Silver nanoparticles reduce ACE2 expression via changing mitochondrial function in human fibroblast-like lung cell and periodontal ligament fibroblast cells

Silver nanoparticles reduce ACE2 expression via changing mitochondrial function in human fibroblast-like lung cell and periodontal ligament fibroblast cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Silver nanoparticles reduce ACE2 expression via changing mitochondrial function in human fibroblast-like lung cell and periodontal ligament fibroblast cells Shosei Takahashi, Kazuo Tomita, Kento Igarashi, Yoshikazu Kuwahara, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4760785/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Silver nanoparticles (AgNPs) have demonstrated antibacterial properties and are widely recognized as one of the most prominent types of nanoparticles. Recent studies have highlighted their effectiveness against coronaviruses. However, the detailed molecular mechanisms underlying the action of AgNPs on viruses and their impacts on the human body remain to be fully elucidated. Thus, we attempt to delineate the preventive effects of AgNPs against SARS-CoV-2 infection. Our findings indicate that treatment with AgNPs reduces ACE2 expression, a key receptor for SARS-CoV-2 particularly in normal oral and lung cells. Additionally, we observed a decrease in the binding affinity of the spike protein to the cell after AgNP treatment. Through western blot analysis, we identified the involvement of the AKT and/or mTOR signaling pathway in this process. Since AKT and mTOR signaling have been reported to affect mitochondrial function, we investigated the effects of AgNP treatment on mitochondria. As a result, we found the localization of AgNPs within mitochondria. Furthermore, it was accompanied by an increase in mitochondrial Fe 2+ and reactive oxygen species levels, ultimately resulting in mitochondrial dysfunction. Our results underscore the remarkable efficacy of AgNP treatment in preventing coronavirus infections. Silver nanoparticle SARS-CoV-2 ACE2 mitochondria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Nanoparticles are structures with a size ranging from 1 to 100 nm. The technology for researching, developing, and controlling nanoparticles is called nanotechnology, a concept introduced by Richard Feynman at the annual meeting of the American Physical Society held at the California Institute of Technology in 1959 [ 1 ]. Nanotechnology has attracted widespread recognition for its applications in aerospace engineering, medical healthcare, and consumer products [ 2 , 3 ]. Among all nanoparticles, silver nanoparticles (AgNPs) are currently estimated to be the most commercially utilized, particularly in a diverse array of biomedical and consumer products, owing to their broad-spectrum antimicrobial activity [ 4 ]. While silver is generally safe for human consumption, with its use in food products, it is recognized that AgNPs can effectively combat microorganisms in liquids containing approximately 1 µg/ml (1 ppm) of ions, a phenomenon known as “trace metal action“ [ 5 , 6 ]. Furthermore, AgNPs exhibit high diffusivity and stability in liquids. These nanoparticles increase the concentration of silver ions per mass of silver, thereby augmenting their antimicrobial potency. Silver ions exert their antimicrobial effects by denaturing proteins, generating reactive oxygen species (ROS), and damaging DNA upon internalization into the cells of bacteria, fungi, protozoa, and viruses [ 6 , 7 ]. Studies have also demonstrated the effectiveness of AgNPs in inhibiting extracellular SARS-CoV-2, the severe acute respiratory syndrome coronavirus 2 [ 8 ]. AgNPs have been observed to enter cells via endocytosis and localize with mitochondria [ 9 – 11 ]. However, the intricate molecular mechanisms underlying the actions of AgNPs within cells or against SARS-CoV-2 yet to be fully elucidated. COVID-19 is the new infectious respiratory disease in Wuhan, China in December 2019 [ 12 ]. SARS-COV-2, the virus that causes COVID-19 exploits angiotensin-converting enzyme 2 (ACE2) as a cell surface receptor for its spike protein. SARS-COV-2 also exploits transmembrane protease, serine 2 (TMPRSS2) as an essential enzyme for viral cleavage and cellular entry through endocytosis from the cell surface [ 13 – 15 ]. Following SARS-CoV-2 infection, an average incubation period of four to five days precedes the onset of symptoms, with an additional five to six days needed for the viral load to peak [ 16 ]. A strong immune response typically inactivates SARS-CoV-2 and clears infected cells, thereby preventing pulmonary vascular damage and mitigating disease progression [ 16 ]. However, inadequate immune responses may lead to pyroptosis, an inflammatory form of cell death, exacerbating symptoms by up-regulating protease expression and oxidative stress, both crucial for SARS-CoV-2 propagation [ 17 ]. It has been reported that one of the factors that control ACE2 expression is AKT [ 18 ]. It has also been reported mTOR signaling is involved in the control of ACE2 expression because cancer patients receiving mTOR inhibitors as anticancer drugs are lower ACE2 expression [ 19 ]. mTOR has been shown to regulate mitochondria, and the PI3K/Akt/mTOR pathway [ 20 ]. On the other hand, SARS-CoV-2 RNA has been observed to localize to mitochondria after infection, down-regulating mitochondrial membrane potential (Ψm) and inducing mitophagy [ 21 ]. Mitochondria serve as the cell’s powerhouses and as significant sources of ROS and play a critical role in intracellular iron utilization [ 22 , 23 ]. They regulate various forms of cell death, including apoptosis, autophagy, and ferroptosis [ 24 – 26 ]. The generation of mitochondrial ROS stems from electron leakage within the electron transport chain, with approximately 1–3% of electrons typically leaking and reacting with oxygen to produce superoxide [ 27 ]. Superoxide is then neutralized by superoxide dismutase, forming hydrogen peroxide (H 2 O 2 ), a type of ROS. When combined with Fe 2+ , H 2 O 2 generates highly reactive hydroxyl radicals (ཥOH) via the Fenton reaction. This process has dual implications: it can negatively impact the plasma membrane, leading to cell death and exerting bactericidal effects against invading microorganisms [ 28 ]. Additionally, studies have indicated that ACE2 knockdown impairs mitochondrial respiration and reduces ATP production in kidney cells [ 27 ]. Thus, it is suggested that mitochondrial function plays a pivotal role in ACE2-mediated SARS-CoV-2 infection and that AgNP may suppress SARS-CoV-2 infection. However, it remains unclear whether AgNP treatment effectively reduces ACE2 expression and lowers the risk of infection. Therefore, we investigated the cellular response to AgNP treatment, primarily focusing on mitochondrial function, and explored its potential impact on SARS-CoV-2 infection using normal lung cell, which can cause severe pneumonia due to infection, and normal oral cavity cell, which is one of the entry points for infection. Materials and methods Cell culture Human fibroblast-like lung cell (VA-13) were provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. Human Periodontal Ligament Fibroblasts (HPLF) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). VA-13 cells were cultured in RPMI 1640 (Fujifilm Wako, Osaka, Japan) supplemented with 5% fetal bovine serum (FBS; Thermo Fisher Scientific, Massachusetts, USA). HPLF cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS. Silver nanoparticles (AgNP) treatment AgNP were obtained from Pikasshu Co. Ltd (Kumamoto, Japan), with an average diameter of approximately 40 nm. The AgNP solution was prepared by diluting the stock solution from 10,000 µg/ml to 100 µg/ml using milli-Q water (Merck KGaA, Darmstadt, Germany). Subsequently, the appropriate concentration of AgNP was added to the culture medium. Cell viability The effect of AgNP on cell proliferation was assessed using the water-soluble tetrazolium salt assay with Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan), following the manufacturer’s instructions. Cells in the exponential growth phase were seeded in a 96-well plate (5×10 3 per well) for 24 h before experimentation. Subsequently, the cells were treated with AgNP at concentrations of 0.01, 0.1, 1, 10, and 100 µg/ml in RPMI 1640 medium and incubated for 2 h. After incubation, the medium was replaced, and cells were further incubated for 46 h. Following this, 10 µl of CCK-8 reagent was added to each well, and the absorbance of the wells was measured using a microplate reader (Multiskan FC; Thermo Fisher Scientific). Quantitative PCR (qPCR) Total RNA extraction from cells was performed using ISOGEN (Nippon Gene, Toyama, Japan), followed by cDNA synthesis using ReverTra Ace (TOYOBO CO Ltd., Osaka, Japan) according to the manufacturer’s instructions. qPCR analysis was performed using an equivalent of 1 ng of total RNA, as previously described [ 29 ]. The qPCR reaction was conducted using the CFX Duet Real-Time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with THUNDERBIRD qPCR mix (TOYOBO). β-actin served as the internal control. The PCR protocol consisted of an initial denaturation step (95°C, 3 min), followed by 40 amplification cycles (95°C for 10 s, 60°C for 30 s). Each experiment was performed in triplicate, and gene expression levels relative to β-actin were determined using the ΔΔ Ct method. Primer sequences are provided below. ACE2 F: 5ʹ-GGGATCAGAGATCGGAAGAAGAAA-3ʹ ACE2 R: 5ʹ-AGGAGGTCTGAACATCATCAGTG-3ʹ TMPRSS2 F: 5ʹ-AATCGGTGTGTTCGCCTCTAC-3ʹ TMPRSS2 R: 5ʹ-CGTAGTTCTCGTTCCAGTCGT-3ʹ β-actin F: 5ʹ-AGAGCTACGAGCTGCCTGAC-3ʹ β-actin R: 5ʹ-AGCACTGTGTTGGCGTACAG-3ʹ SARS-CoV-2 spike Protein binding assay The interaction between the cell surface receptor and the SARS-CoV-2 spike protein was observed using a BZ-8000 fluorescence microscope after the fluorescent labeling of the spike protein. The RBD-S1 spike protein (SARS-CoV-2 spike protein) His Tag (HEK293) (Aviscera Bioscience, Inc., Santa Clara, CA, USA) was labeled with fluorescein using the Fluorescein Labeling Kit (Dojindo) in accordance with the manufacturer’s instructions. After a 2-h treatment with AgNPs, spike protein with FITC in Hank’s balanced salt solution (HBSS) (250 ng/ml) was applied to the cells in glass-bottomed dishes and incubated for 10 min at 37°C. Subsequently, the cells were washed twice with HBSS, and the spike protein with FITC was visualized using a fluorescence microscope equipped with a PlanFluor ELWD DM 20×/0.45 NA lens and a GFP-BP filter. Western blotting After treatment with 0.1 µg/ml of AgNP for 2 h, cells were harvested and incubated with cell lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 1 mM sodium fluoride, 1 mM sodium vanadate, and 1 mM phenylmethylsulfonyl fluoride. Equal amounts of protein (20 µg per lane) from each cell lysate were separated by SDS-polyacrylamide gel electrophoresis using 7.5% (for mTOR) or 10% (for pAKT T308 and ACE2) polyacrylamide gels under reducing conditions. The separated proteins were then transferred onto a polyvinylidene difluoride membrane. Following blocking with 5% skim milk in PBST (blocking solution), the membranes were incubated overnight at 4°C with specific primary antibodies (ACE2: 28868-1-AP, Proteintech, Rosemont, IL, USA; mTOR: 2983, pAKT T308 : 13038, Cell Signaling Technology) diluted 1:1000 in blocking solution. After washing five times with PBST, the membranes were incubated with ECL™ anti-rabbit IgG, horseradish peroxidase linked whole antibody (NA934V: Global Life Sciences Technologies Japan K.K., Tokyo, Japan; diluted 1:2000) at room temperature for 2 h. After another five washes with PBST, immunoreactive proteins were visualized using ImmunoStar Zeta (Fujifilm Wako) and imaged with a ChemiDoc XRS Plus instrument (Bio-Rad Laboratories). Antiβ-actin antibody (4970L: Cell Signaling Technology; diluted 1:2000) was used as a loading control. Detection of AgNPs in mitochondria The scattered light imaging method [ 30 ] was employed to detect AgNPs. Cells were treated with 50 nM MitoTracker Red CMXRos (Thermo Fisher Scientific) and 0.1 µg/ml AgNP in HBSS for 30 min at 37°C. Subsequently, the medium containing MitoTracker Red CMXRos and AgNPs was aspirated, and cells were washed with HBSS to remove any residual dye and nanoparticles. The colocalization of AgNP and MitoTracker Red CMXRos within the cells was visualized using a BZ-8000 fluorescence microscope (Keyence Corporation, Osaka, Japan) equipped with a PlanApo 20×/0.75 NA lens, GFP-BP, and Texas Red filters. Detection of mitochondrial Fe 2+ , ROS, and membrane potential Mitochondrial Fe 2+ , mtROS, and Ψm were assessed using Mito-FerroGreen (Dojindo), mitoSOX (Thermo Fisher Scientific), and JC-10™ (Thermo Fisher Scientific), respectively. Following a 2-h treatment with AgNPs, cells in glass-bottomed dishes were exposed to 5 µM Mito-FerroGreen, 5 µM mitoSOX, or 2 µM JC-10 in HBSS (084-08345: Fujifilm Wako) for 30 min at 37°C. After the incubation period, cells were washed twice with fresh HBSS. Fluorescence images were captured using a BZ-8000 fluorescence microscope (Keyence Corporation, Osaka, Japan) equipped with a PlanFluor ELWD DM 20×/0.45 NA lens, GFP-BP, and Texas Red filters. Fluorescence intensity was quantified using ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/ , 1997–2012). Endocytosis inhibitor assay To inhibit endocytosis of AgNP, Pitstop2 (Selleck Biotech, Kanagawa Japan) and Dynasore (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were treated to VA-13 and HPLF. Briefly, cells in glass-bottomed dishes were treated with 20 µM Pitstop2 or 20 µM Dynasore for 15 min, then treated with 0.1 µg/ml of AgNP for 2 h. After the AgNP treatment, cells were exposed to 5 µM mitoSOX in HBSS (084-08345: Fujifilm Wako) for 30 min at 37°C. Fluorescence images were captured using a BZ-8000 fluorescence microscope as described above. Statistical analysis Student’s t -test was conducted following the F test to compare the two groups. For comparisons involving three or more groups, one-way ANOVA with Scheffe’s F test was conducted using Statcel4 software (OMS publishing, Saitama, Japan). A significance level of P < 0.05 was adopted. The results are presented as means ± standard errors of the mean. Results The cell viability by AgNP treatment No significant change in cell viability was observed with AgNP treatment at concentrations blow 0.1 µg/ml. However, upon exposure to 1 µg/ml of AgNP, cells exhibited a decreased cell survival rate after 48 h (Fig. 1 a). Microscopic examination at this time point revealed that the cells appeared swollen, resembling balloons (Fig. 1 b). ACE2 gene expression was decreased by AgNP treatment in normal cells We examined the cellular response to 0.1 µg/ml of AgNP treatment, a concentration that did not significantly affect cell viability across both cell lines. Following treatment, the expressions of ACE2 and TMPRSS2 genes were down-regulated in both cell lines (Fig. 2 ). These findings suggest that AgNP treatment reduces the susceptibility of cells to coronavirus infection. AgNP treatment reduced the binding of spike protein to cells To assess the spike protein’s binding capability to the cell surface, we performed the SARS-CoV-2 spike protein binding assay using FITC-conjugated spike protein. A decreased FITC signal was noted following treatment with 0.1 µg/ml of AgNP (Fig. 3 ). Protein expression change after AgNP treatment To elucidate the molecular mechanism underlying AgNP treatment, we investigated protein expression following exposure to AgNP (Fig. 4 ). In VA13 cells, the expression of mTOR was down-regulated by AgNP treatment. In HPLF cells, the phosphorylation of AKT at T308 was promoted. ACE2 expression was down-regulated upon treatment with 0.1 µg/ml of AgNP in both cell lines. Treated AgNPs localize mainly in mitochondria We employed the scattered light imaging method [ 38 ] to elucidate the subcellular localization of AgNP. This technique allows for real-time, label-free detection of AgNP in living cells. Through this approach, we successfully identified AgNP in mitochondria following AgNP treatment (Figs. 5 a–c). Additionally, we observed green dot-like fluorescence when AgNP were immobilized in polyacrylamide gel and examined in a glass-bottomed dish. Importantly, this green fluorescence was not evident in the absence of AgNP administration (see Supplemental Fig. 1), confirming its association with AgNP. Furthermore, no green fluorescence was observed under identical conditions when cells were treated solely with MitoTracker, indicating that this fluorescence does not arise from MitoTracker Red leakage (Figs. 5 d–f). Effects of AgNP treatment on mitochondria Having observed the localization of AgNP in mitochondria, we examined their impact on mitochondrial function. Consequently, we noted a substantial increase in mitochondrial Fe 2+ levels and mtROS after AgNP treatment. Additionally, AgNP treatment led to a significant down-regulation of Ψm (Fig. 6 ). Effect of endocytosis inhibitors on mitoSOX expression after AgNP treatment We investigated the effect of endocytosis inhibitors After AgNP treatment using Pitstop2 and Dynasore. As a result, both inhibitors effectively inhibited the mtROS generation after AgNP treatment (Fig. 7 ). This result indicates that AgNPs enter cells by endocytosis and influence at least the generation of mtROS. Discussion In this study, we demonstrated that AgNPs exert effects on mitochondria and suppress ACE2 expression, which is particularly evident in normal cells. Our findings suggest the potential of AgNP in preventing SARS-CoV-2 infection. However, further investigations are needed to determine the optimal concentration and size of AgNP for prevention and their impact on coronavirus proliferation after infection. For example, it has been reported that the cytotoxicity may vary for different cell types and different AgNP particle sizes [ 8 , 31 ]. In conjunction with previous research, our study underscores the preventive efficacy of AgNP against coronavirus infections. Therefore, practices such as handwashing, gargling, and using AgNP-containing products such as toothpaste and toothbrushes could be highly effective in preventing viral infections. ACE2 was originally discovered as a membrane-bound peptidase that degrades ANG II to Ang-(1–7) in the cell. ANG II binds to the ANG II type 1 receptor, stimulating inflammation, fibrosis, oxidative stress, and an increase in blood pressure. Ang-(1–7) binds to the Mas receptor, exerting anti-inflammatory and antifibrotic effects, stimulating nitric oxide release, and reducing blood pressure. One of the factors that control ACE2 expression is AKT signaling. AKT, a serine/threonine kinase, plays important roles in multiple cellular processes, including glucose metabolism, and is known to inhibit AMPK [ 32 ]. AMPK is activated in response to a shortage of intracellular energy and regulates metabolism [ 33 ]. Additionally, AMPK has been reported to phosphorylate and stabilize ACE2 [ 34 ]. Therefore, in HPLF cells, AKT activation via phosphorylation inhibits AMPK, which likely prevents the stabilization of ACE2 and reduces its expression. AgNPs are primarily internalized into cells via endocytosis, where they are metabolized within intracellular lysosomes [ 35 – 38 ]. Subsequently, AgNPs leak from lysosomes as silver ions, eventually localizing to mitochondria, where they induce ROS generation in a dose-dependent manner. Combined with our findings, it is plausible that silver ions transfer ions to iron in mitochondria, promoting ROS production through the Fenton reaction. Silver ions transferred to mitochondria have been reported to affect the electron transport chain, leading to decreased oxygen consumption [ 36 ] and mitochondrial dysfunction, ultimately reducing membrane potential [ 39 ]. While prior studies have indicated that decreased ACE2 expression correlates with mitochondrial dysfunction, the exact relationship between mitochondrial status and ACE2 expression remains unclear [ 27 ]. Our study revealed that AgNPs internalized into cells are translocated to mitochondria, leading to decreased ACE2 expression in VA-13, and HPLF cells. This suggests that mitochondrial status may indeed influence ACE2 expression. Moreover, phosphorylation of AKT in mitochondria, potentially regulated by AgNP treatment, may play a role in modulating ACE2 expression. It has been reported that AKT and prohibitin 1 (PHB1), a constituent protein of mitochondria, are phosphorylated and form a complex in mitochondria [ 40 ]. Additionally, PHB2 has been shown to bind directly to AKT [ 41 ]. These findings suggest that AgNP treatment induces AKT phosphorylation in the mitochondria, subsequently regulating the expression of ACE2. On the other hand, it has been reported that cancer patients receiving mTOR inhibitors as anticancer drugs are less likely to contract COVID-19 due to lower ACE2 expression [ 19 ]. mTOR has been shown to regulate mitophagy, mitochondria, and the PI3K/Akt/mTOR pathway [ 20 ], indicating a close interrelation among these processes. Further investigations are needed to elucidate the mechanisms underlying AgNPs’ ability to mitigate coronavirus infections, AgNPs appear to provide a beneficial effect by preventing SARS-CoV-2 infection of cells by down-regulating ACE-2 receptors. However, AgNPs can themselves be cytotoxic by causing mitochondrial dysfunction as shown in this study and other studies [ 31 , 42 ]. SARS-CoV-2 infection can also cause alteration of mitochondrial function [ 43 ]. Hence AgNPs should be used with caution. Use along with mitochondrial protective agents such as N-Acetyl L-Cysteine can help to extract the benefits of AgNPs while minimizing their side effects. Conclusion In this study, we have elucidated that AgNP treatment effectively reduces ACE2 expression and the spike protein’s binding to the cell surface in normal cells. Furthermore, we have demonstrated that AgNPs localize to mitochondria and exhibit varying routes of infection suppression depending on the cell type. A schematic representation of the potential mechanism underlying the inhibition of SARS-CoV-2 infection by AgNP is presented in Fig. 8 . Future investigations should encompass exploring the effects of AgNP treatment on organs such as the heart and kidneys, where ACE2 plays pivotal roles, evaluating the long-term biological impacts of exposure, and assessing potential synergistic effects with other mitochondria-targeting drugs. Nonetheless, the safety profile of silver, widely used in food, offers considerable reassurance regarding its potential to prevent coronavirus infections. Mitochondria-targeted therapeutic and preventive approaches employing AgNPs are promising as effective tools against coronavirus infections. Declarations Ethical Statements: Not applicable. This study only involves in vitro experiments and there was no involvement of Human Participants and/or Animals in it. Competing Interests Shosei Takahashi is an officer of Pikasshu Co., Ltd. and owns stock in Pikasshu Co., Ltd. but receives no compensation as a member of the board of directors. Funding: This work was supported by JSPS KAKENHI: Grant Number 24K13073. Author Contribution S.T. and K.T. designed and carried out experiments, analyzed the data and wrote the manuscript. K.I., Y.K., J.K., N.K., K.T., and A.K. helped perform experiments, finalized results, and reviewed and edited the manuscript. Y.K. designed the experiment, took part in the optimization of the method, and reviewed and edited the manuscript. T.S. designed and supervised the study and reviewed and edited the manuscript. Data availability The materials and data used and/or analyzed in the present study are available from the corresponding author upon reasonable request. References Feynman RP. (1960) There’s plenty of room at the bottom. Eng. Sci. 23(5):22-36. Gerber C, Lang HP. (2006) How the doors to the nano-world were opened. Nat Nanotech 1:3-5. https://doi.org/10.1038/nnano.2006.70. Singh N, Manshian B, Jenkins GJ, Griffiths SM, Williams PM, Maffeis TG, et al. (2009) NanoGenotoxicology: the DNA damaging potential of engineered nanomaterials. Biomaterials. 30(23-24):3891-3914. https://doi.org/10.1016/j.biomaterials.2009.04.009. Durán N, Durán M, de Jesus MB, Seabra AB, Fávaro WJ, Nakazato G. (2016) Silver nanoparticles: A new view on mechanistic aspects on antimicrobial activity. Nanomedicine. 12(3):789-799. https://doi.org/10.1016/j.nano.2015.11.016. Naegeli, von V. Deut. Schr. Schweiz. (1893) Naturforsch. Ges. 33:174-182. Russell AD, Hugo WB. (1994) Antimicrobial activity and action of silver. Prog Med Chem. 31:351-370. https://doi.org/10.1016/s0079-6468(08)70024-9. Galdiero S, Falanga A, Vitiello M, Cantisani M, Marra V, Galdiero M. (2011) Silver nanoparticles as potential antiviral agents. Molecules. 16(10):8894-8918. https://doi.org/10.3390/molecules16108894. Jeremiah SS, Miyakawa K, Morita T, Yamaoka Y, Ryo A. (2020) Potent antiviral effect of silver nanoparticles on SARS-CoV-2. Biochem Biophys Res Commun. 533(1):195-200. https://doi.org/10.1016/j.bbrc.2020.09.018. Wu M, Guo H, Liu L, Liu Y, Xie L. (2019) Size-dependent cellular uptake and localization profiles of silver nanoparticles. Int J Nanomedicine. 14:4247-4259. https://doi.org/10.2147/IJN.S201107. Li J, Zhang B, Chang X, Gan J, Li W, Niu S, et al. (2020) Silver nanoparticles modulate mitochondrial dynamics and biogenesis in HepG2 cells. Environ Pollut. 256:113430. https://doi.org/10.1016/j.envpol.2019.113430. Dey S, Fageria L, Sharma A, Mukherjee S, Pande S, Chowdhury R, Chowdhury S. (2022) Silver nanoparticle-induced alteration of mitochondrial and ER homeostasis affects human breast cancer cell fate. Toxicol Rep. 9:1977-1984. doi: 10.1016/j.toxrep.2022.10.017. Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. (2020) A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 382(8):727-733. https://doi.org/10.1056/NEJMoa2001017. Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 579(7798):270-273. https://doi.org/10.1038/s41586-020-2012-7. Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, et al. (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 181(2):271-280.e8. https://doi.org/10.1016/j.cell.2020.02.052. Azkur AK, Akdis M, Azkur D, Sokolowska M, van de Veen W, Brüggen MC, et al. (2020) Immune response to SARS-CoV-2 and mechanisms of immunopathological changes in COVID-19. Allergy. 75(7):1564-1581. https://doi.org/10.1111/all.14364. Tay MZ, Poh CM, Rénia L, MacAry PA, Ng LFP. (2020) The trinity of COVID-19: immunity, inflammation and intervention. Nat Rev Immunol. 20(6):363-374. https://doi.org/10.1038/s41577-020-0311-8. Delgado-Roche L, Mesta F. (2020) Oxidative Stress as Key Player in Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Infection. Arch Med Res. 51(5):384-387. https://doi.org/10.1016/j.arcmed.2020.04.019. Wang B, Li D, Fiselier A, Kovalchuk I, Kovalchuk O. (2022) New AKT-dependent mechanisms of anti-COVID-19 action of high-CBD Cannabis sativa extracts. Cell Death Discov. 8(1):110. https://doi.org/10.1038/s41420-022-00876-y. Foote MB, White JR, Jee J, Argilés G, Wan JCM, Rousseau B, et al. (2021) Association of Antineoplastic Therapy With Decreased SARS-CoV-2 Infection Rates in Patients With Cancer. JAMA Oncol. 7(11):1686-1691. https://doi.org/10.1001/jamaoncol.2021.3585. Qiao L, Guo Z, Liu H, Liu J, Lin X, Deng H, et al. (2022) Protective Effect of Mitophagy Regulated by mTOR Signaling Pathway in Liver Fibrosis Associated with Selenium. Nutrients. 14(12):2410. https://doi.org/10.3390/nu14122410. Shang C, Liu Z, Zhu Y, Lu J, Ge C, Zhang C, et al. (2022) SARS-CoV-2 Causes Mitochondrial Dysfunction and Mitophagy Impairment. Front Microbiol. 12:780768. https://doi.org/10.3389/fmicb.2021.780768. Indo HP, Davidson M, Yen HC, Suenaga S, Tomita K, Nishii T, et al. (2007) Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage. Mitochondrion. 7;106-118. https://doi.org/10.1016/j.mito.2006.11.026. Andrews NC. (1999) Disorders of iron metabolism. N Engl J Med. 341(26):1986-1995. https://doi.org/10.1056/NEJM199912233412607. Green DR, Reed JC. (1998) Mitochondria and apoptosis. Science. 281; 1309-1312. https://doi.org/10.1126/science.281.5381.1309. Chandel NS. (2014) Mitochondria as signaling organelles. BMC Biol. 12(1):1-7. https://doi.org/10.1186/1741-7007-12-34. Takashi Y, Tomita K, Kuwahara Y, Roudkenar MH, Roushandeh AM, Igarashi K, et al. (2020) Mitochondrial dysfunction promotes aquaporin expression that controls hydrogen peroxide permeability and ferroptosis. Free Radic Biol Med. 161:60-70. https://doi.org/10.1016/j.freeradbiomed.2020.09.027. Boveris A, Chance B. (1973) The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J. 134:707-716. https://doi.org/10.1042/bj1340707. Shi TT, Yang FY, Liu C, Cao X, Lu J, Zhang XL, et al. (2018) Angiotensin-converting enzyme 2 regulates mitochondrial function in pancreatic β-cells. Biochem Biophys Res Commun. 495(1):860-866. https://doi.org/10.1016/j.bbrc.2017.11.055. Tomita K, Kuwahara Y, Takashi Y, Tsukahara T, Kurimasa A, Fukumoto M, et al. (2017) Sensitivity of mitochondrial DNA depleted rho0 cells to H 2 O 2 depends on the plasma membrane status. Biochem Biophys Res Commun. 490(2):330-335. https://doi.org/10.1016/j.bbrc.2017.06.044 Wang F, Chen B, Yan B, Yin Y, Hu L, Liang Y, et al. (2019) Scattered Light Imaging Enables Real-Time Monitoring of Label-Free Nanoparticles and Fluorescent Biomolecules in Live Cells. J Am Chem Soc. 141(36):14043-14047. https://doi.org/10.1021/jacs.9b05894. He J, Ma Y, Niu X, Pei J, Yan R, Xu F, et al. (2024) Silver nanoparticles induce endothelial cytotoxicity through ROS-mediated mitochondria-lysosome damage and autophagy perturbation: The protective role of N-acetylcysteine. Toxicology. 502:153734. https://doi.org/10.1016/j.tox.2024.153734. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR. (2003) Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem. 278(41):39422-394227. https://doi.org/10.1074/jbc.M305371200. Herzig S, Shaw RJ. (2018) AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol. 19(2):121-135. https://doi.org/10.1038/nrm.2017.95. Zhang J, Dong J, Martin M, He M, Gongol B, Marin TL, et al. (2018) AMP-activated Protein Kinase Phosphorylation of Angiotensin-Converting Enzyme 2 in Endothelium Mitigates Pulmonary Hypertension. Am J Respir Crit Care Med. 198(4):509-520. https://doi.org/10.1164/rccm.201712-2570OC. Wu M, Guo H, Liu L, Liu Y, Xie L. (2019) Size-dependent cellular uptake and localization profiles of silver nanoparticles. Int J Nanomedicine. 14:4247-4259. https://doi.org/10.2147/IJN.S201107. Miyayama T, Arai Y, Suzuki N, Hirano S. (2013) Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate. Toxicology. 305:20-29. https://doi.org/10.1016/j.tox.2013.01.004. Miyayama T, Matsuoka M. (2016) Involvement of lysosomal dysfunction in silver nanoparticle-induced cellular damage in A549 human lung alveolar epithelial cells. J Occup Med Toxicol. 11:1. https://doi.org/10.1186/s12995-016-0090-0. Brkić Ahmed L, Milić M, Pongrac IM, Marjanović AM, Mlinarić H, Pavičić I, et al. (2017) Impact of surface functionalization on the uptake mechanism and toxicity effects of silver nanoparticles in HepG2 cells. Food Chem Toxicol. 107(Pt A):349-361. https://doi.org/10.1016/j.fct.2017.07.016. Singh RP, Ramarao P. (2012) Cellular uptake, intracellular trafficking and cytotoxicity of silver nanoparticles. Toxicol Lett. 213(2):249-259. https://doi.org/10.1016/j.toxlet.2012.07.009. Li XH, Chai RR, Chen GW, Zhang LF, Tan-Tai WJ, Shi HJ, et al. (2020) Prohibitin (PHB) interacts with AKT in mitochondria to coordinately modulate sperm motility. Asian J Androl. 22(6):583-589. https://doi.org/10.4103/aja.aja_46_20. Sun L, Liu L, Yang XJ, Wu Z. (2004) Akt binds prohibitin 2 and relieves its repression of MyoD and muscle differentiation. J Cell Sci. 117(Pt 14):3021-3029. https://doi.org/10.1242/jcs.01142. Li J, Zhang B, Chang X, Gan J, Li W, Niu S, et al. (2020) Silver nanoparticles modulate mitochondrial dynamics and biogenesis in HepG2 cells. Environ Pollut. 256:113430. https://doi.org/10.1016/j.envpol.2019.113430. Gao Y, Kok WL, Sharma V, Illsley CS, Hanks S, Tredwin C, et al. (2023) SARS-CoV-2 infection causes periodontal fibrotic pathogenesis through deregulating mitochondrial beta-oxidation. Cell Death Discov. 9(1):175. https://doi.org/10.1038/s41420-023-01474-2. Additional Declarations Competing interest reported. Shosei Takahashi is an officer of Pikasshu Co., Ltd. and owns stock in Pikasshu Co., Ltd. but receives no compensation as a member of the board of directors. Supplementary Files TakahashietalforMolbiolRep2024SupplementalFigures.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4760785","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":336361190,"identity":"897adbe3-9a6f-4b50-bcda-5dd82cce7940","order_by":0,"name":"Shosei Takahashi","email":"","orcid":"","institution":"Kagoshima University Graduate School of Medical and Dental Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shosei","middleName":"","lastName":"Takahashi","suffix":""},{"id":336361193,"identity":"41c67644-7671-4034-9238-01ad2778736a","order_by":1,"name":"Kazuo Tomita","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABNUlEQVRIie2RMUvDQBSAXynEJZg1R7H+hQsBQZD0r1wJpMvVP1ChASGb7ZpM/oWOjtHDZDl1PSdbBKcOEUE6qS8WqTWpdBS5b7i7x9333r07AI3mD2KXAwNoQ1quPgdzOa32fyhGqTBw8Vgj3F7BMt3wu/Ir5Pz0ejJbeL1RfpZPQXre2Lq5vFInmGRHTOHwoqK0DCNQjPn9RN52Q1C+n8THTPAMFTOgQGRFaRvmASrN/kRxJ4Si6VNlUsGNd0zA8RbRJmXYow/zUhn69E6i8oZVrHmt0loqgmFyVJTwaMqp6Eeo2PVVSBRgL0HuJJI7MZM5I3GpjMCN7Cea1vRiC5HdL44G+7u5pEWRDTqWJd0X/gp7Y8ufPZLqi63DAO/zFZQ/Jki46eyKzlrUeN5C0Wg0mv/OB3uydStZCBbXAAAAAElFTkSuQmCC","orcid":"","institution":"Kagoshima University Graduate School of Medical and Dental Sciences","correspondingAuthor":true,"prefix":"","firstName":"Kazuo","middleName":"","lastName":"Tomita","suffix":""},{"id":336361194,"identity":"0145024d-aa05-4cd0-a1fa-c8ca51a62b01","order_by":2,"name":"Kento Igarashi","email":"","orcid":"","institution":"Kagoshima University Graduate School of Medical and Dental Sciences","correspondingAuthor":false,"prefix":"","firstName":"Kento","middleName":"","lastName":"Igarashi","suffix":""},{"id":336361195,"identity":"d508f981-3a3e-4c9d-9ee8-da73ae58a798","order_by":3,"name":"Yoshikazu Kuwahara","email":"","orcid":"","institution":"Tohoku Medical and Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Yoshikazu","middleName":"","lastName":"Kuwahara","suffix":""},{"id":336361199,"identity":"01d09228-a624-416e-92cf-e2a908e503ab","order_by":4,"name":"Junichi Kitanaka","email":"","orcid":"","institution":"Hyogo Medical University","correspondingAuthor":false,"prefix":"","firstName":"Junichi","middleName":"","lastName":"Kitanaka","suffix":""},{"id":336361203,"identity":"05f5e9de-3156-45ed-bd2d-db866379063d","order_by":5,"name":"Nobue Kitanaka","email":"","orcid":"","institution":"Hyogo Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nobue","middleName":"","lastName":"Kitanaka","suffix":""},{"id":336361205,"identity":"5ae28c46-b416-44cb-8762-8e4edc5f10bc","order_by":6,"name":"Koh-ichi Tanaka","email":"","orcid":"","institution":"Hyogo Medical University","correspondingAuthor":false,"prefix":"","firstName":"Koh-ichi","middleName":"","lastName":"Tanaka","suffix":""},{"id":336361206,"identity":"ec9b1717-db4a-4607-b689-5cdfa46b0731","order_by":7,"name":"Akihiro Kurimasa","email":"","orcid":"","institution":"Tohoku Medical and Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Akihiro","middleName":"","lastName":"Kurimasa","suffix":""},{"id":336361209,"identity":"2adc9a43-8adf-46e0-8ad5-a94fb97b6328","order_by":8,"name":"Yoshiaki Kamikawa","email":"","orcid":"","institution":"Kagoshima University Graduate School of Medical and Dental Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yoshiaki","middleName":"","lastName":"Kamikawa","suffix":""},{"id":336361210,"identity":"e8ed457d-0ba3-4493-bf2a-329475708e55","order_by":9,"name":"Tomoaki Sato","email":"","orcid":"","institution":"Kagoshima University Graduate School of Medical and Dental Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tomoaki","middleName":"","lastName":"Sato","suffix":""}],"badges":[],"createdAt":"2024-07-18 07:44:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4760785/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4760785/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62361182,"identity":"672ebcd9-f2f0-4c01-8f93-a70eb13647ee","added_by":"auto","created_at":"2024-08-13 10:12:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1400081,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability and morphology after AgNP treatment. \u003cstrong\u003ea\u003c/strong\u003e: Cell survival ratio. Cells were exposed to varying concentrations of AgNP (0.01, 0.1, 1, 10, and 100 µg/ml), and their survival ratios relative to the control were determined using CCK-8 after 48 h. **: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 as determined by Scheffe’s F test (vs control). \u003cstrong\u003eb\u003c/strong\u003e: Morphology of AgNP-treated cells. Cellular morphology was examined under an optical microscope after a 2-h treatment with AgNP at 1 µg/ml. A balloon-like cellular morphology, which seemed to be necrosis was observed after AgNP treatment.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4760785/v1/3bc9a1ee98da6ab9a8d5dee6.png"},{"id":62361186,"identity":"f7490612-1171-48b5-a99a-441d9209936d","added_by":"auto","created_at":"2024-08-13 10:12:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":277717,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression changes after AgNP treatment. The relative expression levels of \u003cem\u003eACE2\u003c/em\u003e, and \u003cem\u003eTMPRSS2\u003c/em\u003e were measured using quantitative PCR two hours after treatment. Upper row: Relative \u003cem\u003eACE2\u003c/em\u003e expression. Lower row: Relative \u003cem\u003eTMPRSS2\u003c/em\u003eexpression. AgNP treatment significantly reduced \u003cem\u003eACE2\u003c/em\u003e and \u003cem\u003eTMPRSS2\u003c/em\u003eexpression in VA-13 and HPLF cells. **: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 as determined by Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4760785/v1/833759c648465276695c7eda.png"},{"id":62362083,"identity":"c705e297-d6b1-40d5-9ce7-073812248a19","added_by":"auto","created_at":"2024-08-13 10:20:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":959144,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in cell and SARS-CoV-2 spike protein binding after AgNP treatment. The binding of the SARS-CoV-2 spike protein to cell surface receptors was assayed using fluorescently labeled SARS-CoV-2 spike protein. \u003cstrong\u003ea\u003c/strong\u003e: Representative fluorescence images of cells. Upper row: control, lower row: AgNP treatment. \u003cstrong\u003eb, c\u003c/strong\u003e: Changes in relative fluorescent intensity after AgNP treatment. \u003cstrong\u003eb\u003c/strong\u003e: VA-13 cells. \u003cstrong\u003ec\u003c/strong\u003e: HPLF cells. Fluorescence images were captured from three independent dishes for each treatment. Reduced signal intensity was observed after treatment with 0.1 µg/ml of AgNP in VA-13, and HPLF cells. **: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 as determined by Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4760785/v1/de64d21eeae6b07a6e55cd8a.png"},{"id":62362766,"identity":"24020018-a293-492b-a57f-999b20caac8d","added_by":"auto","created_at":"2024-08-13 10:28:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":354561,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blotting. Western blotting of mTOR, pAKT\u003csup\u003eT308\u003c/sup\u003e and ACE2 after AgNP treatment. Twenty micrograms of each cell lysate were separated by SDS-PAGE and transferred to PVDF membranes. In VA-13 cells, the expression of pAKT\u003csup\u003eT308\u003c/sup\u003e was down-regulated by AgNP treatment. In HPLF cells, an up-regulation was observed in the expression of phosphorylation of AKT\u003csup\u003eT308\u003c/sup\u003e. Subsequent analysis revealed that the expression of ACE2 was down-regulated after treatment with 0.1 µg/ml of AgNP in VA-13, and HPLF cells.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4760785/v1/ac8f4468b11f2043df3aecf9.png"},{"id":62361184,"identity":"bdb3a72d-fc8f-4848-9f8c-a8758ff80cc0","added_by":"auto","created_at":"2024-08-13 10:12:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1999373,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of AgNPs in mitochondria.\u003cstrong\u003e \u003c/strong\u003eThe scattered light imaging method was used to detect AgNP using a BZ-8000 fluorescent microscope. MitoTracker Red CMXRos and 0.1 µg/ml AgNPs in HBSS were treated for 30 min in HPLF cells. \u003cstrong\u003ea\u003c/strong\u003e: AgNP detection using the GFP-BP filter. \u003cstrong\u003eb\u003c/strong\u003e: Mitochondrial detection using MitoTracker Red CMXRos. \u003cstrong\u003ec\u003c/strong\u003e: The merged image of a and b. \u003cstrong\u003ed\u003c/strong\u003e: AgNP detection using the GFP-BP filter without AgNP treatment. \u003cstrong\u003ee\u003c/strong\u003e: Mitochondrial detection using MitoTracker Red CMXRos without AgNP treatment. \u003cstrong\u003ef\u003c/strong\u003e: The merged image of d and e. After AgNP treatment, merged AgNP and mitochondria appeared as yellow dots (white arrowhead in \u003cstrong\u003ec\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4760785/v1/3788e01b134ff9294dc07663.png"},{"id":62361190,"identity":"2cf32a94-5815-49ff-8c2f-f29b88150135","added_by":"auto","created_at":"2024-08-13 10:12:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1481641,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of mitochondrial Fe\u003csup\u003e2+\u003c/sup\u003e, ROS (mtROS), and membrane potential (Ψm). Mitochondrial Fe\u003csup\u003e2+\u003c/sup\u003e, mtROS, and Ψm levels were assessed using Mito-FerroGreen, MitoSOX, and JC-10 after a two-hour AgNP treatment. Upper low: Representative fluorescence images and relative fluorescent intensity changes of Fe\u003csup\u003e2+\u003c/sup\u003e after AgNP treatment. Middle row: Representative fluorescence images and relative fluorescent intensity changes of mtROS, Lower row: Representative fluorescence images and relative fluorescent intensity changes of Ψm. Fluorescence images were captured from three separate dishes for each treatment. AgNP treatment led to significant increases in mitochondrial Fe\u003csup\u003e2+\u003c/sup\u003e and mtROS levels and significant decrease in Ψm. **: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by Student’s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4760785/v1/2fabdf4368328fcbb098b713.png"},{"id":62361191,"identity":"0be06376-cc18-4b79-b485-d801002de9d9","added_by":"auto","created_at":"2024-08-13 10:12:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1161070,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of endocytosis inhibitors on the increase of mtROS after AgNP treatment. Endocytosis inhibitors (Pitstop2 and Dynasore; 5µM each) were treated before AgNP treatment. mtROS levels were assessed using MitoSOX after a two-hour AgNP treatment. Enhanced mtROS by AgNP treatment were rescued by endocytosis inhibitor indicating that AgNP enters cells through endocytosis and affects mitochondria.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4760785/v1/c113875c0bf037c73e8d319b.png"},{"id":62362084,"identity":"5c4d5aa8-ebe9-4ea1-8090-48bfabde07b2","added_by":"auto","created_at":"2024-08-13 10:20:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1038903,"visible":true,"origin":"","legend":"\u003cp\u003ePossible mechanism of suppressing SARS-CoV-2 infection by AgNP.\u003cstrong\u003e \u003c/strong\u003eIn normal cells, AgNP treatment reduces ACE2 expression via mitochondrial dysfunction. Upon endocytosis, AgNPs migrate to intracellular organelles, including mitochondria, releasing silver ions. These ions foster the generation of ROS, promoting Fe\u003csup\u003e2+\u003c/sup\u003e production in mitochondria through a Fenton reaction with iron. ROS regulates AKT/mTOR signaling. AKT modulates ACE2 expression and stability in HPLF cells via its phosphorylation. Decrease of mTOR result in ACE2 down-regulation. Refer to the text for further details.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4760785/v1/25f3a02497ac56439f4e2ff6.png"},{"id":64407316,"identity":"09ad81ef-089c-4286-818f-5750fac6509e","added_by":"auto","created_at":"2024-09-12 18:05:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11866688,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4760785/v1/9150ac4c-eb33-439a-8907-92639cc1f8c1.pdf"},{"id":62361189,"identity":"e698d50d-8dfc-4357-90b8-55d982b3b2be","added_by":"auto","created_at":"2024-08-13 10:12:45","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1911621,"visible":true,"origin":"","legend":"","description":"","filename":"TakahashietalforMolbiolRep2024SupplementalFigures.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4760785/v1/7fcf85c9dbc91b272b31a485.pptx"}],"financialInterests":"Competing interest reported. Shosei Takahashi is an officer of Pikasshu Co., Ltd. and owns stock in Pikasshu Co., Ltd. but receives no compensation as a member of the board of directors.","formattedTitle":"Silver nanoparticles reduce ACE2 expression via changing mitochondrial function in human fibroblast-like lung cell and periodontal ligament fibroblast cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNanoparticles are structures with a size ranging from 1 to 100 nm. The technology for researching, developing, and controlling nanoparticles is called nanotechnology, a concept introduced by Richard Feynman at the annual meeting of the American Physical Society held at the California Institute of Technology in 1959 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Nanotechnology has attracted widespread recognition for its applications in aerospace engineering, medical healthcare, and consumer products [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among all nanoparticles, silver nanoparticles (AgNPs) are currently estimated to be the most commercially utilized, particularly in a diverse array of biomedical and consumer products, owing to their broad-spectrum antimicrobial activity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. While silver is generally safe for human consumption, with its use in food products, it is recognized that AgNPs can effectively combat microorganisms in liquids containing approximately 1 \u0026micro;g/ml (1 ppm) of ions, a phenomenon known as \u0026ldquo;trace metal action\u0026ldquo; [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Furthermore, AgNPs exhibit high diffusivity and stability in liquids. These nanoparticles increase the concentration of silver ions per mass of silver, thereby augmenting their antimicrobial potency. Silver ions exert their antimicrobial effects by denaturing proteins, generating reactive oxygen species (ROS), and damaging DNA upon internalization into the cells of bacteria, fungi, protozoa, and viruses [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Studies have also demonstrated the effectiveness of AgNPs in inhibiting extracellular SARS-CoV-2, the severe acute respiratory syndrome coronavirus 2 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. AgNPs have been observed to enter cells via endocytosis and localize with mitochondria [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, the intricate molecular mechanisms underlying the actions of AgNPs within cells or against SARS-CoV-2 yet to be fully elucidated.\u003c/p\u003e \u003cp\u003eCOVID-19 is the new infectious respiratory disease in Wuhan, China in December 2019 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. SARS-COV-2, the virus that causes COVID-19 exploits angiotensin-converting enzyme 2 (ACE2) as a cell surface receptor for its spike protein. SARS-COV-2 also exploits transmembrane protease, serine 2 (TMPRSS2) as an essential enzyme for viral cleavage and cellular entry through endocytosis from the cell surface [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Following SARS-CoV-2 infection, an average incubation period of four to five days precedes the onset of symptoms, with an additional five to six days needed for the viral load to peak [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A strong immune response typically inactivates SARS-CoV-2 and clears infected cells, thereby preventing pulmonary vascular damage and mitigating disease progression [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, inadequate immune responses may lead to pyroptosis, an inflammatory form of cell death, exacerbating symptoms by up-regulating protease expression and oxidative stress, both crucial for SARS-CoV-2 propagation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt has been reported that one of the factors that control ACE2 expression is AKT [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It has also been reported mTOR signaling is involved in the control of ACE2 expression because cancer patients receiving mTOR inhibitors as anticancer drugs are lower ACE2 expression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. mTOR has been shown to regulate mitochondria, and the PI3K/Akt/mTOR pathway [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. On the other hand, SARS-CoV-2 RNA has been observed to localize to mitochondria after infection, down-regulating mitochondrial membrane potential (Ψm) and inducing mitophagy [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Mitochondria serve as the cell\u0026rsquo;s powerhouses and as significant sources of ROS and play a critical role in intracellular iron utilization [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. They regulate various forms of cell death, including apoptosis, autophagy, and ferroptosis [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The generation of mitochondrial ROS stems from electron leakage within the electron transport chain, with approximately 1\u0026ndash;3% of electrons typically leaking and reacting with oxygen to produce superoxide [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Superoxide is then neutralized by superoxide dismutase, forming hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), a type of ROS. When combined with Fe\u003csup\u003e2+\u003c/sup\u003e, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e generates highly reactive hydroxyl radicals (ཥOH) via the Fenton reaction. This process has dual implications: it can negatively impact the plasma membrane, leading to cell death and exerting bactericidal effects against invading microorganisms [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Additionally, studies have indicated that ACE2 knockdown impairs mitochondrial respiration and reduces ATP production in kidney cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus, it is suggested that mitochondrial function plays a pivotal role in ACE2-mediated SARS-CoV-2 infection and that AgNP may suppress SARS-CoV-2 infection. However, it remains unclear whether AgNP treatment effectively reduces ACE2 expression and lowers the risk of infection. Therefore, we investigated the cellular response to AgNP treatment, primarily focusing on mitochondrial function, and explored its potential impact on SARS-CoV-2 infection using normal lung cell, which can cause severe pneumonia due to infection, and normal oral cavity cell, which is one of the entry points for infection.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHuman fibroblast-like lung cell (VA-13) were provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. Human Periodontal Ligament Fibroblasts (HPLF) were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). VA-13 cells were cultured in RPMI 1640 (Fujifilm Wako, Osaka, Japan) supplemented with 5% fetal bovine serum (FBS; Thermo Fisher Scientific, Massachusetts, USA). HPLF cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSilver nanoparticles (AgNP) treatment\u003c/h2\u003e \u003cp\u003eAgNP were obtained from Pikasshu Co. Ltd (Kumamoto, Japan), with an average diameter of approximately 40 nm. The AgNP solution was prepared by diluting the stock solution from 10,000 \u0026micro;g/ml to 100 \u0026micro;g/ml using milli-Q water (Merck KGaA, Darmstadt, Germany). Subsequently, the appropriate concentration of AgNP was added to the culture medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell viability\u003c/h2\u003e \u003cp\u003eThe effect of AgNP on cell proliferation was assessed using the water-soluble tetrazolium salt assay with Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan), following the manufacturer\u0026rsquo;s instructions. Cells in the exponential growth phase were seeded in a 96-well plate (5\u0026times;10\u003csup\u003e3\u003c/sup\u003e per well) for 24 h before experimentation. Subsequently, the cells were treated with AgNP at concentrations of 0.01, 0.1, 1, 10, and 100 \u0026micro;g/ml in RPMI 1640 medium and incubated for 2 h. After incubation, the medium was replaced, and cells were further incubated for 46 h. Following this, 10 \u0026micro;l of CCK-8 reagent was added to each well, and the absorbance of the wells was measured using a microplate reader (Multiskan FC; Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative PCR (qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA extraction from cells was performed using ISOGEN (Nippon Gene, Toyama, Japan), followed by cDNA synthesis using ReverTra Ace (TOYOBO CO Ltd., Osaka, Japan) according to the manufacturer\u0026rsquo;s instructions. qPCR analysis was performed using an equivalent of 1 ng of total RNA, as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The qPCR reaction was conducted using the CFX Duet Real-Time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with THUNDERBIRD qPCR mix (TOYOBO). β-actin served as the internal control. The PCR protocol consisted of an initial denaturation step (95\u0026deg;C, 3 min), followed by 40 amplification cycles (95\u0026deg;C for 10 s, 60\u0026deg;C for 30 s). Each experiment was performed in triplicate, and gene expression levels relative to β-actin were determined using the \u003csub\u003eΔΔ\u003c/sub\u003eCt method. Primer sequences are provided below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eACE2 F: 5ʹ-GGGATCAGAGATCGGAAGAAGAAA-3ʹ\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eACE2 R: 5ʹ-AGGAGGTCTGAACATCATCAGTG-3ʹ\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section4\"\u003e \u003ch2\u003eTMPRSS2 F: 5ʹ-AATCGGTGTGTTCGCCTCTAC-3ʹ\u003c/h2\u003e \u003cp\u003eTMPRSS2 R: 5ʹ-CGTAGTTCTCGTTCCAGTCGT-3ʹ\u003c/p\u003e \u003cp\u003eβ-actin F: 5ʹ-AGAGCTACGAGCTGCCTGAC-3ʹ\u003c/p\u003e \u003cp\u003eβ-actin R: 5ʹ-AGCACTGTGTTGGCGTACAG-3ʹ\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSARS-CoV-2 spike Protein binding assay\u003c/h2\u003e \u003cp\u003eThe interaction between the cell surface receptor and the SARS-CoV-2 spike protein was observed using a BZ-8000 fluorescence microscope after the fluorescent labeling of the spike protein. The RBD-S1 spike protein (SARS-CoV-2 spike protein) His Tag (HEK293) (Aviscera Bioscience, Inc., Santa Clara, CA, USA) was labeled with fluorescein using the Fluorescein Labeling Kit (Dojindo) in accordance with the manufacturer\u0026rsquo;s instructions. After a 2-h treatment with AgNPs, spike protein with FITC in Hank\u0026rsquo;s balanced salt solution (HBSS) (250 ng/ml) was applied to the cells in glass-bottomed dishes and incubated for 10 min at 37\u0026deg;C. Subsequently, the cells were washed twice with HBSS, and the spike protein with FITC was visualized using a fluorescence microscope equipped with a PlanFluor ELWD DM 20\u0026times;/0.45 NA lens and a GFP-BP filter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eAfter treatment with 0.1 \u0026micro;g/ml of AgNP for 2 h, cells were harvested and incubated with cell lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 1 mM sodium fluoride, 1 mM sodium vanadate, and 1 mM phenylmethylsulfonyl fluoride. Equal amounts of protein (20 \u0026micro;g per lane) from each cell lysate were separated by SDS-polyacrylamide gel electrophoresis using 7.5% (for mTOR) or 10% (for pAKT\u003csup\u003eT308\u003c/sup\u003e and ACE2) polyacrylamide gels under reducing conditions. The separated proteins were then transferred onto a polyvinylidene difluoride membrane. Following blocking with 5% skim milk in PBST (blocking solution), the membranes were incubated overnight at 4\u0026deg;C with specific primary antibodies (ACE2: 28868-1-AP, Proteintech, Rosemont, IL, USA; mTOR: 2983, pAKT\u003csup\u003eT308\u003c/sup\u003e: 13038, Cell Signaling Technology) diluted 1:1000 in blocking solution. After washing five times with PBST, the membranes were incubated with ECL\u0026trade; anti-rabbit IgG, horseradish peroxidase linked whole antibody (NA934V: Global Life Sciences Technologies Japan K.K., Tokyo, Japan; diluted 1:2000) at room temperature for 2 h. After another five washes with PBST, immunoreactive proteins were visualized using ImmunoStar Zeta (Fujifilm Wako) and imaged with a ChemiDoc XRS Plus instrument (Bio-Rad Laboratories). Antiβ-actin antibody (4970L: Cell Signaling Technology; diluted 1:2000) was used as a loading control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetection of AgNPs in mitochondria\u003c/h2\u003e \u003cp\u003eThe scattered light imaging method [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] was employed to detect AgNPs. Cells were treated with 50 nM MitoTracker Red CMXRos (Thermo Fisher Scientific) and 0.1 \u0026micro;g/ml AgNP in HBSS for 30 min at 37\u0026deg;C. Subsequently, the medium containing MitoTracker Red CMXRos and AgNPs was aspirated, and cells were washed with HBSS to remove any residual dye and nanoparticles. The colocalization of AgNP and MitoTracker Red CMXRos within the cells was visualized using a BZ-8000 fluorescence microscope (Keyence Corporation, Osaka, Japan) equipped with a PlanApo 20\u0026times;/0.75 NA lens, GFP-BP, and Texas Red filters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDetection of mitochondrial Fe\u003csup\u003e2+\u003c/sup\u003e, ROS, and membrane potential\u003c/h2\u003e \u003cp\u003eMitochondrial Fe\u003csup\u003e2+\u003c/sup\u003e\u003csub\u003e,\u003c/sub\u003e mtROS, and Ψm were assessed using Mito-FerroGreen (Dojindo), mitoSOX (Thermo Fisher Scientific), and JC-10\u0026trade; (Thermo Fisher Scientific), respectively. Following a 2-h treatment with AgNPs, cells in glass-bottomed dishes were exposed to 5 \u0026micro;M Mito-FerroGreen, 5 \u0026micro;M mitoSOX, or 2 \u0026micro;M JC-10 in HBSS (084-08345: Fujifilm Wako) for 30 min at 37\u0026deg;C. After the incubation period, cells were washed twice with fresh HBSS. Fluorescence images were captured using a BZ-8000 fluorescence microscope (Keyence Corporation, Osaka, Japan) equipped with a PlanFluor ELWD DM 20\u0026times;/0.45 NA lens, GFP-BP, and Texas Red filters. Fluorescence intensity was quantified using ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rsb.info.nih.gov/ij/\u003c/span\u003e\u003cspan address=\"http://rsb.info.nih.gov/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, 1997\u0026ndash;2012).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEndocytosis inhibitor assay\u003c/h2\u003e \u003cp\u003eTo inhibit endocytosis of AgNP, Pitstop2 (Selleck Biotech, Kanagawa Japan) and Dynasore (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) were treated to VA-13 and HPLF. Briefly, cells in glass-bottomed dishes were treated with 20 \u0026micro;M Pitstop2 or 20 \u0026micro;M Dynasore for 15 min, then treated with 0.1 \u0026micro;g/ml of AgNP for 2 h. After the AgNP treatment, cells were exposed to 5 \u0026micro;M mitoSOX in HBSS (084-08345: Fujifilm Wako) for 30 min at 37\u0026deg;C. Fluorescence images were captured using a BZ-8000 fluorescence microscope as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStudent\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was conducted following the F test to compare the two groups. For comparisons involving three or more groups, one-way ANOVA with Scheffe\u0026rsquo;s \u003cem\u003eF\u003c/em\u003e test was conducted using Statcel4 software (OMS publishing, Saitama, Japan). A significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was adopted. The results are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the mean.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eThe cell viability by AgNP treatment\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eNo significant change in cell viability was observed with AgNP treatment at concentrations blow 0.1 \u0026micro;g/ml. However, upon exposure to 1 \u0026micro;g/ml of AgNP, cells exhibited a decreased cell survival rate after 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Microscopic examination at this time point revealed that the cells appeared swollen, resembling balloons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eACE2 gene expression was decreased by AgNP treatment in normal cells\u003c/h2\u003e \u003cp\u003eWe examined the cellular response to 0.1 \u0026micro;g/ml of AgNP treatment, a concentration that did not significantly affect cell viability across both cell lines. Following treatment, the expressions of ACE2 and TMPRSS2 genes were down-regulated in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings suggest that AgNP treatment reduces the susceptibility of cells to coronavirus infection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAgNP treatment reduced the binding of spike protein to cells\u003c/h2\u003e \u003cp\u003eTo assess the spike protein\u0026rsquo;s binding capability to the cell surface, we performed the SARS-CoV-2 spike protein binding assay using FITC-conjugated spike protein. A decreased FITC signal was noted following treatment with 0.1 \u0026micro;g/ml of AgNP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression change after AgNP treatment\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanism underlying AgNP treatment, we investigated protein expression following exposure to AgNP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In VA13 cells, the expression of mTOR was down-regulated by AgNP treatment. In HPLF cells, the phosphorylation of AKT at T308 was promoted. ACE2 expression was down-regulated upon treatment with 0.1 \u0026micro;g/ml of AgNP in both cell lines.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTreated AgNPs localize mainly in mitochondria\u003c/h2\u003e \u003cp\u003eWe employed the scattered light imaging method [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] to elucidate the subcellular localization of AgNP. This technique allows for real-time, label-free detection of AgNP in living cells. Through this approach, we successfully identified AgNP in mitochondria following AgNP treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026ndash;c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, we observed green dot-like fluorescence when AgNP were immobilized in polyacrylamide gel and examined in a glass-bottomed dish. Importantly, this green fluorescence was not evident in the absence of AgNP administration (see Supplemental Fig.\u0026nbsp;1), confirming its association with AgNP. Furthermore, no green fluorescence was observed under identical conditions when cells were treated solely with MitoTracker, indicating that this fluorescence does not arise from MitoTracker Red leakage (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed\u0026ndash;f).\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003eEffects of AgNP treatment on mitochondria\u003c/h2\u003e \u003cp\u003eHaving observed the localization of AgNP in mitochondria, we examined their impact on mitochondrial function. Consequently, we noted a substantial increase in mitochondrial Fe\u003csup\u003e2+\u003c/sup\u003e levels and mtROS after AgNP treatment. Additionally, AgNP treatment led to a significant down-regulation of Ψm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eEffect of endocytosis inhibitors on mitoSOX expression after AgNP treatment\u003c/h2\u003e \u003cp\u003eWe investigated the effect of endocytosis inhibitors After AgNP treatment using Pitstop2 and Dynasore. As a result, both inhibitors effectively inhibited the mtROS generation after AgNP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This result indicates that AgNPs enter cells by endocytosis and influence at least the generation of mtROS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrated that AgNPs exert effects on mitochondria and suppress ACE2 expression, which is particularly evident in normal cells. Our findings suggest the potential of AgNP in preventing SARS-CoV-2 infection. However, further investigations are needed to determine the optimal concentration and size of AgNP for prevention and their impact on coronavirus proliferation after infection. For example, it has been reported that the cytotoxicity may vary for different cell types and different AgNP particle sizes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In conjunction with previous research, our study underscores the preventive efficacy of AgNP against coronavirus infections. Therefore, practices such as handwashing, gargling, and using AgNP-containing products such as toothpaste and toothbrushes could be highly effective in preventing viral infections.\u003c/p\u003e \u003cp\u003eACE2 was originally discovered as a membrane-bound peptidase that degrades ANG II to Ang-(1\u0026ndash;7) in the cell. ANG II binds to the ANG II type 1 receptor, stimulating inflammation, fibrosis, oxidative stress, and an increase in blood pressure. Ang-(1\u0026ndash;7) binds to the Mas receptor, exerting anti-inflammatory and antifibrotic effects, stimulating nitric oxide release, and reducing blood pressure. One of the factors that control ACE2 expression is AKT signaling. AKT, a serine/threonine kinase, plays important roles in multiple cellular processes, including glucose metabolism, and is known to inhibit AMPK [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. AMPK is activated in response to a shortage of intracellular energy and regulates metabolism [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, AMPK has been reported to phosphorylate and stabilize ACE2 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, in HPLF cells, AKT activation via phosphorylation inhibits AMPK, which likely prevents the stabilization of ACE2 and reduces its expression.\u003c/p\u003e \u003cp\u003eAgNPs are primarily internalized into cells via endocytosis, where they are metabolized within intracellular lysosomes [\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Subsequently, AgNPs leak from lysosomes as silver ions, eventually localizing to mitochondria, where they induce ROS generation in a dose-dependent manner. Combined with our findings, it is plausible that silver ions transfer ions to iron in mitochondria, promoting ROS production through the Fenton reaction. Silver ions transferred to mitochondria have been reported to affect the electron transport chain, leading to decreased oxygen consumption [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and mitochondrial dysfunction, ultimately reducing membrane potential [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhile prior studies have indicated that decreased ACE2 expression correlates with mitochondrial dysfunction, the exact relationship between mitochondrial status and ACE2 expression remains unclear [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Our study revealed that AgNPs internalized into cells are translocated to mitochondria, leading to decreased ACE2 expression in VA-13, and HPLF cells. This suggests that mitochondrial status may indeed influence ACE2 expression. Moreover, phosphorylation of AKT in mitochondria, potentially regulated by AgNP treatment, may play a role in modulating ACE2 expression. It has been reported that AKT and prohibitin 1 (PHB1), a constituent protein of mitochondria, are phosphorylated and form a complex in mitochondria [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additionally, PHB2 has been shown to bind directly to AKT [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. These findings suggest that AgNP treatment induces AKT phosphorylation in the mitochondria, subsequently regulating the expression of ACE2. On the other hand, it has been reported that cancer patients receiving mTOR inhibitors as anticancer drugs are less likely to contract COVID-19 due to lower ACE2 expression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. mTOR has been shown to regulate mitophagy, mitochondria, and the PI3K/Akt/mTOR pathway [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], indicating a close interrelation among these processes. Further investigations are needed to elucidate the mechanisms underlying AgNPs\u0026rsquo; ability to mitigate coronavirus infections, AgNPs appear to provide a beneficial effect by preventing SARS-CoV-2 infection of cells by down-regulating ACE-2 receptors. However, AgNPs can themselves be cytotoxic by causing mitochondrial dysfunction as shown in this study and other studies [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. SARS-CoV-2 infection can also cause alteration of mitochondrial function [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Hence AgNPs should be used with caution. Use along with mitochondrial protective agents such as N-Acetyl L-Cysteine can help to extract the benefits of AgNPs while minimizing their side effects.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we have elucidated that AgNP treatment effectively reduces ACE2 expression and the spike protein\u0026rsquo;s binding to the cell surface in normal cells. Furthermore, we have demonstrated that AgNPs localize to mitochondria and exhibit varying routes of infection suppression depending on the cell type. A schematic representation of the potential mechanism underlying the inhibition of SARS-CoV-2 infection by AgNP is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Future investigations should encompass exploring the effects of AgNP treatment on organs such as the heart and kidneys, where ACE2 plays pivotal roles, evaluating the long-term biological impacts of exposure, and assessing potential synergistic effects with other mitochondria-targeting drugs. Nonetheless, the safety profile of silver, widely used in food, offers considerable reassurance regarding its potential to prevent coronavirus infections. Mitochondria-targeted therapeutic and preventive approaches employing AgNPs are promising as effective tools against coronavirus infections.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eEthical Statements:\u003c/h2\u003e\n\u003cp\u003eNot applicable. This study only involves \u003cem\u003ein vitro\u003c/em\u003e experiments and there was no involvement of Human Participants and/or Animals in it.\u003c/p\u003e\n\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eShosei Takahashi is an officer of Pikasshu Co., Ltd. and owns stock in Pikasshu Co., Ltd. but receives no compensation as a member of the board of directors.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI: Grant Number 24K13073.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eS.T. and K.T. designed and carried out experiments, analyzed the data and wrote the manuscript. K.I., Y.K., J.K., N.K., K.T., and A.K. helped perform experiments, finalized results, and reviewed and edited the manuscript. Y.K. designed the experiment, took part in the optimization of the method, and reviewed and edited the manuscript. T.S. designed and supervised the study and reviewed and edited the manuscript.\u003c/p\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n \u003ch2\u003eData availability\u003c/h2\u003e\n \u003cp\u003eThe materials and data used and/or analyzed in the present study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFeynman RP. (1960) There\u0026rsquo;s plenty of room at the bottom. Eng. Sci. 23(5):22-36.\u003c/li\u003e\n\u003cli\u003eGerber C, Lang HP. (2006) How the doors to the nano-world were opened. Nat Nanotech 1:3-5. https://doi.org/10.1038/nnano.2006.70.\u003c/li\u003e\n\u003cli\u003eSingh N, Manshian B, Jenkins GJ, Griffiths SM, Williams PM, Maffeis TG, et al. (2009) NanoGenotoxicology: the DNA damaging potential of engineered nanomaterials. Biomaterials. 30(23-24):3891-3914. https://doi.org/10.1016/j.biomaterials.2009.04.009.\u003c/li\u003e\n\u003cli\u003eDur\u0026aacute;n N, Dur\u0026aacute;n M, de Jesus MB, Seabra AB, F\u0026aacute;varo WJ, Nakazato G. (2016) Silver nanoparticles: A new view on mechanistic aspects on antimicrobial activity. Nanomedicine. 12(3):789-799. https://doi.org/10.1016/j.nano.2015.11.016.\u003c/li\u003e\n\u003cli\u003eNaegeli, von V. Deut. Schr. Schweiz. (1893) Naturforsch. Ges. 33:174-182.\u003c/li\u003e\n\u003cli\u003eRussell AD, Hugo WB. (1994) Antimicrobial activity and action of silver. Prog Med Chem. 31:351-370. https://doi.org/10.1016/s0079-6468(08)70024-9.\u003c/li\u003e\n\u003cli\u003eGaldiero S, Falanga A, Vitiello M, Cantisani M, Marra V, Galdiero M. (2011) Silver nanoparticles as potential antiviral agents. Molecules. 16(10):8894-8918. https://doi.org/10.3390/molecules16108894.\u003c/li\u003e\n\u003cli\u003eJeremiah SS, Miyakawa K, Morita T, Yamaoka Y, Ryo A. (2020) Potent antiviral effect of silver nanoparticles on SARS-CoV-2. Biochem Biophys Res Commun. 533(1):195-200. https://doi.org/10.1016/j.bbrc.2020.09.018.\u003c/li\u003e\n\u003cli\u003eWu M, Guo H, Liu L, Liu Y, Xie L. (2019) Size-dependent cellular uptake and localization profiles of silver nanoparticles. Int J Nanomedicine. 14:4247-4259. https://doi.org/10.2147/IJN.S201107.\u003c/li\u003e\n\u003cli\u003eLi J, Zhang B, Chang X, Gan J, Li W, Niu S, et al. (2020) Silver nanoparticles modulate mitochondrial dynamics and biogenesis in HepG2 cells. Environ Pollut. 256:113430. https://doi.org/10.1016/j.envpol.2019.113430.\u003c/li\u003e\n\u003cli\u003eDey S, Fageria L, Sharma A, Mukherjee S, Pande S, Chowdhury R, Chowdhury S. (2022) Silver nanoparticle-induced alteration of mitochondrial and ER homeostasis affects human breast cancer cell fate. Toxicol Rep. 9:1977-1984. doi: 10.1016/j.toxrep.2022.10.017.\u003c/li\u003e\n\u003cli\u003eZhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. (2020) A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 382(8):727-733. https://doi.org/10.1056/NEJMoa2001017.\u003c/li\u003e\n\u003cli\u003eZhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. (2020) A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 579(7798):270-273. https://doi.org/10.1038/s41586-020-2012-7.\u003c/li\u003e\n\u003cli\u003eHoffmann M, Kleine-Weber H, Schroeder S, Kr\u0026uuml;ger N, Herrler T, Erichsen S, et al. (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 181(2):271-280.e8. https://doi.org/10.1016/j.cell.2020.02.052. \u003c/li\u003e\n\u003cli\u003eAzkur AK, Akdis M, Azkur D, Sokolowska M, van de Veen W, Br\u0026uuml;ggen MC, et al. (2020) Immune response to SARS-CoV-2 and mechanisms of immunopathological changes in COVID-19. Allergy. 75(7):1564-1581. https://doi.org/10.1111/all.14364.\u003c/li\u003e\n\u003cli\u003eTay MZ, Poh CM, R\u0026eacute;nia L, MacAry PA, Ng LFP. (2020) The trinity of COVID-19: immunity, inflammation and intervention. Nat Rev Immunol. 20(6):363-374. https://doi.org/10.1038/s41577-020-0311-8.\u003c/li\u003e\n\u003cli\u003eDelgado-Roche L, Mesta F. (2020) Oxidative Stress as Key Player in Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Infection. Arch Med Res. 51(5):384-387. https://doi.org/10.1016/j.arcmed.2020.04.019.\u003c/li\u003e\n\u003cli\u003eWang B, Li D, Fiselier A, Kovalchuk I, Kovalchuk O. (2022) New AKT-dependent mechanisms of anti-COVID-19 action of high-CBD Cannabis sativa extracts. Cell Death Discov. 8(1):110. https://doi.org/10.1038/s41420-022-00876-y.\u003c/li\u003e\n\u003cli\u003eFoote MB, White JR, Jee J, Argil\u0026eacute;s G, Wan JCM, Rousseau B, et al. (2021) Association of Antineoplastic Therapy With Decreased SARS-CoV-2 Infection Rates in Patients With Cancer. JAMA Oncol. 7(11):1686-1691. https://doi.org/10.1001/jamaoncol.2021.3585.\u003c/li\u003e\n\u003cli\u003eQiao L, Guo Z, Liu H, Liu J, Lin X, Deng H, et al. (2022) Protective Effect of Mitophagy Regulated by mTOR Signaling Pathway in Liver Fibrosis Associated with Selenium. Nutrients. 14(12):2410. https://doi.org/10.3390/nu14122410.\u003c/li\u003e\n\u003cli\u003eShang C, Liu Z, Zhu Y, Lu J, Ge C, Zhang C, et al. (2022) SARS-CoV-2 Causes Mitochondrial Dysfunction and Mitophagy Impairment. Front Microbiol. 12:780768. https://doi.org/10.3389/fmicb.2021.780768.\u003c/li\u003e\n\u003cli\u003eIndo HP, Davidson M, Yen HC, Suenaga S, Tomita K, Nishii T, et al. (2007) Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage. Mitochondrion. 7;106-118. https://doi.org/10.1016/j.mito.2006.11.026.\u003c/li\u003e\n\u003cli\u003eAndrews NC. (1999) Disorders of iron metabolism. N Engl J Med. 341(26):1986-1995. https://doi.org/10.1056/NEJM199912233412607.\u003c/li\u003e\n\u003cli\u003eGreen DR, Reed JC. (1998) Mitochondria and apoptosis. Science. 281; 1309-1312. https://doi.org/10.1126/science.281.5381.1309.\u003c/li\u003e\n\u003cli\u003eChandel NS. (2014) Mitochondria as signaling organelles. BMC Biol. 12(1):1-7. https://doi.org/10.1186/1741-7007-12-34.\u003c/li\u003e\n\u003cli\u003eTakashi Y, Tomita K, Kuwahara Y, Roudkenar MH, Roushandeh AM, Igarashi K, et al. (2020) Mitochondrial dysfunction promotes aquaporin expression that controls hydrogen peroxide permeability and ferroptosis. Free Radic Biol Med. 161:60-70. https://doi.org/10.1016/j.freeradbiomed.2020.09.027.\u003c/li\u003e\n\u003cli\u003eBoveris A, Chance B. (1973) The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J. 134:707-716. https://doi.org/10.1042/bj1340707.\u003c/li\u003e\n\u003cli\u003eShi TT, Yang FY, Liu C, Cao X, Lu J, Zhang XL, et al. (2018) Angiotensin-converting enzyme 2 regulates mitochondrial function in pancreatic \u0026beta;-cells. Biochem Biophys Res Commun. 495(1):860-866. https://doi.org/10.1016/j.bbrc.2017.11.055.\u003c/li\u003e\n\u003cli\u003eTomita K, Kuwahara Y, Takashi Y, Tsukahara T, Kurimasa A, Fukumoto M, et al. (2017) Sensitivity of mitochondrial DNA depleted rho0 cells to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e depends on the plasma membrane status. Biochem Biophys Res Commun. 490(2):330-335. https://doi.org/10.1016/j.bbrc.2017.06.044\u003c/li\u003e\n\u003cli\u003eWang F, Chen B, Yan B, Yin Y, Hu L, Liang Y, et al. (2019) Scattered Light Imaging Enables Real-Time Monitoring of Label-Free Nanoparticles and Fluorescent Biomolecules in Live Cells. J Am Chem Soc. 141(36):14043-14047. https://doi.org/10.1021/jacs.9b05894.\u003c/li\u003e\n\u003cli\u003eHe J, Ma Y, Niu X, Pei J, Yan R, Xu F, et al. (2024) Silver nanoparticles induce endothelial cytotoxicity through ROS-mediated mitochondria-lysosome damage and autophagy perturbation: The protective role of N-acetylcysteine. Toxicology. 502:153734. https://doi.org/10.1016/j.tox.2024.153734.\u003c/li\u003e\n\u003cli\u003eKovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR. (2003) Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem. 278(41):39422-394227. https://doi.org/10.1074/jbc.M305371200.\u003c/li\u003e\n\u003cli\u003eHerzig S, Shaw RJ. (2018) AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol. 19(2):121-135. https://doi.org/10.1038/nrm.2017.95.\u003c/li\u003e\n\u003cli\u003eZhang J, Dong J, Martin M, He M, Gongol B, Marin TL, et al. (2018) AMP-activated Protein Kinase Phosphorylation of Angiotensin-Converting Enzyme 2 in Endothelium Mitigates Pulmonary Hypertension. Am J Respir Crit Care Med. 198(4):509-520. https://doi.org/10.1164/rccm.201712-2570OC.\u003c/li\u003e\n\u003cli\u003eWu M, Guo H, Liu L, Liu Y, Xie L. (2019) Size-dependent cellular uptake and localization profiles of silver nanoparticles. Int J Nanomedicine. 14:4247-4259. https://doi.org/10.2147/IJN.S201107.\u003c/li\u003e\n\u003cli\u003eMiyayama T, Arai Y, Suzuki N, Hirano S. (2013) Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate. Toxicology. 305:20-29. https://doi.org/10.1016/j.tox.2013.01.004.\u003c/li\u003e\n\u003cli\u003eMiyayama T, Matsuoka M. (2016) Involvement of lysosomal dysfunction in silver nanoparticle-induced cellular damage in A549 human lung alveolar epithelial cells. J Occup Med Toxicol. 11:1. https://doi.org/10.1186/s12995-016-0090-0.\u003c/li\u003e\n\u003cli\u003eBrkić Ahmed L, Milić M, Pongrac IM, Marjanović AM, Mlinarić H, Pavičić I, et al. (2017) Impact of surface functionalization on the uptake mechanism and toxicity effects of silver nanoparticles in HepG2 cells. Food Chem Toxicol. 107(Pt A):349-361. https://doi.org/10.1016/j.fct.2017.07.016.\u003c/li\u003e\n\u003cli\u003eSingh RP, Ramarao P. (2012) Cellular uptake, intracellular trafficking and cytotoxicity of silver nanoparticles. Toxicol Lett. 213(2):249-259. https://doi.org/10.1016/j.toxlet.2012.07.009.\u003c/li\u003e\n\u003cli\u003eLi XH, Chai RR, Chen GW, Zhang LF, Tan-Tai WJ, Shi HJ, et al. (2020) Prohibitin (PHB) interacts with AKT in mitochondria to coordinately modulate sperm motility. Asian J Androl. 22(6):583-589. https://doi.org/10.4103/aja.aja_46_20.\u003c/li\u003e\n\u003cli\u003eSun L, Liu L, Yang XJ, Wu Z. (2004) Akt binds prohibitin 2 and relieves its repression of MyoD and muscle differentiation. J Cell Sci. 117(Pt 14):3021-3029. https://doi.org/10.1242/jcs.01142.\u003c/li\u003e\n\u003cli\u003eLi J, Zhang B, Chang X, Gan J, Li W, Niu S, et al. (2020) Silver nanoparticles modulate mitochondrial dynamics and biogenesis in HepG2 cells. Environ Pollut. 256:113430. https://doi.org/10.1016/j.envpol.2019.113430.\u003c/li\u003e\n\u003cli\u003eGao Y, Kok WL, Sharma V, Illsley CS, Hanks S, Tredwin C, et al. (2023) SARS-CoV-2 infection causes periodontal fibrotic pathogenesis through deregulating mitochondrial beta-oxidation. Cell Death Discov. 9(1):175. https://doi.org/10.1038/s41420-023-01474-2.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Silver nanoparticle, SARS-CoV-2, ACE2, mitochondria","lastPublishedDoi":"10.21203/rs.3.rs-4760785/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4760785/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSilver nanoparticles (AgNPs) have demonstrated antibacterial properties and are widely recognized as one of the most prominent types of nanoparticles. Recent studies have highlighted their effectiveness against coronaviruses. However, the detailed molecular mechanisms underlying the action of AgNPs on viruses and their impacts on the human body remain to be fully elucidated. Thus, we attempt to delineate the preventive effects of AgNPs against SARS-CoV-2 infection. Our findings indicate that treatment with AgNPs reduces ACE2 expression, a key receptor for SARS-CoV-2 particularly in normal oral and lung cells. Additionally, we observed a decrease in the binding affinity of the spike protein to the cell after AgNP treatment. Through western blot analysis, we identified the involvement of the AKT and/or mTOR signaling pathway in this process. Since AKT and mTOR signaling have been reported to affect mitochondrial function, we investigated the effects of AgNP treatment on mitochondria. As a result, we found the localization of AgNPs within mitochondria. Furthermore, it was accompanied by an increase in mitochondrial Fe\u003csup\u003e2+\u003c/sup\u003e and reactive oxygen species levels, ultimately resulting in mitochondrial dysfunction. Our results underscore the remarkable efficacy of AgNP treatment in preventing coronavirus infections.\u003c/p\u003e","manuscriptTitle":"Silver nanoparticles reduce ACE2 expression via changing mitochondrial function in human fibroblast-like lung cell and periodontal ligament fibroblast cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-13 10:12:40","doi":"10.21203/rs.3.rs-4760785/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f3036b2e-a705-4cc3-a3cf-6e560e2aca92","owner":[],"postedDate":"August 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-26T11:53:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-13 10:12:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4760785","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4760785","identity":"rs-4760785","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}