Table I
Primer sequences.
| Gene name | Forward primer | Reverse primer |
|---|---|---|
| TNF-α | CGCTGAGGTCAATCTGC | GGCTGGGTAGAGAATGGA |
| Caspase-9 | AGCGATTCTGCCTTTCAC | TGGAGATTTTGTGGTCAGC |
| mTOR | CTGGGGCTGCTTTCTGT | ACGGTTTTCTGCCTCTTGT |
| AMPK | CATCCCCAAACCTGTCC | ACAAGCCCCGAACAAAA |
Fig. 1.
Effect of NAC on the viability and cytokine levels of RAW264.7 cells during incubation with MA.
(A) Different groups of RAW264.7 cells were treated with MA (50 μg/ml) for 6, 12, 24, and 36 h and then analyzed for viability with MTT. Changes in the optical density (OD) values at 560 nm were recorded with a microplate reader. (B) The cell culture supernatants were collected after 24 h in the MA group and NAC + MA group; the levels of secreted cytokines in the samples were quantified. The graphical representation shows the relative concentration of the secreted IL-6. (Mean ± standard deviation, n = 3 experiments, *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test).
Fig. 2.
Effects of NAC on apoptosis-related factors in RAW264.7 cells during incubation with MA.
The MA group was treated with MA (50 μg/ml) for 24 h. MA + NAC group was pre-treated with NAC at a concentration of 600 mg/ml for 2 h before treatment with MA. The results are representative of three separate experiments. (A) Representative Western blot of BAX in RAW264.7 treated with MA or MA + NAC (n = 3 experiments, *p < 0.05, **p < 0.01 by image J). (B) Quantification of BAX levels, as represented in A it was quantified by densitometry and standardized to the β-actin level. (C, D) The levels of TNF-α and Casp9 mRNA was standardized by the double delta CT method. (Mean ± standard deviation, n = 3 experiments, *p < 0.05, **p < 0.01 by Student’s t-test).
Fig. 3.
Effects of NAC on autophagy-related factors in RAW264.7 cells during incubation with MA.
The MA group was treated with MA (50 μg/ml) for 24 h. MA + NAC group was pre-treated with NAC at a concentration of 600 mg/ml for 2 h before treatment with MA. (A) Representative Western blot of beclin-1 and LC3 in RAW264.7 macrophages treated with MA or MA + NAC (n = 3 experiments). (B, C) Quantification of beclin-1(b) and LC3(c) levels, as represented in A. (D, E) The levels of mTOR and AMPK mRNA was quantified by densitometry, and standardized to the β-actin level. (Mean ± standard deviation, n = 3 experiments, *p < 0.05, **p < 0.01 by Student’s t-test).
Fig. 4.
Morphological changes in the lung tissue (H&E staining) in ICR mice treated with MA and induced by NAC.
Representative photomicrographs illustrated characteristic lesions in 8 week-old C57BL/6 mice. The lung tissues, which had not been lavaged, were fixed with 4% paraformaldehyde solution and routinely processed for pathological slices. The morphological change of the lung tissue was observed after H&E staining. (A) The control group. (B) The NAC group. (C) The MA group. (D) The MA + NAC group. (Scale bar 50 μm).