Cov nyhuv ntawm Cistanches Deserticola Extract Rau Kev Raug Mob Ntawm Luav RTECs Los Ntawm H2O2

Abstract: Luav lub raum tubular epithelial hlwb raug coj mus rau hauv vitro los kawm txog cov txiaj ntsig ntawm H2O2 ntawm cell prolif eration thiab Cistanche deserticola extract ntawm tubular epithelial hlwb raug mob los ntawm H2O2. Cell morphology tau siv los soj ntsuam cov txiaj ntsig ntawm H2O2 ntawm kev loj hlob ntawm luav raum tubular epithelial hlwb los ntawm MTT txoj kev thiab kev tiv thaiv ntawm 2,20 thiab 200 mg·L － 1 C． deserticola dej extract thiab cawv extract tau kawm los ntawm LDH tso tawm. Cov txiaj ntsig tau pom tias tus naj npawb ntawm morphological hloov pauv ntawm cov hlwb tau maj mam nce ntxiv. Tus naj npawb ntawm cov cell txuas tau maj mam txo. Tus nqi inhibition ntawm cell proliferation thiab LDH tso tawm tau nce ntxiv nrog rau kev nce siab ntawm H2O2. High koob C. deserticola extract yuav txo tau qhov inhibiting nyhuv ntawm TECs proliferation los ntawm H2O2 (P ＜ 0． 01). Cov dej ex traction thiab cawv extract ntawm C. deserticola tuaj yeem txo qis LDH tso tawm (P ＜ 0. 01). H2O2 muaj qee qhov cuam tshuam rau cov tshuaj tiv thaiv kev loj hlob ntawm TECs ntawm luav, thaum C. deserticola extract muaj kev tiv thaiv ntawm TECs raug mob los ntawm H2O2

Cov lus tseem ceeb: Cistanches deserticola; lub raum tubular epithelial cell; cell puas preface

Cistanche deserticola, tseem hu ua suab puam ginseng yog qhuav, scaly, mesophyll qia ntawm Cistanche deserticola YC Ma los yog Cistanche tubulosa (Schenk) Xav hauv tsev neeg Orobaceae. Nws muaj cov teebmeem ntawm tonifying raum yang, muaj txiaj ntsig zoo thiab cov ntshav, moistening plab hnyuv, thiab tshem tawm cem quav. Kev lig kev cai siv rau lub raum yang deficiency, essence thiab ntshav deficiency, impotence thiab infertility, lub duav thiab lub hauv caug mob, tsis muaj zog ntawm cov leeg thiab pob txha, thiab plab hnyuv dryness thiab cem quav, nws yog feem ntau siv tonifying raum yang tshuaj nyob rau hauv keeb kwm.. 

Cistanche deserticola

H2O2, raws li ib daim ntawv ntawm reactive oxygen hom, muaj ib tug muaj zog nyhuv ntawm inducing lipid peroxidation thiab muaj peev xwm hnov ​​mob nrog yuav luag txhua cellular tivthaiv, ua rau saw lipid peroxidation, thiab ua rau cell apoptosis thiab ua tsis taus pa [2,3]. Qhov kev sim tsuas yog tshawb xyuas cov txiaj ntsig ntawm Cistanche deserticola extract ntawm H2O2-induced proliferation inhibition thiab raug mob ntawm luav raum tubular epithelial hlwb thiab ua ntej tshawb nrhiav nws cov kev tiv thaiv ntawm lub raum tubular epithelial hlwb.

1 Cov khoom siv thiab cov txheej txheem

1.1 Kev sim tshuaj

Cistanche deserticola ma tau yuav los ntawm Sichuan Chuannan Pharmaceutical Co., Ltd. (batch naj npawb 151201).

Suab puam nyob cistanche

1.2 Tsiaj

New Zealand dawb luav, txiv neej, hnub nyoog 20 hnub, yuav los ntawm lub hauv paus kev sim ntawm Guizhou Livestock Research Institute, nrog rau tsiaj daim ntawv tso cai naj npawb SCXK (Guizhou) 2012-001.

1.3 Reagents

DMEM/F-12 (1:1), Gbico Corporation; Fetal bovine serum, Zhejiang Tianhang Biotechnology Co., Ltd; Dimethyl sulfoxide (DMSO), Beijing Dingguo Changsheng Biotechnology Co., Ltd; H2O2, Sichuan Jinshan Pharmaceutical Co., Ltd. Trypsin, Amresco Company; Thiazole xiav (MTT), Genview; Nonradioactive cytotoxicity kuaj cov khoom, Promega Corporation; Anhydrous ethanol, Tianjin Fuyu Fine Chemical Co., Ltd.

1.4 Cov cuab yeej

CO2 incubator, Hom 3111, Thermo Fisher Scientific; Enzyme marker, 1510 yam, Thermo Fisher Scientific; Ultra huv workbench, SW-CJ-1FD, Suzhou Purification Equipment Co., Ltd; Inverted fluorescence microscope, Ningbo Shunyu; Rotating evaporator, RE-52AA, Shanghai Yarong Biochemical Instrument Factory; Lub tshuab ziab khaub ncaws, LGJ-12, Beijing Songyuan Huaxing Technology Development Co., Ltd.

1.5 Method

1. 5.1 Kev npaj ntawm Cistanche deserticola extract

Siv cov tshuaj ntsuab qhuav qhuav ntawm Cistanche deserticola thiab zom rau hauv me me. Ua kom raug hnyav 100 g ntawm txhua cov hmoov thiab ntxiv 10 npaug ntawm cov dej distilled thiab 75 feem pua ​​​​ethanol kom sov thiab reflux rau extraction 3 zaug. Thawj zaug yog 3 teev, thiab lub sij hawm thib ob yog 1 5 teev, lub sij hawm thib peb rau 1 teev, sib koom ua ke 3 filtrates, txo siab thiab mloog zoo rau cov extract, xam cov extract yield li 22 30 feem pua, ethanol extract 21 70 feem pua ​​, volatile residual cawv, lub freeze-drying mechanism kom tau dej thiab cawv rho tawm los ntawm Cistanche deserticola, kaw, qhuav, thiab khaws cia nyob rau hauv ib qho chaw tsaus rau yav tom ntej siv.

1. 5. 2 Cell Culture

Tsim ib lub raum tubular epithelial cell qauv siv txoj kev piav qhia los ntawm Zhao Dan li al. [4], cais thiab kab lis kev cai thawj lub raum tubular epithelial hlwb los ntawm 20- hnub qub luav, thiab tos kom lub hlwb loj hlob mus rau ntau tshaj 90 feem pua ​​ntawm kab lis kev cai tais, thiab ces zom lawv nrog trypsin. Siv DMEM/F-12 ua tiav kab lis kev cai nruab nrab uas muaj 1 feem pua ​​​​fetal bovine serum ntawm tus nqi ntawm 1 ob × 104 hlwb raug inoculated ntawm 96 lub phaj zoo thiab kab lis kev cai ntawm 37 degree thiab 5 feem pua ​​CO2.

1. 5. 3 Tsim kom muaj ib tug luav raum tubular epithelial cell raug mob qauv induced los ntawm 3 H2O2

Xa mus rau 1 5. 2 Inoculate hlwb, thiab tom qab 48 teev ntawm cov kab lis kev cai ruaj khov, siv H2O2 rau kev puas tsuaj. Teem ib pab pawg sim thiab pab pawg tswj hwm dawb huv, nrog 4 rov ua dua hauv txhua pab pawg, rov ua peb zaug. Cov pab pawg sim: Hloov cov kab lis kev cai ua tiav nrog qhov sib txawv ntawm H2O2, nrog H2O2 concentrations ntawm 550, 500, 450, 400, thiab 350, raws li μ Mol · L-1, pawg tswj dawb paug: Hloov cov kab lis kev cai ua tiav, txuas ntxiv cog qoob loo rau 24 teev nyob rau hauv qhov xwm txheej ntawm 37 degree thiab 5 feem pua ​​​​CO2, ua qhov kev tshuaj xyuas microscopic, thiab sau cov duab. MTT [5] txoj kev siv los ntsuas qhov kev loj hlob ntawm cov hlwb tom qab qhov kev txiav txim ntawm H2O2, thiab tus nqi absorbance (OD tus nqi) yog ntsuas ntawm 490 nm. Siv cov khoom siv uas tsis yog-radioactive cytotoxicity detection los txiav txim qhov kev hloov hauv cell LDH tso tawm tom qab kev kho H2O2, LDH tso tawm (feem pua)=supernatant LDH tus nqi / (supernatant LDH tus nqi ntxiv rau lysate LDH tus nqi) × 100 feem pua ​​. AO/EB fluorescence staining [6]: Siv 100 feem μ G / mL AO tov thiab 100 μ 100 g / mL EB tov txhua μ L. Sib tov zoo thiab ntxiv 10 rau txhua lub qhov dej μ L. Tam sim ntawd muab tso rau ntawm qhov sib piv ntawm theem inverted. fluorescence microscope rau kev soj ntsuam, tswj kev soj ntsuam kev pom tsis hloov hauv 5 feeb, thiab sau cov duab hauv qab lub teeb xiav ntawm 450 ± 10 nm thiab lub teeb ntsuab ntawm 532 ± 10 nm, raws li. Siv cov txheej txheem sau cov fluorescence rau fluorescence synthesis kom tau cov duab fluorescence staining.

1. 5. 4 Cov nyhuv ntawm 4 Cistanche extract hmoov ntawm H2O2-induced puas rau luav raum tubular epithelial hlwb

Cistanche hmoov

Nyem qhov no mus saib Cistanche rau raum kab mob khoom

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Raws li 1 5. 2 Inoculate cells, thiab tom qab 24 teev ntawm cov kab lis kev cai ruaj khov, ntxiv 200 ul ntawm cov kab lis kev cai tiav uas muaj Cistanche deserticola extract rau txhua qhov dej los tiv thaiv cov hlwb rau 24 teev. Teem ib pab pawg sim, ib pab pawg qauv, thiab pab pawg tswj hwm dawb paug, nrog peb rov ua dua ib pab pawg. Kev sim pab pawg thiab cov qauv: hloov nrog 400 μ Ua tiav kab lis kev cai nruab nrab nrog mol · L-1 H2O2; Pab pawg tswj tsis pub muaj: ua tiav kab lis kev cai nruab nrab. Txuas ntxiv cog qoob loo rau 24 teev. Kev loj hlob ntawm tes raug ntsuas los ntawm kev siv MTT txoj kev, thiab kev hloov pauv hauv LDH tso tawm ntawm tes tau ntsuas los ntawm kev ntsuas cov khoom siv uas tsis yog-radioactive cytotoxicity.

1.6 Kev ua lej

Cov ntaub ntawv tau tshwm sim hauv x ± s siv SPSS 19 0 statistical software tau siv rau kev txheeb cais. Ib qho kev tsom xam ntawm qhov sib txawv tau siv rau kev sib piv ntawm pab pawg, thiab LSD txoj kev siv rau kev sib koom ua ke ntau qhov sib piv, nrog P<0 05 indicating statistical significance.

2 Kev Tshawb Fawb thiab Kev Tshawb Fawb

2.1 Morphological hloov ntawm luav raum tubular epithelial hlwb induced los ntawm H2O2

Tom qab cov luav lub raum tubular epithelial hlwb raug kho nrog ntau qhov sib txawv ntawm H2O2 rau 24 teev, morphology ntawm luav lub raum tubular epithelial hlwb raug soj ntsuam nyob rau hauv lub tshuab ntsuas, raws li qhia hauv daim duab 1. Nrog rau qhov nce ntawm H2O2 concentration, cov xov tooj ntawm tes. Nrog rau kev hloov morphological maj mam nce, cov xov tooj ntawm cov hlwb adhering rau parietal cell maj mam txo, qee lub hlwb tau ntev dua, cell gloss txo, thiab qhov loj me me. Cov concentration ntawm H2O2 mus txog 550 μ Mol · L-1, axons ploj, cell membranes rupture, cell shrinkage, cell gaps nce, thiab qhov khoob tshwm sim ntawm thaj ua rau lub hlwb.

Fig. 1 Cov nyhuv ntawm H2O2 concentration ntawm morphology ntawm luavRTEC (100 ×)

2.2 Cov nyhuv ntawm 2.2 H2O2 ntawm kev loj hlob ntawm luav raum tubular epithelial hlwb

Siv 350, 400, 450, 500, thiab 550 raws li μ Mol · L-1 H2O2 kho luav raum tubular epithelial hlwb rau 24 teev, thiab qhov nqus tau raug ntsuas los ntawm MTT txoj kev. Cov txiaj ntsig tau pom tias qhov sib txawv ntawm H2O2 muaj kev cuam tshuam rau kev loj hlob ntawm luav raum tubular epithelial hlwb, nrog rau IC50 tus nqi ntawm 439 07 μ mol·L － 1, 500 μ Cov nyhuv inhibitory ntawm H2O2 nrog mol·L -1 ntawm kev loj hlob ntawm tes tuaj yeem ncav cuag 50 feem pua ​​​​tom qab 24 teev, yog li qhov kev xav tau no tau raug xaiv rau kev tshawb fawb tom ntej.

2.3 Cov nyhuv ntawm 2.3 H2O2 ntawm kev tso tawm LDH hauv luav raum tubular epithelial hlwb

Tom qab 24 teev ntawm H2O2 kev kho mob ntawm luav lub raum tubular epithelial hlwb, OD qhov tseem ceeb ntawm LDH cov tshuaj tiv thaiv hauv cell supernatant thiab cell lysate raug ntsuas los ntawm kev siv cov kev tshawb nrhiav tsis yog radioactive cytotoxicity, thiab kev tso tawm ntawm LDH los ntawm cov hlwb raug xam. Cov txiaj ntsig tau pom nyob rau hauv Table 1. Qhov sib txawv ntawm H2O2 tuaj yeem ua rau nce LDH tso tawm ntawm luav lub raum tubular epithelial hlwb, thiab qhov siab dua H2O2 concentration, qhov ntau dua LDH tso tawm hauv qhov concentration-dependent. H2O2 ntawm qhov siab ntawm 400 mmol · L-1 ua rau cov cell LDH tso tawm ntau tshaj li 50 feem pua, ua rau cov cell puas.

Tab. 1 Cov teebmeem ntawm H2O2 ntawm LDH tso tawm los ntawm luav RTECs

Lus Cim: Piv nrog rau pawg tswj hwm dawb paug, * P<0 05; ＊＊ P ＜ 0. 01

2.4 AO/EB fluorescence staining tshwm sim ntawm luav lub raum tubular epithelial hlwb kho nrog 2.4 H2O2

Tom qab cov luav lub raum tubular epithelial hlwb raug kho nrog H2O2 rau 24 teev, cov hlwb tau stained nrog AO/EB fluorescence, raws li qhia hauv daim duab 2. Nws tau txiav txim ua ntej tias cov txheej txheem ntawm H2O2 ntawm luav lub raum tubular epithelial hlwb feem ntau yog rau ua rau necrosis. Kev sib txawv ntawm H2O2 tuaj yeem ua rau ntau qhov sib txawv ntawm necrosis hauv luav lub raum tubular epithelial hlwb, nrog H2O2 concentrations tsawg dua 400 μ Ntawm mol · L-1, cov chromatin ntawm cov nucleus shrinks rau hauv lub voj voog nrog cov xim ntsuab, uas feem ntau yog txiav txim raws li thaum ntxov apoptosis. Nrog rau qhov nce ntawm H2O2 concentration, nws yog ntau tshaj 450 μ Ntawm mol · L-1, cov hlwb pom cov txiv kab ntxwv-liab liab tsis nyob ib puag ncig hlaws, thiab tus naj npawb ntawm cov hlwb necrotic nce nyob rau hauv ib tug concentration-dependent yam.

2.5 Cov nyhuv ntawm 2.5 Cistanche deserticola ntawm H2O2-induced proliferation ntawm luav raum tubular epithelial hlwb

Kev Tshawb Fawb Txog Cistanche deserticola

Cov ciaj sia taus ntawm luav lub raum tubular epithelial hlwb nyob rau hauv ntau qhov sib txawv (2, 20, 200 mg! L-1) ntawm cov dej thiab cawv rho tawm los ntawm Cistanche deserticola pom tsis muaj qhov sib txawv tseem ceeb piv rau cov cell cultured hauv ib txwm muaj yam tsis muaj kev cuam tshuam. Raws li pom nyob rau hauv daim duab 3, piv nrog rau cov qauv pab pawg neeg, cov high-concentration pawg ntawm cawv thiab dej rho tawm los ntawm Cistanche deserticola txo cov inhibitory nyhuv ntawm H2O2 ntawm lub proliferation ntawm luav raum tubular epithelial hlwb (P.<0 01).

Fig. 2 AO/EB staining of luavRTECs kho los ntawm qhov sib txawv ntawm H2O2 (100 ×)

Lus Cim: Piv nrog rau pawg qauv, * P<0 05,＊＊P ＜ 0. 01

Fig. 3 Cov nyhuv ntawm C. deserticola ntawm inhibition ntawm H2O2 - induced luavRTECs proliferation

2.6 Cov nyhuv ntawm 2.6 Cistanche deserticola ntawm H2O2 induced puas rau luav raum tubular epithelial hlwb

Cov txiaj ntsig ntawm qhov cuam tshuam ntawm kev tso tawm ntawm LDH hauv cov hlwb tau pom hauv daim duab 4. Qhov siab, nruab nrab, thiab qis concentration pawg ntawm ethanol extract thiab dej extract ntawm Cistanche deserticola ua rau txo qis LDH tso tawm hauv luav raum tubular epithelial hlwb tom qab H2O2. kev kho mob dua li hauv pawg qauv (P<0 01), the results are concentration-dependent, with higher concentrations leading to lower cell LDH release.

Lus Cim: Piv nrog rau pawg qauv, * P<0 05,＊＊P ＜ 0. 01

Fig. 4 Cov nyhuv ntawm C. deserticola ntawm H2O2 - induced LDH tso tawm ntawm luavRTECs

3 Kev sib tham

Cistanche deserticola, lub npe hu ua "desert ginseng", muaj txiaj ntsig zoo heev thiab yog cov tshuaj suav tshuaj tonic nto moo. "Shennong Materia Medica Classic" [7] piav qhia cov tshuaj qib siab tshaj plaws xws li: "Shangyao ... feem ntau txhawb nqa lub neej kom teb rau saum ntuj ceeb tsheej, tsis muaj tshuaj lom, thiab yuav tsum tau noj ntau zaus yam tsis ua mob rau tib neeg ..." Cistanche deserticola tau teev tseg raws li cov tshuaj qib siab tshaj plaws, nrog rau lub suab qab zib thiab kub me ntsis. Cov "Cov Ntawv Teev Cov Kws Kho Mob" [8] taw qhia tias Cistanche deserticola yog "qaub, ntsev, thiab tsis muaj tshuaj lom". Tom qab ntawd, cov ntaub ntawv ntawm qhov xwm txheej, saj, thiab meridian ntawm Cistanche deserticola nyob rau hauv ntau yam dynasties ntawm Suav tshuaj ntsuab yog zoo ib yam. Nws qhov xwm txheej yog sov me ntsis, thiab nws saj yog qab zib, qaub, thiab ntsev, uas belongs rau lub raum thiab cov hnyuv loj meridians. Kev lig kev cai siv rau lub raum yang tsis muaj peev xwm, lub duav thiab lub hauv caug mob, thiab cov leeg thiab pob txha tsis muaj zog, nws cov tshuaj pharmacological thiab cov txheej txheem ib txwm ua rau kev tshawb fawb. Cov kev tshawb fawb tshuaj niaj hnub no tau pom tias Cistanche deserticola tuaj yeem tiv thaiv kev laus, txhim kho kev kawm thiab kev nco, thiab tswj kev tiv thaiv kab mob. Nws yog dav siv nyob rau hauv kev kho mob cov tshuaj ntawm tsoos suav tshuaj, tsoos suav tshuaj patent tshuaj, thiab yooj yim npaj thiab kev kho mob cov khoom.

Cistanche ntxiv nyob ze kuv

Oxidative kev nyuaj siab yog qhov tseem ceeb inducement ntawm qee cov kab mob loj, uas yog vim qhov tsis txaus ntawm kev tsim thiab tshem tawm cov pa oxygen reactive hauv lub cev, ua rau qee qhov kev puas tsuaj oxidative rau histiocytes. Yog li ntawd, kev tshawb fawb tob txog cov txheej txheem ntawm kev ntxhov siab tuaj yeem nrhiav txoj hauv kev tsim nyog los tiv thaiv, txhim kho lossis txawm tias tshem tawm cov kab mob chim siab. Kev ntxhov siab oxidative tuaj yeem ua rau ntau yam cell thiab protein peroxidation hauv lub cev [9], thiab H2O2 raug xaiv los ua ib qho ntawm cov khoom siv exogenous rau kev puas tsuaj hauv kev tshawb fawb. Nws muaj cov txiaj ntsig zoo ntawm inducing lipid peroxidation, ua rau cov saw lipid peroxidation cov tshuaj tiv thaiv, thiab ua rau cell apoptosis thiab ua haujlwm tsis zoo. Tam sim no, kev tshawb fawb txog nephrotoxicity ntawm cov tshuaj exogenous no thiab nws cov nyhuv antagonistic feem ntau yog nyob ntawm kev tsim cov qauv tsiaj, thiab muaj qee cov ntaub ntawv qhia txog kev tsim cov qauv hauv vitro rau kev tshawb fawb txog. Piv nrog rau tsiaj cov qauv ntawm nephrotoxicity, cov qauv hauv vitro nephrotoxic cell muaj qhov zoo xws li lub hom phiaj meej ntawm qhov kev txiav txim, lub voj voog tshuaj luv luv, thiab noj tsawg.

Suav tshuaj ntsuab cistanche

Kawm txog cov qauv hauv vitro nephrotoxic cell induced los ntawm exogenous tshuaj los ntawm kev hloov hauv cell morphology, inhibitory cuam tshuam ntawm exogenous tshuaj ntawm cell proliferation, thiab kev hloov hauv cell LDH tso tawm. Qhov sib txawv ntawm H2 O2 muaj qhov cuam tshuam loj rau kev hloov morphological ntawm cov hlwb. Raws li qhov concentration ntawm cov tshuaj exogenous nce, nws pom tias tus naj npawb ntawm cell deformations nce. MTT assay yog ib txoj hauv kev uas feem ntau siv los kuaj xyuas cov cell nyob. Hauv kev sim qhov sib txawv ntawm Cistanche deserticola tiv thaiv luav lub raum tubular epithelial hlwb los ntawm H2O2 kev puas tsuaj, qhov sib txawv ntawm Cistanche deserticola txo qis tus nqi inhibitory ntawm H2O2 ntawm luav lub raum tubular epithelial hlwb, thiab nws yog concentration nyob ntawm seb. Thaum lub cell membrane puas lawm, intracellular LDH yuav raug tso tawm mus rau hauv kab lis kev cai nruab nrab, LDH cov ntsiab lus nyob rau hauv lub cell kab lis kev cai nruab nrab yog ib qho tseem ceeb qhia rau kev kuaj cell tuag. Txoj kev tshawb no pom tias qib LDH hauv cell kab lis kev cai nruab nrab ntawm pawg qauv nrog H2O2- ua rau muaj kev puas tsuaj rau luav raum tubular epithelial hlwb nce ntxiv, thaum qib LDH hauv pawg sim tau kho nrog ntau qhov sib txawv ntawm Cistanche deserticola. txo nyob rau hauv ib tug concentration-dependent yam.Cov txiaj ntsig kev tshawb fawb qhia tias tsim kom muaj tus qauv ntawm H2O2-induced kev puas tsuaj rau luav lub raum tubular epithelial hlwb thiab ua ntej tshawb nrhiav kev tiv thaiv ntawm Cistanche deserticola extract ntawm lub raum tubular epithelial hlwb, tsis tsuas yog lays lub hauv paus rau kev tshawb fawb ntawm cell puas tshwm sim. los ntawm oxidative kev nyuaj siab tab sis kuj muab cov ntaub ntawv yooj yim rau kev txhim kho thiab kev siv ntawm Cistanche deserticola.

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