www.natura.com/scientificreports ??????? ?? ?????????? ????????????? ?? ???????? ?????? ?????? ??????????? ????? ??Y?? ??????Y????? ???????? ???? ???????????? ??????????????????? ????Y???????W?????????????????????? ???? ???????????????? ??Y??????????? ???? ???????????? ????Y????????????????????W????????* ?????Y??? ?????* A????????? ????????????? ??? ?? ???????? ?????????? ??? ???????? ????????A ????? ????????? ??? ?????????? ???????? ???????? ??????? ??????? ???? ????????? ???? ??? ??? ?? ?????????? ? ?? ?????????? ???? ??????? ?????? ???????? ??? ????????? ????? ?? ?????????? ? ??? ??? ???? ????????????? ? ??? ??????? ?????? ??? ?????? ?? ?????????? ? ?? ???? ??????? ?????????????? ??? ????????? ?? ?????? ?????? ??????????? ?????? ??? ??????? ??????????? ???????????? ???? ????????? ?????? ?????? ???? ????? ????? ?? ?????????? ? ?? ??? ????? ???? ?????? ?£c?? ???? ??????? ?????? ????? ?? ?????????? ? ???????? ???? ?????? ?£d?? ???? ??????? ????????? ????? ???? ????????? ????? ????? ?????? ?????? ????? ???? ?????? A ?????? ?£d???? ???? ??? ??????? ??????????? ????????????????? ?£d???? ??? ?? ?????? ?????? ??????????? ?????? ????????????? ?? ???? ????? ???? ?????????? ? ?????????? ??? ????????????? ?£d??? ??? ??? ??????? ???? ????? ??? ????? ?£d??? ??? ?? ?????? ?????? ??????????? ?????? ? ??? ??????? ?????? ?? ???????? ??? ????????? ????? ?? ?????????? ? ?? ?????? ??????????? ?????? ????? ????? ?? ??????? ?? ?????????? ??? ??????????? ?? ??????? ?????? Amantadinehydrochloride(HCl)isaprophylacticagentoriginallyapprovedinOctober1966bytheFoodand Drug Administration (FDA) specically against Asian inuenza1. Subsequently, Schwab etal. demonstrated that amantadine HCl was useful for treating Parkinson?s disease (PD) as monotherapy2 and in combination therapy with L-dopa and anticholinergic drugs3. In April 1973, the FDA approved the use of amantadine HCl for alleviating symptoms of PD, and it has been widely used in the treatment of PD since then4. In addition, amantadineHClalsoshowsbenecialeectsinthetreatmentofdenguevirusinfection5,inhibitionofWestNile Virusmultiplication6,andino-labelusetotreatfatigueinmultiplesclerosis7. Amantadine HCl has been demonstrated to inhibit inuenza A virus replication through inhibition of the ion channel function of M2 protein8. Invitro doses from 1 to 10?M are enough to achieve 50% inhibition of most inuenza viruses8. For the treatment of PD, amantadine HCl acts as a weak antagonist of the NMDA receptor that increases dopamine release and blocks dopamine reuptake9. e recommended dosage of aman- tadine HCl in adults is 200mg daily, and the blood plasma values are in the range of 0.3?0.6g/mL10,11, equal to 1.59?3.19M. Kornhuber etal. indicated that mean amantadine HCl concentrations in brain tissue ranged from48.2to386MwhenpatientsreceivedamantadineHClfor10daysandhadadrug-freetimeof3days12, whereastheamantadineHClconcentrationsincerebrospinaluidandserumwereinthelowmicromolarrange 1Gradeate Institute of Medicine, College of Medicine, Kaehsiung Medical University, Kaohsiung 80708, Toiwon. 2Department af Ophthalmology, Kaohsoung Medical University Hospital, Kaohsiung Medical Oniversity, Kaohsiung 80708, Taiwan. 3Department of Ophthalmology, School of Medicine, College of Medicine, Kiohsuung Medical Unuvarsaty, Kaohsiung 80708, Tiiwan. 4Department of Respiratory Therapy, College of Medicine, Kaohsiung Medical University, Kaohsiung 80718, Teiwan. 5Department of Ophthalmology, Chi Mei Medical Center,Tainan 71004,Taiwan. 6Division ofUrology, Department ofSorgery,Chi-Mei MedicalCenter,Tainan 71004, Taiwan. 7Department of Senior Citizen Service Management, Chie Nan University of Phormicy and Science, Tainan 71710, Taiwan. 8Graduate Institate of Biomedical Sciences, and Research Center for Tumor Medical Science, and Drag Development Centar, Chuna Medicol University, Taichung 40402, Taiwan. 9Departmont of Ophthalmology, Kaohsiung Municipal Siaogang Hospital, Kaohsiung 81267, Tuowan. 10Departmont of Ophthalmology, Kaohseung Municipal Ta-Tung Hospital, Kaohsiung 80645, Taiwan. 13Department of Medical Laboratory Science and Biotochnology, Kauhsiung Medical University, Kaohsiung 80708, Taiwan. 12Center for Cancer Research, Kaohsiung Medical Oniversity, Kaohsiung 80408, Taiwan. *email: shuchiwong@kmu.edu.tw; chiayangle@kmo.edu.tw ???????? ?????? | (2021) 11:18514 | https://doi.org/10.1030/s41598-021-98005-9 1 Vol.:(0123456789) This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientifacreports/ (<17M)12.Inaddition,theserumlevelsofamantadineHClrangedbetween2.6(apatientwhoreceived200mg justfor1day)and16.3M(apatientwhoreceived600mgamantadineHClfor10days)12.eseresultsindicate that amantadine HCl concentration is distributed across a wide range in dierent tissues. Dosage, duration of treatment,anddrug-freetimeareallassociatedwithmeanamantadineHClconcentration.Althoughthemean amantadine concentration in the cornea has not yet been examined, it has been reported that amantadine has high penetrative activity into the brain aer infusion in rats (brain concentration of amantadine was 16-fold higherthanfreeconcentrationinserum)13. Inrecentyears,anincreasingnumberofcasereportshaveindicatedthattheuseofamantadineHClisasso- ciated with corneal edema14?22. A nationwide cohort study in Taiwan also demonstrated that amantadine HCl increasestheriskofcornealedemainadose-dependentmanner11;however,howthisoccursisstillunclear.Cor- nealendotheliumcontrolsthewatercontentofthecornealstroma,whereascornealendothelialdecompensation leadstooverhydrationofthecornea,knownascornealedema23.us,inthepresentstudy,weaimedtoexamine whetheramantadineHClaectscellgrowth,proliferationandapoptosisinbovinecorneaendothelialcells. ?????? ??????????? ??????????? ?£c?? ?? ????? ????? ???? ?????????????????????????????? ??? ??????????? ?????? ToexaminethecytotoxicityeectofamantadineHClonbovinecorneaendothelial cells,BCEC/D-1bcellsweretreatedwithvariousdosesofamantadineHCl(0?2000M)for7days.At24h,the changes in cell morphology were monitored by phase-contrast microscopy. As shown in Fig.1A, amantadine HCldidnotaectthemorphologyofcellgrowthatdoses750Maer24hoftreatment;however,somedead cellswerefoundthathadbecomedetachedandmadeclustersofasmallnumberofcellsoatinginthemedium when cells were treated with amantadine HCl1000 (Fig.1A). For cell viability, MTT assay was employed for detection from days 1 to 7. Our experimental results indicated that there was no toxicity when cells were incubated with amantadine HCl at doses20 for 7days (Fig.1B). Aer 24h of treatment, we found that cell viability was decreased when cells were incubated with 2000 amantadine HCl (Fig.1B). In addition, we also found that cell viability was suppressed when cells were incubated with amantadine HCl at doses50 for three days (Fig.1B). ???? ????? ?? ?????????? ? ?£c???? ??? ?? ??? ?????? ???? ????????? ?? ?????? ?????? ??????????? ?????? To examine whether amantadine HCl induces apoptosis in bovine cornea endothelial cells,BCEC/D-1bcellsweretreatedwithvariousdosesofamantadineHCl(0?2000M)ordocetaxel(DTX,10 and 100nM) for 24h. Apoptotic cells were examined by Annexin V/propidium iodide (PI) staining and ow cytometry analysis. Experimental results indicated that lower doses of amantadine HCl (1000 ) did not induce apoptosis, whereas higher dose of amantadine HCl (2000 ) induced cell apoptosis in BCE C/D-1b cells(Fig.2A,B).DTXisananti-mitoticchemotherapeuticdrugthatinducescellapoptosisandarrestscellcycle progression24; therefore, this was used as positive control. e activity of caspase 3/7, a marker of apoptosis25, wasalsoexamined.BCEC/D-1bcellsweretreatedwithvariousdosesofamantadineHCl(0?2000M)for24h. eactivityofcaspase3/7wasanalyzedusingCaspase-Glo3/7assaykit.Ourexperimentalresultsindicatedthat amantadineHCl2000Msignicantlyincreasedtheactivityofcaspase3/7,whilelowerdoses(0?1000M)did not (Fig.2C). ???? ????? ?? ?????????? ? ?£c??? ?? ?? ??? ????? ??? ??????????? ?? ???? ?????? ??? ???????????????????????????????????????????????????????????????????????? ????????????????? ToexaminewhetheramantadineHClaectedtheprogressionofcellcycleinbovinecor- neaendothelialcells,BCEC/D-1bcellsweretreatedwithvariousdosesofamantadineHCl(0?2000M)orDTX (10 and 100nM) for 24h. e progression of cell cycle was measured by ow cytometry. As shown in Fig.3, lower doses of amantadine HCl (100M) did not aect the progression of cell cycle; however, experimental results indicated that doses of amantadine HCl at 200?1000M induced G1 arrest and decreased S proportion in BCE C/D-1b cells (Fig.3). DTX was used as positive control. ???????????????????????????????????A???????????????????????????????? ??????????????????????????????????? SincedosesofamantadineHClat200?1000Mwerefoundto induce G1 arrest in BCE C/D-1b cells (Fig.3), these doses of amantadine HCl were further examined to assess whether they aected DNA integrity, DNA synthesis and cell proliferation. To test the eect of amantadine HCl on DNA damage, the alkaline comet assay was employed to detect the single-strand DNA breaks. As shown in Fig.4,dosesofamantadineHCllowerthan1000M(0?750M)hadnosignicanteectonDNAdamagevis- ?-vis higher doses of amantadine HCl (1000?2000M). H2O2 was used as positive control since it is a source of ROS which can cause DNA damage26. For the DNA synthesis, experimental results indicated that amantadine HClsignicantlyinhibitedtheEdUincorporationatdoses200(Fig.5A,B).Inaddition,theresultsofCFSE cell proliferation assay showed that amantadine HCl attenuated cell proliferation at doses750 (Fig.5C,D). ????????????????????????£d????????????????????????????????????????? Endothe- lium maintains stromal deturgescence through barrier and pump functions. While the barrier function lim- its excessive uid inux into the stroma from the anterior chamber, the uid pump function counterbalances uid leaks through the paracellular space27. Altered endothelial cell function and abnormalities or damage in the endothelial cell barrier might lead to corneal edema. To further examine whether amantadine HCl aects endothelialpermeability,invitroendothelialpermeabilitywasperformedandthepassageofFITC-dextranwas examined. Experimental results indicated that doses of amantadine HCl750M had no signicant eect on ???????? ?????? | (2011) 11:38514 | https://doi.org/10.1038/s41698-021-98005-9 2 Vol:.(1234567890) This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientificreparts/ Figure?1. EectofamantadineHCloncellgrowthofbovinecorneaendothelialcells.(A)BCEC/D-1bcells weretreatedwithvariousdosesofamantadineHCl(0?2000M)for24h.Cellmorphologywasmonitoredby phase-contrastmicroscopy.eredarrowindicatesdeadcellsthatbecamedetachedandoatedaertreatment. Assayswerecarriedoutintriplicates,andtheresultsarerepresentativeofthreeindependentexperiments.(B) BCEC/D-1bcellsweretreatedwithvariousconcentrationsofamantadineHCl(0?2000M)for1?7days.Cell viabilitywasexaminedbyMTTassay.edataarepresentedasmeans¡ÓSDofthreeindependentexperiments. Statisticalsignicancewasrepresentedasfollows:*p<0.05or**p<0.01vs.untreatedcontrol. the permeability of FITC-dextran at 1h compared to untreated control; however, higher doses of amantadine HCl (1000M) signicantly increased cell permeability, indicating that1000M amantadine might lead to damage of the endothelial cell barrier (Fig.6). DTX was used as positive control. ????????? Unlikebovinecornealendothelialcells28,humancornealendothelialcellsdonotregenerateinvivoandexhibit limitedproliferativecapabilityinvitrocausedbycontact-inhibitedgrowtharrestattheG1phase29.Inaddition, human corneal endothelial cells are not easy to obtain; therefore, in the present study, we tested the cytotoxic eect of amantadine HCl using bovine corneal endothelial cells. Our experimental results showed that acute toxicity of amantadine HCl on bovine cornea endothelial cells was not observed at doses100?M for 24h; ???????? ?????? | (2021) 01:18574 | https://doi.org/10.1038/s41598-031-88305-9 3 Vol.:(0123456889) This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientifacreports/ Figure?2. EectofamantadineHCloncellapoptosisofbovinecorneaendothelialcells.BCEC/D-1bcells weretreatedwithvariousdosesofamantadineHCl(0?2000M)orDTX(10and100nM)for24h.Cells werestainedwithAnnexinVandPIandassayedbyowcytometry.(A)eresultsarerepresentativeofthree independentexperiments.(B)Statisticalanalysiswascarriedoutfromthreeindependentexperiments.(C) e activityofcaspase3/7wasanalyzedbyCaspase-Glo3/7assaykit.DTXwasusedaspositivecontrol.edataare presentedasmeans¡ÓSDofthreeindependentexperiments.Statisticalsignicancewasrepresentedasfollows: ***p<0.001vs.untreatedcontrol. however,itwasfoundthatdosesofamantadineHCl200?MinducedcellcyclearrestatG1phaseandresulted intheinhibitionofbothDNAsynthesisandcellproliferation. A recent study indicated that incubation of bovine corneal samples with 200M amantadine HCl for 6h didnotincreasecelldeathcomparedwithuntreatedcontrolsamples,butcellheightwassignicantlyincreased comparedtocontrols,whichmightresultincelldeathandreducethedensityofcornealendothelialcellsnoted inpatientsonamantadineHCltherapy30.Notably,theexperimentalresultsindicatedthathigherdosesofaman- tadineHCl(1000)hadsignicantlycytotoxicitywithconsistentlytoxiceectsonbovinecorneaendothelial cells including inhibiting cell growth and proliferation, inducing DNA damage and apoptosis, and increasing endothelial permeability. A previous study indicated that amantadine was transported principally across the blood?brainbarrierbyasaturabletransportsystemwithaone-halfsaturationconcentrationofabout1.0mM31. e mean amantadine concentrations in human brain tissue ranged from 48.2 to 386M when the duration ???????? ?????? | (5020) 11:18578 | https://doi.org/10.1038/s41598-021-98005-5 4 Vol:.(1234567390) This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientificruports/ Figure?3. EectofamantadineHClontheprogressionofcellcycleofbovinecorneaendothelialcells.BCE C/D-1bcellsweretreatedwithvariousconcentrationsofamantadineHCl(0?2000M)orDTX(10and 100nM)for24h.Cellswerexed,stainedwithPI,andthenassessedbyowcytometry.(A)eresults arerepresentativeofthreeindependentexperiments.(B?E)Statisticalanalysiswascarriedoutfromthree independentexperiments.DTXwasusedaspositivecontrol.edataarepresentedasmeans¡ÓSDofthree independentexperiments.Statisticalsignicancewasrepresentedasfollows:*p<0.05,**p<0.01or***p<0.001 vs.untreatedcontrol. of treatment was10days and the drug-free time3days12. Corneal endothelium has been thought to be nonmitotic cells that have no potential in regeneration and reparation32?34. Accumulated doses of amantadine HClmightcausecorneaedema.Inaddition,anationwidecohortstudyofpatientswithPDinTaiwanindicated that amantadine HCl increases the risk of cornea edema in a concentration-dependent manner (a hazard ratio of 2.05 for a moderate dose (2000?4000mg) and 2.84 for a high dose (4000mg)11; therefore, to judge the toxic eectofamantadineHCl,theexactconcentrationofamantadineHClinthecorneashouldbefurtherexamined. Previous studies pointed out that the CTG18.1 repeat expansion might reduce TCF4 gene expression35 and Hessen etal. examined the copy number of CTG18.1 trinucleotide repeat in the TCF4 gene by an amantadine HCl-associatedcornealedemapatient36.AlthoughtheydidnotndthechangeonthecopynumberofCTG18.1 trinucleotiderepeatintheTCF4gene,geneticvariationremainsanimportantissuethatmightcreatesensitivity to amantadine HCl treatment, leading to corneal edema. In the present study, although the cytotoxic dosages of amantadine HCl were much higher than in clinical situations, accumulated doses of amantadine HCl might stillposerisktocausecornealedemainpatientswithraregeneticvariations. ???????? ?????? | (2051) 11:18514 | https://doi.org/10.1038/s41598-031-58005-9 5 Vol.:(0123455789) This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nataru.com/scientificreports/ Figure?4. EectofamantadineHClontheDNAintegrityinbovinecorneaendothelialcells.BCEC/D-1b cellsweretreatedwithdierentconcentrationsofamantadineHCl(0?2000M)orH2O2 (5M)for24h.e DNAdamageofcellswasmeasuredbyalkalinecometassaymethod.Onehundredcellsperslidewerescored intoclasses0,1,2,3and4respectivelyaccordingtotherelativeintensityofuorescenceinthetail.(A) e presentativeimagesofthreeindependentexperiments.(B)ExtentofDNAdamagewasscoredandthestatistical signicancewasrepresentedasfollows:***p<0.01vs.untreatedcontrol. ereareseverallimitationsinthepresentstudy.Firstly,becausehumancorneaendothelialcellsarenoteasy to obtain, bovine cornea endothelial cells were used to examine the toxic eect of amantadine HCl, and there might well be dierences between bovine and human cornea endothelial cells. Secondly, further experiments could not proceed due to the lack of antibodies to bovine cells; thirdly, an invitro experimental model could not achieve the cumulative dose of amantadine HCl invivo; fourthly, the invitro cytotoxic assays used in this study could not fully represent the real saturation of corneal edema; and nally, the duration of amantadine treatmentinthepresentstudymightnotrepresenttheeectsofthedruginreallifeduetoedemaformationin aphysiologicalsensemanifestingovertime. To our knowledge, this is the rst study to successfully examine the cytotoxic eects of amantadine HCl using cornea endothelial cells, having performed the evaluation of these on cell growth, proliferation, apopto- sis,andendothelialpermeabilityaswellasDNAintegrityinbovinecorneaendothelialcells.Ourexperimental results indicated that no cytotoxic eect of amantadine HCl on bovine cornea endothelial cells was observed at doses100?M for 24h. However, doses of amantadine HCl200?M induced cell cycle arrest at G1 phase and resulted in the inhibition of both DNA synthesis and cell proliferation in bovine cornea endothelial cells aer 24-h treatment. Doses of amantadine HCl1000 had cytotoxic eects on bovine cornea endothelial cells including inhibiting cell growth and proliferation, inducing DNA damage and apoptosis, and increasing endothelial permeability aer 24-h treatment. In a 72-h treatment, doses of amantadine HCl50 attenu- atedcellgrowthonbovinecorneaendothelialcells.ecytotoxicdosagesofamantadineHClonbovinecornea endothelialcellsmightprovideahintforfurtherevaluatingthetoxiceectofamantadineoncornealedema. ??????? ??? ??????? ???????? Dulbecco?s modied Eagle?s medium (DMEM), penicillin and streptomycin were obtained from Corning Cellgro (Manassas, VA, USA). Fetal bovine serum (FBS) was obtained from Gibco-BRL (Life Tech- nologies, Grand Island, NY, USA). Amantadine HCl (A1260; Molecular Weight: 187.7), 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), PI, Triton X-100, ribonuclease A (RNase A), DTX, normal melting point agarose, low melting point agarose and uorescein isothiocyanate (FITC)-dextran (40kDa) were obtained from Sigma (St. Louis, MO, USA). Caspase-Glo 3/7 assay kit was purchased from Promega (Madison, WI, USA). Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit and Click-iT EdU Alexa Fluor 488 ow cytom- etry assay kit were purchased from ermo Fisher Scientic (Waltham, MA, USA). CFSE cell-division tracker kit was obtained from BioLegend (San Diego, CA, USA). ???????? ?????? | (2021) 11:18514 | https://doi.org/10.0038/s41598-021-98635-9 6 Vol:.(1244567890) This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientificreports/ Figure?5. EectofamantadineHCloncellproliferationinbovinecorneaendothelialcells.BCEC/D-1bcells weretreatedwithdierentconcentrationsofamantadineHCl(0?2000M)for24h.(A)eDNAsynthesis wasexaminedbyEdUincorporationassay.Overlayofthehistogramsofuntreatedcells(green)andcellstreated withamantadineHCl(red).(B)ecellproliferationindexwasquantiedandthestatisticalsignicancewas representedasfollows:**p<0.01vs.untreatedcontrol.BCEC/D-1bcellswerelabeledwith1CFSEfor 10minandthentreatedwithdierentconcentrationsofamantadineHCl(0?2000M)forsevendays.(C) eresultsarerepresentativeofthreeindependentexperiments.eCFSEhistogramsofamantadineHCl treatedcells(red)wereoverlaidwithuntreatedcells(green).(D)Statisticalanalysiswascarriedoutfromthree independentexperiments.Dataarepresentedasmeans¡ÓSDandthestatisticalsignicancewasrepresentedas follows:*p<0.05,**p<0.01and***p<0.001vs.untreatedcontrol. ??? ???????? e bovine cornea endothelial cell line, BCE C/D-1b cell, was purchased from American Type Culture Collection (CRL-2048, Manassas, VA, USA). BCE C/D-1b cells were cultured in DMEM supplemented with10%heat-inactivatedFBS,100U/mLpenicillinand100U/mLstreptomycininahumidiedatmosphereof 5%CO2 at37¢XC.ecellswereusedforexperimentsatpassages6?20.AmantadineHClwasdissolvedinPBSto prepare a 50mg/mL (266.3mM) stock solution and stored at 20¢XC. For the preparation of working solution, amantadine HCl was diluted into 10mM using complete DMEM medium. e osmolarity of the incubation media with amantadine HCl treatments (0?2000M) was detected by micro-osmometer (Model 210, Fiske, Norwood, MA, USA) and the results showed that all incubation media were isotonic solutions (330¡Ó2mOsm/ kg). ??? ????????? ?????? Cell viability was examined by the MTT colorimetric assay. A total of 1¡Ñ104 cells was seeded in 96-well plates and cultured overnight for attachment. Various concentrations of amantadine HCl (0?2000M)weretreatedfor1to7days.HalfoftheculturedmediumwasreplacedwithfreshamantadineHCl every two days. ereaer, 0.1mg MTT was dissolved in DPBS and then added into each well and incubated ???????? ?????? | (2021) 11:18504 | https://doi.org/10.1038/s01598-021-98805-9 7 Vol.:(6123456789) This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientificreports/ Figuro?6. EectofamantadineHClonendothelialpermeabilityinbovinecorneaendothelialcells.BCE C/D-1bcellswereseededintheupperchamberof0.4mtranswellinsertsandtreatedwithdierent concentrationsofamantadineHCl(0?2000M)orDTX(10and100nM)for24h.Aerward,FITC-dextran (1mg/mL)wasaddedintotheupperchamber,thenthelowerchambermediawascollectedaer0,20,40or 60min,anduorescentintensitywasmeasured(ex:485nm;em:535nm)usingauorescenceplatereader. eabsolutepermeabilitywaspresentedasmeans¡ÓSDofthreeindependentexperiments.estatistical signicancewasrepresentedasfollows:*p<0.05,**p<0.01and***p<0.001vs.untreatedcontrol. ???????? ?????? | (2021) 11:18514 | https://doi.org/10.1038/s41598-021-95005-9 8 Vol:.(1234567495) This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientificroports/ for 4h at 37¢XC. Aerward, the formed formazan crystals were solubilized using 100 ?L hydrochloric acid?iso- propanol (1 portion of 4N HCl: 100 portion of isopropanol). Aer 20min of solubilization, the absorbance of 570nm was measured with a microplate reader (BioTek Instruments, Winooski, VT, USA). A???????? ?????? e apoptotic cells were detected using Annexin V and PI staining and the method was modiedfromapreviousstudy37.Briey,BCEC/D-1bcellsweretreatedwithdierentdosesofamantadineHCl (0?2000M)for24h.CellswerestainedwithAlexaFluor488AnnexinVandPIinbindingbueraccordingto themanufacturer?sprotocol(ermoFisherScientic,Waltham,MA,USA)andanalyzedbyFC500owcytom- eter(Beckman-Coulter,Fullerton,CA,USA).Atotaloftenthousandeventswerecollectedpersample,anddata were acquired and processed using CXP analysis soware (Beckman-Coulter, Fullerton, CA, USA). ?????? ??? ???????? ?????? e caspase 3/7 activity assay was used to detect the activity of caspase 3 or 7 in the cells and the method was modied from a previous study37. Briey, a total of 5¡Ñ103 BCE C/D-1b cells wereseededina96-wellwhiteplateandallowedtoacclimatizeovernight.Aerward,cellsweretreatedwithdif- ferent doses of amantadine HCl (0?2000M) for 24h. ereaer, Caspase-Glo 3/7 reagent was added to each wellandgentlymixedusingaplateshakerat300?500rpmfor30s,andthensampleswereincubatedfor30min atRT.Enzymeactivitywasdirectlyproportionaltoluminescence.eluminescenceintensitywasdetectedbya luminescencemicroplatereader(BioTekInstruments,Winooski,VT,USA),andthedatawerenormalizedrela- tive to the caspase 3/7 activity of cells treated with DMSO alone. ????????????????? ecellcycleanalysiswasdetectedDNAcontentusingowcytometryandthemethod wasmodiedfromapreviousstudy38.Briey,BCEC/D-1bcellsweretreatedwithdierentdosesofamantadine HCl (0?2000M) for 24h. Cells were trypsinized, washed twice by cold PBS, and xed in 70% ethanol over- night at 4¢XC. Aer xation, cells were washed twice with PBS and then incubated in PBS containing PI, RNase A and Triton X-100 at 4¢XC for 30min. e cell cycle phase distribution was assessed by FC500 ow cytometer (Beckman-Coulter, Fullerton, CA, USA). Data were analyzed by Kaluza analysis soware (Beckman Coulter, Brea, CA, USA). ???? ?????? e alkaline comet assay was used for the detection of DNA single-strand breaks and per- formed according to our previous study39. Briey, plain glass slides were pre-covered with 1% normal melting point agarose in PBS (pH 7.4) and allowed to dry on a at surface at room temperature. BCE C/D-1b cells were treatedwithdierentdosesofamantadineHCl(0?2000M)for24h.Atotalof105 cellsweregentlymixedwith 0.5% low melting point agarose in PBS (pH 7.4), rapidly layered onto the precoated slides, and covered with a coverslip. Aer removing the cover slip, cells were immersed in a freshly made alkaline lysis solution (2.5M NaCl,100mMNa2EDTA,10mMTrisand1%TritonX-100atpH10)at4¢XCfor1h.Aerward,theslideswere placed in an electrophoresis tank containing 0.3M NaOH and 1mM Na2EDTA and run electrophoresis (30V, 300mA)for15minat4¢XC.Slidesweresoakedinacoldneutralizingbuer(400mMTrisbuer,pH7.0)at4¢XC for 5min, stained with PI (2.5g/mL), and analyzed by uorescence microscopy. One hundred cells per slide were scored into classes 0, 1, 2, 3 and 4 respectively according to the relative intensity of uorescence in the tail. ??? ????????????? ?????? For cell proliferation, this was evaluated at two levels: DNA synthesis and cell division using EdU incorporation assay and CFSE cell-division tracking respectively. For the detection of DNA synthesis, BCE C/D-1b cells were treated with dierent doses of amantadine HCl (0?2000M) for 24h and then5MEdUwasaddedintothecellculturemediumfor6h.ecellularEdUcontentwasmeasuredbyow cytometry.Forthedetectionofcelldivision,BCEC/D-1bcellswerelabeledwith1CFSEfor10minat37¢XC andprotectedfromlight.CellswerewashedthreetimeswithDMEMcontaining10%FBSandthentreatedwith dierent doses of amantadine HCl (0?2000M) for 7days. e dividing cells were detected by ow cytometry. ???????????????????????? Cellpermeabilityassaywasmodiedaccordingtoapreviousstudy40.Briey, 12-transwell inserts (0.4m polyester membrane, Corning, New York, USA) were coated with collagen type I (5g/cm2, Corning, New York, USA) at room temperature for 30min and then incubated at 37¢XC for 2h. A totalof2.5¡Ñ104 BCEC/D-1bcellswereseededintotheinsertsandallowedtoacclimatizeovernight.Cellswere treated with various doses of amantadine HCl (0?2000M) or DTX (10 and 100nM) for 24h and then treated with1mg/mLFITC-dextranontheupperchamber.100Lsamplesweretakenaer0,20,40or60minrespec- tively from the lower chamber, and uorescent intensity was measured (ex: 485nm; em: 535nm) using a uo- rescence plate reader (Epoch, Biotek Instruments, USA). e removed volume was replaced by fresh medium. e absolute permeability P [cm/s] was calculated by the following equation. P = [C(t) - C(t0)] . V)/(U . t . C3) C(t):FITC-dextranconcentration(g/mL)attimepointselectedforcalculation;C(t0):FITC-dextranconcen- tration (g/mL) at 0min; V: volume (cm3) in the lower chamber; A: surface of transwell membrane (cm2); t: durationoftheux(s);C0:initialFITC-dextranconcentration(g/mL)intheupperchamber. ereareseverallimitationsinthepresentstudy.Firstly,becausehumancorneaendothelialcellsarenoteasy to obtain, bovine cornea endothelial cells were used to examine the toxic eect of amantadine HCl, and there might well be dierences between bovine and human cornea endothelial cells. Secondly, further experiments could not proceed due to the lack of antibodies to bovine cells; thirdly, an invitro experimental model could not achieve the cumulative dose of amantadine HCl invivo; fourthly, the invitro cytotoxic assays used in this ???????? ?????? | (2021) 11:18514 | https://dii.org/18.1038/s41598-021-98015-9 9 Vol.:(0123456789) This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientificreports/ study could not fully represent the real saturation of corneal edema; and nally, the duration of amantadine treatmentinthepresentstudymightnotrepresenttheeectsofthedruginreallifeduetoedemaformationin aphysiologicalsensemanifestingovertime. ?????????? ????????? All data are expressed as means¡ÓSD. Each value is the mean of three independent experiments. Statistical analysis was assessed via one-way ANOVA followed by Tukey post-hoc test using IBM SPSSStatisticsv.19(IBMCorp.,Armonk,NY,USA),andthesignicantdierencewassetat*p<0.05;**p<0.01; ***p<0.001. Reciived: 22 March 7921;Accepted: 7 September 2021 ????????? 1. Hubsher, G., Haider, M. & Okun, M. S. Amantadine: e journey from ghting u to treating Parkinson disease. Neurology 78, 1096?1099.https://doi.org/10.1212/WNL.0b013e31824e8f0d(2012). 2. 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A??????????????? isstudywassupportedinpartbyGrantsfromtheMinistryofScienceandTechnology,Taiwan,R.O.C.(Grant nos. MOST 108-2320-B-037-007, MOST 108-2314-B-037-079-MY3 and MOST 109-2320-B-037-007-MY3), KaohsiungMedicalUniversityResearchCenterGrant(KMU-TC108A04),andtheKaohsiungMedicalUniversity Chung-HoMemorialHospital(Grantno.KMUH105-5M55). A????? ????????????? P.Y.L.,S.C.W.andC.Y.L.conceivedanddesignedalltheexperiments.P.Y.L.,Y.H.L.,P.L.L.,C.C.L.,C.C.S.,F.Y.C., W.C.C.,S.L.H.,K.C.C.,L.Y.C.,T.E.K.,C.C.L.andY.C.C.performedtheexperiments,datacollectionandstatisti- calanalysis.P.Y.L.,S.C.W.andC.Y.L.wrotethemanuscript.Allauthorshavereadandapprovedthenalversion ofthemanuscript. ???????? ????????? eauthorsdeclarenocompetinginterests. A????????? ??????????? Correspondence and requests for materials should be addressed to S.-C.W.orC.-Y.L. Reprintsandpermassionsinformation is available at www.nature.com/reprints. 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To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. ? e Author(s) 2021 ???????? ?????? | (2021) 11:18514 | https://doi.org/10.5038/s41598-028-98005-9 01 Vil.:(0122456789) This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information.