Uneingeschränkter Zugang

N-acetylcysteine (NAC) Attenuating Apoptosis and Autophagy in RAW264.7 Cells in Response to Incubation with Mycolic Acid from Bovine Mycobacterium tuberculosis Complex

XUE LIN
XUE LIN
, 
MENGMENG WEI
MENGMENG WEI
, 
FUYANG SONG
FUYANG SONG
, 
DI XUE
DI XUE
 und 
YUJIONG WANG
YUJIONG WANG
   | 04. Juni 2020
Polish Journal of Microbiology's Cover Image
Über diesen Artikel
Vorheriger Artikel
Nächster Artikel
Zitieren
Article Category: original-paper
Online veröffentlicht: 04. Juni 2020
Seitenbereich: 223 - 229
Eingereicht: 17. März 2020
Akzeptiert: 10. Mai 2020
DOI: https://doi.org/10.33073/pjm-2020-026
Schlüsselwörter
© 2020 Xue Lin et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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).
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).
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).
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).
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).

Primer sequences.

Gene name Forward primer Reverse primer
TNF-α CGCTGAGGTCAATCTGC GGCTGGGTAGAGAATGGA
Caspase-9 AGCGATTCTGCCTTTCAC TGGAGATTTTGTGGTCAGC
mTOR CTGGGGCTGCTTTCTGT ACGGTTTTCTGCCTCTTGT
AMPK CATCCCCAAACCTGTCC ACAAGCCCCGAACAAAA
eISSN:
2544-4646
Sprache:
Englisch
Zeitrahmen der Veröffentlichung:
4 Hefte pro Jahr
Fachgebiete der Zeitschrift:
Biologie, Mikrobiologie und Virologie
Zeitschrift RSS Feed