R. Vesicarius L. Exerts Nephroprotective Effects Tawm tsam Cisplatin-induced Oxidative Stress
Mar 26, 2022
Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com
Md. Mahmud Hasan1, Feem ntau. Sayla Tasmin2, Ahmed M. El-Shahawi3, Mona M. Elseehy4, Md. Abu Reza ib1thiab Ariful Haque2*
Abstract
Keeb kwm:Cisplatin yog ib qho tshuaj tiv thaiv kab mob zoo heev, tab sis nws txoj kev siv tau poob qis heev vim yog mob nephrotoxicity. R. vicarious L. yog ib tsob ntoo nplooj uas muaj pov thawj muaj cov tshuaj tiv thaiv angiogenic, tiv thaiv kev mob, tiv thaiv proliferative, hepatoprotective, thiab nephroprotective peev xwm. Yog li, txoj kev tshawb no tau tsim los tshuaj xyuas nws cov tshuaj methanol extract (RVE) kom muaj txiaj ntsig nephroprotective.
Txoj kev:Feem ntau, hauv vitro, cov haujlwm antioxidant ntawm RVE tau lees paub raws li 2, 2-diphenyl-1-picrylhydrazyl (DPPH) dawb radical scavenging aptitude. Tom qab ntawd, Swiss Albino txiv neej nas tau kho nrog cisplatin (2.5 mg / kg) rau 5 hnub ua tiav txhawm rau ua rau muaj nephrotoxicity. Rov qab los ntawm nephrotoxicity raug tshuaj xyuas los ntawm kev kho tsiaj nrog RVE (25, 50, thiab 100 mg / kg) intraperitoneally (ip) rau 5 hnub sib law liag. Tom qab ua tiav kev kho mob, cov nas tau muab txi thiab muab lub raum sau. Ib feem ntawm nws yog homogenized hauv sodium phosphate tsis rau kev ntsuas malondialdehyde (MDA) qib, lwm qhov yog siv los ntsuas cov noob (NQO1, p53, thiab Bcl-2) qhia. Ntxiv mus, hydrogen peroxide (H2O2) neutralizing muaj peev xwm ntawm RVE raug soj ntsuam nyob rau hauv HK{15}} hlwb hauv vitro. Thaum kawg, bioactive phytochemicals hauv RVE tau txiav txim siab siv roj chromatography-mass spectrometry (GC-MS).
Cov txiaj ntsig:RVE pom nyob rau hauv vitro antioxidant kev ua si nyob rau hauv ib koob tshuaj-dependent zam nrog 37.39 ± 1.89 ug / mL IC50 tus nqi.
Kev kho mob nrog RVE zoo kawg nkaus (p < {{0}}}05)="" txo="" mda="" cov="" ntsiab="" lus="" hauv="" cov="" ntaub="" so="" ntswg.="" tsis="" tas="" li="" ntawd,="" qhov="" kev="" qhia="" ntawm="" nqo,="" p53,="" thiab="" bcl-2="" noob="" tau="" txo="" qis="" (p=""><0.05) txo="" qis="" hauv="" cov="" koob="" tshuaj="" raws="" li="" kev="" tswj="" hwm="" ntawm="" rve.="" rve="" tseem="" ceeb="" (p="">< 0.05)="" thim="" rov="" qab="" qib="" h2o2="" hauv="" hk-2="" hlwb="" kom="" zoo="" li="" qub.="" los="" ntawm="" gc-ms,="" kaum="" lub="" tebchaw="" suav="" nrog="" peb="" paub="" antioxidants="" "4h-pyran-4-ib,="" 2,="" 3-dihydro-3,5-dihydroxy-6-methyl-"="" ,="" "hexadecanoic="" acid",="" thiab="" "squalene"="" tau="" kuaj="" pom.="" cov="" extract="" tau="" nplua="" nuj="" nrog="" alkaloid="">
Xaus:Zuag qhia tag nrho, RVE muaj kev tiv thaiv zoo tiv thaiv cisplatin-vim lub raum puas.
Ntsiab lus:Cisplatin, R. vicarious, nas, raum, HK-2 hlwb, Oxidative stress, NQO1 gene
Cistanche deserticola tiv thaiv kab mob raum, nyem qhov no kom tau txais cov qauv
Taw qhia
Kev ntxhov siab oxidative yog qhov tshwm sim ntawm qhov tsis sib xws ntawm kev tsim cov pa oxygen reactive (ROS) thiab cov txheej txheem tiv thaiv antioxidant tsis tu ncua [1]. Cov tshuaj tiv thaiv biochemical tsis tu ncua, nquag raug rau qhov chaw tsis zoo, thiab kev noj cov tshuaj xenobiotics siab ua rau ROS ntau lawm [1]. ROS cuam tshuam nrog cov cysteine residues ntawm redox-sensitive signaling molecules nrog rau cov kev hloov pauv, protein tyrosine phosphatases, thiab protein kinases; Yog li ntawd, oxidation ntawm thiol pab pawg ntawm cov residues coj mus rau kev hloov pauv ntawm cov proteins tsom, kev ua haujlwm lom neeg, kev ua kom muaj peev xwm, kev tiv thaiv kab mob, thiab ntxiv cell nyob / tuag paradigms [2]. Oxygen-muaj tshuaj lom neeg uas muaj cov khoom reactive hu ua ROS uas suav nrog dawb radicals thiab non-radical molecules xws li superoxide thiab H2O2, feem [3]. Oxidative stress induced los ntawm ROS yog txuas nrog etiology ntawm ntau yam kab mob xws li mob qog noj ntshav. Mob myeloid leukemia (AML) yog mob qog noj ntshav ntawm cov qe ntshav hauv cov pob txha. Cov xwm txheej cellular thiab molecular hauv qab AML suav nrog DNA puas, clonal propagation, nce cell tuag, thiab ntxiv genetic instability, uas yog tshwm sim los ntawm ROS-induced oxidative kev nyuaj siab [4]. Tib neeg lub cev muaj zog tau txais txiaj ntsig nrog ntau lub tswv yim uas tuaj yeem tsim cov tshuaj tiv thaiv antioxidants los tiv thaiv oxidative kev nyuaj siab uas ua rau kev tiv thaiv cov hlwb los ntawm cov tshuaj lom thiab pab tiv thaiv kab mob [5]. Txawm li cas los xij, cov hlwb tsim cov txheej txheem endogenous los tiv thaiv oxidative kev nyuaj siab thiab txuag ROS [6].
NAD(P)H: quinone oxidoreductase 1 (NQO1) yog ib qho flavoenzyme [7] uas tuaj yeem ua rau ob lossis plaub-electron reduction thiab siv cov cuab yeej no los tshem tawm quinines [8]. Nws tuaj yeem tiv thaiv cov hlwb los ntawm kev puas tsuaj oxidative los ntawm kev ua kom redox cycling ib sab thiab los ntawm kev txo qis ntawm cov dawb radicals [8]. Dhau li ntawm xenobiotic detoxification, NQO1 tseem koom nrog hauv superoxide neutralization, kev hloov pauv ntawm p53 proteasomal degradation [9], Bcl -2 inhibition [10], thiab txhim khu kev raug mob ntawm tes [11].
Cisplatin yog thawj Pawg Saib Xyuas Khoom Noj thiab Tshuaj (FDA)-pom zoo platinum-based tshuaj tiv thaiv kab mob [12]. Cisplatin exerts apoptosis los ntawm inducing oxidative kev nyuaj siab thiab overexpression ntawm qog suppressor noob p53 [12]. Ntau yam kev phiv xws li nephrotoxicity, hepatotoxicity, gastrotoxicity, ototoxicity, myelosuppression, thiab neurotoxicity yog tshwm sim los ntawm cisplatin-induced oxidative stress [12]. Cov kev mob tshwm sim no tau ua rau txo qis kev siv cisplatin txawm tias nws muaj txiaj ntsig zoo rau kev tiv thaiv kab mob. Cisplatin paub zoo tias yuav ua rau muaj kev ntxhov siab oxidative thiab suppress NQO1 gene hauv nas raum [13]. Yog li ntawd, kev tshawb nrhiav thiab kev lees paub rau cov txiaj ntsig zoo ntawm cov tshuaj antioxidants tau dhau los ua ib qho kev paub txog. Kev noj cov zaub mov uas tau txais los ntawm cov nroj tsuag antioxidants xws li flavonoids, carotenoids, thiab phenolic tebchaw yuav ua rau muaj kev tiv thaiv kab mob plawv, cataracts, thiab cancer [14].
R. vicarious (Polygonaceae) hu ua "Takpalong/ Chukapalong/Amlabetom" hauv Bengali [15]. Nws loj hlob nyob rau hauv cov suab puam thiab ib nrab suab puam ntawm Asia, Australia, thiab North Africa [16]. Nws yog ib tsob nroj me me uas muaj kev puas tsuaj hauv Bangladesh. Hauv Bangladesh, tib neeg haus tag nrho cov nroj tsuag ua zaub tom qab ua noj nrog ntsev, cov txuj lom sib txawv, thiab roj. Qee zaum, tib neeg tsuas yog siv cov nplooj tshiab hauv cov zaub xam lav sib xyaw ua lwm txoj rau lettuce. Cov nplooj nyoos yog me ntsis qaub, tab sis nws dhau los ua qaub heev tom qab ua noj. Ntxiv mus, cov nplooj me me feem ntau tau muab sib xyaw rau hauv cov ntses tais diav thaum ua noj kom muaj cov kua qaub me me.
Cov nroj tsuag no tau siv los ua zaub thiab tshuaj ntsuab thoob ntiaj teb [17]. Cov nplooj thiab cov noob yog siv los ua tshuaj tua kab mob rau nab thiab scorpion venom, ntsig txog [17]. Hauv kev kho mob pej xeem, R. vicarious tau ntev tau siv los kho cov kab mob siab, kev zom zaub mov tsis zoo, cem quav, kab mob, ntuav, flatulence, mob plawv, mob, mob poov xab, dyspepsia, toothache, bronchitis, hawb pob, scabies, leucoderma, thiab raws li ib tug tshwm sim. laxative, plab, appetizer, tonic, diuretic, thiab analgesic [18] Cov nroj tsuag no suav nrog ntau cov tshuaj lom neeg tseem ceeb suav nrog flavonoids, anthraquinones, carotenoids, vitamins, lipids, thiab organic acids, uas yog lub npe hu ua antioxidant, antimicrobial, thiab anticancer agents [19]. Txhua feem ntawm cov nroj tsuag no muaj quercetin (flavonoids) hauv qhov siab [15]. Cov nroj tsuag no muaj 0.25 mg vitamin A, 1.33 mg vitamin C, 2.37 mg vitamin E [15], 3.38 mg flavonoids, thiab 5.66 mg polyphenols [20] ib 100 g qhuav hnyav.
Shahat thiab cov npoj yaig [21] tau pom cov tshuaj tiv thaiv angiogenic thiab tiv thaiv kev loj hlob ntawm methanol (80 feem pua) extract ntawm R. vicarious aerial feem tiv thaiv kab mob hepatocellular hauv cov qauv nas. Lwm txoj kev tshawb fawb pom nyob rau hauv vitro anti-angiogenic peev xwm ntawm R. vicarious extract [22]. Methanol extract ntawm tag nrho R. vicarious exerts tiv thaiv carbon tetrachloride-induced hepatotoxicity nyob rau hauv vivo [23]. Anti-inflammatory nyhuv nyob rau hauv luav tau tshwm sim los ntawm methanolic nplooj extract ntawm R. vicarious [24]. Ib txoj kev tshawb fawb tsis ntev los no [25] tau tshaj tawm hauv vivo nephroprotective nyhuv ntawm fractionated ethanolic R. vicarious extract tiv thaiv gentamicin thiab potassium dichromate toxicity.
Ua raws li cov ntaub ntawv saum toj no hauv kev txiav txim siab, peb tsom los tshuaj xyuas cov txiaj ntsig ntawm R. vicarious extracts (RVE) hais txog kev rov qab los ntawm cisplatin-induced nephrotoxicity los ntawm kev tswj NQO1 gene qhia hauv tus qauv tsiaj.
cistanche cov txiaj ntsig kev noj qab haus huv: kho cov kab mob raum
Cov ntaub ntawv thiab cov txheej txheem
Tshuaj thiab reagents
Cisplatin thiab 2, 2-diphenyl-1-picrylhydrazyl (DPPH) tau yuav los ntawm SIGMA-ALDRICH (USA).
Creatinine Colorimetric Assay Kit (cov khoom ID - 700,460) tau yuav los ntawm Cayman Chemical (USA). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), thiab tshuaj tua kab mob (10,000 U/mL penicillin thiab 10,000 ug/mL streptomycin) tau muas los ntawm Gibco (Gibco Laboratories, USA). ROS-Glo™ H2O2 Assay kit thiab GoTaq® qPCR Master Mix tau txais los ntawm Promega (USA). Cov khoom siv thim rov qab TIAN- tsab ntawv M-MLV tau yuav los ntawm TIANGEN (Tuam Tshoj) thiab primers los ntawm IDT (Integrated DNA Technologies, Malaysia). Tag nrho lwm yam tshuaj thiab reagents siv nyob rau hauv qhov kev sim no yog ntawm analytical qib.
Cog qauv sau thiab extract npaj
Cov nroj tsuag tshiab R. vicarious tau yuav los ntawm lub khw hauv zos ntawm Sonadighi, Rajshahi, Bangladesh. Cov qauv cog tau txheeb xyuas thiab lees paub los ntawm Dr. Ahmad Humayan Kabir, Department of Botany, University of Rajshahi, Bangladesh. Ib qho piv txwv hauv qab daim npav no. 00095 tau muab khaws cia rau hauv herbarium ntawm Department of Botany, University of Rajshahi. Cov av ntawm cov nroj tsuag tau ntxuav, qhuav ntawm 37 degree, hauv av rau cov hmoov ntxhib siv lub tshuab ziab khaub ncaws, thiab muab cia rau hauv lub thawv ntim ntawm 4 degree. Cov hmoov zoo (10 g) yog yaj hauv methanol (500 mL). Cov ntsiab lus tau sonicated (Son-prep 150, Suav teb) ntawm 20 kHz rau 10 min. Kev lim dej ntawm cov extract tau ua los ntawm kev siv iav Fiber Lim ntawv (Macherey NAGEL, GmBH, German) nrog DURAN Lim Apparatus (German). Thaum kawg, cov filtrate tau concentrated siv lub tshuab ziab khaub ncaws (VirTis Benchtop Pro, SP SCIENTIFIC, USA). Lub extract tau thaum kawg lub npe RVE.
Kev ntsuam xyuas kev ua haujlwm hauv vitro antioxidant
In-vitro antioxidant muaj peev xwm ntawm RVE tau ua raws li kev khawb ntawm DPPH raws li tau piav qhia yav dhau los [26] nrog kev hloov kho me ntsis. DPPH radical scavenging peev xwm ntawm RVE tau soj ntsuam raws li hloov cov xim liab ntawm DPPH rau xim daj. Cov tshuaj tiv thaiv sib xyaw hauv txhua lub raj micro-centrifuge (2 mL) muaj 950 μL methanolic tov ntawm DPPH radicals (0.1 mM) thiab 50 μL RVE los ntawm tsib qhov sib txawv (200, 500, 1000, 2000, thiab 4000 ug / mL methanol) ua kom qhov kawg ntawm 10, 25, 50, 100 thiab 200 ug / mL. Lwm lub raj uas muaj 50 μL methanol thiab 950 μL methanolic tov ntawm DPPH tau khaws cia raws li kev tswj hwm. Cov hlab ntsha tau muab tso rau hauv qhov chaw tsaus rau 30 feeb. Lub absorbance ntawm cov khoom sib tov tau coj los ntawm 517 nm siv GENESYS 10S UV-VIS spectrophotometer (Thermo SCIENTIFIC, USA). Thaum kawg, qhov feem pua ntawm cov kev ua si ntawm radical scavenging (RSA) raug xam raws li qhov hloov pauv ntawm DPPH siv cov qauv hauv qab no feem pua RSA=[(ADPPH − ARVE)/ADPPH] × 100 qhov twg ADPPH yog qhov absorbance ntawm DPPH tov. (tswj) thiab ARVE yog qhov nqus ntawm RVE cov tshuaj. Qhov concentration ntawm RVE ua rau 50 feem pua RSA tau hu ua IC50 tus nqi thiab tau suav nrog siv daim duab tso rau feem pua RSA tiv thaiv qhov sib txawv RVE ntau siv.
Kev sim tsiaj txhu thiab kev sim tsim
Txiv neej Swiss Albino nas muaj hnub nyoog 42 hnub (30-32 g lub cev hnyav) tau acclimatized rau 1 lub lis piam ua ntej pib qhov kev sim hauv ib chav (kub ntawm 25 ± 2 degree thiab ~ 50 feem pua cov av noo, 12 h tsaus / lub teeb voj voog). Cov dej haus thiab zaub mov tau muab tso rau hauv libitum.
Cov nas tau muab cais ua yim pawg (n {{0}}). Thawj pawg (tswj) tau kho nrog 0.2 mL ntawm 0.9 feem pua NaCl. Plaub pawg tom ntej no tau kho nrog cisplatin ntawm 2.5 mg / kg rau 5 hnub ntawm lub sijhawm 24 teev. Tom qab kev tswj hwm cisplatin, ib pab pawg (ib pab pawg thib ob) raug tso tseg yam tsis muaj kev kho mob ntxiv thiab raug xaiv raws li pawg tswj hwm kev ntxhov siab. Pab pawg thib peb, plaub, thiab thib tsib tau kho ntxiv nrog RVE ntawm 25, 50, thiab 100 mg / kg, raws li 5 hnub. Ntxiv peb pawg tau kho nrog RVE nkaus xwb ntawm 25, 50, thiab 100 mg / kg, raws li 5 hnub. Cisplatin thiab RVE tau yaj hauv dej distilled. Tag nrho cov kev kho mob tau muab intraperitoneally. Tom qab 24 teev ntawm kev kho mob kawg, cov tsiaj tau euthanized tom qab lub ncauj tsev menyuam dislocation [25]. Tom qab ntawd lub peritoneum tau qhib nrog txiab, cov ntshav tau sau tom qab lub plawv dhia, thiab lub raum tau sau los ntawm kev siv forceps. Ntshav tau raug kuaj xyuas qib ntawm creatinine hauv cov ntshav. Lub raum tau raug ntsuas los ntsuas cov qib malondialdehyde thiab cov noob qhia.
Kev ntsuas ntshav creatinine
Serum creatinine tau ntsuas siv Creatinine Colorimetric Assay Kit-700,460 (Cayman Chemical, USA) raws li cov chaw tsim khoom raws tu qauv muab nrog cov khoom siv.
Kev ntsuas ntawm lub raum lipid peroxidation
Malondialdehyde (MDA) yog ib qho khoom kawg ntawm lipid per- oxidation hauv lub raum cov ntaub so ntswg thiab feem ntau raug ntsuas raws li qhov ntsuas ntawm ROS ntau lawm. Txawm li cas los xij, qib MDA tau ntsuas hauv lub raum cov ntaub so ntswg raws li kev tshawb fawb ua ntej [27]. Thaum xub thawj, lub raum cov ntaub so ntswg yog homogenized nyob rau hauv sodium phosphate buffer (0.1 M, pH 7.4). Cov tshuaj tiv thaiv muaj xws li 0.8% thiobarbituric acid (1.5 mL), 8.1% SDS (200 μL), 20 feem pua (pH 3.5) acetic acid (1.5 mL), thiab dH2O (600 μL) tau ntxiv rau 100 μL ntawm homogenized cov ntaub so ntswg, thiab qhov sib tov yog ces incubated ntawm 95 degree rau 1 h. Tom qab txias, cov sib tov tau centrifuged ntawm 10, 000 g rau 10 min ntawm 4 degree thiab qhov absorbance ntawm supernatant yog ntsuas ntawm 532 nm nrog tus qauv 1, 1, 3, 3- tetra methoxy propane. Tus nqi ntawm tag nrho cov protein tau ntsuas siv cov khoom siv Bradford Protein Assay (BIO-RAD, Tebchaws Asmeskas), thiab los ntawm kev sib piv nrog cov qauv bovine serum albumin (BSA). Qhov kev siv ntawm lipid peroxides tau qhia raws li nanomoles (nM) ntawm MDA ib milligram (mg) ntawm cov protein.
Real-time polymerase chain reaction (real-time PCR)
PCR lub sijhawm tiag tiag tau ua raws li tau piav qhia yav dhau los [28, 29]. Tag nrho RNA los ntawm lub raum cov ntaub so ntswg raug rho tawm siv TRIzol® reagent (Invitrogen) raws li cov txheej txheem muab los ntawm cov chaw tsim khoom. Tom qab ntawd cov RNA rho tawm (1 ug) tau hloov mus rau cDNA. Ua ntej, 2 μL random hexamer (10 μM), 2 μL dNTPs (10 mM), 1 ug RNA, thiab nuclease-free HO mus txog 15 μL tau noj thiab incubated rau 5 min ntawm 70 degree. Qhov sib tov tau muab tso rau hauv dej khov rau 2 min. Tom qab ntawd 4 μL ntawm 1st strand buffer (5x) thiab 1 μL M-MLV thim rov qab transcriptase tau ntxiv rau hauv txhua lub raj thiab incubated rau 10 min thiab 50
min ntawm 25 degree thiab 42 degree, feem. Thaum kawg, M- MLV thim rov qab-transcriptase enzyme yog inactivated los ntawm incubating sib tov ntawm 95 degree rau 5 min. Cov khoom sib xyaw ua ke cDNA tau raug rau PCR lub sijhawm tiag tiag kom muaj nuj nqis ntawm NQO1, p53, thiab Bcl-2 gene qhia siv cov primers tshwj xeeb (Table 1 ). Txhua qhov tshuaj tiv thaiv (10 μL) tau ua hauv triplicate suav nrog 5 μL GoTaq qPCR Master Mix (2x) (Promega, USA), 0.5 μL (10 mM) ntawm txhua tus primer, 3 μL nuclease-dawb dej, thiab 1 μL cDNA hauv 48-cov tshuaj tiv thaiv zoo. Thermal cycling tau ua los ntawm kev siv lub sijhawm tiag tiag PCR tshuab (Eco™ Real-Time PCR System, Illumine®, USA) nrog rau kev caij tsheb kauj vab: 95 degree rau 10 min, ua raws li 40 lub voj voog ntawm 95 degree rau 30 s, 50 degree rau 30s, thiab 72 degree rau 25s. Qhov tshwj xeeb ntawm PCR cov tshuaj tiv thaiv tau lees paub los ntawm kev tshuaj xyuas qhov nkhaus ntawm 95 degree rau 15 s, 45 degree rau 15 s, thiab 95 degree rau 15 s. Qhov tshwj xeeb ntawm PCR cov tshuaj tiv thaiv tau lees paub los ntawm kev tshuaj xyuas qhov nkhaus ntawm 95 degree rau 15 s, 45 degree rau 15 s, thiab 95 degree rau 15 s. Qhov txheeb ze ntawm cov noob qhia tau ua tiav siv endogenous GAPDH noob raws li kev tswj raws li txoj kev ΔΔCq.
Cell kab lis kev cai thiab kev kho mob
Tib neeg lub raum proximal tubule epithelial cell kab, HK-2 hlwb, tau khaws cia hauv DMEM ntxiv nrog 10 feem pua FBS thiab tshuaj tua kab mob (50 U/mL ntawm penicillin thiab 50 ug/mL streptomycin) nyob rau hauv lub incubator nrog 5 feem pua CO2 thiab 95 feem pua ntawm cov av noo ntawm 37 degree.
Kev ntsuam xyuas H2O2
H2O2 qib hauv HK-2 hlwb tau kwv yees los ntawm kev siv ROS- Glo™ H2O2 Assay kit (Promega, USA) raws li cov txheej txheem muab los ntawm cov chaw tsim khoom. HK-2 hlwb (1000 hlwb) hauv 70 μL DMEM tau muab tso rau hauv qhov dej ntawm 96-zoo microtiter phaj. Tom qab tso cai txuas ntawm cov hlwb ntawm phab ntsa, 10 μL DMEM los ntawm qhov dej ntawm microtiter phaj tau hloov nrog 10 μL cisplatin (25 μM hauv DMEM) thiab khaws cia hauv qhov cub rau 12 teev. Tom qab ntawd, 10 μL RVE tau ntxiv los ua qhov kawg ntawm 125, 250, thiab 500 ug / mL hauv DMEM thiab incubated rau 12 teev ntxiv. Tom qab ntawd, 20 μL H2O2 Substrate Solution thiab 100 μL ROS-Glo™ Detection Solution tau ntxiv rau txhua qhov dej. Cov tshuaj tiv thaiv yog incubated nyob rau hauv chav tsev kub rau 20 min. Thaum kawg, luminescence tau ntsuas los ntawm kev siv GloMax Luminometer (Promega, USA).
GC-MS tsom xam
GC-MS tsom xam ntawm RVE (kua kua hauv methanol) tau ua raws li tau piav qhia yav dhau los [30] siv GCMS- QP2020 (SHIMADZU) suav nrog tus qauv pib (AOC{ {5}}s), qhov pib-injector (AOC-20}i), thiab Gas Chromatograph (GC-2010 Plus) cuam tshuam rau Mass Spectrometer nruab nrog SH-Rxi{{1{{ 37}}}}}Sil MS capillary kem (30 m × 0.25 μm ID × 0.25 μm DF). Cov cab kuj roj Helium tau khaws cia ntawm tus nqi ntws tsis tu ncua ntawm 1.72 mL / min, thiab qhov ntim ntawm 5 μL tau raug nrog 10: 1 sib faib. Qhov ntsuas kub ntawm lub tshuab txhaj tshuaj tau khaws cia ntawm 220 degree, qhov kub ntawm ion yog 280 degree, qhov cub kub tau programmed los ntawm 80 degree (tuav rau 2 min), nrog rau qhov nce ntawm 5 degree / min mus rau 150 degree (tuav lub sijhawm 5.0. min), ces 5 degree / min rau 280 degree, xaus nrog 8 min isothermal ntawm 280 degree . Mass spectra raug coj ntawm 1.5 kV nrog lub sijhawm ntsuas ntawm 0.5 s thiab cov qauv tau khiav ntawm thaj tsam ntawm 45-350 m / z. Cov kuab tshuaj ncua yog los ntawm 0 mus rau 3 min, thiab tag nrho GC-MS khiav lub sij hawm yog 55 min. Qhov txheeb ze concentration ntawm cov tshuaj tau kuaj pom los ntawm kev sib piv nws qhov nruab nrab qhov siab tshaj plaws rau tag nrho cheeb tsam. Kev txhais lus ntawm huab hwm coj-spectrum hauv GC-MS tau ua los ntawm National Institute Standard thiab Technology (NIST) cov ntaub ntawv suav nrog NIST08, NIST08s, thiab NIST14.
Kev txheeb cais
Cov kev txheeb xyuas txheeb cais tau ua los ntawm ANOVA ua raws li Dunnett's T3 xeem siv IBM SPPS (version 20) software. Cov ntaub ntawv yog qhia raws li txhais tau tias ± tus qauv sib txawv (SD). Qhov sib piv tseem ceeb tau txiav txim siab ntawm p<0.05. all="" of="" the="" graphs="" were="" prepared="" using="" microsoft="" excel="" (version="">
Cov txiaj ntsig
Kev ntsuam xyuas kev ua haujlwm hauv vitro antioxidant
Txawm hais tias yav dhau los RVE tau tshaj tawm tias muaj kev ua haujlwm antioxidant, peb tau kuaj xyuas cov haujlwm antioxidant ntawm peb cov extract siv DPPH dawb radical scavenging peev. RVE neutralized DPPH koob tshuaj-raws li (Daim duab 1). RVE tau nthuav tawm ntau yam hauv vitro antioxidant kev ua haujlwm thiab suav IC50 tus nqi ntawm RVE yog 37.39 ± 1.89 ug / mL.
Kev ntsuas ntshav creatinine
Qib creatinine hauv cov nas tau nce siab (p < {{0}}}.05)="" nce="" ntxiv="" tom="" qab="" kev="" tswj="" hwm="" cisplatin="" (table="" 2).="" kev="" kho="" mob="" nrog="" rve="" zoo="" kawg="" nkaus="" (p=""><0.05) ameliorated="" creatinine="" theem="" nyob="" rau="" hauv="" ib="" koob="" tshuaj="" raws="" li="" kev="" zam="" (table="">
Kev ntsuas ntawm lub raum lipid peroxidation
Piv rau kev tswj, cisplatin considerably (p < {{0}}}.05)="" augmented="" mda="" cov="" ntsiab="" lus="" nyob="" rau="" hauv="" lub="" raum="" ntaub="" so="" ntswg="" ntawm="" nas="" (table="" 3).="" nyob="" rau="" hauv="" sib="" piv,="" rve="" kev="" kho="" mob="" considerably="" (p=""><0.05) rov="" qab="" mda="" rau="" yuav="" luag="" ib="" txwm="" nyob="" rau="" hauv="" ib="" koob="" tshuaj="" raws="" li="" kev="" zam="" (table="">
Real-time PCR
Cisplatin ntau heev (p < {{0}}.05)="" txo="" nqo1="" mrna="" kev="" qhia="" los="" ntawm="" 0.15-fold="" thiab="" nce="" p53="" thiab="" bcl-2="" mrna="" qhia="" los="" ntawm="" 24="" thiab="" 4.2-fold,="" raws="" li="" (fig.="" 2).="" rve="" muaj="" txiaj="" ntsig="" zoo="" (p="">< 0.05)="" txo="" nqo1,="" p53,="" thiab="" bcl-2="" mrna="" qhia="" nyob="" rau="" hauv="" koob="" tshuaj="" raws="" li="" kev="" zam="" (daim="" duab="" 2).="" piv="" rau="" tib="" pawg="" cisplatin-kho,="" nqo1="" mrna="" qhia="" tau="" nce="" 3.57,="" 6.36,="" thiab="" 9.28-fold="" ntawm="" 25,="" 50,="" thiab="" 100="" mg/kg="" rve,="" feem="" (fig.="" 2a).="" ib="" zaug="" ntxiv,="" p53="" mrna="" qhia="" tau="" txo="" qis="" los="" ntawm="" 0.63,="" 0.46,="" thiab="" 0.21-fold="" ntawm="" 25,="" 50,="" thiab="" 100="" mg/kg="" rve,="" raws="" li="" (fig.="" 2b).="" bcl-2="" mrna="" qhia="" kuj="" raug="" txo="" los="" ntawm="" 0.71,="" 0.40,="" thiab="" 0.32-fold="" ntawm="" 25,="" 50,="" thiab="" 100="" mg/kg="" rve,="" raws="" li="" (fig.="" 2c).="" tab="" sis,="" tsis="" muaj="" qhov="" tseem="" ceeb="" (p=""> 0.05) kev hloov pauv tau pom nyob rau hauv kev qhia theem ntawm NQO1, p53, thiab Bcl-2 noob vim kev kho mob nrog RVE (Fig. 3).
H2O2ntsuas ntsuas
Hauv kev ntsuas H2O2 ntsuas, H2O2theem tau suav tias yog proportionate rau luminescence. Kev tswj hwm ntawm cisplatin ntau heev (p < {{{0}}}="" 055)="" nce="" qib="" h2o2="" los="" ntawm="" 2.2-fold="" (fig.="" 4).="" kev="" kho="" mob="" nrog="" rve="" txo="" qis="" heev="" (p="">< 0="" 05)="" h2o2="" qib="" los="" ntawm="" 0.25,="" 0.38,="" thiab="" 0.49-fold="" ntawm="" 125,="" 250,="" thiab="" 500="" ug="" ml,="" sib="">
GC-MS tsom xam
Tag nrho ntawm 10 lub tebchaw (Table 4 thiab Fig. 5) suav nrog "Isoborneol, pentamethyldisilanyl ether (sesquiterpene alcohol)", "Thymine (pyrimidine nucleobase)", "4H-Py-ran-4-ib, 2, {{ 8}}dihydro-3, 5-dihydroxy-6-methyl- (saponin), "Hexadecanoic acid, methyl ester (fatty acid methyl ester)", "9, 12-} Octadecadienoic acid, methyl ester (fatty acid methyl ester), "9-Octadecenoic acid (Z)-, methyl ester (fatty acid methyl ester)," Methyl stearate (fatty acid methyl ester), "Disooctyl phthalate (ester), "13-Docosenamide, (Z)- (alkaloid)", thiab "Squalene (tri-terpene)" raug kuaj pom hauv RVE.
cistanche tubolosa cov txiaj ntsig: txo cov ntshav lipids
Kev sib tham
Ntuj thiab hluavtaws antioxidants tau kawm tiav thiab qhia tau hais tias ua haujlwm rau kev tiv thaiv lossis kev ua kom muaj toxicity hauv tsiaj physiology [31]. Cov tshuaj tiv thaiv antioxidant yog cov khoom tsim los ntawm cov tshuaj synthesis lossis los ntawm kev rho tawm los ntawm cov khoom noj ntuj, tab sis cov no tsis zoo ib yam li cov tshuaj antioxidants muaj nyob hauv cov zaub mov [5]. Yog li ntawd, cov kev xav tau sib cais nyob rau lub sijhawm los yog tsis yog hluavtaws antioxidants muab cov txiaj ntsig zoo rau kev noj qab haus huv xws li ntuj antioxidants [32]. Qhov kev xav tau los txo qis kev siv cov tshuaj tua kab mob antioxidant thiab nrhiav lwm txoj hauv kev, pheej yig, txuas ntxiv dua tshiab, ntuj tsim, thiab tejzaum nws muaj kev nyab xeeb ntawm cov khoom siv zoo antioxidants.
Ib qho ntawm cov txheej txheem tseem ceeb tshaj plaws yog cov khoom siv hluav taws xob nuclear erythroid-2 cuam tshuam txog -2 (Nrf2) txoj hauv kev uas feem ntau tiv thaiv cov hlwb los ntawm oxidative kev ntxhov siab vim los ntawm exogenous lossis endogenous stressors [27]. Cov tshuaj tiv thaiv antioxidants ua rau muaj kev qhia ntawm Nrf2, uas txuas ntxiv mus rau hauv lub nucleus thiab khi rau cov tshuaj tiv thaiv kab mob antioxidant (ARE) uas provokes qhia theem II detoxifying thiab antioxidant gene NQO1 [33, 34]. NQO1 yog dav thiab sib txawv hauv cov ntaub so ntswg tshwj xeeb. NQO1 yog cytosolic antioxidant flavoprotein uas catalyzes lub 2-electron txo ntawm quinones rau hydroquinones, uas ua rau detoxification ntawm electrophiles thiab cia siab tias yuav tau txais redox cycling [35]. Raws li kev tshawb fawb yav dhau los [36], -lapachone activates NQO1, uas ntxiv rau nce intracellular NAD ntxiv rau qib thiab tiv thaiv lub raum ntawm cisplatin-induced mob hnyav.
Cisplatin paub txog kev puas tsuaj hauv glomerular filtration membrane los ntawm oxidative kev nyuaj siab, o, thiab apoptosis uas tag nrho ua rau txo glomerular pom tus nqi thiab poob ntawm ib txwm membrane permeability [37]. Yog li, qib creatinine hauv cov ntshav tau nce ntxiv. Serum creatinine yog ib qho ntawm lub raum kev ua haujlwm ntawm lub raum. Kev kho cisplatin nce MDA cov ntsiab lus hauv cov ntaub so ntswg uas yog cov khoom lag luam thib ob ntawm lipid peroxidation thiab daim ntawv tshaj tawm no tsis tu ncua nrog cov kev tshawb fawb yav dhau los [27, 35, 37, 38]. Kev kho mob nrog RVE txo qis MDA cov ntsiab lus hauv lub raum. Nyob rau tib lub sijhawm, qib creatinine kuj tau txo qis uas qhia txog cov txiaj ntsig zoo ntawm RVE.
Tsis tas li ntawd, NQO1 kev qhia tau txo qis, thiab p53 thiab Bcl -2 kev qhia tau nce ntxiv tom qab kis rau cisplatin. Hais txog NQO1 thiab p53 qhia hauv vivo, peb cov txiaj ntsig zoo ib yam nrog kev kawm ua ntej [13]. Ib txoj kev tshawb fawb tsis ntev los no [39] tau pom tias cisplatin txo qis kev qhia ntawm Bcl -2 hauv lub raum ntawm nas, tab sis kuj ceeb tias peb pom cov lus qhia siab. Qhov sib txawv no yuav yog qhov sib txawv ntawm koob tshuaj [40] raws li Mohamed thiab cov npoj yaig [39] siv 8 mg / kg rau 12 hnub, thaum peb siv 2.5 mg / kg tsuas yog 5 hnub. Lwm txoj kev tshawb fawb [41] tau hais tias cisplatin tuaj yeem ua rau kev nthuav qhia ntawm Bcl -2 ntawm ib koob thaum nws tsis yog-cytotoxic. Ntxiv dua thiab, nce Bcl -2 qhia sensitizes hlwb ntawm oxidative kev nyuaj siab [40].
Txawm li cas los xij, kev qhia ntawm p53 yog qhov qis ntawm ib txwm muaj lub cev tab sis xav tias yuav tsum tau tswj hwm ib zaug kho nrog cisplatin vim tias tus neeg saib xyuas platinum-based chemotherapeutic activates p53 dependent apoptotic pathway. Thaum peb kho cov nas nrog cisplatin, p53 tau nce ntxiv hauv cov nas hauv lub raum piv rau kev tswj hwm. Lwm qhov proto-oncogene Bcl-2 kuj tseem cuam tshuam nrog NQO1 qhia qib. Ua raws li cov lus qhia p53, peb kuj pom tias Bcl - 2 qib tau nce nrog kev kho cisplatin, tab sis rov them rov qab zoo dua thaum kho nrog RVE. Qhov no yog ua tau, nyob rau hauv teb rau oxidative kev nyuaj siab, lub p53 noob tau txais activated thiab ua rau ntes cell voj voog, senescence, los yog apoptosis [6]. Nrog detoxification, NQO1 overexpression feem ntau suav hais tias muaj feem cuam tshuam nrog apoptosis hauv cov qog nqaij hlav cancer [10], txawm tias lub hauv paus txheej txheem ntawm apoptosis thiab overexpression ntawm NQO1 tseem muaj teeb meem. Ntxiv mus, nyob rau hauv hepatocellular carcinoma, NQO1 overexpression de- creases Bcl{18}} qhia [10]. Proto-oncogene Bcl- 2 kuj muaj p53 zoo li kev sib raug zoo nrog NQO1 qhia. p53 yog ib qho kev hloov pauv tshwj xeeb uas tau txais los ntawm ntau hom cellular stress [42], whereas Bcl -2 overexpression ua raws li mitochondrial pore-stabilizer los pab txhawb cytochrome-c tso tawm thaum oxidative stress [12]. Hauv peb cov ntaub ntawv, peb tau tshawb xyuas ob qho tib si ntawm cov lus teb cov noob nrog kev kho cisplatin hauv cov ntaub so ntswg hauv lub raum thiab pom lawv qhov kev nthuav qhia ntxiv. Kev kho RVE tau thim rov qab qhov kev qhia ntawm p53 thiab Bcl-2 mus rau yuav luag ib txwm nyob rau hauv koob tshuaj raws li kev zam. Hom Bcl no -2 qhia abrogation kuj tau qhia siv ROS scavenger Trolox [43]. Ntxiv mus, qib H2O2 kuj tau nce ntxiv hauv HK-2 hlwb vim kev kho nrog cisplatin hauv vitro. Tom qab kev kho mob nrog RVE, qib H2O2 tau rov qab zoo li qub. Qhov no tej zaum yog vim cov nyhuv ntawm RVE kev kho mob uas nce NQO1 qhia [44], uas tiv thaiv oxidative kev nyuaj siab [6].
GC-MS chromatogram tau lees paub tias muaj kaum lub tebchaw hauv RVE. Ntawm cov no, "4H-Pyran- 4-ib, 2, 3-dihydro-3, 5-dihydroxy-6-methyl-", "Hexadecanoic acid", thiab " Squalene" yog cov tshuaj antioxidants paub zoo [45–47]. Peb lub tebchaw no tag nrho tuaj yeem ua rau muaj kev sib koom ua ke nephroprotective nyhuv. Cov kev tshawb fawb yav dhau los kuj tau tshaj tawm txog kev induction ntawm NQO1 qhia los ntawm vitamin A [48], vitamin C [49], vitamin E [50], flavonoids [51], thiab polyphenols [50]. Yog li ntawd, daim ntawv qhia no qhia elucidating seb cov extract tshwj xeeb no muaj dab tsi ntawm vitamin A, vitamin C, vitamin E, flavonoids, thiab polyphenol, los yog tsis.
Xaus
Qhov kev tshawb pom tag nrho qhia tias RVE muaj txiaj ntsig zoo hauv kev tiv thaiv lub raum los ntawm kev puas tsuaj los ntawm cisplatin. Yog li ntawd, nws yog ib qho tseem ceeb rau elucidate lub ntsiab lus teb lub luag hauj lwm rau mitigating cisplatin-induced nephrotoxicity uas yuav ua tau zoo rau tib neeg daim ntawv thov.
Kev lees paub
Cov kws sau ntawv ua tsaug rau Bangladesh Council of Scientific thiab Industrial Research (BCSIR), Rajshahi ceg rau kev muab kev pab cuam hauv chav kuaj kom muaj nuj nqis RNA.
Cov neeg sau ntawv pab txhawb
MMH ua qhov kev sim tsim, kev sim, kev txheeb xyuas cov ntaub ntawv thiab kev npaj, sau ntawv sau thiab kho, sau ntawv kho thiab sau ntawv; MST & MME ua qhov kev sim thiab kev tshuaj xyuas cov ntaub ntawv; AME & MAR pab txhawb kev saib xyuas thiab kev pab; AH yog lub luag haujlwm rau kev tsim cov tswv yim, kev saib xyuas, cov peev txheej, thiab kev kho cov ntawv sau. Txhua tus kws sau ntawv
tshuaj xyuas cov ntawv sau. Cov kws sau ntawv tau nyeem thiab pom zoo cov ntawv sau kawg.
Nyiaj txiag
Txoj haujlwm tam sim no tau txais nyiaj los ntawm Taif University Cov Kws Tshawb Fawb Txhawb Txoj Haujlwm tus lej (TURSP - 2020/75), Taif University, Taif, Saudi Arabia.
Muaj cov ntaub ntawv thiab cov ntaub ntawv.
Tag nrho cov ntaub ntawv ntsig txog muaj thiab tuaj yeem muab tau raws li qhov kev thov rau tus neeg sau ntawv.
Cov lus tshaj tawm
Kev pom zoo ntawm kev ncaj ncees thiab kev tso cai los koom
Kev coj ncaj ncees los ua qhov kev sim no tau pom zoo los ntawm Institutional Animal, Medical Ethics, Biosafety, thiab Biosecurity Committee (IAMEBBC), Institute of Biological Sciences (IBSc), University of Rajshahi, thiab tau muab nyob rau hauv daim ntawv teev npe 31/{{1} }IAMEBBC/IBSc. Txhua txoj hauv kev tau ua raws li cov lus qhia thiab cov cai uas tau muab los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees saum toj no. Txoj kev tshawb no tau ua tiav raws li ARRIVE (Kev Tshawb Fawb Tsiaj: Kev Qhia Txog Hauv Vivo Kev sim) cov lus qhia. "Kev tso cai los koom nrog" tsis muaj feem xyuam rau txoj kev tshawb no.
Sau Paub meej
1Molecular Biology thiab Protein Science Laboratory, Department of Genetic Engineering thiab Biotechnology, Kws qhia ntawv ntawm Lub Neej thiab Ntiaj Teb Sciences, University of Rajshahi, Rajshahi 6205, Bangladesh. 2Molecular Pathology Laboratory, Lub Tsev Kawm Ntawv ntawm Biological Sciences, University of Rajshahi, Rajshahi 6205, Bangladesh. 3 Department of Biotechnology, College of Science, Taif University, PO Box 11099, Taif 21944, Saudi Arabia. 4 Department of Genetics, Kws Qhia Ntawv ntawm Kev Ua Liaj Ua Teb, Alexandria University, Alexandria 21545, Egypt.
Cov ntaub ntawv
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