Aristolochic Acid Induces Renal Fibrosis Thiab Senescence hauv nas
Mar 23, 2022
Abstract:Lub raum yog ib qho ntawm cov kabmob uas muaj feem cuam tshuam rau lub hnub nyoog txog kev puas tsuaj. Feem ntau, lub raum kev laus yog nrog rau lub raum fifibrosis, uas yog qhov kawg ntawm txoj kev mob ntev.kab mob raum. Aristolochic acid (AA), tus neeg sawv cev nephrotoxic, ua rau AA nephropathy (AAN), uas yog tus cwj pwm los ntawm kev mob raum fifibrosis thiab kev ua haujlwm tsis zoo. Txawm hais tias lub raum fifibrosis yog txuam nrog raum kev laus, seb AA induces lub raum laus tseem tsis meej. Lub hom phiaj ntawm txoj kev tshawb fawb tam sim no yog txhawm rau tshawb xyuas qhov muaj peev xwm siv AAN ua tus qauv ntawm lub raum kev laus. Ntawm no, peb tau tshuaj xyuas qhov muaj feem cuam tshuam txog ntawm AAN qauv los ntawm kev tswj hwm AA mus rau C57BL / 6 nas. Piv nrog rau kev tswj hwm, pawg AA pom tau tias muaj kev lausraumphenotypes, xws li lub raum atrophy, lub raum ua haujlwm poob qis, thiab tubulointerstitial fifibrosis. Tsis tas li ntawd, AA txhawb nqa cellular senescence tshwj xeeb hauvlub raumthiab nce lub raum p16 mRNA kev qhia thiab senescence-associated -galactosidase kev ua haujlwm. Tsis tas li ntawd, AA-kho nas tau pom qhov sib thooj ntawm tubular mitochondrial abnormalities, nrog rau cov kab mob oxygen reactive. Klotho, antiaging gene, kuj tseem txo qis hauv covlub raumntawm AA-kho nas. Ua ke, cov txiaj ntsig ntawm txoj kev tshawb fawb tam sim no qhia tau tias AA hloov pauv qhov cuam tshuam txog kev laus, thiab lub raum fifibrosis muaj feem cuam tshuam rau lub raum laus.
Ntsiab lus:mob raum mob; raum fifibrosis; aristolochic acid; kev laus; cellular senescence
CISTANCHE yuav txhim kho rau lub raum/ raum mob
Taw qhiaNrog rau kev ua neej tsis tu ncua ntawm tib neeg, ntau tus tib neeg raug kev txom nyem los ntawm hnub nyoog cuam tshuam.Lub raumfeem ntau cuam tshuam los ntawm hnub nyoog txog cov ntaub so ntswg puas, thiab qhov tshwm sim ntawm mob ntevmob raumnce nrog hnub nyoog [1]. Lub raum aging accelerates tag nrho cov laus, uas ua rau ib tug luv luv lifespan. Yog li ntawd, kev tshawb fawb txog kev laus lub raum yog qhov tsim nyog, txawm hais tias tsim nyog tsiaj qauv tsis tau tsim tag nrho. Lub raum laus yog nrog los ntawm ntau yam kev hloov pauv pathological, nrog rau lub raum atrophy, glomerulosclerosis, thiab tubulointerstitial fifibrosis [2]. Lub raum tubulointerstitial fifibrosis yog qhov kawg ntawm txoj hauv kev hauv ntau hom kab mob raum, uas qhia tias lub raum fifibrosis yog ze rau cov neeg laus.raum. Hauv kev tshawb fawb kev laus, nas yog cov cuab yeej txhim khu kev qha vim tias lawv cov caj ces sib thooj rau tib neeg, muaj peev xwm los tswj cov genome, thiab qhov tseeb tias lawv nthuav tawm cov phenotypes zoo sib xws rau tib neeg thaum lawv lub neej [3]. Kev soj ntsuam ntev siv cov nas inbred yog qhov zoo tagnrho raws li tus qauv ntawm kev laus, txawm hais tias nws siv sijhawm los ua raws cov nas rau lawv lub neej tag nrho. Tsis tas li ntawd, muaj ntau yam hloov pauv hloov pauv nas qauv ntawm kev laus. Klotho-tsis muaj nas yog ib qho qauv ntawm kev laus ntxov ntxov uas siv rau hauv kev tshawb fawb kev laus. Txawm li cas los xij, hauv cov qauv no, lub raum ua haujlwm tsis raug cuam tshuam, thiab ob peb lub cev uas tsis yog lub raum tsis zoo [4]. Yog li, tus qauv nas no yuav tsis tsim nyog rau kev tshawb fawb txog kev laus lub raum.Aristolochic acid (AA) kev tswj hwm ua rau muaj qee yam mob raum raug mob hu ua AA nephropathy (AAN), uas yog tus cwj pwm los ntawm kev sib txuam ntau ntawm fibrosis [5,6]. AA yog tshuaj lom rau lub raum tubular epithelium vim nws txhawb kev tsim cov DNA adducts hauv lub raum cov ntaub so ntswg [7]. AA tsis zoo li yuav cuam tshuam rau cov kabmob uas tsis yog cov zis thiab tej zaum yuav muaj txiaj ntsig zoo rau kev soj ntsuam ntawmraum- kev hloov kho tshwj xeeb. Yog li, AA feem ntau yog siv los tsim cov qauv ntawm lub raum fifibrosis hauv nas [8,9]. Txawm li cas los xij, nws tseem tsis tau paub meej tias qhov kev hloov pauv ntawm AA yog cuam tshuam nrog kev laus hauv lublub raum. Hauv txoj kev tshawb no, peb tau tshuaj xyuas cov hnub nyoog ntsig txog phenotypes thiab molecular yam hauv AAN hauv cov nas.
Cov txiaj ntsig 2.1. Kev tswj hwm AA tau ua rau poob ceeb thawj, lub raum Atrophy, thiab kev poob qis hauv lub raum ua haujlwmLub cev hnyav (BW) thiab systolic ntshav siab (BP) tau zoo ib yam ntawm cov tsheb-tswj thiab AA pawg. BW hauv pawg tswj hwm lub tsheb tau nce tsis tu ncua dhau 8 lub lis piam. Txawm li cas los xij, qhov hnyav nce tau cuam tshuam los ntawm AA cov thawj coj, thiab AA pawg tau txo qis BW ntawm 4 thiab 8 lub lis piam tom qab pib ntawm AA cov thawj coj (Daim duab 1A). Tsis muaj qhov sib txawv tseem ceeb hauv systolic BP lossis lub plawv dhia ntawm lub tsheb-tswj thiab AA pawg (Daim duab 1B, C). Lub plawv hnyav / BW piv tsis pom qhov txawv ntawm cov pab pawg ntawm 8 lub lis piam tom qab pib ntawm AA cov thawj coj (Daim duab 1D), qhovraumQhov hnyav / BW piv tau qis dua hauv AA pawg ntawm 8 lub lis piam tom qab pib ntawm AA cov thawj coj (Daim duab 1E). Peb tom ntej no tau tshuaj xyuas lub raum ua haujlwm hauv lub tsheb-tswj thiab AA pawg. Plasma creatinine thiab urea nitrogen (UN) ntau dua hauv AA pawg ntau dua li pawg tswj hwm tsheb ntawm 8 lub lis piam tom qab pib ntawm AA cov thawj coj. Tsis tas li ntawd, AA kev kho mob txo qis creatinine clearance piv nrog rau pawg tswj tsheb ntawm 8 lub lis piam tom qab pib ntawm AA cov thawj coj (Daim duab 1F-H).
2.2. AA Administration Induced Overt Tubulointerstitial Fibrosis thiab Tseem Ceeb Upregulation ntawm Fibrosis-Related Gene Expression nyob rau hauv lub raumKev tshuaj xyuas histological siv cov tshuaj pleev xim rau lub caij nyoog acid-Schiff (PAS) qhia tias thaj chaw glomerular tau txo qis hauv pawg AA piv nrog pawg tswj tsheb (Daim duab 2A). Kev nthuav dav tubulointerstitial fifibrosis, soj ntsuam siv Masson's trichrome (MT) staining, tau pom nyob rau hauv pawg AA (Daim duab 2B), nrog rau kev tswj hwm ntawm mRNA qib ntawm lub raum fifibrosis-txog cov noob, collagen I thiab III, thiab kev hloov pauv kev loj hlob ( TGF)- (Figure 2C–E).
2.3.AA Kev Tswj Xyuas Kev Ua Haujlwm Ceev Cellular Senescence, Mitochondrial Dysfunction, thiab Tshaj Tawm Ntawm Cov Kab Mob Oxy/gen (ROS) hauv lub raumTxhawm rau soj ntsuam cellular senescence, peb tau tshuaj xyuas lub raum mRNA qhia ntawm p53, p21, p16, thiab glutaminase (GLS), Pab pawg AA pom tau tias muaj ntau dua mRNA qib ntawm cov noob caj noob ces hauv cov noob caj noob ces.lub raum(Daim duab 3A-D). Tsis tas li ntawd, cov senescence-associated -galactosidase(SA- -gal) staining siv nyob rau hauv lublub raumtau nce hauv pawg AA thiab yuav luag tsis tuaj nyob hauv pawg tswj hwm tsheb (Daim duab 3E).Hauv pawg AA, electron microscopy qhia qhov ploj ntawm mitochondrial cristae, mitochondrial fragmentation, cytoplasmic vacuolization, thiab autolysosomes nyob rau hauv proximal tubular cells, concomitant nrog rau downregulation of BCL2/adenovirus E1B 19-kDa interacting protein 3(Bnip3), a mitochondria-related gene (Figure 3FG). Lub raum mRNA qhia ntawm Nox2. ib feem ntawm nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, tau nce ntau hauv pawg AA piv nrog pawg tswj tsheb (Daim duab 3H). Tsis tas li ntawd, kev tshuaj ntsuam xyuas sab hnub poob tau qhia tias lub raum 4-hydroxy-2- nominal (4-HNE) qib tau nce siab hauv pawg AA (Daim duab 3I). Cov txiaj ntsig no tau qhia tias AA cov thawj coj ua rau cov cellular senescence, mitochondrial tsis ua haujlwm, thiab ROS tsub zuj zuj hauvlub raum.
2.4. AA txo lub raum Klotho Protein nthuav qhiaPeb tau tshuaj xyuas lub raum qhia txog kev tiv thaiv cov proteins hauv lub tsheb-tswj thiab AA pawg. Klotho kev qhia tau txo qis hauv pawg AA piv nrog pawg tswj tsheb (Daim duab 4A), thaum lub raum qhia ntawm nicotinamide phosphoribosyltransferase (NAMPT) thiab sirtuin1 (SIRT1) tau zoo sib xws ntawm cov pab pawg (Daim duab 4B,C).
3. Kev sib thamRau peb txoj kev paub, qhov kev tshawb fawb tam sim no yog thawj zaug tsom mus rau kev ntsuam xyuas ntawm lub raum kev laus-txuas hloov pauv hauv AAN siv cov cim ntawm kev laus, xws li p16 thiab SA- -gal kev ua si. Kev kho AA txhawb nqa lub raum atrophy, tubulointerstitial fibrosis, thiab lub raum ua haujlwm tsis zoo, uas tau nrog los ntawm kev txhim kho lub raum p16 mRNA thiab SA- -gal-positive staining. Tsis tas li ntawd, pawg AA tau qhia txog cov yam ntxwv ntawm lub raum kev laus txog cov txheej txheem xws li mitochondrial abnormalities, nce oxidative kev nyuaj siab, thiab downregulation ntawm antiaging noob, Klotho. Ua ke, peb cov kev soj ntsuam pom tau hais tias kev tswj hwm AA ntev ib nrab ua rau lub raum laus.
Cellular senescence yog qhov raug ntes mus tas li ntawm cell proliferation nyob rau hauv teb rau ntau yam stressors, xws li DNA puas, thiab pab mus rau kev laus thiab kev laus cov kab mob. Kev qhia ntawm p16, cyclin-dependent kinase inhibitor, muaj feem cuam tshuam nrog kev laus hauv ntau yam kabmob, suav nrog raum. Kev txwv caloric nce lifespan los ntawm inhibiting pl6 qhia [10]. Tsis tas li ntawd, kev tshem tawm p16-qhia cov hlwb hauv INK-ATTAC cov nas los ntawm kev txhaj tshuaj AP20187, FK506-binding protein dimerizer [11], ua rau txo qis ntawm lub raum laus phenotypes, xws li glomerular sclerosis thiab lub raum. kev ua haujlwm poob qis. Yog li ntawd, p16 yog ib qho tseem ceeb tshaj plaws uas cuam tshuam txog kev laus. Senescent hlwb tuaj yeem kuaj pom siv SA- -gal staining, uas qhia tau tias muaj zog -galactosidase kev ua haujlwm ntawm pH 6.0 [12], thiab yog siv dav siv biomarker ntawm senescent thiab hnub nyoog hlwb. Lub raum mRNA qhia ntawm pl16 nce hauv cov laus naslub raumthiab yog nrog los ntawm kev nce SA- -gal kev ua haujlwm hauv lub raum epithelium [13I. Hauv txoj kev tshawb fawb tam sim no, peb tau pom tias muaj zog rau lub raum p16 mRNA qhia thiab SA- -gal-staining, qhia tias AA ua rau lub raum tsis zoo. GLS tsis ntev los no tau tshaj tawm tias yog qhov tseem ceeb rau kev ciaj sia ntawm cov hlwb senescent los ntawm kev txhim kho glutaminolysis thiab intracellular pH neutralization [14]. Inhibition ntawm GLS-raws li glutaminolysis nyob rau hauv cov nas muaj hnub nyoog tau pom tias tshem tawm cov hlwb senescent thiab ameliorate hnub nyoog ntsig txog lub cev tsis ua haujlwm. Nyob rau hauv txoj kev tshawb no, upregulation ntawm lub raum GLS mRNA qhia lub raum tsub zuj zuj ntawm senescent hlwb nyob rau hauv pab pawg neeg. Yog li, kev tswj hwm AA ntev ua rau cov cellular senescence hauv ob lub raum.
CISTANCHE yuav txhim kho lub raum / raum ua haujlwm
Ntxiv nrog rau cellular senescence, mitochondrial dysfunction yog ib qho tseem ceeb mechanism nyob rau hauv lub hnub nyoog ntawm cov ntaub so ntswg puas thiab yog nrog los ntawm ROS tsub zuj zuj [15,16. Mitochondrial dysfunction drives thiab tswj cellular senescence [17] whereas cellular senescence pab ncaj qha rau mitochondrial tsis ua hauj lwm [18]. Bnip3, nyob rau hauv feem ntau nyob rau hauv lub sab nrauv mitochondrial membrane, yuav ua si lub luag hauj lwm nyob rau hauv cov kev cai ntawm mitophagy nyob rau hauv kab lis kev cai raum proximal tubular hlwb teb rau oxidative kev nyuaj siab thiab hypoxia. Hauv cov nas muaj hnub nyoog, kev txwv caloric nce autophagy hauv tubular hlwb, los ntawm kev tswj hwm ntawm Bnip3, thiab txhim kho lub hnub nyoog cuam tshuam ntawm lub raum ua haujlwm [19]. Nyob rau hauv txoj kev tshawb no, electron microscopy qhia txog cov yam ntxwv ntawm mitochondrial abnormalities uas tej zaum yuav cuam tshuam los ntawm txo lub raum Bnip3 qhia los yog cellular senescence. Mitochondria yog lub ntsiab intracellular qhov chaw ntawm ROS. Txhawm rau ntsuas qhov cuam tshuam ntawm mitochondrial abnormalities ntawm lub raum ROS, peb tau tshuaj xyuas lub raum pob ntawm 4-HINE thiab ua kom pom AA-induced ROS tsub zuj zuj hauv lub raum.lub raum.NADPH oxidases yog qhov tseem ceeb ntawm ROS. Thaum lub sijhawm mob hnyav ntawm AAN hauv cov nas, lub raum mRNA qhia ntawm Nox2 tau nce siab, thiab kev muaj nitric oxide raug txo, uas ua rau muaj kev pheej hmoo ntawm hypoxia thiab ischemia [20. Nyob rau hauv cov laus naslub raum, tsub zuj zuj ntawm ROS yog nrog los ntawm kev nthuav qhia ntawm Nox2 [21]. Cov kev tshawb pom no qhia tias AA tuaj yeem ua rau muaj hnub nyoog ntsig txog pheno-hom los ntawm ROS tsub zuj zuj los ntawm mitochondrial dysfunction thiab upregulation ntawm NADPH oxidases.
Renal Klotho gene qhia tau txo qis hauv pawg AA, qhov kev qhia ntawm NAMPT thiab SIRTl zoo ib yam ntawm cov pab pawg. Klotho yog ib qho tshuaj tiv thaiv kab mob uas encodes ib leeg-pass transmembrane protein uas ua raws li kev tiv thaiv kev laus. Cov txheej txheem kev laus hauv Klotho-tsis muaj nas zoo ib yam li tib neeg, suav nrog lub neej luv dua, muaj menyuam tsis taus, arteriosclerosis, tawv nqaij atrophy, osteoporosis, thiab emphysema [22]. Hypomorphic mutant Klotho nas kuj tau nthuav tawm cov phenotype ntawm kev laus zuj zus, uas tau rov qab los ntawm p16 ablation [23]. Txij li thaum Klotho yog ib qho tseem ceeb hauv kev laus, Klotho noob hloov cov nas tau siv dav hauv kev tshawb fawb txog kev laus. Txawm li cas los xij, Klotho gene-modified nas tej zaum yuav tsis tsim nyog rau kev tshawb fawb ntawm lub raum kev laus vim qhov kev laus ntawm cov nas no thiab qhov tseeb tias lub raum ua haujlwm tsis raug kuaj pom (piv txwv li, qib creatinine). Hauv qhov sib piv, tus qauv AAN nas tuaj yeem siv los ua tus qauv tsim tshuaj ntawm lub raum kev laus rau kev tshawb fawb, vim nws muaj ntau yam zoo, xws li cov txheeb ze ntawm kev siv tau yooj yim thiab muaj peev xwm kwv yees qhov cuam tshuam ntawm kev cuam tshuam ntawm lub raum ua haujlwm poob. .
Txoj kev tshawb no muaj qee qhov kev txwv. Peb soj ntsuam lub raum kev laus, tsom feem ntau ntawm cellular senescence, mitochondrial morphology, thiab kev laus hais txog cov noob qhia. Txawm li cas los xij, cov txheej txheem ntawm kev laus yog qhov nyuaj thiab tsis to taub. Tsis tas li ntawd, txawm hais tias Klotho gene qhia tau txo qis hauv cov lus teb rau AA, peb tsis tuaj yeem ua rau muaj kev sib raug zoo nrog cov kev hloov pauv hauv AAN. Cov kev tshawb fawb ntxiv yuav tsum tau txiav txim siab txog lub luag haujlwm ntawm ntau lub molecules koom nrog hauv kev txhim kho AAN. Txawm li cas los xij, txoj kev tshawb fawb tam sim no muab kev nkag siab tshiab rau kev siv Avan ua qauv ntawm lub raum kev laus. Nws yog ib qho tseem ceeb kom nco ntsoov tias phenotypic hloov nyob rau hauv lubraummuaj kev cuam tshuam nrog lifespan. Txawm tias covraumyog yooj yim cuam tshuam los ntawm kev laus-kawg hloov pauv, inhibition ntawm lub raum aging yuav lengthen tag nrho lifespan. Yog li, kev tshawb fawb ntxiv ntawm lub raum kev laus siv cov qauv ntawm AAN tuaj yeem ua rau muaj kev noj qab haus huv ntxiv rau yav tom ntej.
4. Cov ntaub ntawv thiab cov txheej txheem4.1. Tsiaj txhuTxoj kev tshawb no tau ua raws li National Institutes of Health cov lus qhia rau kev siv cov tsiaj sim. Txhua qhov kev sim tsiaj tau pom zoo los ntawm Pawg Saib Xyuas Tsiaj Studies ntawm Yokohama City University (tus naj npawb pom zoo: FA20-027) thiab tau ua raws li ARRIVE cov lus qhia. Kev siv zog ua kom txo qis cov tsiaj txhu siv thiab lawv cov kev txom nyem. Cov nas tau nyob hauv qhov chaw tswj hwm hauv qab 12-h lub teeb / 12-h lub voj voog tsaus ntuj ntawm qhov kub ntawm 25 ° C. Cov nas tau nkag mus rau zaub mov thiab dej dawb.
CISTANCHE yuav txhim kho lub raum / raum kab mob
Yim-lub lim tiam-laus txiv neej C57BL/6 nas tau yuav los ntawm Charles River Laboratories (Wilmington, MA, USA) thiab muab rau lub tsheb-tswj lossis AA pawg tom qab 1 lub lis piam ntawm acclimatization, nas tau intraperitoneally tswj nrog tsheb lossis AA (Sigma -Aldrich, St.Louis, MO, USA), uas tau yaj hauv ib qho me me ntawm dimethyl sulfoxide. Ua ntej txoj kev tshawb fawb tam sim no, peb tau ua qhov kev tshawb fawb ua ntej uas siv ntau txoj cai. Thawj cov txheej txheem suav nrog kev tswj hwm ntawm AA (3 mg / kg) rau C57BL / 6 nas intraperitoneally ob zaug hauv ib lub lis piam rau 4 lub lis piam yam tsis muaj sijhawm hloov kho; nyob rau hauv cov txheej txheem no, AA ua rau mob raum mob, xws li dilated proximal tubules nrog poob ntawm txhuam ciam teb (Cov duab ntxiv S1). Hauv cov txheej txheem thib ob, C57BL / 6 nas tau muab tshuaj nrog AA (2.5 mg / kg) intraperitoneally ib zaug hauv ib lub lis piam rau 4 lub lis piam tom qab los ntawm kev hloov kho lub sijhawm rau 4 lub lis piam; plasma creatinine nyob rau hauv cov nas no yog 0.19 ± {{20}}}080 mg/dL piv rau 0.18 ± 0.010 mg/dL (tswj tsheb-tswj piv rau AA, feem; txhais tau tias ± tus qauv yuam kev ntawm qhov nruab nrab (SEM), p=0 86, unpaired Student's t-test, n=4 ib pab pawg) thiab plasma UN yog 31.3 ± 5.95 mg/dL piv 30.4 ± 3.87 mg/dL (tswj tsheb-tswj piv rau AA, feem; txhais tau tias ± SEM, p=0.90, unpaired Student's t-test, n=4 ib pab). Yog li ntawd, peb tau txiav txim siab tias cov txheej txheem no tsis haum rau lub raum raug mob qauv vim tias AA tsis ua rau lub raum ua haujlwm tsis zoo. Nyob rau hauv txoj cai thib peb, C57BL / 6 nas tau muab tshuaj nrog AA (3 mg / kg) intraperitoneally ob zaug hauv ib lub lis piam rau 10 lub lis piam yam tsis muaj sijhawm hloov kho; nyob rau hauv cov nas no, cov neeg tuag nyob rau hauv pawg AA yog 83 feem pua (n=6), whereas tsis muaj nas nyob rau hauv lub tsheb-tswj pab pawg neeg tuag (n=4). Hauv cov txheej txheem no, peb tsis tuaj yeem ntsuas qhov ntsuas lub raum phenotype raug ntxias los ntawm AA vim tias muaj kev tuag ntau. Hauv cov txheej txheem thib plaub, C57BL / 6 nas tau muab tshuaj nrog AA (3 mg / kg) intraperitoneally ob zaug hauv ib lub lis piam rau 4 lub lis piam tom qab hloov kho lub sijhawm rau 4 lub lis piam; Cov kab mob raum ntev, xws li tubulointerstitial fifibrosis, thiab lub raum tsis ua haujlwm tau pom nyob rau hauv cov nas no [9]. Yog li ntawd, raws li cov txiaj ntsig ntawm qhov kev tshawb fawb ua ntej no, peb tau txais txoj cai thib plaub los ua cov kev sim hauv kev tshawb fawb tam sim no.
4.2.BP Kev ntsuasSystolic BP thiab lub plawv dhia tau ntsuas los ntawm kev piav qhia yav dhau los tail-cuff txoj kev (BP-Monitor MK-2000; Muromachi Kikai Co., Tokyo, Nyiv) [24,25]. Txhua qhov kev ntsuas tau ua nyob nruab nrab ntawm 9:00 thiab 14:00. Tsawg kawg yog 10 qhov ntsuas tau ua nyob rau hauv txhua tus nas, thiab tus nqi nruab nrab yog siv rau kev tshuaj xyuas.
4.3. Real-Time Quantitatiose Reverse-Transcription PCR Analysis Tag Nrho RNA tau muab rho tawm los ntawm lub raum cov ntaub so ntswg siv ISOGEN (Nippon Gene, Tokyo, Nyiv), thiab ntxiv DNA (cDNA) tau tsim los siv SuperScript IⅢFirst-Strand System (Invitrogen, Waltham, MA, USA). Kev txheeb xyuas qhov rov qab-txhim kho PCR ntawm lub sijhawm tiag tiag tau ua tiav siv CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA), thiab cov khoom siv rov qab rov qab tau tsim nrog TaqMan PCR Master Mix thiab kev cai TaqMan sojntsuam (Applied Biosystems, Waltham, MA, USA), raws li tau piav ua ntej [26]. TaqMan sojntsuam tawm tsam cov noob caj noob ces tau siv: collagen I(Mm00801666_g1), collagen II (Mm01254476_}m1), TGF- (Mm01178819 m1), p53(Mm00441964 g1), p21( Mm04205640 g1).p16(Mm00494449 m1)GLS (Mm01257297_}m1), Bnip3 (Mm01275600_}g1), thiab Nox2 (Mm00627011_}m1).mRNA theem normalized rau cov ntawm 18S ribosomal RNA.
4.4. Western Blot Analysis Protein qhia tau txheeb xyuas los ntawm kev txheeb xyuas sab hnub poob ntawm cov ntaub so ntswg homogenates uas tau piav qhia yav dhau los [27,28]. Tag nrho cov protein rho tawm tau npaj los ntawm cov ntaub so ntswg siv sodium dodecyl sulfate-muaj cov qauv tsis. Cov protein concentration ntawm txhua tus qauv raug ntsuas los ntawm kev siv cov khoom siv Detergent-Compatible Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA) thiab NanoDrop Ib (Thermo Fisher Scientific, Waltham, MA, USA), nrog rau cov bovine serum albumin ua cov txheem. Qhov sib npaug ntawm cov protein rho tawm los ntawm cov qauv ntaub so ntswg tau muab faib rau ntawm 5-20 feem pua polyacrylamide gels (ATTO Corp., Tokyo, Nyiv). Cov protein sib cais tau raug xa mus rau polyvinylidene difluoride membranes siv Semi-Dry Transfer System (ATTO Corp., Tokyo, Nyiv). Membranes raug thaiv rau 1 h ntawm chav tsev kub nrog phosphate-buffered saline uas muaj 5 feem pua skim-mis nyuj hmoov. Membranes tau incubated nrog thawj cov tshuaj tiv thaiv tawm tsam Klotho (ab181373 1:1000; Abcam, Cambridge, UK), NAMPT(sc-67020 1:5000; Abcam), SIRT1(07-131 1:1000, MilliporeSigma , Burlington, MA, USA), 4-HINE(MHN-100P 1:1000; JaICA, Fukuroi, Nyiv), thiab glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118, 1: 2000; Cell Signaling Technology, Danvers, MA, USA). Membranes raug ntxuav thiab incubated nrog lwm cov tshuaj tiv thaiv rau 60 min ntawm chav tsev kub. Cov chaw rau cov tshuaj tiv thaiv-antigen cov tshuaj tiv thaiv tau pom los ntawm kev siv cov tshuaj tiv thaiv kab mob ua kom zoo dua qub (Merck, Kenilworth, NJ, USA) GAPDH tau siv los tswj kev thauj khoom. Cov duab tau txheeb xyuas ntau yam siv ChemiDoc Touch (Bio-Rad Laboratories, Hercules, CA, USA).
4.5. Histological Analysis tau ua raws li tau piav qhia yav dhau los [29]. Lub raum cov ntaub so ntswg los ntawm nas tau kho nrog 4 feem pua ntawm paraformaldehyde thiab tom qab ntawd muab tso rau hauv paraffin. Ntu (4 um tuab) tau pleev xim nrog PAS thiab MT stains. Txhawm rau ntsuas thaj tsam glomerular, 50 glomeruli ib tus nas raug ntsuas thiab ntsuas qhov nruab nrab. Tag nrho cov duab tau txais los ntawm BZ-9000 microscope (Keyence Corp., Osaka, Nyiv).
CISTANCHE yuav txhim kho lub raum / raum mob ntshav qab zib
4.6. SA- -al Staining Lub raum cov ntaub so ntswg los ntawm nas tau khov sai heev thiab mounted nyob rau hauv ib qho Optimal Cutting Temperature compound (Sakura FinetekJapan, Co., Ltd., Tokyo, Nyiv).Sections (4 μm tuab) tau npaj siv lub cryostat (HM550-VPD; Thermo Fisher Scientific) thiab mounted rau iav slides. SA- -gal kev ua ub no tau ntsuas los ntawm kev siv cov khoom siv kuaj pom qhov tsis hnov tsw (Bio Vision Inc., Milpitas, CA, USA) raws li cov chaw tsim khoom cov txheej txheem. Cov qauv tau pom nyob rau hauv qhov ci ci ntawm × 100 magnification siv BZ-9000microscope (Keyence Corp.. 4.7.Electron MicroscopyElectron microscopy tsom xam tau ua raws li tau piav qhia yav dhau los [30]. Cov nas raug tshuaj loog nrog isoflurane thiab perfused los ntawm txoj cai aortic arch nrog heparinized (5 U / mL) physiological saline thiab 2.5 feem pua glutaraldehyde hauv 0.1 mol / L phosphate tsis nyob ntawm pH 7.4. Cov qauv rau kis tau tus mob electron microscopy tau immersed nyob rau hauv 1 feem pua osmium tetroxide rau 2 h, serially dehydrated nyob rau hauv ethanol, thiab embedded nyob rau hauv ib tug Epon sib tov. UlItrathin seem tau stained nrog uranyl acetate thiab lead citrate thiab tshuaj xyuas siv Hitachi H-7500 kis tau tus mob electron microscope ua haujlwm ntawm 80 kV (Hitachi, Ltd., Tokyo, Nyiv). Cov seem tau pom ntawm × 5000 magnification thiab yees duab siv lub koob yees duab them nyiaj sib txuas.
4.8.Biochemical AnalysisCov ntshav kuaj tau sau los ntawm kev mob plawv hauv lub xeev pub. Tag nrho cov ntshav kuaj tau centrifuged ntawm 3000 rpm (MR-150; Tomy Seiko Co., Ltd., Tokyo, Nyiv) rau 10 min ntawm 4 degree kom cais cov ntshav. Cov txiaj ntsig ntshav plasma tau khaws cia ntawm -80 degree . Plasma creatinine, UN, thiab qib urinary creatinine tau ntsuas siv Hitachi 7180 autoanalyzer (Hitachi, Ltd., Tokyo, Nyiv).
4.9. Kev txheeb cais Kev txheeb xyuas txheeb cais tau ua tiav siv GraphPad Prism software (GraphPad Software Inc., San Diego, CA, USA). Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias ± SEM. Cov tub ntxhais kawm tsis tau ua ke qhov f-test yog siv los sib piv qhov sib txawv ntawm cov tsheb-tswj thiab AA pawg.p<0.05 was="" considered="" statistically="">
5. Cov lus xausAA kev tswj hwm ua rauraumsenescence, nrog rau tubulointerstitial fibrosis thiab lub raum atrophy. Cov txiaj ntsig tau qhia tias lub raum fibrosis muaj feem cuam tshuam nrog rau lub raum kev laus thiab tias AAN yuav muaj txiaj ntsig zoo li araum- cov qauv kev laus tshwj xeeb.