Li Zhengjuan, Wang Shuoshi, Li Liping
Department of Obstetrics, Shenzhen People's Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, China.
Department of Obstetrics and Gynecology, The Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou, China.
Front Cell Dev Biol. 2022 Mar 9;10:810282. doi: 10.3389/fcell.2022.810282. eCollection 2022.
Advanced oxidation protein products (AOPPs), the novel marker of oxidative stress, have been found to be elevated in preeclampsia (PE). To date, the effect of AOPPs on the senescence of trophoblast cells is still unclear. In this study, we investigated whether AOPPs promoted the senescence of trophoblast cells and explored the underlying mechanisms of AOPPs-induced aging process which may facilitate the progression of PE. The trophoblast cell line HTR-8/SV neo cells were cultured in the presence of PBS, AOPPs, AOPPs plus an anti-oxidant N-acetyl-L-cysteine (NAC). In some experiments, cells were pre-treated with rapamycin (an activator of autophagy), 3-MA (an inhibitor of autophagy), or cyclic pifithrin-α (PFT-α, an antagonist of p53), and then treated with AOPPs. Cellular senescence was analyzed by measuring the levels of senescence-associated β-galactosidase (SA β-Gal), senescence-associated heterochromatin foci (SAHF), mitochondrial membrane potential (ΔΨm), and cell cycle. Cell autophagic flux was analyzed by measuring tandem fluorescence-tagged LC3 reporter (mCherry-EGFP-LC3). Levels of p53, phosphorylated p53 (-p53), p21, BECN1, p62, -mTOR and -p70S6K were measured by western blot. Treatment with AOPPs significantly increased the levels of SA β-Gal and SAHF, the percentage of cells in the G0/G1 phase, and decreased cell ΔΨm compared with the control group. Co-treatment with NAC and AOPPs significantly reversed AOPPs-induced senescence. Pre-treatment with rapamycin or 3-MA significantly inhibited or promoted AOPPs-induced senescence, respectively. In addition, administration of AOPPs significantly decreased the numbers of mCherryEGFP autophagosomes and mCherryEGFP autolysosomes in cells compared with cells treated with PBS. Furthermore, AOPPs significantly increased the levels of proteins -p53, p21, -mTOR and -p70S6K compared with the control group. Pre-treatment with rapamycin or PFT-α significantly down-regulated the levels of SA -Gal, SAHF, p-p53, p21, autophagy related protein p62, the percentage of cells in the G0/G1 phase, and significantly up-regulated ΔΨm, autophagy related protein BECN1, autophagosomes and autolysosomes compared with cells only treated with AOPPs. AOPPs may induce trophoblast cell senescence by inhibiting the autophagy process in a p53/mTOR/p70S6K-dependent pathway.
晚期氧化蛋白产物（AOPPs）是氧化应激的新型标志物，已发现在子痫前期（PE）中升高。迄今为止，AOPPs对滋养层细胞衰老的影响仍不清楚。在本研究中，我们调查了AOPPs是否促进滋养层细胞衰老，并探索了AOPPs诱导衰老过程的潜在机制，这可能会促进PE的进展。将滋养层细胞系HTR-8/SV neo细胞在PBS、AOPPs、AOPPs加抗氧化剂N-乙酰-L-半胱氨酸（NAC）存在的情况下进行培养。在一些实验中，细胞先用雷帕霉素（自噬激活剂）、3-甲基腺嘌呤（3-MA，自噬抑制剂）或环磷酰胺-α（PFT-α，p53拮抗剂）预处理，然后用AOPPs处理。通过测量衰老相关β-半乳糖苷酶（SA β-Gal）、衰老相关异染色质灶（SAHF）、线粒体膜电位（ΔΨm）和细胞周期来分析细胞衰老。通过测量串联荧光标记的LC3报告基因（mCherry-EGFP-LC3）来分析细胞自噬通量。通过蛋白质印迹法测量p53、磷酸化p53（-p53）、p21、BECN1、p62、-mTOR和-p70S6K的水平。与对照组相比，用AOPPs处理显著增加了SA β-Gal和SAHF的水平、G0/G1期细胞的百分比，并降低了细胞ΔΨm。NAC与AOPPs共同处理显著逆转了AOPPs诱导的衰老。用雷帕霉素或3-MA预处理分别显著抑制或促进了AOPPs诱导的衰老。此外，与用PBS处理的细胞相比，给予AOPPs显著减少了细胞中mCherryEGFP自噬体和mCherryEGFP自溶酶体的数量。此外，与对照组相比，AOPPs显著增加了-p53、p21、-mTOR和-p70S6K蛋白的水平。与仅用AOPPs处理的细胞相比，用雷帕霉素或PFT-α预处理显著下调了SA -Gal、SAHF、p-p53、p21、自噬相关蛋白p62、G0/G1期细胞的百分比，并显著上调了ΔΨm、自噬相关蛋白BECN1、自噬体和自溶酶体。AOPPs可能通过p53/mTOR/p70S6K依赖性途径抑制自噬过程来诱导滋养层细胞衰老。
