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Supjaroen P, Niamsi W, Thummarati P, Laiwattanapaisal W. An In Vitro Cell Model of Intestinal Barrier Function Using a Low-Cost 3D-Printed Transwell Device and Paper-Based Cell Membrane. Int J Mol Sci 2025; 26:2524. [PMID: 40141167 PMCID: PMC11941856 DOI: 10.3390/ijms26062524] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2024] [Revised: 02/27/2025] [Accepted: 03/10/2025] [Indexed: 03/28/2025] Open
Abstract
Current in vitro methods for intestinal barrier assessment predominantly utilize two-dimensional (2D) membrane inserts in standard culture plates, which are widely recognized for their inability to replicate the microenvironment critical to intestinal barrier functionality. Our study focuses on creating an alternative method for intestinal barrier function by integrating a 3D-printed transwell device with a paper-based membrane. Caco-2 cells were grown on a Matrigel-modified paper membrane, in which the tight junction formation was evaluated using TEER measurements. Neutrophil-like dHL-60 cells were employed for neutrophil extracellular trap (NET) formation experiments. Furthermore, intestinal barrier dysfunction was demonstrated using NET-isolated and Staurosporine interventions. Intestinal barrier characteristics were investigated through immunofluorescence staining of specific proteins and scanning electron microscopy (SEM). Our paper-based intestinal barrier exhibited an increased resistance in a time-dependent manner, consistent with immunofluorescence images of Zonulin Occludens-1 (ZO-1) expression. Interestingly, immunofluorescence analysis revealed changes in the morphology of the intestinal barrier and the formation of surface villi. These disruptions were found to alter the localization of tight junctions, impacting epithelial polarization and surface functionality. Moreover, we successfully demonstrated the permeability of a paper-based intestinal barrier using FITC-dextran assay. Hence, the 3D-printed transwell device integrated with a paper membrane insert presents a straightforward, cost-effective, and sustainable platform for an in vitro cell model to evaluate intestinal barrier function.
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Affiliation(s)
- Pitaksit Supjaroen
- Graduate Program in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand; (P.S.)
| | - Wisanu Niamsi
- Graduate Program in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand; (P.S.)
| | - Parichut Thummarati
- Centre of Excellence for Biosensors and Bioengineering (CEBB), Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand;
- Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand
| | - Wanida Laiwattanapaisal
- Centre of Excellence for Biosensors and Bioengineering (CEBB), Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand;
- Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand
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Rembiałkowska N, Kucharczyk J, Radzevičiūtė-Valčiukė E, Novickij V, Tonci M, Dündar A, Kulbacka J, Szlasa W. Enhancing lung cancer growth inhibition with calcium ions: Role of mid- and high-frequency electric field pulses. Biomed Pharmacother 2024; 181:117691. [PMID: 39557010 DOI: 10.1016/j.biopha.2024.117691] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2024] [Revised: 11/09/2024] [Accepted: 11/12/2024] [Indexed: 11/20/2024] Open
Abstract
Calcium electroporation (CaEP) involves the combination of calcium ions with electroporation, which is induced by pulsed electric fields (PEFs). This study explores the application of high-frequency unipolar nanosecond pulsed electric fields (nsPEFs: 8-14 kV/cm, 200 ns, 10 kHz, 100 kHz, 1 MHz repetition frequency pulse bursts, n = 100) and their potential in inhibiting lung cancer cell growth. As a reference, standard microsecond range parametric protocols were used (100 µs x 8 pulses). Methods included cell permeability quantification through Yo-Pro-1 uptake, cell viability assays, immunofluorescence studies for apoptosis and EMT markers, analysis of cell death types depending on repetition frequency pulse bursts. We determined the susceptibility of human lung cancer to electric pulses, characterized the efficacy of CaEP, and investigated cell death types depending on repetition frequency pulse bursts. We have shown that adding calcium ions to the applied nsPEF protocol increases cytotoxicity. Additionally, the use of these electroporation parameters can modulate key cellular processes, such as the epithelial-mesenchymal transition and apoptosis, as indicated by changes in the expression of markers such as E-cadherin, N-cadherin, BCL-2, and p53. Changes in cell morphology over time were observed using holotomographic microscopy. The study provides insights into the modulation of key cellular processes, indicating that nsPEF technology could improve the outcomes of conventional cancer treatments through enhanced efficacy and potentially mitigating drug resistance mechanisms. The promising results advocate for further research to optimize nsPEF protocols for clinical application, highlighting the potential of electrical fields in advancing cancer therapy.
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Affiliation(s)
- Nina Rembiałkowska
- Wroclaw Medical University, Faculty of Pharmacy, Department of Molecular and Cellular Biology, Wroclaw, Poland.
| | - Julia Kucharczyk
- The Students' Research Group, UMW, SKN No. 148, Wroclaw Medical University, Faculty of Pharmacy, Department of Molecular and Cellular Biology, Wroclaw, Poland.
| | - Eivina Radzevičiūtė-Valčiukė
- Vilnius Gediminas Technical University, Faculty of Electronics, Vilnius, Lithuania; State Research Institute Centre for Innovative Medicine, Department of Immunology and Bioelectrochemistry, Vilnius, Lithuania.
| | - Vitalij Novickij
- Vilnius Gediminas Technical University, Faculty of Electronics, Vilnius, Lithuania; State Research Institute Centre for Innovative Medicine, Department of Immunology and Bioelectrochemistry, Vilnius, Lithuania.
| | - Margherita Tonci
- Faculty of Medicine and Surgery, University of Pisa, Pisa, Italy.
| | - Ata Dündar
- Faculty of Medicine, Istanbul University Cerrahpaşa, Istanbul, Turkey.
| | - Julita Kulbacka
- Wroclaw Medical University, Faculty of Pharmacy, Department of Molecular and Cellular Biology, Wroclaw, Poland; State Research Institute Centre for Innovative Medicine, Department of Immunology and Bioelectrochemistry, Vilnius, Lithuania.
| | - Wojciech Szlasa
- Wroclaw Medical University, Faculty of Pharmacy, Department of Molecular and Cellular Biology, Wroclaw, Poland; Medical University Hospital, Wroclaw, Poland.
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Lin JJ, Ning T, Jia SC, Li KJ, Huang YC, Liu Q, Lin JH, Zhang XT. Evaluation of genetic response of mesenchymal stem cells to nanosecond pulsed electric fields by whole transcriptome sequencing. World J Stem Cells 2024; 16:305-323. [PMID: 38577234 PMCID: PMC10989289 DOI: 10.4252/wjsc.v16.i3.305] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/13/2023] [Revised: 01/31/2024] [Accepted: 02/28/2024] [Indexed: 03/25/2024] Open
Abstract
BACKGROUND Mesenchymal stem cells (MSCs) modulated by various exogenous signals have been applied extensively in regenerative medicine research. Notably, nanosecond pulsed electric fields (nsPEFs), characterized by short duration and high strength, significantly influence cell phenotypes and regulate MSCs differentiation via multiple pathways. Consequently, we used transcriptomics to study changes in messenger RNA (mRNA), long noncoding RNA (lncRNA), microRNA (miRNA), and circular RNA expression during nsPEFs application. AIM To explore gene expression profiles and potential transcriptional regulatory mechanisms in MSCs pretreated with nsPEFs. METHODS The impact of nsPEFs on the MSCs transcriptome was investigated through whole transcriptome sequencing. MSCs were pretreated with 5-pulse nsPEFs (100 ns at 10 kV/cm, 1 Hz), followed by total RNA isolation. Each transcript was normalized by fragments per kilobase per million. Fold change and difference significance were applied to screen the differentially expressed genes (DEGs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to elucidate gene functions, complemented by quantitative polymerase chain reaction verification. RESULTS In total, 263 DEGs were discovered, with 92 upregulated and 171 downregulated. DEGs were predominantly enriched in epithelial cell proliferation, osteoblast differentiation, mesenchymal cell differentiation, nuclear division, and wound healing. Regarding cellular components, DEGs are primarily involved in condensed chromosome, chromosomal region, actin cytoskeleton, and kinetochore. From aspect of molecular functions, DEGs are mainly involved in glycosaminoglycan binding, integrin binding, nuclear steroid receptor activity, cytoskeletal motor activity, and steroid binding. Quantitative real-time polymerase chain reaction confirmed targeted transcript regulation. CONCLUSION Our systematic investigation of the wide-ranging transcriptional pattern modulated by nsPEFs revealed the differential expression of 263 mRNAs, 2 miRNAs, and 65 lncRNAs. Our study demonstrates that nsPEFs may affect stem cells through several signaling pathways, which are involved in vesicular transport, calcium ion transport, cytoskeleton, and cell differentiation.
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Affiliation(s)
- Jian-Jing Lin
- Department of Sports Medicine and Rehabilitation, Peking University Shenzhen Hospital, Shenzhen 518036, Guangdong Province, China
| | - Tong Ning
- Institute of Medical Science, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250033, Shandong Province, China
| | - Shi-Cheng Jia
- Department of Sports Medicine and Rehabilitation, Peking University Shenzhen Hospital, Shenzhen 518036, Guangdong Province, China
| | - Ke-Jia Li
- Department of Biomedical Engineering, Institute of Future Technology, Peking University, Beijing 100871, China
| | - Yong-Can Huang
- Shenzhen Engineering Laboratory of Orthopaedic Regenerative Technologies, Peking University Shenzhen Hospital, Shenzhen 518036, Guangdong Province, China
| | - Qiang Liu
- Arthritis Clinical and Research Center, Peking University People's Hospital, Beijing 100044, China
| | - Jian-Hao Lin
- Arthritis Clinical and Research Center, Peking University People's Hospital, Beijing 100044, China
| | - Xin-Tao Zhang
- Department of Sports Medicine and Rehabilitation, Peking University Shenzhen Hospital, Shenzhen 518036, Guangdong Province, China.
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Li X, Hu L, Naeem A, Xiao S, Yang M, Shang H, Zhang J. Neutrophil Extracellular Traps in Tumors and Potential Use of Traditional Herbal Medicine Formulations for Its Regulation. Int J Nanomedicine 2024; 19:2851-2877. [PMID: 38529365 PMCID: PMC10961241 DOI: 10.2147/ijn.s449181] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2023] [Accepted: 02/28/2024] [Indexed: 03/27/2024] Open
Abstract
Neutrophil extracellular traps (NETs) are extracellular fibers composed of deoxyribonucleic acid (DNA) and decorated proteins produced by neutrophils. Recently, NETs have been associated with the development of many diseases, including tumors. Herein, we reviewed the correlation between NETs and tumors. In addition, we detailed active compounds from traditional herbal medicine formulations that inhibit NETs, related nanodrug delivery systems, and antibodies that serve as "guiding moieties" to ensure targeted delivery to NETs. Furthermore, we discussed the strategies used by pathogenic microorganisms to evade NETs.
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Affiliation(s)
- Xiang Li
- National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Chinese Medicine, Nanchang, Jiangxi, 330006, People’s Republic of China
| | - Lei Hu
- National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Chinese Medicine, Nanchang, Jiangxi, 330006, People’s Republic of China
| | - Abid Naeem
- Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Chinese Medicine, Nanchang, Jiangxi, 330004, People’s Republic of China
- School of Life Science, Advanced Research Institute of Multidisciplinary Science, School of Medical Technology, Key Laboratory of Molecular Medicine and Biotherapy, Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, Beijing Institute of Technology, Beijing, 100081, People’s Republic of China
| | - Shanghua Xiao
- Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Chinese Medicine, Nanchang, Jiangxi, 330004, People’s Republic of China
| | - Ming Yang
- Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Chinese Medicine, Nanchang, Jiangxi, 330004, People’s Republic of China
| | - Hongming Shang
- Department of Biochemistry & Chemical Biology, Vanderbilt University, Nashville, TN, USA
| | - Jing Zhang
- National Pharmaceutical Engineering Center for Solid Preparation in Chinese Herbal Medicine, Jiangxi University of Chinese Medicine, Nanchang, Jiangxi, 330006, People’s Republic of China
- Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Chinese Medicine, Nanchang, Jiangxi, 330004, People’s Republic of China
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Wang Y, Ma R, Huang Z, Zhou Y, Wang K, Xiao Z, Guo Q, Yang D, Han M, Shen S, Qian J, Gao X, Liu Z, Zhou L, Yin S, Zheng S. Investigation of lethal thresholds of nanosecond pulsed electric field in rabbit VX2 hepatic tumors through finite element analysis and verification with a single-needle bipolar electrode: A prospective strategy employing three-dimensional comparisons. Comput Biol Med 2024; 168:107824. [PMID: 38086143 DOI: 10.1016/j.compbiomed.2023.107824] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2023] [Revised: 11/16/2023] [Accepted: 12/04/2023] [Indexed: 01/10/2024]
Abstract
Pulsed electric field has emerged as a promising modality for the solid tumor ablation with the advantage in treatment planning, however, the accurate prediction of the lesion margin requires the determination of the lethal electric field (E) thresholds. Herein we employ the highly repetitive nanosecond pulsed electric field (RnsPEF) to ablate the normal and VX2 tumor-bearing livers of rabbits. The ultrasound-guided surgery is operated using the conventional double- and newly devised single-needle bipolar electrodes. Finite element analysis is also introduced to simulate the E distribution in the practical treatments. Two- and three-dimensional investigations are performed on the image measurements and reconstructed calcification models on micro-CT, respectively. Specially, an algorithm considering the model surface, volume and shape is employed to compare the similarities between the simulative and experimental models. Blood vessel injury, temperature and synergistic efficacy with doxorubicin (DOX) are also investigated. According to the three-dimensional calculation, the overall E threshold is 4536.4 ± 618.2 V/cm and the single-needle bipolar electrode is verified to be effective in tissue ablation. Vessels are well preserved and the increment of temperature is limited. Synergy of RnsPEF and DOX shows increased apoptosis and improved long-term tumor survival. Our study presents a prospective strategy for the evaluation of the lethal E threshold, which can be considered to guide the future clinical treatment planning for RnsPEF.
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Affiliation(s)
- Yubo Wang
- Key Laboratory of Multi-Organ Transplantation Research (Ministry of Health), First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, 310003, China
| | - Rongwei Ma
- Institute of Industrial Ecology and Environment, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, Zhejiang Province, 310003, China
| | - Zhiliang Huang
- Department of Ultrasound, Shulan Hospital, Hangzhou, Zhejiang Province, 310003, China
| | - Yuan Zhou
- Key Laboratory of Multi-Organ Transplantation Research (Ministry of Health), First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, 310003, China
| | - Ke Wang
- College of Computer Science and Technology, China University of Minning and Technology, Xuzhou, Jiangsu Province, 221008, China
| | - Zhoufang Xiao
- College of Computer Science and Technology, Hangzhou Dianzi University, Hangzhou, Zhejiang Province, 310003, China
| | - Qiang Guo
- Department of Ultrasound, Shulan Hospital, Hangzhou, Zhejiang Province, 310003, China
| | - Dezhi Yang
- Department of Ultrasound, Shulan Hospital, Hangzhou, Zhejiang Province, 310003, China
| | - Mingchen Han
- College of Computer Science and Technology, China University of Minning and Technology, Xuzhou, Jiangsu Province, 221008, China
| | - Shuwei Shen
- College of Computer Science and Technology, Hangzhou Dianzi University, Hangzhou, Zhejiang Province, 310003, China
| | - Junjie Qian
- Key Laboratory of Multi-Organ Transplantation Research (Ministry of Health), First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, 310003, China
| | - Xingxing Gao
- Key Laboratory of Multi-Organ Transplantation Research (Ministry of Health), First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, 310003, China
| | - Zhen Liu
- Institute of Industrial Ecology and Environment, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, Zhejiang Province, 310003, China
| | - Lin Zhou
- Key Laboratory of Multi-Organ Transplantation Research (Ministry of Health), First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, 310003, China
| | - Shengyong Yin
- Key Laboratory of Multi-Organ Transplantation Research (Ministry of Health), First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, 310003, China.
| | - Shunsen Zheng
- Key Laboratory of Multi-Organ Transplantation Research (Ministry of Health), First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, 310003, China; Department of Ultrasound, Shulan Hospital, Hangzhou, Zhejiang Province, 310003, China.
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Jia E, Zhu H, Geng H, Zhong L, Qiu X, Xie J, Xiao Y, Jiang Y, Xiao M, Zhang Y, Wei J, Tang D, Zhang J. The Inhibition of Osteoblast Viability by Monosodium Urate Crystal-Stimulated Neutrophil-Derived Exosomes. Front Immunol 2022; 13:809586. [PMID: 35655781 PMCID: PMC9152014 DOI: 10.3389/fimmu.2022.809586] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2021] [Accepted: 04/22/2022] [Indexed: 12/27/2022] Open
Abstract
Background and Objective Bone erosion is common in patients with gout. The role of neutrophil-derived exosomes in gouty bone erosion remains elusive. This study aimed to investigate the functions of the neutrophil-derived exosomes in the development of bone erosion in gout. Methods Neutrophil-derived exosomes were collected and assessed by transmission electron microscopy and nanoparticle tracking analysis. Cell counting kit-8 assay was applied to evaluate cell viability, and cell apoptosis was assessed by flow cytometry. In addition, quantitative Real-time PCR and Western blotting were used to determine the expression levels of alkaline phosphatase (ALP), osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL). Neutrophil-derived exosomes were tagged with PKH67. The miRNA expression profiles of exosomes and human fetal osteoblasts (hFOB) were compared using high-throughput sequencing. Functional miRNAs transfected into hFOB after co-incubation with exosomes were selected and validated by preliminary qPCR. Results Neutrophil-derived exosomes were stimulated by monosodium urate (MSU). The exosomes could inhibit the viability of the hFOB, and the expression levels of ALP and OPG were down-regulated, while the expression level of RANKL was up-regulated. However, there was no significant difference in the viability of osteoclasts and the expression of nuclear factor of activated T cells 1. Exosomes were observed in the cytoplasm under a confocal microscopy, confirming that exosomes could be taken up by hFOB. In total, 2590 miRNAs were found, of which 47 miRNAs were differentially expressed. Among the delivered miRNAs, miR-1246 exhibited the highest level of differential expression. The viability of hFOB was reduced by miR-1246 mimics and increased by miR-1246 inhibitors. There was no significant difference in hFOB apoptosis rate between the miR-1246 mimic and miR-1246 inhibitor group. MiR-1246 overexpression decreased the expression levels of ALP and OPG, whereas increasing the expression level of RANKL. In contrast, miR-1246 inhibitor increased the expression levels of ALP and OPG, while decreasing the expression level of RANKL. Neutrophil-derived exosomes stimulated by MSU could increase the expression of miR-1246. Conclusion Neutrophil-derived exosomes stimulated by MSU could inhibit the viability of osteoblasts.
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Affiliation(s)
- Ertao Jia
- The Department of Rheumatology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, China.,The Department of Rheumatology, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
| | - Haiqiong Zhu
- Shenzhen Traditional Chinese Medicine Hospital Affiliated to Nanjing University of Chinese Medicine, Shenzhen, China
| | - Hongling Geng
- The Department of Gynecology, Guangdong Provincial Hospital of Chinese Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China
| | - Li Zhong
- The Department of Rheumatology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, China.,The Department of Rheumatology, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
| | - Xia Qiu
- The Department of Rheumatology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, China.,The Department of Rheumatology, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
| | - Jingjing Xie
- The Department of Rheumatology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, China.,The Department of Rheumatology, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
| | - Yuya Xiao
- The Department of Rheumatology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, China.,The Department of Rheumatology, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
| | - Yubao Jiang
- The Department of Rheumatology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, China.,The Department of Rheumatology, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
| | - Min Xiao
- The Department of Rheumatology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, China.,The Department of Rheumatology, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
| | - Yanying Zhang
- The Department of Rheumatology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, China.,The Department of Rheumatology, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
| | - Jiaxin Wei
- The Department of Rheumatology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, China.,The Department of Rheumatology, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
| | - Dabin Tang
- The Department of Rheumatology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, China.,The Department of Rheumatology, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
| | - Jianyong Zhang
- The Department of Rheumatology, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, China.,The Department of Rheumatology, The Fourth Clinical Medical College of Guangzhou University of Chinese Medicine, Shenzhen, China
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Kasprzycka W, Trębińska-Stryjewska A, Lewandowski RB, Stępińska M, Osuchowska PN, Dobrzyńska M, Achour Y, Osuchowski ŁP, Starzyński J, Mierczyk Z, Trafny EA. Nanosecond Pulsed Electric Field Only Transiently Affects the Cellular and Molecular Processes of Leydig Cells. Int J Mol Sci 2021; 22:ijms222011236. [PMID: 34681896 PMCID: PMC8541366 DOI: 10.3390/ijms222011236] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2021] [Revised: 10/13/2021] [Accepted: 10/15/2021] [Indexed: 12/11/2022] Open
Abstract
The purpose of this study was to verify whether the nanosecond pulsed electric field, not eliciting thermal effects, permanently changes the molecular processes and gene expression of Leydig TM3 cells. The cells were exposed to a moderate electric field (80 quasi-rectangular shape pulses, 60 ns pulse width, and an electric field of 14 kV/cm). The putative disturbances were recorded over 24 h. After exposure to the nanosecond pulsed electric field, a 19% increase in cell diameter, a loss of microvilli, and a 70% reduction in cell adhesion were observed. Some cells showed the nonapoptotic externalization of phosphatidylserine through the pores in the plasma membrane. The cell proportion in the subG1 phase increased by 8% at the expense of the S and G2/M phases, and the DNA was fragmented in a small proportion of the cells. The membrane mitochondrial potential and superoxide content decreased by 37% and 23%, respectively. Microarray’s transcriptome analysis demonstrated a negative transient effect on the expression of genes involved in oxidative phosphorylation, DNA repair, cell proliferation, and the overexpression of plasma membrane proteins. We conclude that nanosecond pulsed electric field affected the physiology and gene expression of TM3 cells transiently, with a noticeable heterogeneity of cellular responses.
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Affiliation(s)
- Wiktoria Kasprzycka
- Biomedical Engineering Centre, Institute of Optoelectronics, Military University of Technology, 00-908 Warsaw, Poland; (W.K.); (A.T.-S.); (R.B.L.); (M.S.); (P.N.O.); (M.D.); (Ł.P.O.); (Z.M.)
| | - Alicja Trębińska-Stryjewska
- Biomedical Engineering Centre, Institute of Optoelectronics, Military University of Technology, 00-908 Warsaw, Poland; (W.K.); (A.T.-S.); (R.B.L.); (M.S.); (P.N.O.); (M.D.); (Ł.P.O.); (Z.M.)
| | - Rafał Bogdan Lewandowski
- Biomedical Engineering Centre, Institute of Optoelectronics, Military University of Technology, 00-908 Warsaw, Poland; (W.K.); (A.T.-S.); (R.B.L.); (M.S.); (P.N.O.); (M.D.); (Ł.P.O.); (Z.M.)
| | - Małgorzata Stępińska
- Biomedical Engineering Centre, Institute of Optoelectronics, Military University of Technology, 00-908 Warsaw, Poland; (W.K.); (A.T.-S.); (R.B.L.); (M.S.); (P.N.O.); (M.D.); (Ł.P.O.); (Z.M.)
| | - Paulina Natalia Osuchowska
- Biomedical Engineering Centre, Institute of Optoelectronics, Military University of Technology, 00-908 Warsaw, Poland; (W.K.); (A.T.-S.); (R.B.L.); (M.S.); (P.N.O.); (M.D.); (Ł.P.O.); (Z.M.)
| | - Monika Dobrzyńska
- Biomedical Engineering Centre, Institute of Optoelectronics, Military University of Technology, 00-908 Warsaw, Poland; (W.K.); (A.T.-S.); (R.B.L.); (M.S.); (P.N.O.); (M.D.); (Ł.P.O.); (Z.M.)
| | - Yahia Achour
- Faculty of Electronics, Military University of Technology, 00-908 Warsaw, Poland; (Y.A.); (J.S.)
| | - Łukasz Paweł Osuchowski
- Biomedical Engineering Centre, Institute of Optoelectronics, Military University of Technology, 00-908 Warsaw, Poland; (W.K.); (A.T.-S.); (R.B.L.); (M.S.); (P.N.O.); (M.D.); (Ł.P.O.); (Z.M.)
| | - Jacek Starzyński
- Faculty of Electronics, Military University of Technology, 00-908 Warsaw, Poland; (Y.A.); (J.S.)
| | - Zygmunt Mierczyk
- Biomedical Engineering Centre, Institute of Optoelectronics, Military University of Technology, 00-908 Warsaw, Poland; (W.K.); (A.T.-S.); (R.B.L.); (M.S.); (P.N.O.); (M.D.); (Ł.P.O.); (Z.M.)
| | - Elżbieta Anna Trafny
- Biomedical Engineering Centre, Institute of Optoelectronics, Military University of Technology, 00-908 Warsaw, Poland; (W.K.); (A.T.-S.); (R.B.L.); (M.S.); (P.N.O.); (M.D.); (Ł.P.O.); (Z.M.)
- Correspondence:
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8
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Yang Q, Kajimoto S, Kobayashi Y, Hiramatsu H, Nakabayashi T. Regulation of Cell Volume by Nanosecond Pulsed Electric Fields. J Phys Chem B 2021; 125:10692-10700. [PMID: 34519209 DOI: 10.1021/acs.jpcb.1c06058] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Stimulation of cells by nanosecond pulsed electric fields (nsPEFs) has attracted attention as a technology for medical applications such as cancer treatment. nsPEFs have been shown to affect intracellular environments without significant damage to cell membranes; however, the mechanism underlying the effect of nsPEFs on cells remains unclear. In this study, we constructed electrodes for applying nsPEFs and analyzed the change in volume of a single cell due to nsPEFs using fluorescence and Raman microscopy. It was shown that the direction of the change depended on the applied electric field; expansion due to the influx of water was observed at high electric field, and cell shrinkage was observed at low electric field. The change in cell volume was correlated to the change in the intracellular Ca2+ concentration, and nsPEFs-induced shrinking was not observed when the Ca2+-free medium was used. This result suggests that the cell shrinkage is related to the regulatory volume decrease where the cell adjusts the increase in intracellular Ca2+ concentration, inducing the efflux of ions and water from the cell.
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Affiliation(s)
- Qi Yang
- Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
| | - Shinji Kajimoto
- Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980-8578, Japan.,JST PRESTO, Kawaguchi, Saitama 332-0012, Japan
| | - Yuki Kobayashi
- Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
| | - Hirotsugu Hiramatsu
- Department of Applied Chemistry and Institute of Molecular Science, National Yang Ming Chiao Tung University, 1001, Ta-Hsueh Road, Hsinchu 30010, Taiwan.,Center for Emergent Functional Matter Science, National Yang Ming Chiao Tung University, Hsinchu 30010, Taiwan
| | - Takakazu Nakabayashi
- Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
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9
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Cichon I, Ortmann W, Kolaczkowska E. Metabolic Pathways Involved in Formation of Spontaneous and Lipopolysaccharide-Induced Neutrophil Extracellular Traps (NETs) Differ in Obesity and Systemic Inflammation. Int J Mol Sci 2021; 22:ijms22147718. [PMID: 34299338 PMCID: PMC8303382 DOI: 10.3390/ijms22147718] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2021] [Revised: 07/14/2021] [Accepted: 07/15/2021] [Indexed: 02/07/2023] Open
Abstract
Obesity manifests itself with low-grade chronic inflammation that shapes immune responses during infection. Albeit obese individuals are at risk of higher mortality due to comorbidities, they are better protected from systemic inflammation. Recently, we showed that in the vasculature of obese mice kept on high-fat diet (HFD), neutrophils produce less neutrophil extracellular traps (NETs) than in lean controls (normal diet, ND). NETs are used by neutrophils to counteract severe infection, but they also cause collateral damage. Hardly anything is known about metabolic requirements for their formation, especially in the context of obesity and/or sepsis. Thus, we aimed to study the immunometabolism of NET formation by application of ex vivo neutrophil analyses (Seahorse analyzer, selective inhibitors, confocal imaging) and intravital microscopy. The obtained data show that glycolysis and/or pentose phosphate pathway are involved in NETs release by ND neutrophils in both physiological and inflammatory conditions. In contrast, such cells of septic HFD mice utilize these routes only to spontaneously cast NETs, while after secondary ex vivo activation they exhibit so called "exhausted phenotype", which manifests itself in diminished NET release despite high glycolytic potential and flexibility to oxidize fatty acids. Moreover, impact of ATP synthase inhibition on NET formation is revealed. Overall, the study shows that the neutrophil potential to cast NETs depends on both the metabolic and inflammatory state of the individual.
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10
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Allawadhi P, Khurana A, Allwadhi S, Navik US, Joshi K, Banothu AK, Bharani KK. Potential of electric stimulation for the management of COVID-19. Med Hypotheses 2020; 144:110259. [PMID: 33254561 PMCID: PMC7481069 DOI: 10.1016/j.mehy.2020.110259] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Revised: 08/28/2020] [Accepted: 09/05/2020] [Indexed: 12/15/2022]
Abstract
The COVID-19 pandemic is the most devastating health emergency that humans have seen over the past century. The war against the disease has been handicapped by unavailability of effective therapeutic options. Till date, there is no clinically approved vaccine or drug for the treatment of COVID-19, and the ongoing search to find a novel therapy is progressing at pandemic pace. Herein, we propose a novel hypothesis based on sound research evidence that electric stimulation can be a potential adjuvant to the currently used symptomatic therapies and antiviral drugs. Based on preclinical evidence, we propose that electric stimulation can improve respiratory functions, inhibit SARS-CoV-2 growth, reduce pain, boost immunity and improve the penetration of antiviral drugs. We envisage that our hypothesis, if used clinically as an adjuvant, may significantly improve the therapeutic outcomes of the current treatment regimen being used around the globe for the management of COVID-19.
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Affiliation(s)
- Prince Allawadhi
- Department of Pharmacy, Vaish Institute of Pharmaceutical Education and Research (VIPER), PBDS-UHS, Rohtak-124001, Haryana, India
| | - Amit Khurana
- Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc), PVNRTVU, Rajendranagar, Hyderabad-500030, Telangana, India; Centre for Biomedical Engineering (CBME), Indian Institute of Technology (IIT), Delhi-110016, India.
| | - Sachin Allwadhi
- Department of Computer Science and Engineering, University Institute of Engineering and Technology (UIET), Maharshi Dayanand University, Rohtak-124001, Haryana, India
| | - Uma Shanker Navik
- Department of Pharmacology, School of Basic and Applied Sciences, Central University of Punjab, Bathinda-151001, Punjab, India
| | - Kamaldeep Joshi
- Department of Computer Science and Engineering, University Institute of Engineering and Technology (UIET), Maharshi Dayanand University, Rohtak-124001, Haryana, India
| | - Anil Kumar Banothu
- Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc), PVNRTVU, Rajendranagar, Hyderabad-500030, Telangana, India
| | - Kala Kumar Bharani
- Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science (CVSc), PVNRTVU, Rajendranagar, Hyderabad-500030, Telangana, India.
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11
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Shin SH, Jeong J, Kim JH, Sohn KY, Yoon SY, Kim JW. 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Mitigates Monosodium Urate (MSU)-Induced Acute Gouty Inflammation in BALB/c Mice. Front Immunol 2020; 11:710. [PMID: 32395118 PMCID: PMC7196669 DOI: 10.3389/fimmu.2020.00710] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2019] [Accepted: 03/30/2020] [Indexed: 12/25/2022] Open
Abstract
Acute gouty arthritis is an auto-inflammatory disease caused by the deposition of monosodium urate (MSU) crystals in joints or tissues. Excessive neutrophil recruitment into gouty lesions is a general clinical sign and induces a pain phenotype. Attenuation of successive periods of neutrophil infiltration might be a beneficial approach to achieve therapeutic efficacy. In this study, the activity of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) in attenuation of excess neutrophil infiltration was assessed in gout-induced lesions of BALB/c mice. Neutrophil infiltration in MSU-induced gouty lesions was analyzed using immunohistochemical staining. ELISA and RT-PCR were used to measure attenuation of expression of the major neutrophil chemoattractant, CXC motif chemokine ligand 8 (CXCL8), in a PLAG-treated animal model and in cells in vitro. The animal model revealed massive increased neutrophil infiltration in the MSU-induced gouty lesions, but the PLAG-treated mice had significantly reduced neutrophil numbers in these lesions. The results also indicated that the MSU crystals stimulated a damage-associated molecular pattern that was recognized by the P2Y6 purinergic receptor. This MSU-stimulated P2Y6 receptor was destined to intracellular trafficking. During intracellular endosomal trafficking of the receptor, endosome-dependent signaling provided expression of CXCL8 chemokines for neutrophil recruitment. PLAG accelerated initiation of the intracellular trafficking of the P2Y6 receptor and returning the receptor to the membrane. This process shortened the intracellular retention time of the receptor anchoring endosome and subsequently attenuated endosome-dependent signaling for CXCL8 expression. These study results suggested that PLAG could be used for resolution of acute inflammation induced in gout lesions.
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Affiliation(s)
- Su-Hyun Shin
- Division of Systems Biology and Bioengineering, Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea.,Department of Functional Genomics, University of Science and Technology, Daejeon, South Korea
| | - Jinseon Jeong
- Division of Global New Drug Development, ENZYCHEM Lifesciences, Seoul, South Korea
| | - Joo Heon Kim
- Department of Pathology, EulJi University School of Medicine, Daejeon, South Korea
| | - Ki-Young Sohn
- Division of Global New Drug Development, ENZYCHEM Lifesciences, Seoul, South Korea
| | - Sun Young Yoon
- Division of Global New Drug Development, ENZYCHEM Lifesciences, Seoul, South Korea
| | - Jae Wha Kim
- Division of Systems Biology and Bioengineering, Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea.,Department of Functional Genomics, University of Science and Technology, Daejeon, South Korea
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