Fig 1.
MCL treatment did not affect BV2 cell viability.
(A) BV2 cells were treated with the indicated doses of MCL for 24 h, then the cytotoxicity of MCL was measured by CCK-8 assay. The cell viability result was normalized to BV2 cells without MCL treatment for each other. Data were presented as means ± SD of three independent experiments. (B) BV2 cells were treated with indicated doses of MCL for 24 h. Then, the cells were stained with FITC-Phalloidin, green; and nuclei, Hoechst33258, blue; representative images by immunofluorescence were shown, scale bar = 50 μm.
Fig 2.
MCL inhibited LPS-induced iNOS and COX-2 expression, and NO production in BV2 cells.
(A) BV2 cells were pretreated with MCL (1, 5, and 10 μM) for 1 h and then incubated with LPS (1 μg/ml) for 24 h. The NO concentration in supernatant was determined using Griess reagent. (B) In a parallel experiment, cell lysates were subjected to Western blot analysis and immunoblotted with the indicated proteins. (C) BV2 cells were pretreated with MCL (10 μM) for 1 h and then incubated with LPS (1 μg/ml) for 6 h. Then, mRNA was extracted and the mRNA level of iNOS was evaluated by RT-PCR. (D) BV2 cells were pretreated with MCL (10 μM) for 1 h and then incubated with LPS (1 μg/ml) for 24 h. The indicated proteins were evaluated by Western blot. (E) BV2 cells were pretreated with MCL (10 μM) for 1 h and then incubated with LPS (1 μg/ml) for 6 h. Then, mRNA was extracted and the mRNA level of COX-2 was evaluated by RT-PCR. Data were presented as means ± SD of three independent experiments. **p < 0.01 and ***p < 0.001 vs. LPS alone.
Fig 3.
MCL decreased LPS-induced pro-inflammatory cytokines production.
(A) and (B) BV2 cells were pretreated with MCL (10 μM) for 1 h and then incubated with LPS (1 μg/ml) for 12 h. The secretory levels of TNF-α and IL-6 in supernatants were measured using ELISA. (C), (D) and (E) BV2 cells were treated with MCL (10 μM) for 1 h and followed by LPS treatment for 6 h. Then, mRNA was extracted and the mRNA level of TNF-α (C), IL-6 (D) and IL-1β(E) was evaluated by RT-PCR. Data were presented as means ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. LPS alone.
Fig 4.
MCL repressed LPS-induced NF-κB activity.
(A) and (B) BV2 cells were pretreated with MCL (10 μM) for 1 h and then added LPS (1 μg/ml) for 30 min. Total cell lysates were subjected to Western blot analysis using antibodies against phospho-IκBα (A) or total form of IκBα (B). (C) Cells were treated similar to (A). The cytosolic and nuclear extracts were subjected to Western blot analysis for the indicated proteins. (D) BV2 cells were treated with LPS (1 μg/ml) for 1 h after pretreatment with MCL (10 μM) for 1 h. Then, cells were stained with NF-κB subunit p65 antibody, green; and nuclei, DAPI, blue; scale bar = 20 μm, representative images by immunofluorescence were shown. (E) NF-κB luciferase reporter vector was co-transfected with Renilla vector in BV2 cells. 24 h later, cells were treated with MCL (10 μM) for 1h then followed by LPS (1 μg/ml) incubation for 6 h. Then, cell lysates were collected, and NF-κB luciferase activities against Renilla luciferase activities were measured by the double-luciferase assay system. Data were presented as means ± SD of three independent experiments. **p < 0.01 vs. LPS alone.
Fig 5.
MCL suppressed LPS-induced MAPKs and Akt activities.
(A) and (B) BV2 cells were treated with MCL (10 μM) for 1 h then followed by LPS (1 μg/ml) addition for 30 min. Then, total cell lysates were subjected to Western blot analysis using antibodies against phospho- or total forms of JNK, p38, ERK, and Akt.
Fig 6.
MCL promoted Nrf2 and HO-1 expression.
(A) and (B) BV2 cells were treated with the indicated doses of MCL for 12 h. Then, total cell lysates were subjected to Western blot analysis using antibody against HO-1 (A); or total RNA was extracted and the mRNA level of HO-1 was evaluated by RT-PCR (B). (C) and (D) BV2 cells were treated with MCL (10 μM) for the indicated times (C) or treated with various doses of MCL for 2 h (D). Nuclear and cytosolic fractions were isolated and subjected to Western blot analysis using antibody against Nrf2. Data were presented as means ± SD of three independent experiments. ***p < 0.001 vs. control group.
Fig 7.
DMAMCL alleviated LPS-induced IL-6 expression in the cortex and hippocampus in mice.
Mice were administered with 100 mg/kg DMAMCL by gavage for five consecutive days. On the fifth day, mice were challenged with saline or 0.33 mg/kg LPS i.p for 3 h. Then, the cortex and hippocampus were collected and total RNAs were extracted. IL-6 (A) and TNF-α (B) mRNA levels were determined by RT-PCR. Data were presented as means ± SD (n = 6). *p < 0.05 vs. LPS alone.