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PM 2.5 induces oxidative/antioxidant system imbalance and excessive release of reactive oxygen species (ROS) and produces toxic effects and irreversible damage to the genetic material including chromosomes and DNA. Biochanin A (BCA), an isoflavone with strong antioxidant activity, effectively intervenes against PM 2.5 -induced oxidative damage. The X-ray repair cross-complementary protein 1 (XRCC1)/BER pathway involves DNA damage repair caused by oxidative stress. This paper aims to explore the mechanism of BCA alleviating oxidative DNA damage caused by PM 2.5 by establishing the in vitro cell model based on CRISPR/Cas9 technology and combining it with mechanism pathway research. The results showed that PM 2.5 exposure inhibited the expression of BER and NER pathway proteins and induced the overexpression of ERCC1. BCA showed an effective intervention in the toxicity of PM 2.5 in normal cells, rather than XRCC1 knock-out cells. This laid a foundation for further exploring the key role of XRCC1 in PM 2.5 -caused oxidative damage and the BER/DNA damage repair pathway.

Epidemiological studies have shown that exposure to ambient fine particulate matter (PM 2.5 ) is associated with an increased risk for cardiopulmonary diseases. The MEK5/ERK5 and NF-κB signaling pathways are closely related to the regulation of acute pulmonary cell injury (APCI) and may play an important role in the underlying pathophysiological mechanisms. Related studies have shown that Biochanin A (BCA) effectively interferes with APCI, but the underlying mechanism through which this occurs is not fully understood. Previously, based on proteomic and bioinformatic research, we found the indispensable role of MEK5 in mediating remission effects of BCA against PM 2.5 -induced lung toxicity. Therefore, using A549 adenocarcinoma human alveolar basal epithelial cells (A549 cells), we combined western blot and qRT-PCR to study the protective signaling pathways induced by BCA, indicating that MEK5/ERK5 and NF-κB are both involved in mediating APCI in response to PM 2.5 , and MEK5/ERK5 positively activated NF-κB and its downstream cellular regulatory factors. BCA significantly suppressed PM 2.5 -induced upregulation of MEK5/ERK5 expression and phosphorylation and activation of NF-κB. Furthermore, due to the specificity of the MEK5/ERK5 protein structure, the binding sites and binding patterns of BCA and MEK5 were analyzed using molecular docking correlation techniques, which showed that there are stable hydrogen bonds between BCA and the PB1 domain of MEK5 as well as its kinase domain. BCA forms a stable complex with MEK5, which has potential effects on MEKK2/3-MEK5-ERK5 ternary interactions, p62/αPKC-mediated NF-κB regulation, and inhibition of MEK5 target protein phosphorylation. Therefore, our study suggests that MEK5 is an important regulator of intracellular signaling of APCI in response to PM 2.5 exposure. BCA may exert anti-APCI activity by targeting MEK5 to inhibit activation of the MEK5/ERK5/NF-κB signaling pathway.

Haze pollution has become a global environmental problem, subsequently affecting air quality, climate, economy and human health. Notably, PM 2.5 (particulate matter with an aerodynamic diameter less than 2.5 micrometers) significantly accounts for a variety of adverse health effects, in particular pulmonary diseases such as asthma and lung cancer. Clinical diagnosis and medical treatment of the lung damage caused by PM 2.5 still remain significant challenges due to the lack of specific biomarkers and pathways. Here, we established a rat model of nonsurgical intratracheal instillation to investigate PM 2.5 exposure and employed iTRAQ based analytical technique and bioinformatics tools to identify putative biomarkers and pathways. We identified 163 differentially expressed proteins (DEPs). Among these proteins, we screened six DEPs (HMOX1, MP2K5, XRCC1 E9PTZ7, KNT2 and A1AG) as the putative biomarkers, with significant differentially expressed levels (percentage increment > 140%). Pathway analysis indicated that calcium signaling, MAPK and PI3K/AKT might be involved in the process of PM 2.5 -induced lung damage. Western-blotting was used to verify DEPs in the AEC-II cell model for early diagnosis. In summary, our data can serve as fundamental research clues for further studies of PM 2.5 -induced toxicity in the lungs., Competing Interests: The authors declare no competing financial interests., (This journal is © The Royal Society of Chemistry.)