In vitro evaluation of the toxicity and underlying molecular mechanisms of Janus Fe3O4‐TiO2 nanoparticles in human liver cells

Recent studies show that Janus Fe₃O₄‐TiO₂ nanoparticles (NPs) have potential applications as a multifunctional agent of magnetic resonance imaging (MRI) and photodynamic therapy (PDT) for the diagnosis and therapy of cancer. However, little work has been done on their biological effects. To evaluate the toxicity and underlying molecular mechanisms of Janus Fe₃O₄‐TiO₂ nanoparticles, an in vitro study using a human liver cell line HL‐7702 cells was conducted. For comparison, the Janus Fe₃O₄‐TiO₂ NPs parent material TiO₂ NPs was also evaluated. Results showed that both Fe₃O₄‐TiO₂ NPs and TiO₂ NPs decreased cell viability and ATP levels when applied in treatment, but increased malonaldehyde (MDA) and reactive oxygen species (ROS) generation. Mitochondria JC‐1 staining assay showed that mitochondrial membrane permeability injury occurred in both NPs treated cells. Cell viability analysis showed that TiO₂ NPs induced slightly higher cytotoxicity than Fe₃O₄‐TiO₂ NPs in HL7702 cells. Western blotting indicated that both TiO₂ NPs and Fe₃O₄‐TiO₂ NPs could induce apoptosis, inflammation, and carcinogenesis related signal protein alterations. Comparatively, Fe₃O₄‐TiO₂ NPs induced higher signal protein expressions than TiO₂ NPs under a high treatment dose. However, under a low dose (6.25 μg/cm²), neither NPs had any significant toxicity on HL7702 cells. In addition, our results suggest both Fe₃O₄‐TiO₂ NPs and TiO₂ NPs could induce oxidative stress and have a potential carcinogenetic effect in vitro. Further studies are needed to elaborate the detailed mechanisms of toxicity induced by a high dose of Fe₃O₄‐TiO₂ NPs.     

间充质干细胞移植减轻大鼠肺气肿及氧化应激的探讨

目的研究间充质干细胞(MSCs)移植在肺气肿大鼠氧化应激和肺气肿发展过程中的作用。方法 26只雌性SD大鼠随机分为对照组(A组,n=8),肺气肿组(B组,n=8),肺气肿+MSCs移植组(C组,n=10),将B与C组大鼠在香烟烟雾中暴露14周,制作肺气肿大鼠模型,C组大鼠予MSCs移植。完成4次MSCs移植后的2、4周分别处死C组大鼠各1只,观察MSCs在肺气肿大鼠肺部的定植情况;MSCs移植8周后同时处死各组大鼠,观察大鼠肺组织病理形态,并进行定量分析;用硫代戊巴比妥法测定血清和肺组织匀浆中丙二醛(MDA)的水平,用WST-1法测定各组血清和肺组织匀浆中超氧化物歧化酶(SOD)的水平。结果 MSCs移植后2周及4周,C组大鼠肺组织内均可见标记过的MSCs,随着时间的推移,肺组织内MSCs比例逐渐减少。B组和C组大鼠的肺组织呈现肺气肿样改变,B组和C组大鼠肺组织的平均内衬间隔(MLI)高于A组,平均肺泡数(MAN)明显低于A组,B组大鼠肺组织的MLI高于C组,MAN低于C组(P<0.05)。B组和C组大鼠血清及肺组织中MDA高于A组,SOD低于A组,B组血清及肺组织中MDA高于C组,SOD低于C组(P<0.05)。结论 MSCs移植可能通过降低氧化应激水平对大鼠肺气肿发挥治疗作用。     

Concentration‐dependent induction of reactive oxygen species,cell cycle arrest and apoptosis in human liver cells after nickel nanoparticles exposure

Due to advent of nanotechnology, nickel nanoparticles (Ni NPs) are increasingly recognized for their utility in various applications including catalysts, sensors and electronics. However, the environmental and human health effects of Ni NPs have not been fully investigated. In this study, we examined toxic effects of Ni NPs in human liver (HepG2) cells. Ni NPs were prepared and characterized by X‐ray diffraction, transmission electron microscopy and dynamic light scattering. We observed that Ni NPs (size, ～28 nm; concentration range, 25–100 μg/mL) induced cytotoxicity in HepG2 cells and degree of induction was concentration‐dependent. Ni NPs were also found to induce oxidative stress in dose‐dependent manner evident by induction of reactive oxygen species and depletion of glutathione. Cell cycle analysis of cells treated with Ni NPs exhibited significant increase of apoptotic cell population in subG1 phase. Ni NPs also induced caspase‐3 enzyme activity and apoptotic DNA fragmentation. Upregulation of cell cycle checkpoint gene p53 and bax/bcl‐2 ratio with a concomitant loss in mitochondrial membrane potential suggested that Ni NPs induced apoptosis in HepG2 cells was mediated through mitochondrial pathway. This study warrants that applications of Ni NPs should be carefully assessed as to their toxicity to human health. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 137–148, 2015.     

Endoplasmic reticulum stress contributes to arsenic trioxide‐induced intrinsic apoptosis in human umbilical and bone marrow mesenchymal stem cells

Arsenic trioxide is an old drug and has been used for a long time in traditional Chinese and Western medicines. However, the cancer treatment of arsenic trioxide has heart and vascular toxicity. The cytotoxic effects of arsenic trioxide and its molecular mechanism in human umbilical mesenchymal stem cells (HUMSC) and human bone marrow‐derived mesenchymal stem cells (HMSC‐bm) were investigated in this study. Our results showed that arsenic trioxide significantly reduced the viability of HUMSC and HMSC‐bm in a concentration‐ and time‐dependent manner. Arsenic trioxide is able to induce apoptotic cell death in HUMSC and HMSC‐bm, as shown from the results of morphological examination, flow cytometric analyses, DAPI staining and comet assay. The appearance of arsenic trioxide also led to an increase of intracellular free calcium (Ca²⁺) concentration and the disruption of mitochondrial membrane potential (ΔΨm). The caspase‐9 and caspase‐3 activities were time‐dependently increased in arsenic trioxide‐treated HUMSC and HMSC‐bm. In addition, the proteomic analysis and DNA microarray were carried out to investigate the expression level changes of genes and proteins affected by arsenic trioxide treatment in HUMSC. Our results suggest that arsenic trioxide induces a prompt induction of ER stress and mitochondria‐modulated apoptosis in HUMSC and HMSC‐bm. A framework was proposed for the effect of arsenic trioxide cytotoxicity by targeting ER stress. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 314–328, 2016.     

BPA‐toxicity via superoxide anion overload and a deficit in β‐catenin signaling in human bone mesenchymal stem cells

Bisphenol A (BPA), used in the manufacture of products based on polycarbonate plastics and epoxy resins, is well known as an endocrine‐disrupting monomer. In the current study, BPA increased cytotoxicity in hBMSCs in a dose‐ and time‐dependent manner, concomitantly with increased lipid peroxidation. Increased cell death in BPA‐treated cells was markedly blocked by pretreatment with the superoxide dismutase mimetic MnTBAP and MnTMPyP, but not by catalase, glutathione, the glutathione peroxidase mimetic ebselen, the NOS inhibitor NAME, or the xanthine oxidase inhibitor allopurinol. Furthermore, the decline in nuclear β‐catenin and cyclin D1 levels in hBMSCs exposed to BPA was reversed by MnTBAP treatment. Finally, treatment of hBMSCs with the GSK3β inhibitor LiCl₂ increased nuclear β‐catenin levels and significantly attenuated cytotoxicity compared with BPA treatment. Our current results in hBMSCs exposed to BPA suggest that BPA causes a disturbance in β‐catenin signaling via a superoxide anion overload. © 2016 The Authors Environmental Toxicology Published by Wiley Periodicals, Inc. Environ Toxicol 32: 344–352, 2017.     

Neurotoxicity of metal‐containing nanoparticles and implications in glial cells

With the development of nanotechnology, metal‐containing nanoparticles are used widely in the diagnosis, monitoring and treatment of central nervous system (CNS) diseases. The neurotoxicity of these nanoparticles has drawn attention. Glial cells (particularly microglial cells and astrocytes) have important functions in the CNS. Neural disorders are related to functional/histologic damage to glial cells. Dysfunctions of microglial cells or astrocytes injure the brain, and cause the neurodegeneration seen in Alzheimer's disease and Parkinson's disease. We have summarized the route of access of metal‐containing nanoparticles to the CNS, as well as their neurotoxicity and potential molecular mechanisms involved in glial cells. Metal‐containing nanoparticles cross or bypass the blood‐brain barrier, access the CNS and cause neurotoxicity. The potential mechanisms are related to inflammation, oxidative stress, DNA and/or mitochondrial damage and cell death, all of which are mediated by microglial cell activation, inflammatory factor release, generation of reactive oxygen species, apoptosis and/or autophagy in glial cells. Moreover, these processes increase the burden of the CNS and even accelerate the occurrence or development of neurodegenerative diseases. Some important signaling pathways involved in the mechanism of neurotoxicity in glial cells caused by nanoparticles are also discussed.     

Toxicological effects of pet food ingredients on canine bone marrow‐derived mesenchymal stem cells and enterocyte‐like cells

We developed an in vitro method to assess pet food ingredients safety. Canine bone marrow‐derived mesenchymal stem cells (BMSC) were differentiated into enterocyte‐like cells (ELC) to assess toxicity in cells representing similar patterns of exposure in vivo. The toxicological profile of clove leave oil, eugenol, guanosine monophosphate (GMP), GMP + inosine monophosphate, sorbose, ginger root extract, cinnamon bark oil, cinnamaldehyde, thyme oil, thymol and citric acid was assessed in BMSC and ELC. The LC₅₀ for GMP + inosine monophosphate was 59.42 ± 0.90 and 56.7 ± 3.5 mg ml^–1 for BMSC and ELC; 56.84 ± 0.95 and 53.66 ± 1.36 mg ml^–1 for GMP; 0.02 ± 0.001 and 1.25 ± 0.47 mg ml^–1 for citric acid; 0.077 ± 0.002 and 0.037 ± 0.01 mg ml^–1 for cinnamaldehyde; 0.002 ± 0.0001 and 0.002 ± 0.0008 mg ml^–1 for thymol; 0.080 ± 0.003 and 0.059 ± 0.001 mg ml^–1 for thyme oil; 0.111 ± 0.002 and 0.054 ± 0.01 mg ml^–1 for cinnamon bark oil; 0.119 ± 0.0004 and 0.099 ± 0.011 mg ml^–1 for clove leave oil; 0.04 ± 0.001 and 0.028 ± 0.002 mg ml^–1 for eugenol; 2.80 ± 0.11 and 1.75 ± 0.51 mg ml^–1 for ginger root extract; > 200 and 116.78 ± 7.35 mg ml^–1 for sorbose. Lemon grass oil was evaluated at 0.003–0.9 in BMSC and .03‐0.9 mg ml^–1 in ELC and its mechanistic effect was investigated. The gene toxicology studies showed regulation of 61% genes in CYP450 pathway, 37% in cholestasis and 33% in immunotoxicity pathways for BMSC. For ELC, 80% for heat shock response, 69% for beta‐oxidation and 65% for mitochondrial energy metabolism. In conclusion, these studies provide a baseline against which differential toxicity of dietary feed ingredients can be assessed in vitro for direct effects on canine cells and demonstrate differential toxicity in differentiated cells that represent gastrointestinal epithelial cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Silver nanoparticles: evaluation of DNA damage, toxicity and functional impairment in human mesenchymal stem cells

Silver nanoparticles (Ag-NPs) are the most frequent commercialized nanomaterial currently. Due to a distinct lack of information on hazardous properties of Ag-NPs in human cells, a study was conducted to evaluate Ag-NP induced DNA damage, cell death and functional impairment in human mesenchymal stem cells (hMSCs).Initially, Ag-NPs and their cellular distribution were characterized by transmission electron microscopy (TEM). Ag-NPs were exposed to hMSCs for 1, 3 and 24 h. Cytotoxicity was measured by the trypan blue exclusion test and the fluorescein-diacetate test, DNA damage was evaluated by the comet assay and chromosomal aberration test. Cytokine release of IL-6, IL-8 and VEGF was observed using the ELISA technique. Additionally, hMSC migration capability was tested in a transwell system.TEM revealed a Ag-NP distribution to cytoplasm and nucleus. Cytotoxic effects were seen at concentrations of 10 μg/ml for all test exposure periods. Both, comet assay and chromosomal aberration test showed DNA damage after 1, 3, and 24 h at 0.1 μg/ml. A significant increase of IL-6, IL-8 and VEGF release indicates hMSC activation. Migration ability was not impaired at subtoxic concentrations.In conclusion, we demonstrated cyto- and genotoxic potential of Ag-NPs in hMSCs at significantly higher concentrations as compared to antimicrobial effective levels.     

Transplantation of human umbilical mesenchymal stem cells attenuates dextran sulfate sodium‐induced colitis in mice

Ulcerative colitis is a major form of inflammatory bowel disease and increases the risk of the development of colorectal carcinoma. The anti‐inflammatory and immunomodulatory properties of mesenchymal stem cells (MSC) make them promising tools for treating immune‐mediated and inflammatory diseases. However, the lack of robust technique for harvesting and expanding of MSC has hampered the use of bone marrow and umbilical cord blood derived MSC in clinical applications. In the present study, we investigated the intestinal protective effects of Wharton's jelly‐derived umbilical MSC (UMSC) on dextran sulfate sodium‐induced colitis in mice. The severity of colitis in mice was assessed using bodyweight loss, stool consistency, rectal bleeding, colon shortening and haematological parameters. Colonic myeloperoxidase and pro‐inflammatory cytokines levels were also measured. Furthermore, the expression of cyclooxygenase 2 and inducible nitric oxide synthase in the colon were detected. In addition, intestinal permeability and tight junction proteins expressions in the colon were examined as well. The results showed that Wharton's jelly‐derived UMSC significantly diminished the severity of colitis, reduced histolopathological score, and decreased myeloperoxidase activity and cytokines levels. Furthermore, the UMSC markedly decreased the expression of cyclooxygenase 2and inducible nitric oxide synthase in the colon. In addition, transplantation of UMSC reduced intestinal permeability and upregulated the expression of tight junction proteins. These results show that the anti‐inflammation and regulation of tight junction proteins by Wharton's jelly‐derived UMSC ameliorates colitis.     

Cytoprotective effects of endothelin‐1 on mesenchymal stem cells: an in vitro study

Stem cell‐based therapies is a promising approach for regenerative therapy in various diseases. Some obstacles remain to be solved before clinical application of the cell therapy is realized, including increasing the survival of transplanted stem cells, reducing loss of transplanted cells, and maintaining adequate vascular supply. Recently, stem cell preconditioning with chemical and pharmacological agents has been shown to increase therapeutic efficacy. The present study investigated the effect of endothelin‐1 (ET‐1) on survival, angiogenesis, and migration of mesenchymal stem cells (MSCs), in vitro. MSCs were treated with various concentrations of ET‐1 and the expression of cyclooxygenase‐2 (COX‐2), hypoxia‐inducible factor‐1 (HIF‐1), C‐X‐C chemokine receptor type 4 (CXCR4), C‐C chemokine receptor type 2 (CCR2), vascular endothelial growth factor (VEGF), angiopoietin‐2 (Ang‐2), angiopoietin‐4 (Ang‐4) and matrix metalloproteinase‐2 (MMP‐2) were examined. Caspase 3 activity and prostaglandin E2 (PGE2) were determined by ELISA assay. MSCs migration and tube formation potential were assessed using scratch test and three dimensional vessel formation assay. ET‐1 enhanced the MSCs viability. In ET‐1‐ treated MSCs, expression of COX‐2, HIF‐1, CXCR4, CCR2, VEGF, Ang‐2, Ang‐4 and MMP‐2 were increased compared to control groups. Elevation of all these genes were reversed by celecoxib (50 μmol/L), a selective COX‐2 inhibitor. PGE2 generation, MSCs migration and tube formation were enhanced by ET‐1 conditioning, whereas caspase‐3 activity was reduced in these cells, compared to the control group. The results presented here reveal that preconditioning of MSCs with ET‐1 has strong cytoprotective effects through activation of survival signalling molecules and trophic factors.     

胰高血糖素样肽1和Extentin-4体外诱生人骨髓间充质干细胞分化为分泌胰岛素细胞

目的 探讨并优化人骨髓间充质干细胞（hBMSCs）向分泌胰岛素细胞（IPCs）定向分化的条件,为糖尿病替代疗法寻找新出路.方法 hBMSCs复苏培养后,联合应用胰高血糖素样肽1（GLP-1）、Ex-tentin-4等细胞因子通过三步诱导方案进行体外向IPCs诱导分化.应用反转录-聚合酶链反应检测与β细胞发育和功能相关的基因表达;免疫细胞化学、双硫腙染色证实IPCs的生成;电化学发光法检测细胞胰岛素的分泌量.结果 诱导分化18d后,镜下观察到胰岛样细胞团的生成,双硫腙及免疫细胞化学染色胰岛素均呈现阳性反应;诱导分化的细胞在第18天则可以观察到胰十二指肠同源盒基因1、胰岛素的高表达;诱导分化的细胞接受葡萄糖刺激后能够分泌胰岛素,且胰岛素的分泌量随葡萄糖浓度的提高而增加,高糖胰岛素分泌量[（188.7±1.5）mU/L]与低糖胰岛素分泌量[（60.1±0.9）mU/L]相比,差异有统计学意义（P＜0.05）.结论 体外联合应用GLP-l、Extentin-4、尼克酰胺等细胞因子采用三阶段诱导方案可以大大提高胰岛素细胞诱导分化率,并能促进IPCs的成熟和胰岛素的释放.     

脂肪干细胞分化成血管内皮细胞前后miRNA的差异表达

目的：探讨人脂肪间充质干细胞向血管内皮细胞诱导分化前后microRNA （miRNA ）的差异表达,预测其靶基因。方法对人脂肪间充质干细胞诱导成的血管内皮细胞进行鉴定后,提取人脂肪间充质干细胞向血管内皮细胞诱导分化前后的 miRNA ,微阵列芯片检测表达谱的变化,对有显著差异的miRNA进行实时荧光定量PCR验证,通过TargetScan、Miranda、PITA 、RNAhybrid和microTar五个数据库预测靶基因。结果筛选、验证出四个显著差异表达的miRNA。其中,miR‐29b‐3p和miR‐5096为显著上调,miR‐143‐3p和 miR‐145‐5p为显著下调。预测到 OCT4、SOX2、KLF4、TGFB2、IGF1、TAF11、TMEM169、UHRF1和OSBPL6等相关靶基因。结论人脂肪间充质干细胞向血管内皮细胞诱导分化前后存在显著差异性表达的miRNA ,提示这些miRNA在其分化中有重要的调控作用。     

Behaviour and ultrastructure of human bone marrow-derived mesenchymal stem cells immobilised in alginate-poly-l-lysine-alginate microcapsules

Abstract

Context: Human bone marrow mesenchymal stem cells (hBM-MSCs) show a great promise for the treatment of a variety of diseases. Despite the previous trials to encapsulate hBM-MSCs in alginate-poly-l-lysine-alginate (APA) systems, the various changes that follow immobilisation have not been ascertained yet. Objective: Determine the various consequences derived from entrapment on cell behaviour, putting special emphasis on the ultrastructure. Methods: hBM-MSCs were immobilised in APA microcapsules to further characterise their viability, metabolic activity, proliferation, VEGF-secretability, and morphology. Results: The VEGF produced by monolayer hBM-MSCs increased significantly 1 d post-encapsulation, and was maintained for at least 4 weeks. TEM imaging of cells revealed well preserved ultrastructure indicating protein synthesis and high metabolic activity. Conclusion: Although APA microencapsulation did not support 100% of fully viable hBM-MSCs for long-term cultures, it was conceived to enhance both VEGF secretion and metabolic activity while not losing their stemness characteristics.     

Epigallocatechin‐3‐gallate augments therapeutic effects of mesenchymal stem cells in skin wound healing

In non‐healing wounds, mesenchymal stem cell (MSC)‐based therapies have the potential to activate a series of coordinated cellular processes, including angiogenesis, inflammation, cell migration, proliferation and epidermal terminal differentiation. As pro‐inflammatory reactions play indispensable roles in initiating wound repair, sustained and prolonged inflammation exhibit detrimental effects on skin wound closure. We investigated the feasibility of using an antioxidant agent epigallocatechin‐3‐gallate (EGCG), along with MSCs, to improve wound repair through their immunomodulatory actions. In a rat model of wound healing, a single dose of EGCG at 10 mg/kg increased the efficiency of MSC‐induced skin wound closure. Twenty days after the wound induction, MSC treatment significantly enhanced the epidermal thickness, which was further increased by EGCG administration. Consistently, the highest extent of growth factors upregulation for neovascularization induction was seen in the animals treated by both MSCs and EGCG, associated with a potent anti‐scarring effect throughout the healing process. Finally, expression levels of pro‐inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β) and IL‐6, in the wound area were reduced by MSCs, and this reduction was further potentiated by EGCG co‐administration. EGCG, together with MSCs, can promote skin wound healing likely through their combinational effects in modulating chronic inflammation.     

Protective effects of hepatocyte growth factor gene overexpression against hydrogen peroxide‐induced apoptosis in mesenchymal stem cells

Hepatocyte growth factor (HGF) has recently been reported to exhibit antioxidant and antiapoptotic effects. Therefore, we investigated the effect of overexpression of HGF gene in H₂O₂‐treated mesenchymal stem cells (MSCs). HGF‐overexpression increased the cell viability from 50% to 84%, decreased the population of apoptotic cells from 20% to 16%, and decreased the intracellular reactive oxygen species (ROS) levels from 127% to 100% in cells treated with H₂O₂. HGF suppression decreased the cell viability from 58% to 36%, increased the population of apoptotic cells from 23 to 81%, and increased the intracellular ROS levels from 181% to 240% in cells exposed to H₂O₂. HGF‐overexpression also reduced the expression levels of proapoptotic proteins in MSCs treated with H₂O₂. Phosphorylation of extracellular signal‐regulated kinases, c‐Jun amino‐terminal kinases, and p38, which was induced by H₂O₂, decreased in MSCs overexpressing the HGF gene. Taken together, our results suggest that HGF has a protective effect on H₂O₂‐induced apoptosis in MSCs.