Cistanche Glycoside Upregulates Bmi1 Thiab Txhawb Kev Loj Hlob Ntawm Mouse Spermatogonial Stem Cells Los Ntawm Akt Signaling Pathway

Lub hom phiaj: Tshawb xyuas cov txiaj ntsig txhawb nqa thiab cov txheej txheem cuam tshuam ntawm Cistanche glycosides ntawm kev loj hlob ntawm nas spermatogonial qia hlwb. Txoj kev: MTT assay thiab EdU cell proliferation assay tau siv los txheeb xyuas cov txiaj ntsig ntawm Cistanche glycoside ntawm lub peev xwm loj hlob ntawm nas spermatogonial qia hlwb; Western blot txoj kev siv los txheeb xyuas qhov qhia ntawm Bmi1, p-Akt, thiab Akt proteins los ntawm cistanche glycoside; Qhov kev sim RT-PCR tau kuaj pom cov txiaj ntsig ntawm cov noob muaj feem cuam tshuam, thiab thaum kawg tau qhia txog kev tswj hwm kev sib raug zoo ntawm p-Akt thiab Bmi1 los ntawm inhibitors thiab siRNA. Qhov tshwm sim: Piv nrog rau pawg tswj hwm, 0.5-1 µ M Cistanche glycoside txhawb kev loj hlob ntawm nas spermatogonial qia hlwb (P<0.05); Compared with the control group, the expression levels of p-Akt and Bmi1 proteins in the 0.5-1 µ M Cistanche glycoside treated group increased (P<0.05); Akt inhibitors downregulated the levels of p-Akt and Bmi proteins (P<0.05); SiBmi1 had no significant effect on Akt mRNA levels (P>0.05). Xaus: Cistanche glycoside activates Akt signaling pathway thiab upregulates Bmi1 qhia, txhawb kev loj hlob ntawm nas spermatogonial qia hlwb.

Ntsiab lus: Cistanche glycosides; nas spermatogonial qia hlwb; Bmi1; cell proliferation

Kev xeeb tub tau dhau los ua teeb meem thoob ntiaj teb uas cuam tshuam txoj kev loj hlob ntawm tib neeg lub neej [1-2]. Raws li kev txheeb cais kev sib kis, kwv yees li 15 feem pua ​​-20 feem pua ​​​​ntawm cov khub niam txiv thoob ntiaj teb raug kev txom nyem los ntawm cov kab mob infertility, nrog rau txiv neej ua tsis taus pa ua rau ntau tshaj 50 feem pua ​​​​ntawm cov tsos mob ntxiv lawm tshob [3]. Yog li ntawd, nrhiav kev kho mob zoo rau txiv neej infertility tau dhau los ua ib qho kev tshawb fawb kub nyob rau hauv kev ua me nyuam kev noj qab haus huv ob lub teb chaws thiab thoob ntiaj teb.

Cistanche txiv neej cov txiaj ntsig-Tonifying raum

Nyem qhov no mus saib Cistanche rau raum kab mob khoom

【Nug ntxiv】 Email: cindy.xue@wecistanche.com / Whats App: 0086 18599088692 / Wechat: 18599088692

Lub polycomb protein inhibitory complex 1 (PRC1) muaj xws li B cell-specific Moroni nas leukemia kab mob sib koom ua ke ntawm qhov chaw 1 (Bmi1) tuaj yeem tiv thaiv kev laus ntxov ntxov thiab tswj tus kheej lub peev xwm rov ua dua tshiab ntawm qia hlwb [4]. Protein kinase B (Akt) yog koom nrog hauv kev loj hlob ntawm tes thiab kev sib txawv [5], thiab qhib Akt koom nrog ntau yam kev ua ub no tseem ceeb [6-7].

Cistanche glycoside I (C27H30O11, qhov hnyav molecular: 530.52) yog ib qho hydrolyzate ntawm Cistanche glycoside cais los ntawm cov tshuaj suav tshuaj Cistanche glycoside, uas muaj ntau yam tshuaj thiab kev ua haujlwm lom neeg [8-9]. Raws li cov lus ceeb toom, Cistanche glycoside tej zaum yuav muaj qee yam kev kho mob ntawm pob txha pob txha [10]. Txawm li cas los xij, qhov cuam tshuam ntawm Cistanche glycoside I ntawm txiv neej kev ua haujlwm tsis tau meej meej. Spermatogenic stem cells (SSCs) yog pawg phev qub tshaj plaws uas muaj peev xwm rov ua dua tshiab thiab sib txawv ntawm tus kheej, ua lub luag haujlwm tsis tuaj yeem hloov pauv hauv kev tswj hwm txiv neej kev ua haujlwm. Yog li, txoj kev tshawb no siv nas SSCs ua tus qauv hauv vitro los tshawb txog cov txiaj ntsig ntawm Cistanche glycoside ntawm kev muaj peev xwm ntawm nas SSCs thiab lawv cov txheej txheem ntawm kev ua.

Suab puam ginseng-Tonifying raum

1 Cov khoom siv thiab cov txheej txheem

1.1 Cell Culture

Hauv txoj kev tshawb no, nas spermatogonial qia hlwb (C18-4) tau siv. Cov hlwb tau los ntawm Xib Fwb He Zuping cov kev tshawb fawb pab pawg thiab tau sib cais, huv si, thiab txheeb xyuas raws li cov lus qhia tshwj xeeb. Tus nas spermatogonial qia hlwb tau ntxiv 10 feem pua ​​​​fetal bovine serum (Gibco) thiab 1 feem pua ​​​​penicillin / streptomycin hauv DMEM / F12 (Gibco), thiab kab lis kev cai hauv nruab nrab (37 degree, 5 feem pua ​​CO2)

1.2 MTT kev sim

Inoculate C18-4 nrog rau qhov ntau ntawm 5000 cell ib qhov dej rau hauv 96-zoo phaj kom paub meej tias cov cell tsis sib xws. Tom qab cell adhesion, xaiv cov tshuaj xav tau thiab cov tshuaj concentration rau kev kho mob. Tom qab 72 teev ntawm kev quav yeeb tshuaj, 50 µ L ntawm cov tshuaj thiazole xiav (MTT) tau ntxiv rau hauv lub qhov dej uas cov hlwb raug inoculated, thiab 96 lub qhov dej tau muab tso rau hauv lub cell incubator rau 4-5 teev. Tom qab incubation, muab pov tseg cov kab lis kev cai tshuaj los ntawm 96 lub pas dej, ntxiv 150 µ L ntawm dimethyl sulfoxide (DMSO) rau txhua lub qhov dej, thiab thaum kawg ntsuas qhov nqus tau siv cov enzyme-linked immunosorbent assay (Biotek) ntawm lub wavelength ntawm 490 nm.

Cistanche hmoov-Tonifying raum

1.3 EdU cell proliferation

Ua tib zoo inoculate cells mus rau {{0}} lub phaj zoo (6.0 × 103/well), tom qab cov hlwb ua raws li phab ntsa, ntxiv cov tshuaj rau 24 teev raws li qhov kev xav tau, thiab tom qab ntawd siv 100 μ LDMEM/ F12 muaj 20 µ L EdU ua qhov hloov pauv rau thawj kab lis kev cai nruab nrab thiab kev loj hlob ntawm cov hlwb raug kuaj raws li EdU cov lus qhia cov khoom siv kuaj.

1.4 Western blot

Cov qauv ua ntej khaws cia ntawm tes tau muab faib los ntawm SDS PAGE gel thiab tom qab ntawd xa mus rau PVDF membrane tom qab electrophoresis. Nws raug kaw nrog 5 feem pua ​​​​skimmed mis nyuj rau 1 teev. Nws tau ua ke nrog cov tshuaj tiv thaiv tshwj xeeb ntawm 4 degree thaum hmo ntuj: luav anti-nas p-Akt antibody (1 ∶ 2000) (CST), Akt antibody (1 ∶ 1000) (CST), GAPDH (1 ∶ 5000) (Abcam), Bmi1 (1 ∶ 5000) (Abcam). Hnub ob, khi nrog luav thib ob antibody ntawm chav tsev kub rau 1 teev. Cov protein sib raug tau raug kuaj pom siv Chemiluminescence Liquid (ECL) cov khoom siv (Bio-Rad), thiab thaum kawg, cov tshuaj tiv thaiv antigen-antibody tau soj ntsuam siv ChemiDoc system (Bio-Rad).

1.5 TSI-PCR

Cov reagent siv nyob rau hauv tag nrho cov RNA extraction txheej txheem yog TRIzol reagent (Invitrogen), thiab cDNA yog tau raws li cov lus qhia ntawm thawj strand cDNA reverse transcription kit (Vazyme). RT PCR tau ua tiav siv TaqMan gene qhia Master Mix (Bio-Rad) raws li cov lus qhia, siv ΔΔ Ct txoj kev suav cov kev hloov pauv ntawm cov txheeb ze ntau ntawm mRNA qhia, nrog tus nqi ntawm 2 − ΔΔ Ct. Primer sequence table hauv (Table 1)

1.6 siRNA kev hloov pauv

Lub siRNA ntawm Bmi 1 mRNA tau yuav los ntawm Ribobio thiab cov npe ua ntu zus yog nyob rau hauv Table 2. Cov kev hloov pauv reagent Lipofectamine 6000 (Invitrogen) tau siv. Cells (2.0 × 105/well) tau coj mus rau hmo ntuj hauv DMEM/F 12 ntxiv nrog 10 feem pua ​​​​fetal bovine serum rau ntawm 6-zoo phaj. Raws li cov chaw tsim khoom cov lus qhia, Lipofectamine 6000 tau sib xyaw ua ke nrog kev tswj siRNA thiab Bmi1 siRNA, raws li, thiab hloov mus rau 5 teev hauv kab lis kev cai nruab nrab yam tsis muaj fetal bovine serum. Tom qab ntxuav nrog PBS, thawj kab lis kev cai nruab nrab tau hloov nrog 10 feem pua ​​​​fetal bovine serum DMEM / F12. Tom qab 24 teev, cov hlwb raug sau los txheeb xyuas qhov kev hloov pauv ntawm ntau yam sib xws ntawm cov protein thiab cov noob.

Table 1 RT PCR amplified primer sequences

Table 2 siRNA Bmi1 sequence

1.7 Kev Tshawb Fawb Txog Kev Tshawb Fawb

Tag nrho cov ntaub ntawv tau txheeb xyuas siv SPSS 16.0 thiab GraphPad Prism 8 software. Raws li qhov sib txawv ntawm qhov txiaj ntsig ntawm ob lossis ntau pawg, Cov Tub Ntxhais Kawm t-test thiab ib-txoj kev ANOVA xeem tau raug xaiv los sib piv. Qhov sib txawv ntawm P<0.05 was considered statistically significant.

2 Kev tshwm sim

2.1 Cov teebmeem ntawm Cistanche glycosides ntawm spermatogonial qia hlwb

Txhawm rau txiav txim siab seb Epimedium glycoside kuv puas muaj cov txiaj ntsig zoo ntawm nas spermatogonial qia hlwb, peb pom los ntawm MTT kev sim uas Cistanche glycoside Kuv tuaj yeem txhawb nqa kev loj hlob ntawm nas spermatogonial qia hlwb ntawm qhov ntau ntawm {{0}}.5 thiab 1 µ M (Daim duab 1A). Tom ntej no, txhawm rau txhawm rau txheeb xyuas cov txiaj ntsig zoo ntawm Cistanche glycoside I ntawm nas spermatogonial qia hlwb, peb tau lees paub ntxiv los ntawm EdU cell proliferation thwmsim uas Cistanche glycoside Kuv tuaj yeem txhawb kev loj hlob ntawm nas spermatogonial qia hlwb ntawm qhov ntau ntawm 0.5 thiab 1 µ M. (Daim duab 1B).

Suab puam nyob cistanche-Tonifying raum

2.2 Cistanche glycoside upregulates kev qhia ntawm Bmi1

Cov kev tshawb fawb soj ntsuam yav dhau los tau pom tias Bmi1 tau qhia ntau heev hauv cov nqaij mos piv rau lwm cov ntaub so ntswg hauv nruab nrog cev, thiab feem ntau yog qhia nyob rau hauv lub nucleus ntawm nas spermatogonial qia hlwb. Cov kev tshawb fawb tsis ntev los no tau pom tias Bmi1 yog qhov tseem ceeb hauv kev tswj cov kab mob spermatogonial qia hlwb [12]. Yog li ntawd, peb xav tias Cistanche glycoside Kuv muaj kev tswj hwm ntawm Bmi1, yog li txhawb kev loj hlob ntawm nas spermatogonial qia hlwb. Western blot cov txiaj ntsig tau pom tias Cistanche glycoside Kuv tau txhim kho qhov kev qhia ntawm Bmi1 protein ntau (Daim duab 2).

2.3 Cistanche glycoside activates Akt signaling txoj kev

Raws li cov lus ceeb toom, phosphorylated Akt yog ze ze rau kev loj hlob ntawm tes thiab kev sib txawv [13]. Yog li, peb tau tshawb xyuas cov txiaj ntsig ntawm icariin I ntawm Akt phosphorylation. Peb pom tau tias Cistanche glycosides ho upregulated Akt phosphorylation qhia ntawm concentrations ntawm 0.5 thiab 1 µ M (Daim duab 3).

2.4 Inhibition ntawm Akt downregulation ntawm Bmi1

Raws li cov ntaub ntawv tshawb fawb yav dhau los [14], peb tau xaiv Akt inhibitor (MK-2206) ntawm qhov concentration ntawm 10 (µ M) thiab pom tias inhibiting p-Akt ho inhibit Bmi1. Cov txiaj ntsig no qhia tau tias Bmi1 qhia tau tswj hwm los ntawm Akt (Daim duab 4)

Daim duab 1: Cov nyhuv ntawm Cistanche glycoside I ntawm nas spermatogonial qia hlwb

A. MTT kev soj ntsuam tau siv los txheeb xyuas cov txiaj ntsig ntawm Cistanche glycoside I kho rau 3 hnub ntawm kev muaj peev xwm ntawm nas spermatogonial qia hlwb; B. EdU cell proliferation sim tau siv los kuaj xyuas qhov kev loj hlob ntawm nas spermatogonial qia hlwb tom qab 3 hnub ntawm kev kho mob nrog Cistanche glycosides, nrog rau daim ntawv txheeb xyuas kev txheeb xyuas; *P<0.05, * * P<0.01 compared to the control group, n=3.

Daim duab 2: Cov nyhuv ntawm Cistanche glycoside I ntawm Bmi1 protein

Western blot assay tau siv los txheeb xyuas cov txiaj ntsig ntawm Cistanche deserticola glycoside I kho ntawm kev qhia ntawm Bmi1 hauv nas spermatogonial qia hlwb tom qab 24 teev, thiab daim ntawv qhia kev txheeb cais kuj tau nthuav tawm * P<0.05, compared to the control group, n=3.

Daim duab 3: Cov nyhuv ntawm Cistanche glycoside I ntawm Akt signaling pathway

Western blot assay tau siv los txheeb xyuas qhov kev qhia ntawm Akt thiab Akt phosphorylation hauv nas spermatogonial qia hlwb tom qab 24 teev ntawm kev kho mob nrog Cistanche Cistanche glycoside I, thiab daim ntawv qhia kev txheeb cais kuj tau nthuav tawm ** P<0.01, compared to the control group, n=3.

Daim duab 4: Cov nyhuv ntawm Akt inhibitor ntawm Bmi1. Western blot sim tau siv los txheeb xyuas qhov kev qhia ntawm phosphorylated Akt thiab Bmi1 hauv nas spermatogonial qia hlwb tom qab 24 teev ntawm kev kho Akt inhibitor, thiab daim duab statistical tau nthuav tawm * P<0.05 * * P<0.01, compared to the control group, n=3.

2.5 Silence ntawm Bmi1 tsis cuam tshuam rau Akt qhia

Txhawm rau ua kom muaj txiaj ntsig ntxiv rau kev sib raug zoo ntawm cov dej ntws-down ntws ntawm Bmi1 thiab Akt, peb tau tso tseg Bmi1 thiab pom cov kev hloov pauv hauv cell Akt qhia. Ua ntej, siRNAs ntawm qhov sib txawv ntawm Bmi1 tau kis mus rau hauv nas hlwb los tshawb txog lawv txoj kev hloov pauv. Qhov kev sim RT PCR tau pom tias siBmi1-1, siBmi1-2, thiab siBmi1-3 tag nrho txo ​​qhov kev qhia ntawm Bmi1, nrog siBmi1-2 thiab siBmi1-3 muaj cov teebmeem zoo dua (Daim duab 5A). Western blot tau lees paub ntxiv tias ob qho tib si siBmi1-2 thiab siBmi1-3 tuaj yeem txo Bmi1 protein ntau ntau (Daim duab 5B), yog li peb xaiv siBmi1-2 thiab siBmi{16}} rau kev tshawb fawb tom ntej. Interestingly, RT-PCR qhia tau hais tias kev ntsiag to Bmi1 tsis cuam tshuam rau Akt qhia (Daim duab 5C). Cov txiaj ntsig saum toj no qhia tias Akt nyob rau sab sauv ntawm Bmi1.

Daim duab 5: Qhov cuam tshuam ntawm kev ntsiag to Bmi1 ntawm Akt

A. Kev tshuaj xyuas RT-PCR ntawm qhov cuam tshuam ntawm peb qhov kev ntsiag to ntawm kev qhia ntawm Bmi1 noob; B. Western blot sim tau siv los kuaj xyuas cov txiaj ntsig ntawm peb qhov sib txawv siRNA sib txawv ntawm Bmi1 protein silencing piv rau siCtrl, thiab kev txheeb xyuas txheeb cais tau ua; C. RT-PCR tsom xam ntawm cov nyhuv ntawm kev ntsiag to Bmi1 ntawm Akt gene ** P<0.01, * * * P<0.001 compared to the control group, n=3.

3 Kev sib tham

Infertility cuam tshuam txoj kev loj hlob ntawm tib neeg lub neej, nrog rau txiv neej infertility accounting rau 50 feem pua ​​​​ntawm cov neeg mob infertility. Nrog rau kev txhim kho txuas ntxiv ntawm kev kho mob thev naus laus zis, kev kho mob theem ntawm txiv neej infertility tau zoo tuaj. Txawm li cas los xij, cov kev kho mob phais uas twb muaj lawm, xws li kev pab cuam kev ua me nyuam, muaj qhov tsis zoo xws li tus nqi siab thiab tsis tshua muaj kev vam meej thiab muaj ntau yam me me ntawm cov neeg tau txais txiaj ntsig. Lawv tsim nyog rau cov neeg mob uas tau ua tiav azoospermia thaum lub sijhawm kuaj cov phev micro phev rov qab, muaj cov kab mob ntawm caj ces, thiab tsis kam txais kev kho mob rau ntau yam laj thawj [16]. Yog li ntawd, ob qho tib si hauv tsev thiab thoob ntiaj teb tau ua kom muaj kev nyab xeeb thiab txhim khu kev qha txoj kev kho mob rau txiv neej infertility, thiab kev kho tshuaj tau dhau los ua qhov kev tshawb fawb hotspot nyob rau hauv thaj tsam ntawm txiv neej infertility [17]. Cov kev kho mob uas twb muaj lawm tau pom tias cov tshuaj suav tshuaj hauv Suav teb muaj kev ua tau zoo thiab kev nyab xeeb hauv kev kho cov txiv neej infertility [18].

Cistanche deserticola ua tau zoo-Tonifying raum

Cistanche glycoside yog lub ntsiab lus kub hauv kev kho cov txiv neej infertility hauv cov tshuaj suav tshuaj hauv xyoo tas los. Nws cov ntsiab lus tseem ceeb, Cistanche glycoside, muaj ntau yam kev ua haujlwm tshuaj, suav nrog kev txhim kho lub cev tsis muaj zog [19], txhim kho txiv neej kev ua haujlwm ntawm kev loj hlob [20], antioxidants [21], thiab tiv thaiv pob txha [22]. Txhawm rau kom paub meej ntxiv txog cov txheej txheem ntawm kev ua ntawm Cistanche glycoside tom qab nws nkag mus rau tib neeg lub cev, txoj kev tshawb no xaiv Cistanche glycoside, hydrolyzate ntawm Cistanche glycoside, ua cov khoom tshawb fawb los tshawb txog cov txheej txheem tshwj xeeb uas cov hydrolyzate exerts nws cov tshuaj pharmacological. Ua ntej, peb pom tias Cistanche glycoside kuv tuaj yeem txhawb kev loj hlob ntawm nas spermatogonial qia hlwb. Nws yog ib qho tsim nyog sau cia tias Cistanche glycoside kuv tsis tau txhawb nqa kev loj hlob ntawm nas spermatogonial qia hlwb nyob rau hauv ib qho concentration-dependent yam, qhia tias Cistanche glycoside kuv muaj ntau npaum li cas-kev nyab xeeb. Tom ntej no, peb pom tias Cistanche glycoside Kuv tau txhim kho qib kev qhia ntawm Bmi1 protein ze ze rau txiv neej kev ua haujlwm. Tsis ntev los no, cov kws tshawb fawb tau pom tias qhov tsis muaj Bmi1 tuaj yeem cuam tshuam loj rau kev ua haujlwm ntawm cov txiv neej nas, feem ntau tshwm sim hauv oligospermia thiab cov qe menyuam loj [23]. Lub caij no, lwm txoj kev tshawb fawb los ntawm peb pab pawg tshawb fawb kuj tau lees paub tias inhibiting Bmi1 tuaj yeem cuam tshuam rau kev loj hlob muaj peev xwm ntawm spermatogonial qia hlwb. Yog li, peb xav tias Cistanche glycoside I txhawb kev loj hlob ntawm nas spermatogonial qia hlwb yuav cuam tshuam rau kev tswj hwm Bmi1 kev qhia. Raws li cov lus ceeb toom, p-Akt yog ze ze rau kev loj hlob ntawm tes thiab kev sib txawv [14]. Yog li ntawd, peb tau sim cov kev hloov pauv ntawm p-Akt protein tshwm sim los ntawm Cistanche glycoside I thiab pom tias Cistanche glycoside kuv kuj upregulated p-Akt. Thaum kawg, peb tshawb nrhiav qhov kev sib raug zoo ntawm cov dej ntws thiab qis ntawm Akt thiab Bmi1. Nws yog ib qho tsim nyog sau cia tias muaj qhov sib txawv ntawm cov txiaj ntsig kev tshawb fawb ntawm kev sib raug zoo ntawm cov dej ntws thiab qis ntawm Bmi1 thiab Akt tau txais los ntawm cov chaw soj ntsuam sib txawv [24-25]. Cov txiaj ntsig ntawm txoj kev tshawb no qhia tau hais tias Akt-specific inhibitors downregulate Bmi1 thaum uas ntsiag to Bmi1 tsis cuam tshuam rau Akt mRNA qib, qhia tias Akt tej zaum yuav nce siab ntawm Bmi1, uas yog tswj los ntawm Akt. Hauv kev tshawb fawb txog cov nyhuv ntawm kev ntsiag to Bmi1 ntawm Akt, peb tsuas yog tshawb xyuas nws ntawm mRNA qib. Raws li Bmi1 yog ib qho inhibitor transcription, peb xav tias tej zaum yuav muaj kev cuam tshuam ntawm Bmi1 protein thiab Akt protein. Txawm li cas los xij, tam sim no tsis muaj pov thawj tiav los lees paub qhov kev sib raug zoo ntawm cov dej ntws thiab qis ntawm Akt thiab Bmi1. Yog li ntawd, peb yuav ntxiv thiab txhim kho qhov no yav tom ntej.

Cov teebmeem ntawm Cistanche-Tonifying raum

Hauv cov ntsiab lus, txoj kev tshawb no pom thawj zaug uas Cistanche glycoside Kuv muaj cov txiaj ntsig ntawm kev txhawb nqa kev loj hlob ntawm cov kab mob spermatogonial qia hlwb, nthuav tawm cov txheej txheem tshiab ntawm Cistanche glycoside I txhawb kev loj hlob ntawm spermatogonial qia hlwb ntawm qib cellular, enriching theoretical hauv paus. rau daim ntawv thov ntawm Cistanche glycoside nyob rau hauv kev kho mob ntawm txiv neej infertility.

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