PMID- 38170368 OWN - NLM STAT- MEDLINE DCOM- 20240110 LR - 20240110 IS - 1573-4978 (Electronic) IS - 0301-4851 (Print) IS - 0301-4851 (Linking) VI - 51 IP - 1 DP - 2024 Jan 3 TI - Study of the effect of keap1 on oxidative stress in human umbilical cord mesenchymal stem cells. PG - 67 LID - 10.1007/s11033-023-08997-y [doi] LID - 67 AB - BACKGROUND: HucMSCs had shown promising efficacy in treating childhood diseases, but oxidative stress induced by the poor microenvironment at the site of damage resulted in low cell survival after transplantation, thus preventing the cells from maximizing therapeutic efficacy. Therefore, this study aimed to investigate the role and mechanism of keap1 in oxidative stress injury of human umbilical cord mesenchymal stem cells (hucMSCs), and to provide theoretical support for improving the efficacy of stem cell therapy. METHODS: The hucMSCs were treated with hypoxic low-sugar-free serum (GSDH) to mimic the damaged site microenvironment after implantation. Adenoviral overexpression of keap1 gene of hucMSCs was performed in vitro, and cell proliferation ability was detected by CCK8 assay, crystal violet staining assay, and cell cycle assay. Cellular redox level was assessed by Amplex Red, MDA, and GSH/GSSG kit. Mitochondrial morphology was evaluated by mitotracker Red staining. ATP production was estimated by ATP detection kit. The mRNA and protein expression levels were tested by western blotting and RT-qPCR. RESULTS: GSDH treatment substantially upregulated keap1 expression. Subsequently, we found that overexpression of keap1 notably inhibited cell proliferation and caused cells to stagnate in G1 phase. At the same time, overexpression of keap1 induced the production of large amounts of H(2)O(2) and the accumulation of MDA, but suppressed the GSH/GSSG ratio and the expression of antioxidant proteins NQO1 and SOD1, which caused oxidative stress damage. Overexpression of keap1 induced cells to produce a large number of dysfunctional mitochondria resulting in reduced ATP production. Moreover, Overexpression of keap1 significantly decreased the IKKbeta protein level, while upregulating IkB mRNA levels and downregulating P50 mRNA levels. CONCLUSIONS: Overexpression of keap1 may induce oxidative stress injury in hucMSCs by down-regulating IKKbeta expression and inhibiting NF-kappaB pathway activation. This implies the importance of keap1 in hucMSCs and it may be a potential gene for genetic modification of hucMSCs. CI - (c) 2023. The Author(s). FAU - Deng, Hongrong AU - Deng H AD - Department of Pediatric Research Institute, National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of PediatricsChongqing Key Laboratory of Pediatrics, Chongqing, China. FAU - Chen, Yunxia AU - Chen Y AD - Department of Pediatric Research Institute, National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of PediatricsChongqing Key Laboratory of Pediatrics, Chongqing, China. FAU - Liu, Huiwen AU - Liu H AD - Department of Pediatric Research Institute, National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of PediatricsChongqing Key Laboratory of Pediatrics, Chongqing, China. FAU - Wang, Li AU - Wang L AD - Department of Pediatric Research Institute, National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of PediatricsChongqing Key Laboratory of Pediatrics, Chongqing, China. FAU - Xu, Hao AU - Xu H AD - Department of Pediatric Research Institute, National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of PediatricsChongqing Key Laboratory of Pediatrics, Chongqing, China. AD - Department of Clinical Laboratory, Children's Hospital of Chongqing Medical University, Chongqing, China. FAU - Tan, Bin AU - Tan B AD - Department of Pediatric Research Institute, National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of PediatricsChongqing Key Laboratory of Pediatrics, Chongqing, China. FAU - Yi, Qin AU - Yi Q AD - Department of Pediatric Research Institute, National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of PediatricsChongqing Key Laboratory of Pediatrics, Chongqing, China. FAU - Wang, Rui AU - Wang R AD - Department of Pediatric Research Institute, National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of PediatricsChongqing Key Laboratory of Pediatrics, Chongqing, China. FAU - He, Bolin AU - He B AD - Department of Blood Transfusion, Children's Hospital of Chongqing Medical University, Chongqing, China. FAU - Tian, Jie AU - Tian J AD - Department of Cardiovascular Internal Medicine, Children's Hospital of Chongqing Medical University, Chongqing, China. FAU - Zhu, Jing AU - Zhu J AD - Department of Pediatric Research Institute, National Clinical Research Center for Child Health and Disorders, Children's Hospital of Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders,Chongqing Key Laboratory of PediatricsChongqing Key Laboratory of Pediatrics, Chongqing, China. jingzhu@cqmu.edu.cn. LA - eng GR - 82270271/National Natural Science Foundation of China/ GR - KJQN202300421/Science and Technology Program of Chongqing Municipal Education Commission/ PT - Journal Article DEP - 20240103 PL - Netherlands TA - Mol Biol Rep JT - Molecular biology reports JID - 0403234 RN - 8L70Q75FXE (Adenosine Triphosphate) RN - ULW86O013H (Glutathione Disulfide) RN - BBX060AN9V (Hydrogen Peroxide) RN - EC 2.7.11.10 (I-kappa B Kinase) RN - 0 (Kelch-Like ECH-Associated Protein 1) RN - 0 (NF-E2-Related Factor 2) RN - 0 (RNA, Messenger) RN - 0 (KEAP1 protein, human) SB - IM MH - Child MH - Humans MH - Adenosine Triphosphate MH - Glutathione Disulfide/metabolism MH - *Hydrogen Peroxide/metabolism MH - I-kappa B Kinase/metabolism MH - Kelch-Like ECH-Associated Protein 1/genetics/metabolism MH - *Mesenchymal Stem Cells/metabolism MH - NF-E2-Related Factor 2/genetics/metabolism MH - Oxidative Stress MH - RNA, Messenger/metabolism MH - Umbilical Cord PMC - PMC10764455 OTO - NOTNLM OT - HucMSCs OT - IKKbeta OT - Oxidative stress OT - keap1 COIS- The authors declare no competing interests. EDAT- 2024/01/04 01:18 MHDA- 2024/01/05 06:42 PMCR- 2024/01/03 CRDT- 2024/01/03 12:52 PHST- 2023/08/28 00:00 [received] PHST- 2023/11/06 00:00 [accepted] PHST- 2024/01/05 06:42 [medline] PHST- 2024/01/04 01:18 [pubmed] PHST- 2024/01/03 12:52 [entrez] PHST- 2024/01/03 00:00 [pmc-release] AID - 10.1007/s11033-023-08997-y [pii] AID - 8997 [pii] AID - 10.1007/s11033-023-08997-y [doi] PST - epublish SO - Mol Biol Rep. 2024 Jan 3;51(1):67. doi: 10.1007/s11033-023-08997-y.