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Zirconia nanoparticles (ZrO2-NPs) have been increasingly used in industrial, biomedical and dental materials. Nevertheless, the scientific basis for the toxicological effects of ZrO2-NPs is poorly elucidated, and the understanding of the underlying mechanism is still limited. The hepatic biodistribution and toxicological effects of ZrO2-NPs after a single intravenous administration (20 mg/kg bw) in vivo and the toxicological mechanism toward hepatocytes in vitro were investigated. The liver showed continuous ZrO2-NP accumulation over a 28-d period. Moreover, ZrO2-NPs induced steatosis, functional injury, inflammatory response, and gene alternations in the liver. The main gene altered pathways induced by ZrO2-NP exposure were involved in metabolism, cellular processes, and human diseases. Among these pathways, lipid biosynthesis and metabolism alterations were predominant, and P53 signaling was also identified. Precious gene expression quantification further showed alterations in genes related to lipid biosynthesis and metabolism and cell apoptosis. Meanwhile, the results of the in vitro studies demonstrated ZrO2-NPs induce oxidative stress, lipid accumulation, cell apoptosis and P53-mediated signaling pathway activation in HepG2 cells. This study proves that ZrO2-NPs have impacts on the liver. There is potential concern over the hepatotoxicity of ZrO2-NPs in biomedical applications and occupational exposure through large-scale production.

Small extracellular vesicles (sEVs) are extracellular nanovesicles that contain bioactive proteins, lipids, RNA, and DNA. A variety of biological process is regulated with sEVs. sEVs are an intercellular messenger regulating recipient cell function and play a role in disease initiation and progression. sEVs derived from certain cells, such as mesenchymal stem cells and immune cells, have the potential for clinical therapy as they possess the characteristics of their parental cells. With better understanding of sEVs biogenesis, their transportation properties, extended circulatory capability, and exceptional biocompatibility, sEVs emerge as a potential therapeutic tool in the clinic. Here, we summarize applications of sEVs‐based therapies in different diseases and current knowledge about the strategies in bioengineered sEVs, as well as the challenges for their use in clinical settings., Small extracellular vesicles (sEVs) are extracellular nanovesicles that contain bioactive proteins, lipids, RNA, and DNA. A variety of biological process is regulated with sEVs. sEVs are an intercellular messenger regulating recipient cell function and play a role in disease initiation and progression. sEVs derived from certain cells, such as mesenchymal stem cells and immune cells, have the potential for clinical therapy as they possess the characteristics of their parental cells. With better understanding of sEVs biogenesis, their transportation properties, extended circulatory capability, and exceptional biocompatibility, sEVs emerge as a potential therapeutic tool in the clinic. Here, we summarize applications of sEVs‐based therapy in different diseases and current knowledge about the strategies in bioengineered sEVs, as well as the challenges for their use in clinical settings.

Background Gene therapy is considered a novel way to treat osteosarcoma, and microRNAs are potential therapeutic targets for osteosarcoma. miR-214 has been found to promote osteosarcoma aggression and metastasis. Graphene oxide (GO) is widely used for gene delivery for the distinct physiochemical properties and minimal cytotoxicity. Methods Polyethyleneimine (PEI)-functionalized GO complex was well-prepared and loaded with miR-214 inhibitor at different concentrations. The load efficacy was tested by gel retardation assay and the cy3-labeled fluorescence of cellular uptake. The experiments of wound healing, immunofluorescence staining, Western blot, qRT-PCR and immunohistochemical staining were performed to measure the inhibitory effect of the miR-214 inhibitor systematically released from the complexes against MG63, U2OS cells and xenograft tumors. Results The systematic mechanistic elucidation of the efficient delivery of the miR-214 inhibitor by GO-PEI indicated that the inhibition of cellular miR-214 caused a decrease in osteosarcoma cell invasion and migration and an increase in apoptosis by targeting phosphatase and tensin homolog (PTEN). The synergistic combination of the GO-PEI-miR-214 inhibitor and CDDP chemotherapy showed significant cell death. In a xenograft mouse model, the GO-PEI-miR-214 inhibitor significantly inhibited tumor volume growth. Conclusion This study indicates the potential of functionalized GO-PEI as a vehicle for miRNA inhibitor delivery to treat osteosarcoma with low toxicity and miR-214 can be a good target for osteosarcoma therapy.

Background MicroRNAs (miRNAs) are widely believed to be promising targets for oral squamous cell carcinoma (OSCC) gene therapy. miR-214 has been identified as a promoter of OSCC aggression and metastasis. Methods Graphene oxide-polyethylenimine (GO-PEI) complexes were prepared and loaded with a miRNA inhibitor at different N/P ratios. The transfection efficiency of GO-PEI-inhibitor was tested in Cal27 and SCC9 cells. Moreover, the tumor inhibition ability of GO-PEI-inhibitor was measured in an OSCC xenograft mouse model by intratumoral injection. Results Here, we show that a GO-PEI complex efficiently delivers a miR-214 inhibitor into OSCC cells and controls the intracellular release of the miR-214 inhibitor. These results indicate that the GO-PEI-miR-214 inhibitor complex efficiently inhibited cellular miR-214, resulting in a decrease in OSCC cell invasion and migration and an increase in cell apoptosis by targeting PTEN and p53. In the xenograft mouse model, the GO-PEI-miR-214 inhibitor complex significantly prevented tumor volume growth. Conclusion This study indicates that functionalized GO-PEI with low toxicity has promising potential for miRNA delivery for the treatment of OSCC.

Diabetes and periodontal diseases have a mutual promoting relationship that induces severe tissue damage and cell death. The potential roles of microRNAs (miRNAs) and the type of cell death involved in diabetes-associated periodontitis are obscure. The gingival tissues of patients were obtained and MC3T3-E1 cells were costimulated with high glucose and lipopolysaccharide (LPS). Osseous morphometric analysis was evaluated with micro-CT, and histological characteristics were measured by hematoxylin/eosin and immunohistochemical staining. Cytokine secretion was confirmed by enzyme-linked immunosorbent assay, and reactive oxygen species (ROS) was measured using a DCFH-DA probe kit. Gene expression was measured by real-time quantitative reverse transcription PCR (qRT-PCR), and protein expression was assessed by Western blot and immunofluorescence analysis. The miR-214 level, receptor-interacting serine-threonine protein (RIP) 1, RIP3, and phospho-mixed lineage kinase domain-like (p-MLKL) protein expression were elevated in the inflamed gingival tissues of diabetes-associated periodontitis patients, with activating transcription factor 4 (ATF4) expression showing the opposite effect. The high glucose (22 mM) could not induce significant increase of RIP1, RIP3, and p-MLKL; however, the high glucose and LPS (500-1000 ng/mL) cotreatment resulted in increase in the number of RIP1, RIP3, and p-MLKL in MC3T3-E1 cells. NAC (ROS inhibitor) inhibited RIP1, RIP3, and increased ATF4; however, necrostatin-1 (Nec-1) (RIP1 inhibitor) specifically inhibited the protein expression of RIP1 and RIP3 and had no influence on ATF4. The use of antagomir-214 suppressed the expression of miR-214, RIP1, RIP3, and p-MLKL, but increased ATF4 protein level in glucose and LPS-induced cells. ATF4 knockdown by ATF4 small interfering RNA offset the effect of antagomir-214. RIP1- and RIP3-dependent necroptosis was confirmed in the inflamed gingival tissues of diabetes-associated periodontitis patients and high glucose- and LPS- cotreated cells. It was suggested that miR-214-targeted ATF4 participated in the regulation of necroptosis in vivo and in vitro.

ZrO₂-NPs are widely applied in industry, biomedicine and dentistry, e.g., foundry sands, refractories, ceramics dental prostheses, dental implant coatings and bone defect restorative materials. To date, little information is available on the potential adverse effects and toxic mechanism in human organs associated with exposure to ZrO₂-NPs. The biodistribution of ZrO₂-NPs and the consequent oxidative stress in the spleen, kidney, heart, brain, and lung at six time points after a single injection of ZrO₂-NPs were examined. Histopathological and immunohistochemical changes were also examined. RNA-Seq analysis was conducted in organs with high ZrO₂-NPs accumulations or obvious histopathological changes (brain and spleen). Exposure to the ZrO₂-NPs led to persistent oxidative stress and cell proliferation promotion/inhibition in various organs. RNA-Seq results of the spleen and brain point to significant gene expression changes. Metabolism was identified as leading pathways in the spleen. This study proves ZrO₂-NPs likely have negative impacts on various organs, and exhibit potential disease risks.

The widespread use of nanomaterials (NMs) has raised concerns that exposure to them may introduce potential risks to the human body and environment. The liver is the main target organ for NMs. Hepatotoxic effects caused by NMs have been observed in recent studies but have not been linked to liver disease, and the intrinsic mechanisms are poorly elucidated. Additionally, NMs exhibit varied toxicokinetics and induce enhanced toxic effects in susceptible livers; however, thus far, this issue has not been thoroughly reviewed. This review provides an overview of the toxicokinetics of NMs. We highlight the possibility that NMs induce hepatic diseases, including nonalcoholic steatohepatitis (NASH), fibrosis, liver cancer, and metabolic disorders, and explore the underlying intrinsic mechanisms. Additionally, NM toxicokinetics and the potential induced risks in the livers of susceptible individuals, including subjects with liver disease, obese individuals, aging individuals and individuals of both sexes, are summarized. To understand how NM type affect their toxicity, the influences of the physicochemical and morphological (PCM) properties of NMs on their toxicokinetics and toxicity are also explored. This review provides guidance for further toxicological studies on NMs and will be important for the further development of NMs for applications in various fields.

BackgroundGamma-delta (γδ) T lymphocytes are primed to potently respond to pathogens and transformed cells by recognizing a broad range of antigens. However, adoptive immunotherapy with γδT cells has exhibited mixed treatment responses. Better understanding of γδT cell biology and stratifying healthy donors for allogeneic adoptive therapy is clinically needed to fully realize the therapeutic potential of γδT cells.MethodsWe examine 98 blood samples from healthy donors and measure their expansion capacity after zoledronate stimulation, and test the migration and cytotoxic effector function of expanded γδT cells in 2D culture, 3D tumor spheroid and patient-derived melanoma organoid assays.ResultsWe find that γδT cell expansion capacity is independent of expansion methods, gender, age and HLA type. Basal γδT cell levels in Peripheral blood mononuclear cell (PBMC) correlate well with their expansion, migration and cytotoxic effector capacity in vitro. Circulating γδT cells with lower expression of PD-1, CTLA-4, Eomes, T-bet and CD69, or higher IFN-γ production expand better. γδT cells with central memory and effector memory phenotypes are significantly more abundant in good expanders. A cut-off level of 0.82% γδT cells in PBMC stratifies good versus poor γδT cell expansion with a sensitivity of 97.78%, specificity of 90.48% and area under the curve of 0.968 in a healthy individual. Donors with higher Vδ2 Index Score in PBMC have greater anti-tumor functions including migratory function and cytotoxicity.ConclusionsOur results demonstrate that the interindividual γδT cell functions correlate with their circulating levels in healthy donors. Examination of circulating γδT cell level may be used to select healthy donors to participate in γδT-based immunotherapies.

Background: Zirconia nanoparticles (ZrO2-NPs) have been increasingly used in industrial, biomedical and dental materials. However, the scientific basis for the toxicological effects of ZrO2-NPs is poorly elucidated, and the understanding of the underlying mechanism is still limited. Results: The hepatic biodistribution and toxicological effects of ZrO2-NPs after intravenous administration (20mg/kg bw) in vivo and the toxicological mechanism toward hepatocytes in vitro were investigated. The liver showed continuous ZrO2-NP accumulations liver over a 28-d period. Moreover, ZrO2-NPs induced oxidative stress and increased inflammatory responses and functional injury in the liver. Hepasteatosis and cell death were observed in histopathological and immunohistochemical studies. RNA-seq identified the main pathways involved in the metabolism, cellular process, and human diseases. The RT-qPCR analysis results showed that ZrO2-NP exposure caused the upregulation of P53, Foxo1, Gadd45g, P21, Caspase3, and PPARα and the downregulation of Igfbp2 and Akt in the liver in response to the ZrO2-NP treatment. Meanwhile, the results of the in vitro studies demonstrated that ZrO2-NPs exposure resulted in cytotoxicity in Hepg2 cells in a dose- and time-dependent manner. ZrO2-NPs were proven to induce oxidative stress, lipid accumulation, cell cycle arrest and cell apoptosis to Hepg2 cells. Western-blot analysis further proved the depression of Igfbp2, activation of Akt-mediated signaling pathway and P53-mediated signaling pathway for Hepg2 cells exposure to ZrO2-NPs. Conclusions: This study proves that ZrO2-NPs have negative impacts on the liver and exhibit potential risks for non-alcoholic fatty liver disease (NAFLD). There is potential concern over ZrO2-NPs' hepatoxicity in biomedical applications and occupational exposure through large-scale production.

Supplemental material for Recombinant Human Cytoglobin Prevents Atherosclerosis by Regulating Lipid Metabolism and Oxidative Stress by Lingling Ou, Xin Li, Baihong Chen, Zhenhuang Ge, Junyi Zhang, Ye Zhang, Gaotai Cai, Zhen Li, Ping Wang, Wenqi Dong in Journal of Cardiovascular Pharmacology and Therapeutics

BackgroundGamma delta (γδ) T cells are attractive effector cells for cancer immunotherapy. Vδ2 T cells expanded by zoledronic acid (ZOL) are the most commonly used γδ T cells for adoptive cell therapy. However, adoptive transfer of the expanded Vδ2 T cells has limited clinical efficacy.MethodsWe developed a costimulation method for expansion of Vδ2 T cells in PBMCs by activating γδ T-cell receptor (γδTCR) and Toll-like receptor (TLR) 7/8 using isopentenyl pyrophosphate (IPP) and resiquimod, respectively, and tested the functional markers and antitumoral effects in vitro two-dimensional two-dimensional and three-dimensional spheroid models and in vivo models. Single-cell sequencing dataset analysis and reverse-phase protein array were employed for mechanistic studies.ResultsWe find that Vδ2 T cells expanded by IPP plus resiquimod showed significantly increased cytotoxicity to tumor cells with lower programmed cell death protein 1 (PD-1) expression than Vδ2 T cells expanded by IPP or ZOL. Mechanistically, the costimulation enhanced the activation of the phosphatidylinositol 3-kinase (PI3K)–protein kinase B (PKB/Akt)–the mammalian target of rapamycin (mTOR) pathway and the TLR7/8–MyD88 pathway. Resiquimod stimulated Vδ2 T-cell expansion in both antigen presenting cell dependent and independent manners. In addition, resiquimod decreased the number of adherent inhibitory antigen-presenting cells (APCs) and suppressed the inhibitory function of APCs by decreasing PD-L1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in these cells during in vitro Vδ2 T-cell expansion. Finally, we showed that human Vδ2 T cells can be expanded from PBMCs and spleen of humanized NSG mice using IPP plus resiquimod or ZOL, demonstrating that humanized mice are a promising preclinical model for studying human γδ T-cell development and function.ConclusionsVδ2 T cells expanded by IPP and resiquimod demonstrate improved anti-tumor function and have the potential to increase the efficacy of γδ T cell-based therapies.

A graded glass/graded zirconia (G/Z) system was previously synthesized via the infiltration of a low modulus nanosized glass into a zirconia surface. The bond strength of G/Z to veneering porcelains was demonstrated to be 3-fold higher than in zirconia-based systems. Nevertheless, biocompatibility testing prior to the possible clinical application of G/Z systems is essential. Herein, such biocompatibility testing was performed with L-929 fibroblasts seeded onto G/Z and yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) for 2-72 h. Assessments included an oral mucous membrane irritation test in conjunction with analyses of cell viability, cell morphology, cell cycle, cell apoptosis, oxidative stress responses, inflammatory cytokine expression, and cellular toxicity. Cell viability tests showed no significant decrease in G/Z- and Y-TZP-treated cells over 72 h. Fluorescence and SEM images demonstrated that cell spreading on Y-TZP and G/Z was similar; cells were flattened and well-spread. Oxidative stress data for G/Z- and Y-TZP-treated cells showed no significant difference in ROS production. Cellular toxicity results for G/Z did not elicit significant differences in LDH release compared with Y-TZP over 72 h. G/Z and Y-TZP had no significant differences in IL-1α, IL-8, PGE2, and TNF-α expression and elicited significantly increased IL-8 expression compared with that of the untreated control group. Cells that were cultured with G/Z showed no significant changes in cell cycle distribution compared with Y-TZP or the untreated control group. Cells that were cultured with Y-TZP and G/Z showed no apoptosis compared to untreated controls at 24 and 48 h. According to the oral mucous membrane irritation test, scores for the macroscopic and microscopic observations for both G/Z and Y-TZP sides were 0, demonstrating no consequent irritation. Therefore, the excellent biocompatibility of G/Z indicates that it has potential for future clinical applications.

Graphene-based materials (GBMs) are widely used in many fields, including biomedicine. To date, much attention had been paid to the potential unexpected toxic effects of GBMs. Here, we review the recent literature regarding the impact of GBMs on programmed cell death (PCD). Apoptosis, autophagy, and programmed necrosis are three major PCDs. Mechanistic studies demonstrated that the mitochondrial pathways and MAPKs (JNK, ERK, and p38)- and TGF-β-related signaling pathways are implicated in GBMs-induced apoptosis. Autophagy, unlike apoptosis and necroptosis which are already clear cell death types, plays a vital pro-survival role in cell homeostasis, so its role in cell death should be carefully considered. However, GBMs always induce unrestrained autophagy accelerating cell death. GBMs trigger autophagy through inducing autophagosome accumulation and lysosome impairment. Mitochondrial dysfunction, ER stress, TLRs signaling pathways, and p38 MAPK and NF-κB pathways participate in GBMs-induced autophagy. Programmed necrosis can be activated by RIP kinases, PARP, and TLR-4 signaling in macrophages after GBMs exposure. Though apoptosis, autophagy, and necroptosis are distinguished by some characteristics, their numerous signaling pathways comprise an interconnected network and correlate with each other, such as the TLRs, p53 signaling pathways, and the Beclin-1 and Bcl-2 interaction. A better understanding of the mechanisms of PCD induced by GBMs may allow for a thorough study of the toxicology of GBMs and a more precise determination of the consequences of human exposure to GBMs. These determinations will also benefit safety assessments of the biomedical and therapeutic applications of GBMs.

Hypercholesterolemia is a key factor leading to β-cell dysfunction, but its underlying mechanisms remain unclear. Secretagogin (Scgn), a Ca2+ sensor protein that is expressed at high levels in the islets, has been shown to play a key role in regulating insulin secretion through effects on the soluble N-ethylmaleimide-sensitive factor attachment receptor protein complexes. However, further studies are required to determine whether Scgn plays a role in hypercholesterolemia-associated β-cell dysfunction. The present study investigated the involvement of a microRNA-24 (miR-24)-to-Scgn regulatory pathway in cholesterol-induced β-cell dysfunction. In the present study, MIN6 cells were treated with increasing concentrations of cholesterol and then, the cellular functions and changes in the miR-24-to-Scgn signal pathway were observed. Excessive uptake of cholesterol in MIN6 cells increased the expression of miR-24, resulting in a reduction in Sp1 expression by directly targeting its 3′ untranslated region. As a transcriptional activator of Scgn, downregulation of Sp1 decreased Scgn levels and subsequently decreased the phosphorylation of focal adhesion kinase and paxillin, which is regulated by Scgn. Therefore, the focal adhesions in insulin granules were impaired and insulin exocytosis was reduced. The present study concluded that a miR-24-to-Scgn pathway participates in the mechanism regulating cholesterol accumulation-induced β-cell dysfunction.

Evidence indicates that microRNAs (miRNAs) play vital roles in regulating osteogenic differentiation and bone formation. Methods: Here, we show that a polyethyleneimine (PEI)-functionalized graphene oxide (GO) complex efficiently loaded with the miR-214 inhibitor is assembled into silk fibroin/hydroxyapatite (SF/HAP) scaffolds that spatially control the release of the miR-214 inhibitor. Results: SF/HAP/GO scaffolds with nanosized GO show high mechanical strength, and their hierarchical microporous structures promote cell adhesion and growth. The SF/HAP/GO-PEI scaffolds loaded with mir-214 inhibitor (SF/HAP/GPM) were tested for their ability to enhance osteogenic differentiation by inhibiting the expression of miR-214 while inversely increasing the expression of activating transcription factor 4 (ATF4) and activating the Akt and ERK1/2 signaling pathways in mouse osteoblastic cells (MC3T3-E1) in vitro. Similarly, the scaffolds activated the osteoblastic activity of endogenous osteoblast cells to repair critical-sized bone defects in rats without the need for loading osteoblast cells. Conclusion: This technology is used to increase osteogenic differentiation and mineralized bone formation in bone defects, which helps to achieve cell-free scaffold-based miRNA-inhibitor therapy for bone tissue engineering.

Fibroblast growth factor receptors (FGFRs) have been implicated in the malignant transformation and chemoresistance of epithelial ovarian cancer; however, the underlying molecular mechanisms are poorly understood. Increased sialyltransferase activity that enhances protein sialylation is an important post‑translational process promoting cancer progression and malignancy. In the present study, α2,6‑sialyltransferase (ST6Gal‑I) overexpression or knockdown cell lines were developed, and FGFR1 was examined to understand the effect of sialylation on migration and drug resistance, and the underlying mechanisms. It was identified that cells with ST6Gal‑I overexpression had increased cell viability and migratory ability upon serum deprivation. Moreover, ST6Gal‑I overexpression cells had strong resistance to paclitaxel, as demonstrated by low growth inhibition rate and cell apoptosis level. A mechanistic study showed that ST6Gal‑I overexpression induced high α2,6‑sialylation of FGFR1 and increased the expression of phospho‑ERK1/2 and phospho‑focal adhesion kinase. Further study demonstrated that the FGFR1 inhibitor PD173047 reduced cell viability and induced apoptosis; however, ST6Gal‑I overexpression decreased the anticancer effect of PD173047. In addition, ST6Gal‑I overexpression attenuated the effect of Adriamycin on cancer cells. Collectively, these results suggested that FGFR1 sialylation plays an important role in cell migration and drug chemoresistance in ovarian cancer cells.

Inorganic nanomaterials have been widely applied in biomedicine. However, several studies have noted that inorganic nanoparticles can enter the brain and induce cytoskeletal remodeling, as well as electrophysiological alterations, which are related to neurodevelopmental disorders and neurodegenerative diseases. The toxic effects of inorganic nanomaterials on the cytoskeleton and electrophysiology are summarized in this review. The relationships between inorganic NPs-induced cytoskeletal and electrophysiological alterations in the central nervous system remain obscure. We propose several potential relationships, including those involving N-methyl-D-aspartate receptor function, ion channels, transient receptor potential channels, and the Rho pathway.

Pressure ulcers (PUs) are serious skin injuries whereby the wound healing process is frequently stalled in the inflammatory phase. Myeloid-derived suppressor cells (MDSCs) accumulate as a result of inflammation and promote cutaneous wound healing by mechanisms that are not fully understood. Recently, MDSCs have been shown to differentiate into fibrocytes, which serve as emerging effector cells that enhance cell proliferation in wound healing. We postulate that in wound healing MDSCs not only execute their immunosuppressive function to regulate inflammation but also stimulate cell proliferation once they differentiate into fibrocytes. In the current study, by using full-thickness and PU mouse models, we found that Kruppel-like factor 4 (KLF4) deficiency resulted in decreased accumulation of MDSCs and fibrocytes, and wound healing was significantly delayed. Conversely, KLF4 activation by the plant-derived product Mexicanin I increased the number of MDSCs and fibrocytes and accelerated the wound healing. Collectively, our study revealed a previously unreported function of MDSCs in cutaneous wound healing and identified Mexicanin I as a potential agent to accelerate PU wound healing.

Due to their unique physicochemical properties, graphene-family nanomaterials (GFNs) are widely used in many fields, especially in biomedical applications. Currently, many studies have investigated the biocompatibility and toxicity of GFNs in vivo and in intro. Generally, GFNs may exert different degrees of toxicity in animals or cell models by following with different administration routes and penetrating through physiological barriers, subsequently being distributed in tissues or located in cells, eventually being excreted out of the bodies. This review collects studies on the toxic effects of GFNs in several organs and cell models. We also point out that various factors determine the toxicity of GFNs including the lateral size, surface structure, functionalization, charge, impurities, aggregations, and corona effect ect. In addition, several typical mechanisms underlying GFN toxicity have been revealed, for instance, physical destruction, oxidative stress, DNA damage, inflammatory response, apoptosis, autophagy, and necrosis. In these mechanisms, (toll-like receptors-) TLR-, transforming growth factor β- (TGF-β-) and tumor necrosis factor-alpha (TNF-α) dependent-pathways are involved in the signalling pathway network, and oxidative stress plays a crucial role in these pathways. In this review, we summarize the available information on regulating factors and the mechanisms of GFNs toxicity, and propose some challenges and suggestions for further investigations of GFNs, with the aim of completing the toxicology mechanisms, and providing suggestions to improve the biological safety of GFNs and facilitate their wide application.