Formaldehyde-induced toxicity causes senescence and apoptosis in BMSCs via PI3K/P53 signaling pathways
Authors: She Yali , Yang Jie , Zhu Meng , Liu Zhe , Liu Ben , Ma Xiaojie , Wang Lie , Li Yaling 
Published: July 2023

Abstract
Background
Formaldehyde (FA) has been classified as a human carcinogen by the International Agency for Research on Cancer (IARC) and has toxic effects on various tissues and cells. It is reported that FA can accelerate cellular senescence in mice HT22 cells and induce the apoptosis of BALB/c mice BMSCs. Our previous study has confirmed that FA has genotoxic effects on BMSCs by the formation of DNA-protein crosslinks (DPC), sister chromatid exchange (SCE) and micronucleus (MN). However, whether FA causes apoptosis and senescence effects on human BMSCs has not been fully investigated. The aim of this study is to explore the toxic effects and mechanisms of FA on BMSCs based on senescence and apoptosis.
Method
In this study, Human BMSCs were cultured in vitro and randomly divided into a blank group, a control group and five groups of cells treated with different concentrations (60, 90, 120, 150 and 180 umol/L) of FA. And the cell groups were cultured for 6, 12 and 24 hours. The effect of different concentrations of FA on the viability of human BMSCs was investigated using the MTT assay. Based on the results of MTT assay, we selected the cell group with 120 µmol/L FA for 24 hours for the following experiments. Changes of FA on the morphology of BMSCs were analyzed using the phase-contrast microscope and phalloidin/hoechst33258 staining. We performed bioinformatic analysis on the RNA-Seq data, including differential expression analysis, GO and KEGG analysis in order to further understand the mechanisms of toxicity of FA on BMSCs. The changes in the mRNA and protein expression levels of PIK3CA, Caspase3, Bcl2, P53 and P21 of BMSCs following exposure to FA were detected using qRT-PCR and western blotting.
Result
When FA concentration reached 90 umol/L, the inhibition of proliferation activity of human BMSCs began to appear, and increased with the increase of FA concentration and time. By morphological detection, we observed that BMSCs treated with 120 µmol/L FA became smaller and rounder, of which the cytoskeleton was disordered and the nuclei were pyknotic, dense stained and fragmented, even with apoptotic bodies formed. The results of the bioinformatics analysis showed that 249 differentially expressed mRNAs (DE mRNAs) were identified in the RNA-seq samples, which included 158 upregulated and 91 downregulated mRNAs. And functional enrichment analysis revealed the pathway of accumulation to cellular senescence and apoptosis. Further assays were performed on factors of the PI3K/P53 pathway, which was a pathway related to senescence and apoptosis. The mRNA and protein expression levels of Caspase3, P53 and P21 in the 120 µmol/L FA-treated group were significantly higher than those in the control group, while the mRNA expression levels of PIK3CA and Bcl2 were significantly lower than those in the control group. Treatment with 120 µmol/L FA reduced the protein expression levels of PIK3CA.
Conclusion
FA had toxic effects on human BMSCs and the mechanism might be related to the regulation of PI3K/P53 signaling pathway to promote cellular senescence and apoptosis.