Research Paper Volume 13, Issue 2 pp 2822—2850
Transplanting Rac1-silenced bone marrow mesenchymal stem cells promote neurological function recovery in TBI mice
- 1 Zhejiang Provincial Key Laboratory of Aging and Neurological Disorder Research, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China
- 2 Department of Neurosurgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China
- 3 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
Received: August 6, 2020 Accepted: November 18, 2020 Published: December 19, 2020https://doi.org/10.18632/aging.202334
How to Cite
Copyright: © 2020 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Bone marrow mesenchymal stem cells (BMMSCs)-based therapy has emerged as a promising novel therapy for Traumatic Brain Injury (TBI). However, the therapeutic quantity of viable implanted BMMSCs necessary to initiate efficacy is still undetermined. Increased oxidative stress following TBI, which leads to the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase signaling pathway, has been implicated in accounting for the diminished graft survival and therapeutic effect. To prove this assertion, we silenced the expression of NADPH subunits (p22-phox, p47-phox, and p67-phox) and small GTPase Rac1 in BMMSCs using shRNA. Our results showed that silencing these proteins significantly reduced oxidative stress and cell death/apoptosis, and promoted implanted BMMSCs proliferation after TBI. The most significant result was however seen with Rac1 silencing, which demonstrated decreased expression of apoptotic proteins, enhanced in vitro survival ratio, reduction in TBI lesional volume and significant improvement in neurological function post shRac1-BMMSCs transplantation. Additionally, two RNA-seq hub genes (VEGFA and MMP-2) were identified to play critical roles in shRac1-mediated cell survival. In summary, we propose that knockdown of Rac1 gene could significantly boost cell survival and promote the recovery of neurological functions after BMMSCs transplantation in TBI mice.
TBI: Traumatic brain injury; BMMSCs: Bone marrow mesenchymal stem cells; NADPH: Nicotinamide adenine dinucleotide phosphate; OGD: Oxygen-glucose deprivation; EPS: Endogenous neural stem cells; iPS: Induced pluripotent stem cells; BDNF: Brain-derived neurotrophic factor; GDNF: Glial cell-derived neurotrophic factor; VEGF: Vascular endothelial growth factor; ROS: Reactive oxygen species; FPR: Formyl peptide receptors; AKT: Serine-threonine protein kinase; ATCC: American Type Culture Collection; SD: Sprague Dawley; DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum; PCR: Polymerase chain reaction; CCK-8: Cell counting kit-8; PBS: Phosphate-buffered saline; qRT-PCR: Quantitative reverse transcription-polymerase chain reaction; BSA: Bovine serum albumin; DAPI: 4′,6-diamidino-2-phenylindole; RIPA: Radioimmunoprecipitation assay; PMSF: Phenylmethylsulfonyl fluoride; BCA: Bicinchoninic acid assay; PVDF: Polyvinylidene difluoride; JNK: c-Jun N-terminal Kinase; H&E: Hematoxylin and eosin; mNSS: Modified neurological severity scores; OCT: Optimal cutting temperature; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; FPKM: Fragments per kilobase million; DEGs: Differentially expressed genes; SD: Standard deviation; Bax: BCL-2-Associated X; Bcl-2: b-cell lymphoma-2; N.S.: no significance; DPT: Days post transplantation; TNF: Tumor necrosis factor; HCM: Hypertrophic cardiomyopathy; VEGFA: Vascular endothelial growth factor A; MMP-2: Matrix metalloproteinase 2; CTGF: Connective tissue growth factor; EDN1: Endothelin 1; CCL2: Chemokine (C-C motif) ligand 2; THBS1: Thrombospondin 1; CXCL1: Chemokine (C-X-C motif) ligand 1; TGFβ-2: Transforming growth factor beta 2; GAS6: Growth arrest specific 6; NGF: Nerve growth factor; EPOR: Erythropoietin receptor; FGF-1: Fibroblast growth factor 1; FGF-2: Fibroblast growth factor 2; IL-6: Interleukin 6; IL-18: Interleukin 18; CSF-1: Colony-stimulating factor 1; NT3: Neurotrophin 3; bFGF: Basic fibroblast growth factor; CXCR4: CXC chemokine receptor 4; EGF: Epidermal growth factor.