Cell Journal (Yakhteh)

Number 9, 2023

591-602
Potential Utilisation of Secretome from Ascorbic Acid-Supplemented Stem Cells in Combating Skin Aging: Systematic Review of A Novel Idea

Authors: Komang Ardi Wahyuningsih; Wimpie I Pangkahila; I Wayan Weta Weta; I Gde Raka Widiana; Ida Ayu Ika Wahyuniari

Abstract

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The secretome of stem cells consists of a spectrum of bioactive factors secreted by stem cells grown in culture mediacytokines, chemokines, and growth factors in addition to extracellular vesicles (exosomes and microvesicles). Ease of handling and storage of secretomes along with their bioactivity towards processes in skin aging and customizability makes them an appealing prospective therapy for skin aging. This systematic review aims to investigate the potential usage of ascorbic acid (AA)-supplemented stem cell secretomes (SCS) in managing skin aging. We extracted articles from three databases: PubMed, Scopus, and Cochrane. This review includes in vitro, in vivo, and clinical studies published in English that discuss the correlation of AA-supplemented-SCS with skin aging. We identified 1111 articles from database and non-database sources from which nine studies met the inclusion criteria. However, the study results were less specific due to the limited amount of available research that specifically assessed the effects of AAsupplemented SCS in skin aging. Although further studies are necessary, the AA modification of SCS is a promising potential for improving skin health.

603-612
Gallic Acid Ameliorates Cadmium Effect on Osteogenesis by Activation of Alkaline Phosphatase and Collagen Synthesis

Authors: Mohammad Hossein Abnosi; Javad Sargolzaei; Farshid Nazari

Abstract

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Objective: We previously reported that cadmium (Cd) inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). In addition, gallic acid (GA) improves BMSC differentiation. Here, we aim to study the ability of GA to prevent osteogenic inhibition induced by Cd. Materials and Methods: In this experimental study, BMSCs were extracted and purified from Wistar rats and their viability was determined in the presence of Cd and GA. The results indicated that 1.5 μM Cd and 0.25 μM of GA were appropriate for further investigation. After 20 days in osteogenic medium, matrix production was analysed by alizarin red, calcium content, and alkaline phosphatase (ALP) activity. Osteogenic-related genes and collagen 1A1 (COL1A1) protein expressions were investigated. The preventive effect of GA on oxidative stress and metabolic change induced by Cd was estimated. Results: GA counteracted the inhibitory effect of Cd on matrix production and significantly (P=0.0001) improved the osteogenic differentiation ability of BMSCs. Also, GA prevented the toxic effect of Cd on osteogenic-related gene expressions and nullified the reducing effect of Cd on COL1Al and ALP activity. A significant reduction (P=0.0001) in malondialdehyde and lactic acid concentration showed that GA counteracted both oxidative stress and metabolic changes caused by Cd. Conclusion: GA prevented the toxic effect of Cd, an environmental pollutant and a factor in osteoporosis.

613-624
Bioinformatic Analysis of The Prognostic Value of A Panel of Six Amino Acid Transporters in Human Cancers

Authors: Yaqi Liu; Haijuan Xiong; Chenhui Yan; Yalei Wang; Wenfeng Cao; Shuo Qie

Abstract

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Objective: Solid tumor cells utilize amino acid transporters (AATs) to increase amino acid uptake in response to nutrient-insufficiency. The upregulation of AATs is therefore critical for tumor development and progression. This study identifies the upregulated AATs under amino acid deprived conditions, and further determines the clinicopathological importance of these AATs in evaluating the prognosis of patients with cancers. Materials and Methods: In this experimental study, the Gene Expression Omnibus (GEO) datasets (GSE62673, GSE26370, GSE125782 and GSE150874) were downloaded from the NCBI website and utilized for integrated differential expression and pathway analysis v0.96, Gene Set Enrichment Analysis (GSEA), and REACTOME analyses to identify the AATs upregulated in response to amino acid deprivation. In addition, The Cancer Genome Atlas (TCGA) datasets with prognostic information were assessed and employed to evaluate the association of identified AATs with patients’ prognoses using SurvExpress analysis. Results: Using analysis of NCBI GEO data, this study shows that amino acid deprivation leads to the upregulation of six AAT genes; SLC3A2, SLC7A5, SLC7A1, SLC1A4, SLC7A11 and SLC1A5. GSEA and REACTOME analyses identified altered signaling in cells exposed to amino acid deprivation, such as pathways related to stress responses, the cell cycle and apoptosis. In addition, Principal Component Analysis showed these six AAT genes to be well divided into two distinct clusters in relation to TCGA tumor tissues versus normal counterparts. Finally, Log-Rank analysis confirmed the upregulation of this panel of six AAT genes is correlated with poor prognosis in patients with colorectal, esophageal, kidney and lung cancers. Conclusion: The upregulation of a panel of six AATs is common in several human cancers and may provide a valuable diagnostic tool to evaluate the prognosis of patients with colorectal, esophageal, kidney and lung cancers.

625-632
Protective Effects of Relaxin 2 (RLXH2) against Hypoxia-Induced Oxidative Damage and Cell Death via Activation of The Nrf2/HO-1 Signalling Pathway in Gastric Cancer Cells

Authors: Liguo Wang; Yi Zhou; Hui Lin; Kezhu Hou

Abstract

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Objective: This study aims to investigate the potential role of relaxin, a peptide hormone, in preventing cellular deterioration and death in gastric carcinoma cells under hypoxic conditions. It explores the effects of recombinant relaxin 2 (RLXH2) on growth, cell differentiation, invasive potential, and oxidative damage in these cells. Materials and Methods: In this experimental study, the NCI-N87 cell line was cultured under normal conditions and then subjected to hypoxia using cobalt chloride (CoCl2). The cells were treated with RLXH2, and various assays were performed to assess cellular deterioration, death, and oxidative stress. Western blot and quantitative real time polymerase chain reaction (qRT-PCR) were used to measure the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1, and the translocation of Nrf2 to the nucleus was confirmed through Western blot analysis. Results: This study demonstrates, for the first time, that RLXH2 significantly reduces the formation of reactive oxygen species (ROS) and the release of lactate dehydrogenase (LDH) in gastric cancer cells under hypoxic conditions. RLXH2 also enhances the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT), leading to a decrease in hypoxia-induced oxidative damage. RLXH2 promotes the translocation of Nrf2 to the nucleus, resulting in HO-1 expression. Conclusion: Our findings suggest that RLXH2 plays a significant protective role against hypoxia-induced oxidative damage in gastric carcinoma cells through the Nrf2/HO-1 signalling pathway. This research contributes to a better understanding of the potential therapeutic applications of RLXH2 in gastric cancer treatment.

633-644
Reversing T Cell Exhaustion by Converting Membrane PD-1 to Its Soluble form in Jurkat Cells; Applying The CRISPR/Cas9 Exon Skipping Strategy

Authors: Zeinab Yousefi-Najafabadi; Zohreh Mehmandoostli; Yazdan Asgari; Saeed Kaboli; Reza Falak; Gholam Ali Kardar

Abstract

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Objective: T-cells express two functional forms of the programmed cell death protein 1 (PD-1): membrane (mPD-1) and soluble (sPD-1). The binding of mPD-1 and its ligand (PD-L1) on tumor cells could lead activated lymphocytes toward exhaustion. Selective deletion of the transmembrane domain via alternative splicing of exon-3 in PD-1 mRNA could generate sPD-1. Overexpression of sPD-1 could disrupt the mPD-1/PD-L1 interaction in tumor-specific T cells. We investigated the effect of secreted sPD-1 from pooled engineered and non-engineered T cell supernatant on survival and proliferation of lymphocytes in the tumor microenvironment (TME). Materials and Methods: In this experimental study, we designed two sgRNA sequences upstream and downstream of exon-3 in the PDCD1 gene. The lentiCRISPRv2 puro vector was used to clone the dual sgRNAs and produce lentiviral particles to transduce Jurkat T cells. Analysis assays were used to clarify the change in PD-1 expression pattern in the pooled (engineered and non-engineered) Jurkat cells. Co-culture conditions were established with PD-L1+ cancer cells and lymphocytes. Results: CRISPR/Cas9 could delete exon-3 of the PDCD1 gene in the engineered cells based on the tracking of indels by decomposition (TIDE) and interference of CRISPR edit (ICE) sequencing analysis reports. Our results showed a 12% reduction in mPD-1 positive cell population after CRISPR manipulation and increment in sPD-1 concentration in the supernatant. The increased sPD-1 confirmed its positive effect on proliferation of lymphocytes co-cultured with PDL1+ cancer cells. The survival percent of lymphocytes co-cultured with the pooled cells supernatant was 12.5% more than the control. Conclusion: The CRISPR/Cas9 exon skipping approach could be used in adoptive cell immunotherapies to change PD-1 expression patterns and overcome exhaustion.

645-654
β-Glucan Regulates Lipopolysaccharide Induced Genotoxic Damage to The Liver through The Induction of BRCA1 Protein Expression

Authors: Gözde Aydoğan Kılıç; Mojahed Alsafi

Abstract

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Objective: The present study aims to investigate the role of breast cancer-susceptibility gene 1 (BRCA1) protein in the β-Glucan (βG) molecule mediated regulation of lipopolysaccharide (LPS)-induced liver genotoxicity. Materials and Methods: In this experimental study, totally, 32 male Swiss Albino mice were randomly divided into 4 equal groups: control (C), LPS-administered (LPS), βG-administered (βG) and βG-pre-administered/LPS-administered (βG+LPS). The βG was injected at the dose of 150 mg/kg/day intraperitoneally (i.p.) for 3 days. A single dose of 4 mg/ kg (i.p.) LPS was administered 24 hours after the last βG injection. BRCA1 expression was determined by western blot analysis and confirmed by quantitative immunofluorescence. Proliferating cell nuclear antigen (PCNA), nuclear factor erythroid 2–related factor (Nrf2) and 8-OHdG protein levels were also determined by the immunofluorescence analysis. The alkaline comet assay was performed. superoxide dismutase (SOD), catalase (CAT) and membrane lipid peroxidation were biochemically measured, and light microscopic histology was evaluated. Results: The BRCA1 expression level was significantly decreased in the LPS group. However, in the βG+LPS group, expression of BRCA1 protein was over 2 folds higher than the control. After the LPS induction, the DNA strand breaks, oxidative DNA lesions and abnormal proliferation of the liver cells were almost entirely suppressed in βG preadministrated animals, indicating the BRCA1 mediated ubiquitination of PCNA and activation of the DNA damage repair pathways. Activation of Nrf2 in the βG+LPS group resulted in an increase in the levels of Nrf2 pathway dependent antioxidant enzymes SOD and CAT, prevented the peroxidation of membrane lipids and maintained the histological architecture of the liver. Conclusion: The results manifested that the βG is a strong inducer of the BRCA1 protein expression in the LPSinduced hepatic stress and the protein constitutes the key component of a βG mediated liver protection against an LPS-induced genotoxic and pathological damage.

655-659
Generation of Mouse Model of Hemophilia A by Introducing Novel Mutations, Using CRISPR/Nickase Gene Targeting System

Authors: Mehdi Shamsara; Abbas Jamshidizad; Aidin Rahim-Tayefeh; Maliheh Davari; Ali Rajabi Zangi; Fatemeh Masoumi; Alireza Zomorodipour

Abstract

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Developing mouse models of hemophilia A has been shown to facilitate in vivo studies to explore the probable mechanism(s) underlying the disease and to examine the efficiency of the relevant potential therapeutics. This study aimed to knockout (KO) the coagulation factor viii (fviii) gene in NMRI mice, using CRISPR/Cas9 (D10A/nickase) system, to generate a mouse model of hemophilia A. Two single guide RNAs (sgRNAs), designed from two distinct regions on NMRI mouse FVIII (mFVIII) exon 3, were designed and inserted in the pX335 vector, expressing both sgRNAs and nickase. The recombinant construct was delivered into mouse zygotes and implanted into the pseudopregnant female mice’s uterus. Mutant mice were identified by genotyping, genomic sequencing, and mFVIII activity assessment. Two separate lines of hemophilia A were obtained through interbreeding the offspring of the female mice receiving potential CRISPR-Cas9-edited zygotes. Genomic DNA analysis revealed disruptions of the mfviii gene reading frame through a 22-bp deletion and a 23-bp insertion in two separate founder mice. The founder mice showed all the clinical signs of hemophilia A including; excessive bleeding after injuries, and spontaneous bleeding in joints and other organs. Coagulation test data showed that mFVIII coagulation activity was significantly diminished in the mFVIII knockout (FVIIIKO) mice compared to normal mice. The CRISPR/nickase system was successfully applied to generate mouse lines with the knockout fviii gene. The two novel FVIIIKO mice demonstrated all clinical symptoms of hemophilia A, which could be successfully inherited. Therefore, both of the developed FVIIIKO mouse lines are eligible for being considered as proper mouse models of hemophilia A for in vivo therapeutic studies.

660-664
Age-Related Skin Inflammation in A 2,4-Dintrochlorobenzene-Induced Atopic Dermatitis Mouse Model

Authors: Kyung-Ah Cho; Jiyun Kwon; Hyeon Ju Kim; So-Youn Woo

Abstract

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One of the most affected aspects of the aging process is immunity, with age-related immune system decline being responsible for an increase in susceptibility to infectious diseases and cancer risk. On the other hand, the aging process is accompanied with low-grade pro-inflammatory status. This condition involves a persistent rise in cytokine levels that can activate both innate and adaptive immune systems. Finally, despite the fact that immunological responses to antigenic stimulations decrease with age, the incidence and prevalence of many common autoimmune diseases increase in the elderly population. Overall, the co-existence of a prolonged, low-grade inflammatory status and declining immune activity appears to be a paradoxical phenomenon. This study characterized skin inflammation in mouse dermatitis model of various ages to monitor possible changes of inflammatory responses during aging.