Protective Effect Of Insulin On LPS-induced Injury Of Normal Human Liver Cells

ObjectIntestinal endotoxemia may occur in the course of illness in patients with trauma, sepsis,hepatopathy and other conditions.The major component of Endotoxin is Lipopolysaccharide(LPS) which structure the outer membrane of cell wall of gram-negative bacteria.It has been found that LPS can cause hepatocytes injury by inducing apoptosis of liver cells.Recent studies reported that intensive insulin therapy in critically ill patients decreases the mortality and prevents the incidence of infection and sepsis.However the molecular and cellular mechanisms by which insulin improves survival probability remain unknown.The present study evaluated the effects of insulin on LPS-induced apoptosis in human liver cells(HL7702) and further explored the mechanisms for its anti-apoptotic effect.Method1.MTT assay:To test the cell viability by MTT assay,the cells were divided into control group(0μg/ml LPS) and 10,20,40,80μg/ml LPS groups.HL7702 cells were exposed to various concentrations of LPS for 12,24,36,48h respectively.To test the protective effect of Insulin,different concentrations of Insulin(2.5,5,10,20 IU/ml) protective groups,LPS group(20μg/ml) and control group were set.HL7702 cells were exposed to various concentrations of Insulin for 12,24,36,48h respectively and test the cell viability by MTT assay.2.FCM assay:The percentage of 24 hours apoptosis of each group was examined by flow cytometry with AnnexinV-FITC/PI staining. 3.Real time PCR:Different concentrations of insulin treatment groups,LPS group(20μg/ml) and control group were detected by real time PCR for their mRNA expression levels of bal-2,bax and Caspase-3.Result1.MTT:After exposed to a range of concentrations of LPS for designated periods of time,compared with control group there was a dose and time-dependent decrease in cell viability as measured by MTT assay(P＜0.01).It is observed that incubated with insulin(2.5,5,10,20 IU/ml) caused a significant decrease in the level of cell death compared with that of LPS-treated cells(P＜0.01).2.FCM study indicated that,after being exposed to 20μg/ml LPS for 24 hours,the percentage of apoptotic cells,was increased compared with that of control(28.83±1.32%vs 9.38±0.39%,P＜0.01),Apoptosis ratio was dropped respectively to 23.33±1.15%,20.76±1.21%,14.61±0.91 and 12.30±1.23%with 2.5,5,10,20 IU/ml insulin treatment(P＜0.01).3.Real time PCR results indicated that,incubating cells with 20μg/ml LPS for 24h reduced the mRNA expression level of anti-apoptosis gene bcl-2(0.56±0.12),but increased the mRNA expression level of apoptosis associated gene bax(1.37±0.08) and Caspase-3(0.95±0.10),significantly compared with control group(P＜0.01).Insulin dose-dependently increased the mRNA expression level of gene bcl-2 and reduced the mRNA expression level of gene bax and Caspase-3 in HL7702 cells.After exposure to (2.5,5,10,20 IU/ml) insulin,the mRNA expression level of bcl-2 increased to 0.76±0.10,0.89±0.08,1.01±0.17 and 1.20±0.14,the mRNA expression level of bax decreased to 1.21±0.16,1.02±0.10,0.91±0.06 and 0.78±0.05,and the mRNA expression level of Caspase-3 decreased to 0.81±0.05,0.72±0.04,0.58±0.04 and 0.49±0.05,there were significant difference with these gene expression level of the 20μg/ml LPS group(P＜0.05) unless the 2.5 IU/ml insulin group. Conclusion1.LPS can cause HL7702 cell injury and induce its apoptosis.2.Insulin inhibited HL7702 cell apoptosis induced by LPS at dose-dependent3.The mechanisms of insulin's anti-apoptotic effect maybe included(1) Insulin inhibited the mRNA expression level of apoptosis associated protein Caspase-3 to inhibit HL7702 cell apoptosis by LPS and protected HL7702 cell.(2) Insulin enhanced the mRNA expression level of anti-apoptosis gene bcl-2 and inhibited the mRNA expression level of apoptosis associated gene bax to inhibit HL7702 cell apoptosis by LPS and protected HL7702 cell.