Morphological and biochemical evaluation of RAW 264.7 macrophages after acute and chronic administration of DHEA and AED.
The effect of adrenal hormones namely dehydroepiandrosterone (DHEA, 10-50 ug/day) and androstenedione (AED, 10-50 ug/day), on RAW 264.7 macrophage survival at 24, 48 and 72 hours after lipopolysaccharide (LPS, 2 micrograms/ml) exposure was investigated in an in vitro environment. RAW cells were obtained from American Type Culture Collection and standard laboratory protocols were followed in cells plating (10(6) cells/well), phase terminating, morphological evaluation, and biochemical marker analysis. From physiologic to supraphysiologic doses of DHEA and AED at 24 hours caused increased levels of cellular proteins and cell number without causing any significant (p < 0.05) change in cellular membrane integrity (Maliondialdehyde, MDA) or viability (morphology). At 48 and 72 hours, cells treated with either AED or DHEA did not sustain the increased cellular proliferation as observed at 24 hours and did not significantly differ (p < 0.05) in cellular protein content. RAW 264.7 cells treated with LPS for 30 minutes prior to AED or DHEA exposure caused slight decrease in cell number and cell protein content. The decrease in both cell number and cell protein content were not attributed to increased cell damage or decreases in cell viability due to the fact that the cellular MDA levels were not statistically higher than the control values (p < 0.05). Morphological Evaluations of cells using Image Pro Software, revealed no significant or adverse changes when compared with cells treated with media alone. Dot blot analysis of pro-inflammatory cytokine (TNF alpha) production after LPS treatment was suppressed by DHEA while AED had a minor influence on the responses. These data imply that LPS mediated activation of RAW 264.7 cells can be inhibited by the addition of pharmacological doses of adrenal hormones such as DHEA.
Coleman
T N
TN
University of Mississippi Medical Center, Jackson 39216, USA.
Benghuzzi
H
H
Tucci
M
M
Hughes
J
J
D000276:Adjuvants, Immunologic / Q000008:administration & dosage* / Q000494:pharmacology; D000735:Androstenedione / Q000008:administration & dosage* / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003687:Dehydroepiandrosterone / Q000008:administration & dosage* / Q000494:pharmacology; D007375:Interleukin-1 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008315:Malondialdehyde / Q000378:metabolism; D051379:Mice; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D000276:Adjuvants, Immunologic; D007375:Interleukin-1; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D000735:Androstenedione; D003687:Dehydroepiandrosterone; D008315:Malondialdehyde