Ntu 1: Geraniol Ameliorates Doxorubicin-Mediated raum raug mob los ntawm kev hloov pauv ntawm Antioxidant Status, Inflamation, Thiab Apoptosis: Muaj Peev Xwm Txheej Txheem Ntawm NF-kB Thiab Nrf2/Ho-1

Yog xav paub ntxiv. tiv taujtina.xiang@wecistanche.com

Abstract: Doxorubicin-mediatedraum tsis zooyog ib qho teeb meem loj hauv kev kho mob qog noj ntshav. Raws li, txoj haujlwm no tau tshawb xyuas lub peev xwm ntawm geraniol los hloov doxorubicin-inducedraum puassiv tus qauv nas. Cov nas tau muab faib ua plaub pawg: tswj, doxorubicin (20 mg / kg, intraperitoneal, ip), doxorubicin ntxiv rau 100 mg / kg ntawm geraniol, thiab doxorubicin ntxiv rau 200 mg / kg ntawm geraniol. Ib qho kev txhaj tshuaj doxorubicin ib zaug ua rau lub raum tsis zoo, raws li pom los ntawm kev hloov pauv ntshav creatinine, ntshav urea nitrogen, thiab albumin qhov tseem ceeb; nws kuj ua rau muaj kev hloov pauv hauv lub raum architecture. Tsis tas li ntawd, doxorubicin txhim kho lipid peroxidation thaum txo qis glutathione, catalase kev ua haujlwm, thiab kev qhia ntawm glutathione peroxidase thiab superoxide dismutase. Interestingly, kev kho mob ua ntej nrog geraniol cawm doxorubicin-vim kev hloov pauv hauv lub raum cov tshuaj tiv thaiv kab mob antioxidant, kev ua haujlwm enzymatic, thiab kev qhia ntawm inflammatory thiab apoptosis-mediating genes thiab proteins. Ntxiv mus, kev kho mob prophylactic nrog geraniol khaws cia feem ntau ntawm lub raum histological yam ntxwv nyob rau hauv koob tshuaj. Cov kev tshawb pom no txhawb nqa tias geraniol tuaj yeem tiv thaiv doxorubicin-mediatedraum tsis ua haujlwm. Txawm li cas los xij, kev tshawb fawb ntxiv yog xav tau los qhia meej txog cov txheej txheem ntawm geraniol cov kev tiv thaiv tiv thaiv doxorubicin-mediated raum tsis ua haujlwm.

Ntsiab lusdoxorubicin; mob; oxidative kev nyuaj siab; apoptosis; mob raum; geraniol

1. Taw qhia

Txij li thaum nws tshawb pom, anthracycline tshuaj tiv thaiv kab mob qog noj ntshav doxorubicin (Dox) tau siv dav los tiv thaiv cov qog nqaij hlav thiab hematological malignancies. Ntawm nws cov kev mob tshwm sim feem ntau yog nephrotoxicity, piav qhia tias lub raum tsis ua haujlwm nrog txo qis pom, rov ua dua, thiab tso tawm, uas cuam tshuam nrog kev pheej hmoo tseem ceeb ntawm kev mob thiab kev tuag. Nyob ib ncig ntawm 60 feem pua ​​​​ntawm cov neeg mob qog noj ntshav uas tau txais kev kho mob nephrotoxicity, txwv tsis pub kho cov txiaj ntsig ntawm Dox[5,6]. Txawm hais tias cov txheej txheem tseem ceeb uas ua rau Dox-mediated nephrotoxicity tseem tsis tau paub, cov ntaub ntawv qhia tias cov neeg koom nrog muaj xws lioxidative kev nyuaj siab, mob, thiabapoptosis. Tshwj xeeb, cov pov thawj qhia tias txo Dox-txuasoxidative kev nyuaj siab, mob, thiab cov xwm txheej apoptotic tuaj yeem pab txo cov tshuaj rau lub raum toxicity.

Nuclear factor (erythroid-derived 2)-zoo li 2 (Nrf2) yog ib qho redox-sensitive transcriptional yam nrog antioxidant, anti-inflammatory, thiab cytoprotective peev xwm uas txhawb nqa cellular tiv thaiv systems. Raws li oxidative kev nyuaj siab, Nrf2 tso nws cov cytoplasmic inhibitor protein, uas nkag mus rau hauv lub nucleus thiab activates ob peb genes koom nyob rau hauv antioxidant tiv thaiv, xws li heme oxygenase-1(Ho-1), glutathione peroxidase (GPx), catalase( CAT), thiab superoxide dismutase (SOD) [14-19]. Kev kho mob nrog Dox tau pom tias yuav txo tau ob qho tib si mRNA thiab protein qhia ntawm Nrf2 thiab, yog li ntawd, ntawm cov noob caj noob ces thiab cov protein, ua rau lub raum toxicity.

Nuclear factor-kB (NF-kB) yog ib qho kev hloov pauv uas tswj cov kev qhia ntawm ntau cov noob caj noob ces; Raws li lub hauv paus ntawm NF-kB txoj hauv kev, uas yog qhib los ntawm cov pa oxygen reactive (ROS), nws tuaj yeem ua rau muaj kev mob tshwm sim thiab ua rau cov ntaub so ntswg raug mob [8,22,23]. Ntau cov kev tshawb fawb tau tshaj tawm Dox ua rau mob thiab ua rau muaj cov pro-inflammatory cytokines xws li interleukin-6(IL-6), interleukin-1 beta (L-1 ), thiab qog necrosis factor-alpha(TNF-)[11,13]. Tsis tas li ntawd, nce ROS ntau lawm tom qab Dox-mediated raum kev puas tsuaj tau pom tias ua lub luag haujlwm tseem ceeb hauv kev ua kom lub cev apoptotic txoj hauv kev los ntawm mitochondrial instability. Cov kev txiav txim tseem ceeb ntawm txoj kev no yog mitochondrial-associated proteins Bax thiab Bcl-2; qhov sib npaug ntawm Bcl-2 rau Bax tiv thaiv apoptosis, thaum qhov tsis sib xws ua rau muaj kev nce ntxiv ntawm cov membrane permeability thiab cytochrome c rau hauv cytosol, uas ua rau caspase-9(Casp-9) thiab caspase{ {21}} (Casp-3). Qhov kev ua kom zoo li no feem ntau ua rau DNA tawg thiab tuag ntawm tes.

Nws yog qhov txaus siab rau kev siv tshuaj khomob ntau tshaj plaws ntawm cov tshuaj chemotherapeutic thaum txo lawv cov teebmeem tsis zoo. Raws li, kev tshawb fawb tau ua tiav los tshawb xyuas kev siv ntau tus neeg ua haujlwm nrog Dox los kho nws cov teebmeem tsis zoo [13,30,31]. Herbal extracts thiab lawv cov khoom xyaw bioactive tau ntev tau lees paub rau lawv lub peev xwm los txo cov tshuaj tua kab mob toxicity. Geraniol yog acyclic monoterpene cawv pom nyob rau hauv yuav luag txhua yam tseem ceeb roj, xws li cov qhiav, sawv, txiv kab ntxwv, lavender, thiab txiv qaub [32,33]. Ntau cov kev tshawb fawb tau tshaj tawm tias geraniol muaj ntau yam muaj txiaj ntsig zoo, xws li tshuaj tiv thaiv kab mob, tiv thaiv kab mob cancer [35], tshuaj tiv thaiv kev nyuaj siab [36], tshuaj tiv thaiv kab mob [33], thiab, ntxiv rau, nws tuaj yeem yooj yim mob ntshav qab zib nephropathy [37] .

Rau qhov zoo tshaj plaws ntawm peb txoj kev paub, tam sim no tsis muaj cov ntaub ntawv tshwj xeeb ntawm kev tiv thaiv kev tiv thaiv ntawm geraniol tiv thaiv Dox-mediated nephrotoxicity. Yog li ntawd, peb tau kawm txog cov teebmeem thiab tej yam tshwm sim ntawm kev ua ntawm geraniol ntawm kev raug mob los ntawm Dox los ntawm NF-kB thiab Nrf2/Ho-1 qhia txoj hauv kev hauv ob lub raum ntawm Wistar nas.

2. Cov khoom siv thiab cov txheej txheem

2.1. Tsiaj

Txiv neej Wistar nas tau txais los ntawm King Saud University (KSU) chaw zov tsiaj hauv Riyadh, Saudi Arabia. Cov tsiaj tau khaws cia rau hauv cov xwm txheej tswj hwm, xws li lawv tau khaws cia hauv chav sov (25 ± 1 degree) nrog lub voj voog 12 h lub teeb / tsaus ntuj thiab muaj kev txwv tsis pub nkag mus rau cov dej thiab cov khoom noj khoom haus raws li kev tso cai los ntawm KSU Lub Tsev Haujlwm Saib Xyuas Kev Ncaj Ncees Hauv Zos. (REC) nyob rau hauv daim ntawv tso cai naj npawb KSU-SE-19-122.

2.2. Kev sim tsim

Qhov kev tshawb nrhiav no tau tshuaj xyuas tag nrho ntawm 32 tus txiv neej Wistar nas hnyav 190-210 g, faib ua plaub pawg. Cov nas tau muab ib lub lim tiam los hloov mus rau ib puag ncig ua ntej kev kawm. Cov pab pawg tsheb, tseem hu ua pawg tswj hwm, muaj cov nas uas tau muab cov tshuaj ntawm qhov ncauj ntawm cov ntsev ib txwm. Pawg II muaj cov nas uas tau muab Dox (20 mg / kg ip ib koob tshuaj) rau hnub 17 [11]. Pab pawg ⅢI thiab IV nas tau txais koob tshuaj tiv thaiv kab mob ntawm geraniol (qhov ncauj) ntawm 100 thiab 200 mg / kg, raws li, rau 18 hnub, thiab hnub 17, zoo ib yam, raug rau Dox (20 mg / kg, ip). Hnub tim 18, cov nas tau euthanized siv ketamine/xylazine ua ke hauv ib cheeb tsam tswj. Cov ntshav tau sau thiab muab cais tawm, thiab ob lub raum raug tshem tawm thiab tam sim ntawd flash-krozen hauv kua nitrogen. Kev soj ntsuam biochemical, noob thiab protein qhia tau ua tiav ntawm cov ntaub so ntswg khov. Rau kev kuaj mob histological, cov qauv hauv lub raum tau ntxuav hauv PBS thiab tom qab ntawd khaws cia hauv 4 feem pua ​​​​ formaldehyde tov. Thoob plaws hauv kev tshawb nrhiav, tsis muaj qhov qhia txog kev nyuaj siab lossis kev tuag ntawm tus tsiaj sim.

2.3. Kev txiav txim siab ntawm lub raum ua haujlwm cim

Txhawm rau cais cov ntshav los ntawm cov ntshav sau thaum lub sijhawm txi, cov ntshav kuaj tau centrifuged rau 10 min ntawm 2000 × g hauv lub centrifuge ua ntej txias. Tom qab ntawd cov ntshav tau muab tshuaj ntsuam xyuas kom muaj nuj nqis albumin, ntshav urea nitrogen (BUN), thiab creatinine. Tus nqi ntawmlub raum ua haujlwmCov cim tau suav nrog Siemens Autoanalyzer Dimension RXL MAXTM, Siemens, Washington, DC, USA.

2.4.Eoaluation ntawm Lipid Peroxidation

Lipid peroxidation tau soj ntsuam hauv cov ntaub so ntswg hauv lub raum raws li tau piav qhia yav dhau los los ntawm Ohkawa li al., nrog kev hloov kho me ntsis.

2.5. Quantification ntawm Txo Glutathione

Qib ntawm txo glutathione (GSH) nyob rau hauv lub raum post-mitochondrial supernatant (PMS) tau ntsuas raws li txoj kev tau piav qhia los ntawm Sedlak thiab Lindsay, nrog rau kev hloov kho me me.

2.6. Quantification ntawm Kev Ua Haujlwm ntawm Catalase

Qhov kev ua ntawm CAT hauv lub raum PMS tau txiav txim siab raws li txoj kev piav qhia los ntawm Claiborne, nrog kev hloov kho me me.

2.7. Gene Expression Analysis (RT-qPCR)

Lub TRIzolTMreagent (Thermo Scientific, Waltham, MA, USA) tau siv los rho tag nrho RNA los ntawm lub raum cov ntaub so ntswg raws li cov chaw tsim khoom cov lus qhia. Lub purity thiab kom muaj nuj nqis ntawm rho tawm RNA specimens raug txiav txim siv ib tug NanoDropTM 8000 Spectrophotometer (Thermo Scientific, USA). Cov RNA cais tawm tom qab ntawd thim rov qab mus rau cDNA siv cDNA Synthesis SuperMix (Bimake, Houston, TX, USA), thiab cov lus qhia noob tau suav nrog SYBR Green Master Mix (Bimake, Houston, TX, USA) ntawm Applied Biosystems 7500 Fast Real- Lub Sijhawm PCR System. AACt txoj hauv kev tau siv los txiav txim siab txog qhov sib txheeb ntawm ntau cov noob ntawm cov pab pawg, nrog GAPDH ua haujlwm ua cov noob tu tsev. Table 1 teev cov primer sequences siv nyob rau hauv txoj kev tshawb no (IDT, Leuven, Belgium).

2.8. Immunoblot Analysis

Western blot tsom xam tau ua raws li tau piav qhia los ntawm Alasmari li al. [41]. Luv luv, cais cov proteins (30-50 ug) tau electrophoresed ntawm SDS-PAGE gels thiab tom qab ntawd xa mus rau PVDF daim nyias nyias. Tom qab thaiv nrog 5 feem pua ​​​​ntawm cov mis nyuj qhuav rau 60 feeb, blots tau tshawb xyuas ib hmos ntawm 4 degree nrog cov tshuaj tiv thaiv thawj zaug (tawm tsam Ho-1, Nrf2, TNF-, I6, NF-kB-p65, cleaved-caspase{{ 14}}, Bcl-2, Bax, and GAPDH, dilution 1:1000). Cov membranes tau tom qab ntxuav, ces incubated rau 60 min ntawm chav tsev kub nrog haum HRP-conjugated thib ob antibodies (dilution: 1: 5000). Cov khoom siv ECL reagent thiab gel imaging khoom (Bio-Rad, Hercules, CA, USA) tau siv los kuaj pom muaj cov proteins ntawm daim nyias nyias.

2.9.Histopathology Studies

Lub raum cov ntaub so ntswg tau kho tom qab 4 feem pua ​​formaldehyde, tom qab ntawd ua tiav rau paraffin sectioning thiab staining. Lub microtome tau siv los txiav cov ntu paraffin ntawm qhov tuab ntawm 3 μm. Cov ntu txiav tau raug kho kom tshem tawm cov quav ciab, thiab siv cov tshuaj hematoxylin ntxiv rau eosin (H&E) dye rau kev kuaj histopathological. Lub raum histology cov duab tau txais los ntawm kev siv lub koob yees duab DP72 ua ke rau Olympus BX microscope.

2.10.Data Analysis

Cov ntaub ntawv tau txheeb xyuas nrog lub khoos phis tawj-raws li kev pab cuam Graph Pad Prism 5 (San Diego, CA, USA), thiab cov txiaj ntsig tau nthuav tawm raws li txhais tau tias thiab tus qauv sib txawv (txhais tau tias ± SD). Qhov sib txawv nruab nrab ntawm pab pawg tau raug soj ntsuam siv ib-txoj kev ANOVA ua raws li Tukey qhov kev sib piv, nrog tus nqi p-nqi tsawg dua 0.05 qhia txog qhov tseem ceeb (p<>