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

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

Polish Journal of Microbiology

Volume 69 (2020): Issue 2 (June 2020)

By: XUE LIN, MENGMENG WEI, FUYANG SONG, DI XUE and YUJIONG WANG

Open Access

|Jun 2020

Figures & Tables

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).

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).

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).

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

DOI: https://doi.org/10.33073/pjm-2020-026 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331

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Language: English

Page range: 223 - 229

Submitted on: Mar 17, 2020

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Accepted on: May 10, 2020

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Published on: Jun 4, 2020

Published by: Polish Society of Microbiologists

In partnership with: Paradigm Publishing Services

Publication frequency: 4 issues per year

Keywords:

N-acetylcysteine,

Related subjects:

Microbiology and virology

© 2020 XUE LIN, MENGMENG WEI, FUYANG SONG, DI XUE, YUJIONG WANG, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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