Effect Of RhGH On The LPS Induced U937 Macrophage Apoptosis And NF-κB Expression

Septic shock remains in high mortality despite the recent advance of critical care. It was reported that the development of septic shock is closely related with increased immune cell apoptosis and excessively released inflammatory mediators. Furthermore,cell apoptosis and inflammatory mediators transcription related genes are under the regulation of NF-κB. Francisco et al revealed that NF-κB expression was significantly increased during sepsis, and patient who were died from sepsis had the highest elevated NF-κB activity, therefore, the NF-κB activity and outcome of sepsis is thought to be closely related.Recent study indicated that the recombinant human growth hormone (rhGH) potentially promotes protein synthesis, regulates immune function and reduces stress responses. Our present study demonstrated that the rhGH possesses the ability to inhibit the lipopolysaccharide (endotoxin) induced macrophage apoptosis as well as the NF-κB expression and nucleation, thus provided basis of potential therapeutic use of rhGH in patients with sepsis.AIM The purposes of this study were to demonstrate that the rhGH possesses the ability to inhibit the lipopolysaccharide (endotoxin) induced macrophage apoptosis as well as the NF-κB expression and nucleation.Methods1. U937 cells were cultured on a 2.5 ml 6-well plate until the cell density reached 2×10~6/ml, discard the supernatant and changed with fresh culture media containing LPS (40μg/ml), rhGH(4u/L), or LPS (40μg/ml) +rhGH (4u/L), and an blank control group, each group consisted with 5 repeated wells and cultured in 5% (V/V) CO2 37℃incubator for 16 hours.Cells were analyzed under flowcytometer after centrifuge at 9cm centrifuge radius 1000r/min for 5 min and washed twice by pH 7.2 PBS buffer and diluted to cell density of 1×10~6/ml. The cell apoptosis ratios were represented by percentages of the dead cell/total cell count.2. The cells were cultured and treated as the cells used in the apoptosis assay experiment, the treated culture cells were collected and washed by PBS buffer and diluted and then prepare the smears on the polylysine treated slides and stained according to the manual instruction of NF-κB P65 immunochemistry kit after 4% V/V polymethane 60 min fixation.Results1.the cell apoptosis ratios: The cell apoptosis percentages of the LPS alone treated group were significantly higher than that of the blank control and the group with additional rhGH,P＜0.01. The cell apoptosis percentages of the blank control, the rhGH control, the LPS alone, and the LPS and rhGH group were 3.70+0.66%,3.65+0.66%,9.1+2.40%,and 4.13+0.57% respectively.2. Comparison of cell nuclei positive NF-κB P65 protein cell counts in the 4 experimental groups: The nuclei positive NF-κB P65 protein cell count in the blank control, the rhGH control, the LPS alone, and the LPS and rhGH group were 1.67+0.20%,41.1+10.46%, 1.46+0.41%,and 9.3+1.9% respectively. The percentage of the positive stained cells in LPS alone treated group were significantly higher than that of the blank control and the group with additional rhGH,P＜0.01.Conclusion1. rhGH potentially inhibited the LPS induced macrophage apoptosis.2. rhGH potentially inhibited the LPS induced the activation and nuclear replacement of NF-κB.These results suggest that the protective effect of macrophages against LPS induced apoptosis might be a result of the inhibition of NF-κB activation and nuclear replacement.