Dr Di Xue, Ningxia Key Laboratory of Clinical and Pathogenic Microbiology, General Hospital of Ningxia Medical University, 804 Shengli Road, Yinchuan, Ningxia Hui Autonomous Region 750004, P.R. China
*Contributed equally
Tuberculosis (TB) is a chronic and fatal zoonotic infectious disease caused by
Alveolar macrophages are not only the primary site for
To address this, after incubating THP-1 cells with
THP-1 cells (American Type Culture Collection), a model commonly used for studying the function of macrophages (
An MTT assay was used to assess the viability of cells. THP-1 cells were seeded in a 96-well plate at a density of 5x103 cells/well, and PMA was used to induce cell transformation before being pretreated with various concentrations (0, 50, 100, 150, 200 and 300 µM/ml) of vitamin C for 24 h at 37˚C. Subsequently, cells were treated with lipopolysaccharide (LPS; 1 µg/ml) (Thermo Fisher Scientific, Inc.) for 12 h at 37˚C, and MTT solution (20 µl) was added to each well according to the manufacturer's instructions. The cells were then incubated for 4 h at 37˚C. Finally, DMSO (100 µl) was added to each well, and the absorbance (560 nm) was measured using a microplate reader (Bio-Rad Laboratories, Inc.).
Total protein was extracted from the cells in the different treatment groups [i) Control (untreated) group; ii)
Total RNA was isolated from THP-1 cells using the MiniBEST Universal RNA extraction kit (Takara Bio, Inc.), and RNA was reverse-transcribed to cDNA using a PrimeScript™ RT Reagent kit (Takara Bio, Inc.). The following temperature protocol was used for reverse transcription: 37˚C for 15 min and 85˚C for 5 sec. Subsequently, the obtained cDNA was amplified with qPCR on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using a TB Green Fast qPCR Mix kit (Takara Bio, Inc.). The following thermocycling qPCR conditions were used for amplification: 95˚C for 30 sec; followed by 40 cycles of 95˚C for 5 sec and 65˚C for 30 sec. Relative expression levels were evaluated using the 2-∆∆Cq method and normalized to GAPDH as the internal control (
Cell proliferation was performed using an EdU cell proliferation kit (cat. no. C0075S; Beyotime Institute of Biotechnology). The cells (2x106 cells/well) were seeded into 6-well plates and incubated at 37˚C for 24 h then the cells of different treatment groups were incubated with
The cells (1x106 cells/well) were seeded into 6-well plates and incubated at 37˚C for 24 h, then the cells of different treatment groups were incubated with
The cells (1x106 cells/well) were seeded on coverslips in 6-well plates and incubated at 37˚C for 24 h, then the cells of different treatment groups were incubated with
A total of 16 male BALB/c mice (6-8 weeks old; weight, 20±25 g) were purchased from Jackson ImmunoResearch Laboratories, Inc. All animal experiments and protocols were approved by the Ethical Committee of Ningxia University (Yinchuan, China; specific pathogen free grade; approval no. 2020-024). All animals were housed in a pathogen-free facility (22±2˚C; 50±5% humidity) with a 12-h light/dark cycle, and the mice had
Mice were sacrificed after 21 days by cervical dislocation. The absence of heartbeat and respiration were used as criteria to confirm mouse death. Fresh lung tissue was isolated and washed once with PBS. The tissue was fixed with 4% paraformaldehyde for 20 min at 37˚C and then embedded in paraffin. The embedded tissue was cut into 4-µm thick sections. Finally, the tissue was stained with hematoxylin and eosin at 37˚C for 10 min. The images of stained tissues were then scored by three independent pathologists who were blinded prior to scoring as follows: 1, Normal; 2, slight tissue damage; 3, mild tissue damage; and 4, severe tissue damage (confocal laser scanning microscopy; magnification, x100).
Mice were sacrificed after 21 days. Fresh lung tissue was isolated, fixed overnight with 4% paraformaldehyde (Solarbio, P1110) at 4˚C. Then the samples were dehydrated in ethanol and xylene (70% ethanol 30 min; 80% ethanol 30 min; 95% ethanol 30 min; 100% ethanol 30 min twice; xylene 30 min twice), embedded in paraffin and cut into sections as aforementioned 4-µM thick sections. Lung tissues was transferred to 3-aminopropyl-triethoxysilane-treated microscope slides (ZLI-9001; Zhongshan Company) for immunofluorescence staining. In brief, sections were deparaffinized and rehydrated (xylene 5 min twice; 100% ethanol 3 min twice; 90% ethanol 3 min; 80% ethanol 3 min; 70% ethanol 3 min). After three washes in distilled water, antigen retrieval was performed by microwaving for 15 min in 0.01% sodium citrate buffer (pH 6.0). After three washes in PBS, the lung tissues were treated with 3% H2O2 in PBS for 20 min to quench endogenous peroxidase activity. Nonspecific binding was blocked with 5% BSA in PBS for 15 min at room temperature. Sections were then incubated with primary antibody (NF-κB; 1:200; cat. no. 8242; Cell Signaling Technology, Inc.) overnight at 4˚C. After three washes in PBS, the lung tissue sections were incubated for 20 min at room temperature with horseradish peroxidase-labeled goat anti-rabbit IgG (Zhongshan Company), and rinsed with PBS. The antibody complex was detected using DAB reagent (Zhongshan Company). Finally, the sections were incubated with hematoxylin staining solution (Zhongshan Company) for 20 sec at room temperature and washed once with distilled water. Images were captured using a light microscope (magnification, x100).
All data collected were obtained from at least three independent experiments for each condition. All results were analyzed using GraphPad Prism version 6.0 (GraphPad Software, Inc.) and are presented as the mean ± standard deviation (unless otherwise shown). Unpaired Student's t-test was used to compare differences between two groups, and one-way ANOVA followed by Tukey's post hoc test was used to compare differences between >2 groups. The statistical analysis of the histological score results was conducted using Kruskal-Wallis test followed by Dunn's post hoc test and are presented as median + interquartile range. P<0.05 was considered to indicate a statistically significant difference.
After THP-1 cells were incubated with
Vitamin C exerted a protective effect on the viability of THP-1 cells after LPS stimulation. Thus, whether vitamin C could inhibit the levels of inflammatory factors in THP-1 cells after
To determine whether vitamin C regulated apoptosis of THP-1 cells after
Finally, to confirm the immunosuppressive function of
The pathogenic mechanism of
Vitamin C is an important element in the body and plays a notable role in the immune system. Previous studies have revealed that vitamin C can improve immunity by generating (
Inflammation is a basic pathological process, corresponding to the body's defense response to the stimulation of various pro-inflammatory factors (
Apoptosis is a cellular mechanism of programmed cell death that is tightly regulated by a family of proteases called caspases (
Bcl-2 is the founding member of the Bcl-2 family of apoptosis regulatory proteins that either induce (pro-apoptotic) or inhibit (anti-apoptotic) apoptosis (
Cyt-
In conclusion, the present study demonstrated that the molecular mechanism of vitamin C promoted apoptosis in THP-1 cells infected by
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
YuW designed the project, revised the article and provided technical guidance. DX designed the project, revised the article and coordinated all aspects of the present work. FS and YiW participated in all experiments, performed data analysis, created the figures and wrote the article. XL was responsible for sample preparation and documentation. YiW and FS confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
All animal experiments and experimental protocols were approved by the Ethics Committee of Ningxia University (Yinchuan, China; approval no. 2020-024).
Not applicable.
The authors declare that they have no competing interests.
Effect of vitamin C on the viability of THP-1 cells during incubation with
Effect of
Effect of
Morphological changes of hematoxylin and eosin-stained lung tissues following vitamin C treatment observed in BALB/c mice infected with