Mn-L1 and Mn-L2-inhibited LPS-induced pro-inflammatory gene expression in THP1 human macrophages.

<p><b>(A) Inhibition of pro-inflammatory gene expression in macrophages by Mn-L1 and Mn-L2</b>. THP-1 macrophages were treated for 3 hours with 25 μmol/L of Mn-L1 or Mn-L2, the medium was removed and the cells were incubated one hour with fresh medium containing 100 ng/ml LPS. The mRNA levels were determined by quantitative real time PCR. The values are represented as percentages of the only LPS-stimulated control group. LPS 100% values (mean ± SE) were 5.5 ± 0.9; 63.58 ± 3.7; 74.12 ± 4.4; 13.83 ± 7.7.42 ± 5; and 13.15 ± 4 for GRP78, TNF-α, IL-6, Il-1ß, IL-8 and PTGS2 respectively. <b>(B) Inhibition of the production of pro-inflamatory TNF-α and IL-6 in macrophages by Mn-L1 and Mn-L2</b>. THP-1 macrophages were treated for 3 hours as explained in the figure. After 16 hours’ incubation with 500 ng/ml LPS, TNF-α, and IL-6 protein expression was determined in the cell culture medium. The data of three separate experiments performed in duplicate are shown. * P < 0.01 compared with LPS-treated group.</p

Mn-L1 inhibits LPS cellular signaling in macrophages through the MAP kinase pathway.

<p>Effects of Mn-L1 on the LPS-induced phosphorilation of MAPK kinases. THP-1 macrophages were incubated with Mn-L1 for 3 hours at the concetrations indicated in the figure and then challenged with 500 ng/ml of LPS for 1 hour. The phosphorylated and total ERK, JNK and p38 proteins were determined by Western blotting. ß-actin was used as loading control. Representative blots. (A) Representative blots and (B) densitometric evaluation (n = 3) *P < 0.01 compared to LPS-treated group.</p

Mn-L1 and Mn-L2 anti-inflammatory activity in whole mice.

<p><b>(A) Mn-L1 protected mice from a lethal endotoxemic dose of LPS</b>. Survival data (%) were analyzed by using the Kaplan-Meier method and log rank test. * P < 0.05 versus the LPS-treated group. <b>(B) Inhibition of LPS induction of TNF-α and IL-6 concentration in serum</b>. The graphs show the serum levels of TNF-α (right panel) and IL-6 (left panel) after the different treatments with LPS, Mn-L1 or Mn-L2 as explained under the ordinate axis. Data are presented as mean ± SEM. <b>(C) Mn-L1 attenuates Kupffer cell LPS activation</b>. The panels show the immunohistochemistry for F4/80 and the graph the quantification of positive macrophage area. *P values < 0.05. Data are represented as mean ± SEM. <b>(D) Mn-L1 effect in LPS liver injury</b>. Liver injury was determined by histological examination on H&E-stained sections. Control untreated mice; LPS (mice treated with LPS alone) and LPS + Mn-L1 (mice treated with LPS and Mn-L1).</p