured with high performance liquid chromatography.Results All these two antimicrobial agents were detected in ultrafiltrate,demonstrating hemoconcentration may remove antimicrobial agent.The concentration of plasma antimicrobial agent decreased lineally with the increase of ultrafiltrate volume.At end of balanced ultrafiltration,the concentration of plasma cefotiam was (104.96 ± 44.36) μg/ml,which is about (44.38 ± 7.42) ％ of the initial concentration (238.95 ± 101.12) μg/ml; the concentration of plasma cefmetazole decreased linearly to (25.76 ± 14.78) μg/ml,which is about (49.69 ± 10.49) ％ of the initial concentration (51.49 ± 28.03) μg/ml.The total amount of cefotiam in ultrafiltrate is (27.16 ± 12.17)％ of the total dose administered,whereas cefmetasole in ultrafiltrate is (7.74 ±4.17)％.Conclusion Balanced ultrafiltration may remove antimicrobial agent from serum and has significant influence on plasma concentration of antimicrobial agent.The strategy of surgical prophylaxis should consider this unique technique during extracorporeal circulation.