17β-Estradiol attenuates cytokine-induced nitric oxide production in rat hepatocyte

Abstract

Objective: Nitric oxide (NO) regulation during shock and sepsis is complex. NO production by endothelial NO synthase maintains microvascular perfusion and prevents shock-induced organ injury. However, the overproduction of NO by inducible NO synthase (iNOS) contributes to liver dysfunction after shock and is associated with increased tissue damage and mortality. Estrogen improves organ function and outcome after shock and sepsis, but the mechanism is unknown. We hypothesized that 17β-estradiol would improve organ function by regulating the production of hepatocyte NO.

Methods: Isolated rat hepatocytes were stimulated in vitro with pro-inflammatory cytokines to induce NO synthesis with or without estrogen. Nitrite was detected after 24 hours. INOS protein was determined using Western blot analysis.

Results: Cytokine stimulation increased nitrite and iNOS protein in a dose-dependent manner. The cytokine-induced nitrite increase was significantly decreased by estrogen. iNOS expression was also diminished in cultures with the higher doses of estrogen.

Conclusion: 17β-Estradiol decreases cytokine-stimulated iNOS expression and NO production. The down-regulation of iNOS expression may account for the beneficial role of estrogens in models of sepsis and shock.

Figure 1

Nitrite (NO ⁻₂) production of hepatocytes simultaneously treated with rhIL-1β, rrIFN-□, and different concentrations of E2 in serum-containing medium. Results are the mean + SEM of 3 independent rat experiments performed separately in which NO ⁻₂ was measured from supernatant of hepatocytes cultured in triplicates for each rat experiment. (A) NO ⁻₂ measured from cells stimulated with the cytokines alone minus E2 (3rd column from Y-axis) was used as positive control and all other treatments expressed as a percentage of the control. Asterisk represents a significant (p < 0.05) difference from control cells. (B) Western blot analysis of iNOS protein. A dose-dependent suppression of cytokine-stimulated iNOS protein expression by E2 is demonstrated.

Figure 2

NO ⁻₂ production in cytokine-stimulated hepatocytes cultured in serum-free antibiotic-free medium: (A) Statistically significant suppression of NO ⁻₂ by estradiol begins to occur at the 1uM (p < 0.05) in cells cultured in serum-free, antibiotic-free medium. B. Western blot demonstrating iNOS expression in hepatocytes cultured in serum-free medium. Significant decrease in iNOS protein was noted at 10-25 uM dose of E2 (p<0.05).

Figure 3

Time-course relationship of cytokine-induced NO ⁻₂ production in hepatocyte exposed to two different concentrations of E2 (the two bottom line graphs) versus hepatocytes cultured without E2. Results are the mean + SEM of 3 different rats in which NO ⁻₂ was measured from supernatant of hepatocytes cultured in triplicates. The NO ⁻₂ measured are absolute values in uM.

Figure 4

Line graph demonstrating iNOS mRNA expression in cytokine-stimulated hepatocytes exposed to two different doses of E2 (the two bottom line graphs) versus hepatocytes not exposed to E2 (upper line graph).

Figure 5

Suppression of NO production involves ER-α. Hepatocytes were treated as described in Fig. 1. Concentrations of ER-α ligands were 10uM E2, 10uM PTT, a selective ER-α agonist and 10uM MMP, a specific ER-α antagonist. CM= Cytokine mix of rhIL-1b plus rrIFN-g. DMSO = Vehicle used as negative control.