Lps of escherichia coli

The J774 cells were seeded in pentaplicate in 96-well plates at a density of 10⁵ cells per well. The plates were incubated under standard conditions for 24 h. The cells were treated with LPS of Escherichia coli (E. coli) (10 µg/mL, serotype 055:B5, Sigma-Aldrich Inc., USA) with or without triterpenoids (0.05–1 µM) for an additional 24 h. The production of NO was evaluated by the measurement of the nitrite concentration in a culture medium by using a Griess Reagent System (Promega, Madison, WI, USA), according to the manufacturer’s instructions. The absorbance was measured at 570 nm in a Multiscan RC plate reader (Thermo LabSystems, Helsinki, Finland).

LPS of Escherichia coli (serotype 0127: B8) and methyllycaconitine (MLA), an α7 nAChR antagonist, were purchased from Sigma-Aldrich (St. Louis, MO, USA). PNU120596 and memantine were purchased from Tocris Bioscience (Ellisville, MO, USA). MLA and memantine were dissolved in normal saline (0.9% NaCl), whereas PNU120596 was reconstituted in saline containing 5% dimethyl sulfoxide and 5% Solutol (Sigma, St. Louis, MO, USA). All drugs were injected intraperitoneally in a volume of 10 ml/kg of body weight.

After the basal recording, rats received by venous catheter a bolus injection of S-methylisothiourea (SMT) (3 mg kg^-1, Santa Cruz Biotechnology^®, Texas, TX, United States), a potent inhibitor of iNOS (Szabo et al., 1994 (link); Su et al., 2007 (link)), or physiological saline 0.9% (1 mL kg^-1), the SMT vehicle. After 10 min of recording of SMT or saline effects on blood pressure, a dose of 5 mg kg^-1 of LPS of Escherichia coli (serotype 026:B6, Sigma Chemical Co.) (Mehanna et al., 2007 (link)) was administered as a bolus injection (i.v.). The cardiovascular effects of endotoxin were recorded for 2 h.
At the end of recording, plasma samples were collected for measurement of the nitrite and lipoperoxidation levels (LOOH), the plasma activity of paraoxonase 1 (PON1) and the total antioxidant capacity of plasma. A standard laboratory scale was used to measure body weight, tibia length and uterus weights (Gore et al., 2002 (link); Voltera et al., 2008 (link); Castardo-de-Paula et al., 2017 (link)).