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Download PDF File FOLIA HISTOCHEMICA ORIGINAL PAPER ET CYTOBIOLOGICA Vol. 59, No. 2, 2021 pp. 124–133 Astragalus polysaccharide protects formaldehyde- -induced toxicity by promoting NER pathway in bone marrow mesenchymal stem cells Yali She1, 2*, Xiaowen Zhao1*, Pingfan Wu1, Ling Xue1, Zhe Liu1, Meng Zhu1, Jie Yang1, Yaling Li1, 2 1Department of Pathology, School of Basic Medicine, Gansu University of Chinese Medicine, Lanzhou, Gansu, China 2Provincial-Level Key Laboratory of Molecular Medicine of Major Diseases and Study on Prevention and Treatment of Traditional Chinese Medicine, Gansu University of Chinese Medicine, Lanzhou, Gansu, China *These authors contributed equally to this work. Abstract Introduction. In our previous study, it has been confirmed that formaldehyde (FA) not only inhibits the proliferative activity, but also causes DNA-protein crosslinks (DPCs) formation in bone marrow mesenchymal stem cells (BMSCs). The purpose of this study was to detect the protective effect of astragalus polysaccharide (APS) against the cytotoxicity and genotoxicity of BMSCs exposed to FA, and to explore potential molecular mechanisms of APS activity. Material and methods. Human BMSCs were cultured in vitro and randomly divided into control cells (Ctrl group), FA-treated cells (FA group, 120 μmol/L), and cells incubated with FA and increasing concentra- tions (40, 100, or 400 μg/mL) of APS (FA + APS groups). Cytotoxicity was measured by MTT assay. DNA strand breakage, DNA-protein crosslinks (DPCs), and micronucleus formation were respectively detected by comet assay, KCl-SDS precipitation assay, and micronucleus assay. The mRNA and protein expression level of xeroderma pigmentosum group A (XPA), xeroderma pigmentosum group C (XPC), excision repair cross-complementation group 1 (ERCC1), replication protein A1 (RPA1), and replication protein A2 (RPA2) were all detected by qRT-PCR and Western Blot. Results. Compared with the FA group, the cytotoxicity, DNA strand breakage, DPCs, and micronucleus levels were decreased significantly in FA + APS groups (P < 0.01). Meanwhile, the mRNA and protein expression of XPA, XPC, ERCC1, RPA1, and RPA2 were up-regulated significantly in the FA + APS groups (P < 0.05) with the most prominent effect of the 100 μg/mL APS. Conclusions. Our results suggest that APS can protect the cytotoxicity and genotoxicity of human BMSCs induced by FA. The mechanism may be associated with up-regulated expression of XPA, XPC, ERCC1, RPA1, and RPA2 in the nucleotide excision repair (NER) pathway which promotes DNA damage repair. (Folia Histochemica et Cytobiologica 2021, Vol. 59, No. 2, 124–133) Key words: astragalus polysaccharide; formaldehyde; bone marrow mesenchymal stem cells; toxicity; NER pathway; DNA strand breakage; DNA-protein crosslinks; micronucleus formation Correspondence address: Yaling Li, MD Provincial-Level Key Laboratory of Molecular Medicine of Major Diseases and Study on Prevention and Treatment of Traditional Chinese Medicine, Gansu University of Chinese Medicine, Lanzhou, Gansu, China phone 808-692-1554, fax: 808-692-1973 e-mail: [email protected] This article is available in open access under Creative Common Attribution-Non-Commercial-No Derivatives 4.0 International (CC BY-NC-ND 4.0) license, allowing to download articles and share them with others as long as they credit the authors and the publisher, but without permission to change them in any way or use them commercially. ©Polish Society for Histochemistry and Cytochemistry Folia Histochem Cytobiol. 2021 10.5603/FHC.a2021.0013 www.journals.viamedica.pl/folia_histochemica_cytobiologica ISSN 0239-8508, e-ISSN 1897-5631 Astragalus polysaccharide protects bone marrow mesenchymal stem cells 125 Introduction The present study was performed to characterize the protective effect of APS against the cytotoxicity As one component of bone marrow (BM), bone and genotoxicity of BMSCs exposed to FA. Subse- marrow mesenchymal stem cells (BMSCs) not only quently, we used the qRT-PCR and Western Blot have multi-differentiation potential, but also can to assess the expression of xeroderma pigmentosum regulate the normal proliferation and differentiation group A (XPA), xeroderma pigmentosum group C of hematopoietic stem cells [1, 2]. Thus, if BMSCs (XPC), excision repair cross-complementation group undergo toxic damage, BMSCs will lose their normal 1 (ERCC1), replication protein A1 (RPA1), and repli- control over hematopoietic stem cells which may lead cation protein A2 (RPA2) genes in the NER pathway to myeloid leukemia. in FA- and APS-exposed BMSCs to explore potential Formaldehyde (FA), an environmental pollutant, molecular mechanisms of APS’s activity. is widely present in pollution, tobacco smoke and home accessories [3, 4]. The International Agency Materials and methods for Research on Cancer (IARC) indicated that FA may cause leukemia but the mechanisms remain un- Cell culture. Human BMSCs (Cyagen Biosciences Inc, clear [4]. FA exhibits toxic effects in various types of Santa Clara, CA, US) were cultured in Dulbecco’s Modified mammalian cells, including cultured human myeloid Eagel Media: Nutrient Mixture F-12 1:1 Mixture (D-MEM/ progenitor cells and bone marrow blood progenitor /F12) supplemented with 10% fetal bovine serum for stem cells in vivo [5, 6]. In our previous study, it has been cells, 315 mg/L D-glucose and glutamine (all from Cyagen confirmed that FA induced the excessive DNA-pro- Biosciences, Santa Clara, CA, USA) at 37°C in a humidified tein crosslinks (DPCs) in murine BMSCs [7]. incubator with 5% CO2 (SANYO Electric CO., Ltd, Osaka, The formation of DPCs is related to oxidative Japan). During the logarithmic growth period, the cells were damage caused by FA-induced excessive hydroxyl free detached with 0.25% trypsin-0.02% EDTA. radicals [8–10]. DPCs are the primary DNA damage products induced by FA, and the level of DPCs is Preparation of astragalus polysaccharide (APS) and formal- considered to be a good molecular dosimeter to assess dehyde (FA). The mass fraction of APS (Solarbio Science & FA-related cancer risk [11]. When FA induces DNA Technology Co., Ltd., Beijing, China) was more than 98%. damage, such as DPCs, the cell activates repair mech- APS was dissolved and diluted to the final concentration of anisms to reverse the damage to reestablish stability 40, 100, 400 μg/mL with D-MEM/F12. FA’s (SigmaAldrich, of the genome. The nucleotide excision repair (NER) St. Louis, MO, USA) mass fraction was more than 37%. FA pathway is an important way for DNA damage repair, was diluted with D-MEM/F12 to the final concentration of which mainly repairs the cyclobutane pyrimidine di- 120 μmol/L. mer, DNA adducts, and cross-linking between chains [12, 13]. It was found that the repair of FA-induced Groups. BMSCs were randomly divided into control group, DPCs mainly depends on the NER pathway [14]. FA group, and FA + APS 40, 100 and 400 μg/mL groups. In Astragalus membranaceus, belonging to the the control group, the cells were cultured with D-MEM/F-12. Leguminosae family, is used as a Qi-tonifying herb The cells in the FA group were cultured with 120 μmol/L in traditional Chinese medicine [15]. Astragalus pol- FA of D-MEM/F-12. BMSCs co-cultured with 120 μmol/L ysaccharide (APS), one of the active ingredients of FA and 40, 100, 400 μg/mL APS of D-MEM/F-12 served Astragalus, has been shown to possess antioxidant, as FA + APS 40, 100 and 400 μg/mL groups. FA and APS immunomodulatory, and tumor growth inhibitory were added at the same time and incubated together with properties in modern studies [16–18]. Numerous BMSCs for 24 h. animal and anecdotal human data show that APS has anticancer effects in preclinical studies [19], as well MTT assay. Cell proliferation activity was measured by as can improve myelosuppression induced by cyto- MTT assay. The number of cells was adjusted to 4 × 103 toxic agents and chemopreventive effects [20]. APS per well and inoculated on 96-well plates. Then MTT enhanced the secretion of granulocyte colony-stimu- (Biowest, Loire Valley, France) 20 μL was added into cells lating factor (G-CSF) and maintained the stability of and incubated for 4 h. The supernatant was discarded and the bone marrow hematopoietic microenvironment then DMSO was added before oscillation. The absorbance after chemotherapy [21]. In addition, APS could (A) value was measured at 490 nm with the enzyme marker inhibit ionizing radiation-induced bystander effects (Bio-Rad, Hercules, CA, USA). The cell proliferation rate in BMSCs [22] as well as inhibit apoptosis and senes- of each experimental group was calculated according to cence of BMSCs caused by ferric ammonium citrate the formula: cell proliferation rate = (A experiment — A iron overload [23]. blank)/(A control — A blank) ×100%. ©Polish Society for Histochemistry and Cytochemistry Folia Histochem Cytobiol. 2021 www.journals.viamedica.pl/folia_histochemica_cytobiologica 10.5603/FHC.a2021.0013 ISSN 0239-8508, e-ISSN 1897-5631 126 Yali She et al. Table 1. The sequence of each primer in qRT-PCR mRNA Primer sequence Product length XPA Forward primer 5’-CTACTGGAGGCATGGCTAAT-3’ 132bp Reverse primer 5’-CCATAACAGGTCCTGGTTGA-3’ XPC Forward primer 5’-ACTGCGCAGCCAGAAATCC-3’ 107bp Reverse primer 5’-CTTTGGAGAGAAGGCTCTTC-3’ ERCC1 Forward primer 5’-ATGGACCCTGGGAAGGACA-3’ 133bp Reverse primer 5’-TCTGTGTAGATCGGAATAAGG-3’ RPA1 Forward primer 5’-ATGCAGAAGGGGGATACAA-3’ 145bp Reverse primer 5’-ACTGTGTCGCCAACATGAA-3’ RAP2 Forward primer 5’-ATGACAGCTGCACCCATG-3’ 124bp Reverse primer 5’-TGTTCTGAAAAGATCTCAGG-3’ Comet assay. Comet assay was used to detect DNA strand Japan) under excitation light at 353 nm and emission light breakage. The normal 1% melting point agarose gel (Biow- at 455 nm. The concentrations of free DNA and cross-linked est) was prepared on frosted-glass slides. The cell suspension DNA were quantified according to the standard curve. The was spread onto normal 1% melting point agarose gel after DPCs level was calculated by the following formula: DPCs it was mixed with 1% low melting point agarose (volume = cross-linked DNA concentration/(cross-linked DNA ratio: 1:3) and then immersed in a lysis buffer containing concentration + free DNA concentration) ×100%.
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