microRNA-146a-5p negatively modulates PM2.5 caused inflammation in THP-1 cells via autophagy process

doi:10.1016/j.envpol.2020.115961

Environmental Pollution

Volume 268, Part B, 1 January 2021, 115961

https://doi.org/10.1016/j.envpol.2020.115961 Get rights and content

Highlights

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PM_2.5 increased miR-146a-5p expression level in THP-1 cells.
•
Autophagy regulated cytokine expression caused by PM_2.5.
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miR-146a-5p negatively modulated inflammation via autophagy process.
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IRAK1 and TRAF6 were two important target genes of miR-146a-5P induced by PM_2.5.

Abstract

Ambient fine particulate matter (PM_2.5) can change the expression profile of microRNAs (miRs), which may play important roles in mediating inflammatory responses. The present study attempts to investigate the roles of miR-146a-5p in regulating cytokine expression in a human monocytic leukemia cell line (THP-1). Four types of PM_2.5 extracts obtained from Beijing, China, were subjected to cytotoxic tests in THP-1 cells. These four PM_2.5 extracts included two water extracts collected from non-heating and heating season (WN and WH), and two organic extracts from non-heating and heating season (DN and DH). Firstly, the four PM_2.5 extracts caused cytotoxicity, oxidative stress responses, cytokine gene expressions and interleukin 8 (IL-8) release in THP-1 cells, with WH showing the highest cytotoxicity, WN showing the highest oxidative stress and inflammatory responses. Additionally, we observed expression of miR-146a-5p was significantly increased, with the maximal response of six folds in WN group. Cellular autophagy was initiated by PM_2.5 indicated by related protein and gene expressions. Both RNA interference and autophagy inhibitor were applied to interrupt autophagy process in THP-1 cells. Autophagy dysfunction could alleviate IL-8 expression, suggesting autophagy process regulated cytokine expression and inflammatory response caused by PM_2.5. A chemical inhibitor was applied to inhibit the function of miR-146a-5p, and then the expressions of IL-8 and autophagic genes were significantly aggravated. Meanwhile, two target genes of miR-146a-5p, interleukin-1 associated-kinase-1 (IRAK1) and tumor-necrosis factor receptor-associated factor-6 (TRAF6) were increased dramatically, which also played important roles in regulation of autophagy. These data suggested miR-146a-5p negatively modulated cytokine expression caused by PM_2.5 via autophagy process through the target genes of IRAK1 and TRAF6. Our findings raised the concerns of the changes of miR expression profile and following responses caused by PM_2.5.

Graphical abstract

Section snippets

Author statement

Yu Shang: Conceptualization, Writing - original draft, Writing - review & editing, Visualization. Qianyun Liu: Conceptualization, Writing - review & editing. Lu Wang: Investigation, Visualization. Xinghua Qiu: Resources. Yingjun Chen: Resources. Jing An: Supervision, Writing - review & editing

Materials

The human monocytic leukemia cell line THP-1 was purchased from the Cell Institute of Chinese Academy of Sciences (Shanghai, China). Cell culture related reagents and supplements, RPMI 1640 culture medium, phosphate buffer saline and fetal bovine serum (FBS) were obtained from Sangon Biotech (Shanghai, China). Penicillin, streptomycin, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), phorbol myristate acetate (PMA), and 2′,7′-dichlorodihydrofluoresceindiacetate (DCFH-DA)

Cell proliferation inhibition and oxidative stress

Cytotoxicity was evaluated with MTT method as described above, and the viability of THP-1 cells treated with the four PM_2.5 extracts at different concentrations were showed in Fig. 1A. Compared with control group, a dose-dependent decrease of cell viability was observed after exposed to PM_2.5 extracts for 24 h. No significant difference was existed in viability decrease induced by PM_2.5 extracts collected during heating or non-heating season in Beijing. While, water extracts of PM_2.5 (WH and

Discussion

Our results confirmed and extended previous findings that the exposure of PM_2.5 to in vitro cells could cause oxidative stress, inflammatory response, autophagy, miR-146a-5p up-regulation and finally lead to cell death. Oxidative stress was identified as the first step in the toxic effects caused by PM_2.5. As an enzymatic antioxidant, SOD helps maintain the redox balance by catalyzing the conversion of O²⁻ to H₂O₂. The alteration of SOD activity could be an indicator for oxidative stress

Conclusions

In conclusion, miR-146a-5p is a key factor in modulating IL-8 expression via autophagy process in THP-1 cells induced by PM_2.5 extracts collected in Beijing, China. PM_2.5 in Beijing is a very complicated mixture consisting of various physicochemical properties. It is incredibly challenging to clarify its potential health effects and to reveal the associated toxicological mechanisms. And the complex compositions in PM_2.5 exerted extensive cytotoxic effects in vitro, meanwhile, different

Funding

This work was supported by National Key Research and Development Program of China (No. 2016YFA0602004); National Natural Science Foundation of China (Nos. 41977366, 41877371 and 41561144007); Innovative Research Team in University (No. IRT13078); and Training Plan of Talent Doctors in Yueyang Hospital (RY411.06.42).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Citation Excerpt :
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Fine particulate matter (PM_2.5) exposure is a major threat to public health, and is listed as one of the leading factors associated with global premature mortality. Among the adverse health effects on multiple organs or tissues, the influence of PM_2.5 exposure on cardiovascular system has drawn more and more attention. Although numerous studies have investigated the mechanisms responsible for the cardiovascular toxicity of PM_2.5, the various mechanisms have not been integrated due to the variety of the study models, different levels of toxicity assessment endpoints, etc. Adverse Outcome Pathway (AOP) framework is a useful tool to achieve this goal so as to facilitate comprehensive understanding of toxicity assessment of PM_2.5 on cardiovascular system. This review aims to illustrate the causal mechanistic relationships of PM_2.5-triggered cardiovascular toxicity from different levels (from molecular/cellular/organ to individual/population) by using AOP framework. Based on the AOP Wiki and published literature, we propose an AOP framework focusing on the cardiovascular toxicity induced by PM_2.5 exposure. The molecular initiating event (MIE) is identified as reactive oxygen species generation, followed by the key events (KEs) of oxidative damage and mitochondria dysfunction, which induces vascular endothelial dysfunction via vascular endothelial cell autophagy dysfunction, vascular fibrosis via vascular smooth muscle cell activation, cardiac dysregulation via myocardial apoptosis, and cardiac fibrosis via fibroblast proliferation and myofibroblast differentiation, respectively; all of the above cardiovascular injuries ultimately elevate cardiovascular morbidity and mortality in the general population. As far as we know, this is the first work on PM_2.5-related cardiovascular AOP construction. In the future, more work needs to be done to explore new markers in the safety assessment of cardiovascular toxicity induced by PM_2.5.
Ultrafine black carbon caused mitochondrial oxidative stress, mitochondrial dysfunction and mitophagy in SH-SY5Y cells
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Mitophagy plays key roles in the development of neurodegenerative diseases (Rodolfo et al., 2018; Pradeepkiran and Reddy, 2020). Our previous study showed PM2.5 extracts could induce autophagy, regulating cytokine expression in THP-1 cells (Shang et al., 2021). This study further indicated that uBC, a main component of PM2.5, could result in PINK1/Parkin-dependent mitophagy in SH-SY5Y cells.
Exposure to ambient ultrafine black carbon (uBC, with aerodynamic diameter less than 100 nm) is associated with many neurodegenerative diseases. Oxidative stress is the predominantly reported neurotoxic effects caused by uBC exposure. Mitochondrion is responsible for production of majority of ROS in cells and mitochondrial dysfunction is closely related to adverse nervous outcomes. Mitophagy is an important cellular process to eliminate dysfunctional or damaged mitochondria. However, the mechanisms that modulate mitophagy and mitochondrial dysfunction initiated by uBC remain to be elucidated. The purpose of this study was to investigate how mitochondrial oxidative stress regulated mitochondrial dysfunction and mitophagy in human neuroblastoma cell line (SH-SY5Y) after uBC treatment. RNA interference was further applied to explore the roles of mitophagy in mitochondrial dysfunction. We found uBC triggered cell apoptosis via ROS-mitochondrial apoptotic pathway. The uBC also caused serious mitochondrial damage and respiratory dysfunction, indicated by the abnormalities in mitochondrial division and fusion related proteins, decreased mitochondria number and ATP level. Increased PTEN induced putative kinase 1 (PINK1) and Parkin protein levels and the autolysosome numbers suggested uBC could promote Pink1/Parkin-dependent mitophagy process in SH-SY5Y cells. Mitophagy inhibition could reserve mitochondria number and ATP activity, but not fusion and division related protein levels in SH-SY5Y cells exposed to uBC. Administration of a mitochondria-targeted antioxidant (mitoquinone) significantly eliminated uBC caused apoptosis, mitochondrial dysfunction and mitophagy. Our data suggested mitochondrial oxidative stress regulated uBC induced mitochondrial dysfunction and PINK1/Parkin-dependent mitophagy. PINK1/Parkin-dependent mitophagy probably participated in regulating uBC caused mitochondrial dysfunction but not by controlling mitochondrial fusion and division related proteins. Our results may provide some new insights and evidences to understand the mechanisms of neurotoxicity induced by uBC.
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Zhang et al. proposed that Pb was responsible for greater potential health risks to children, in Taiyuan, China (Zhang et al., 2016a). Our recently research has reported that WN has a stronger ability in initiating anti-oxidation SOD activity in THP-1 cells than WH (Shang et al., 2021). These results suggested that there was close relationship between Ca2+ or Ba2+ concentrations and cytotoxic effect.
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In particular, miR-146a is an important regulator of cellular autophagic events. For example, miR-146a improved cardiomyocyte autophagy [7] and inhibited THP-1 cell autophagy by targeting TRAF6/IRAK1 [8]. Previously, our group demonstrated that knockdown of miR-146a improves Treg autophagy [9].
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^☆: This paper has been recommended for acceptance by Wen Chen.

¹: We announce that Yu Shang and Qianyun Liu contributed equally to this paper and both are first authors.

View full text

microRNA-146a-5p negatively modulates PM2.5 caused inflammation in THP-1 cells via autophagy process☆

Highlights

Abstract

Graphical abstract

Section snippets

Author statement

Materials

Cell proliferation inhibition and oxidative stress

Discussion

Conclusions

Funding

Declaration of competing interest

Toxicol. Vitro

Int. Immunopharm.

Lancet

Chemosphere

Toxicol. Vitro

Environ. Res.

Mutat. Res. Fund Mol. Mech. Mutagen

Sci. Total Environ.

Environ. Pollut.

Int. Immunopharm.

Chemosphere

Toxicol. Vitro

Environ. Pollut.

Environ. Toxicol. Pharmacol.

Biochim. Biophys. Acta Mol. Cell Res.

Mol. Immunol.

Environ. Pollut.

Environ. Pollut.

Environ. Int.

Autophagy core machinery: overcoming spatial barriers in neurons

Journal of Molecular Medicine

MicroRNAs miR-146a/b negatively modulate the senescence-associated inflammatory mediators IL-6 and IL-8

Aging (Albany NY)

Particulate matter air pollution and cardiovascular disease an update to the scientific statement from the American heart association

Circulation

Autophagy protects against active tuberculosis by suppressing bacterial burden and inflammation

Proc. Natl. Acad. Sci. U.S.A.

Autophagy is essential for ultrafine particle-induced inflammation and mucus hyperproduction in airway epithelium

Autophagy

Hypoxia-induced microRNA-146a-5p represses Bcl-2 through Traf6/IRAK1 but not Smad4 to promote chondrocyte autophagy

Biol. Chem.

Inflammasome-independent modulation of cytokine response by autophagy in human cells

PloS One

Airway inflammation in chronic obstructivepulmonary disease (COPD): a true paradox

Expet Rev. Respir. Med.

microRNA-146a-5p negatively modulates PM_2.5 caused inflammation in THP-1 cells via autophagy process☆