MI Chuanliang
School of Laboratory Animal Shandong Laboratory Animal Center,Shandong First Medical University Shandong Academy of Medical Sciences,Ji’nan;School of Laboratory Animal Shandong Laboratory Animal Center,Shandong First Medical University Shandong Academy of Medical Sciences,Ji’nanFU Bin
School of Laboratory Animal Shandong Laboratory Animal Center,Shandong First Medical University Shandong Academy of Medical Sciences,Ji’nanLI Sidi
School of Laboratory Animal Shandong Laboratory Animal Center,Shandong First Medical University Shandong Academy of Medical Sciences,Ji’nanCHEN Zhida
School of Laboratory Animal Shandong Laboratory Animal Center,Shandong First Medical University Shandong Academy of Medical Sciences,Ji’nanGUO Zhongkun
School of Laboratory Animal Shandong Laboratory Animal Center,Shandong First Medical University Shandong Academy of Medical Sciences,Ji’nanWANG Kezhou
School of Laboratory Animal Shandong Laboratory Animal Center,Shandong First Medical University Shandong Academy of Medical Sciences,Ji’nanSchool of Laboratory Animal Shandong Laboratory Animal Center,Shandong First Medical University Shandong Academy of Medical Sciences,Ji’nan
Funded by the Innovation Project of Shandong Academy of Medical Sciences, Jinan Science and Technology Bureau “20 Colleges and Universities”（2021GXRC011）, Shandong Medical and Health Science and Technology Development Plan（2019WS177）, Shandong Province Pig Industry Technology System（SDAIT-08-17）.
Objective To Explore the Adra1a regulation of inflammatory response in primary hepatocytes of LBP knockout mice (Lbp-/-) induced by LPS. Method Using two-step perfusion method to extract primary hepatocytes and constracting an primary hepatocyte inflammation model from WT group and Lbp-/- group. Inhibition of Adra1a expression in Lbp-/- mouse primary hepatocytes by adding inhibitor prazosin and transfecting siRNA .The inhibitor method divided the cells into control group (blank control), LPS group (LPS stimulation), and inhibitor piperazine group (LPS stimulation added after 1 hour of prazosin intervention). The method of siRNA transfection divided primary hepatocytes into four groups: control group (blank control), LPS group (LPS stimulation for 12 hours), negative control group (si-NC interference for 12 hours followed by LPS stimulation for 12 hours), and interference group (si-adra1a interference for 12 hours followed by LPS stimulation for 12 hours). The primary hepatocytes of WT mice were divided into two groups: the control group (blank control) and the LPS group (LPS stimulation for 12 hours). This study used WT type and Lbp-/- type mouse primary liver cells as the research subjects to verify the changes of Adra1a under LPS stimulation using Western blot method. Experimental methods such as CCK8, qPCR, and Western blot were used to verify the improvement of inflammation and survival rate of primary liver cells in Lbp-/- mice treated with prazosin and si-adra1a. Results under LPS stimulation, the expression of Adra1a protein in primary hepatocytes of Lbp-/- mice was significantly increased (P < 0.01), while the wild-type did not change; The cell survival rate of the inhibitor prazosin group and the interference group was significantly increased (P < 0.01; P < 0.05); In the inhibitor prazosin group and interference group, the expression of inflammatory factors TNF-α、IL-1β significantly decreased (P < 0.01). The expression levels of proteins p-p38, p-ERK, and p-JNK related to cell damage and inflammation were also significantly reduced (P < 0.01). Conclusion upregulation of Adra1a expression in Lbp -/- mouse primary liver cells induced by LPS compensates for the role of lipopolysaccharide binding protein (LBP) in conducting injury and inflammatory signals, inhibiting the expression of Adra1a gene can significantly reduce the occurrence of inflammation and cell damage in primary liver cells.
