HydroZitLa Inhibits Calcium Oxalate Pob zeb tsim nyob rau hauv Nephrolithic nas Thiab txhawb nqa lub neej ntev hauv Nematode Caenorhabditis Elegans Part 2

Glycoside ntawm cistanche tuaj yeem ua rau muaj kev ua haujlwm ntawm SOD hauv plawv thiab daim siab cov ntaub so ntswg, thiab txo cov ntsiab lus ntawm lipofuscin thiab MDA hauv txhua cov ntaub so ntswg, tshem tawm ntau yam reactive oxygen radicals (OH-, H₂O₂, thiab lwm yam) thiab tiv thaiv DNA puas. los ntawm OH-radicals. Cistanche phenylethanoid glycosides muaj peev xwm tshem tawm cov dawb radicals, muaj peev xwm txo tau ntau dua li cov vitamin C, txhim kho cov haujlwm ntawm SOD hauv cov phev ncua, txo cov ntsiab lus ntawm MDA, thiab muaj qee yam kev tiv thaiv ntawm cov phev ua haujlwm. Cistanche polysaccharides tuaj yeem txhim kho kev ua haujlwm ntawm SOD thiab GSH-Px hauv erythrocytes thiab ntsws cov ntaub so ntswg ntawm cov nas senescent sim los ntawm D-galactose, nrog rau txo cov ntsiab lus ntawm MDA thiab collagen hauv lub ntsws thiab ntshav, thiab nce cov ntsiab lus ntawm elastin, muaj Cov nyhuv scavenging zoo ntawm DPPH, ncua lub sijhawm ntawm hypoxia hauv cov nas senescent, txhim kho cov haujlwm ntawm SOD hauv cov ntshav, thiab ncua lub physiological degeneration ntawm lub ntsws hauv kev sim cov nas nrog cellular morphological degeneration, kev sim tau pom tias Cistanche muaj peev xwm antioxidant zoo. thiab muaj peev xwm los ua ib qho tshuaj tiv thaiv thiab kho cov kab mob ntawm daim tawv nqaij laus. Nyob rau tib lub sijhawm, echinacoside hauv Cistanche muaj lub peev xwm tseem ceeb txhawm rau txhawm rau DPPH dawb radicals thiab muaj peev xwm tshem tawm cov pa oxygen reactive thiab tiv thaiv dawb radical-induced collagen degradation, thiab kuj muaj kev kho zoo ntawm thymine dawb radical anion puas.

Nyem rau ntawm Cistanche Norge

【Yog xav paub ntxiv:george.deng@wecistanche.com / WhatApp:8613632399501 】

Raws li peb qhov kev tshawb pom tam sim no, HydroZitLa tau siv cov tshuaj tiv thaiv kab mob los ntawm tsawg kawg ob txoj hauv kev. Ua ntej, nws txo cov zis oxalate, txo cov zis CaOx crystallization muaj peev xwm, thiab muab citrate. Qhov thib ob, nws muaj cov tshuaj ntsuab polyphenols uas muaj peev xwm txo qis oxidative kev nyuaj siab thiab mob, yog li inhibiting CaOx lithogenesis. Hauv paus, oxalate yog endogenously synthesized nyob rau hauv lub siab siv glyoxylate raws li ib tug tseem ceeb precursor41. Glyoxylate yog tsim los ntawm glycolate los ntawm glycolate oxidase (GO) enzyme, thiab nws ntxiv hloov mus rau oxalate los ntawm lactate dehydrogenase (LDH). Peb pom tias HydroZitLa txo qis urinary oxalate excretion hauv EG-kho nas. Peb xav tias HydroZitLa inhibited oxalate synthetic txoj hauv lub siab los ntawm downregulation ntawm hepatic enzymes koom nrog hauv glyoxylate metabolism, tshwj xeeb tshaj yog LDH thiab GO. Txoj kev tshawb fawb yav dhau los los ntawm Kailash thiab Varalakshmi tau pom tias txiv tsawb qia extract txo qis hepatic GO thiab LDH qib hauv sodium glycolate-induced hyperoxaluric nas27. Poonguzhali thiab Chegu kuj tau tshaj tawm tias cov khoom siv raw ntawm cov txiv tsawb stems txo cov zis oxalate, glycolic acid, thiab glyoxylic acid hauv glycolate-induced hyperoxaluric nas30. Tsis ntev los no, Patankar et al. pom tau hais tias lawv cov tshuaj ntsuab formulation, hu ua Herbmed Plus (muaj txiv tsawb qia), txo cov theem ntawm LDH kev ua si nyob rau hauv daim siab thiab raum cov ntaub so ntswg42. Txhawm rau txheeb xyuas peb qhov kev xav tau, kev qhia ntawm lub siab thiab lub raum enzymes koom nrog oxalate synthesis hauv EG nas kho nrog HydroZitLa yuav tsum tau tshawb nrhiav ntxiv.

Ntau cov ntaub ntawv pov thawj txhawb nqa tias oxidative kev nyuaj siab, o, thiab lub raum tubular raug mob yog qhov tseem ceeb hauv CaOx lithogenesis3,4,15,43–45. Oxidative stress induced los ntawm oxalate thiab/los yog CaOx crystals ua rau lub raum tubular raug mob uas tsim cov chaw rau crystal nucleation, adhesion, tuav, aggregation, thiab thaum kawg ib tug nidus formation43,46,47. Tsis tas li ntawd, oxidative kev nyuaj siab activates inflammatory cov tshuaj tiv thaiv uas nyob rau hauv lem accelerates lithogenic txheej txheem4,47,48. Yog li, phytomedicine uas muaj cov tshuaj tiv thaiv antioxidant thiab tiv thaiv kab mob yog ib qho kev cog lus rau kev tiv thaiv ntawm lub raum pob zeb tsim24. Aggarwal et al. pom nyob rau hauv EG-induced nephrolithic nas qauv uas bergenin (bioactive phenolic compound cais los ntawm Bergenia ligulata) muaj ib tug muaj zog antipathogenic nyhuv rau mitigate oxidative kev nyuaj siab, ameliorate mitochondrial dysfunction thiab txo tubular raug mob thiab infammation49. Rutin thiab curcumin (zoo-paub hauv paus txawm phytochemicals nrog antioxidant thiab anti-inflammatory zog) tau pom tias inhibit tubulointerstitial puas thiab crystal deposits nyob rau hauv EG-induced nephrolithic nas50. Polyphenols los ntawm cov noob txiv hmab txiv ntoo kuj tau pom tias inhibit intrarenal CaOx crystal deposits nyob rau hauv EG-induced lithiasis nas, plausibly los ntawm lawv cov antioxidant kev ua 51. Ib qho Ayurveda herbal formulation Herbmed tau qhia kom txo tau qhov mob inflammatory thiab crystal deposits nyob rau hauv ob lub raum ntawm EG-induced nephrolithic nas42. Nyob rau hauv txoj kev tshawb no, peb tau pom tias HydroZitLa txo qis intrarenal oxidative kev nyuaj siab, o, thiab CaOx crystal deposits, qhia tias HydroZitLa exerted antioxidant thiab anti-inflammatory ua los tiv thaiv CaOx raum pob zeb tsim.

Ntau cov nroj tsuag polyphenols tau sim ua pov thawj tias muaj txiaj ntsig zoo rau kev ua neej ntev thiab ncua kev laus52. Resveratrol yog tus cwj pwm zoo tshaj plaws ntawm kev ua neej ntev-ntev polyphenolic compound. Nws feem ntau ua los ntawm sirtuin activation thiab oxidative kev nyuaj siab attenuation53. Cov nyhuv ntev ntawm resveratrol kuaj hauv C. elegans qauv sib txawv ntawm cov kev tshawb fawb nrog kev nce hauv nruab nrab lifespan li ntawm 10 mus rau 65 feem pua ​​​​54,55. HydroZitLa tau txuas ntxiv lub neej ntawm cov tsiaj qus-hom C. elegans txog li 34 feem pua ​​​​tsis muaj cov tsos mob ntawm toxicity, qhia tias lub neej ntev-ntev nyhuv ntawm HydroZitLa yog piv nrog cov resveratrol. Kev txwv kev noj haus yog ib qho tseem ceeb paub txog kev ua neej nyob ntev56. Lub pharyngeal twj tso kua mis piv ntawm HydroZitLa worms thiab tswj worms tau muab piv, qhia tias lub neej ncua sij hawm los ntawm HydroZitLa tsis yog los ntawm kev txwv kev noj haus.

HydroZitLa txo qis qhov sib txuam ntawm cov hnub nyoog autofluorescent xim lipofuscin hauv C. elegans, qhia txog kev tiv thaiv kev laus. ROS thiab oxidative kev nyuaj siab yog paub zoo inducers ntawm SIPS, thiab oxidative kev nyuaj siab accelerates lub shortening ntawm telomeres57. Peb tsis ntev los no tau pom tias oxalate, COM, thiab zis los ntawm cov neeg mob uas muaj CaOx pob zeb induced premature senescence, p16 upregulation, thiab telomere attrition nyob rau hauv lub raum tubular hlwb los ntawm oxidative kev nyuaj siab 58. Nyob rau hauv txoj kev tshawb no, peb pom tias HydroZitLa inhibited telomere attrition, p16 upregulation, ntxov ntxov senescence hauv HK-2 hlwb raug H2O2, oxalate, thiab COM. Txawm hais tias cov txheej txheem molecular rau qhov kev tiv thaiv kev laus tsis tau piav qhia meej, HydroZitLa tau pom zoo los tiv thaiv kev laus ntxov ntxov los ntawm inhibition ntawm telomere shortening thiab downregulation ntawm p16 qhia.

Txawm hais tias kev kho tshuaj ntsuab thiab phytomedicines nce zuj zus los ua neeg nyiam thiab lees paub thoob ntiaj teb, cov pov thawj tshawb fawb ntawm kev ua tau zoo thiab kev nyab xeeb yuav tsum tau muab thiab yuav tsum tsis txhob cuam tshuam. Kev sib cuam tshuam tshuaj ntsuab-tshuaj tsis tau kwv yees vim nws muaj ntau yam sib txawv ntawm cov tib neeg, nyob ntawm ntau yam xws li polymorphisms ntawm cytochrome P450 enzyme thiab cov tshuaj thauj khoom, thiab cov kev kho mob uas twb muaj lawm59. Cov kev tsis zoo ntawm cov tshuaj ntsuab thiab tshuaj coadministration yuav tsum tau ua tib zoo saib xyuas los txheeb xyuas qhov muaj peev xwm tshuaj ntsuab-tshuaj cuam tshuam. Cov tshuaj paub zoo tshaj plaws uas cuam tshuam nrog cov tshuaj muaj xws li qej, Baizhi, Danggui, ginseng, ginkgo, licorice, St John's wort, Kava, dub thiab ntev kua txob, Danshen, Huangqin, thiab mis thistle59. Rau peb txoj kev paub, tsis muaj kev phiv loj lossis kev cuam tshuam tshuaj yeeb tshuaj qhia rau cov txiv tsawb qia, npauj npaim xiav pea paj, thiab sappan ntoo. Hauv Thaib teb, txiv tsawb qia yog siv los ua khoom noj. Kev haus ntau dhau (siv ntau dhau) kua txiv tsawb txiv tsawb tuaj yeem ua rau muaj kev fab tshuaj, mob plab, thiab ntuav, yog li kev noj haus tsis txaus. Blue pea paj feem ntau yog siv rau hauv cov zaub mov ua cov xim ntuj, thiab nws kuj yog siv los ua tshuaj yej. Cov txiaj ntsig tsis zoo ntawm kev noj ntau dhau ntawm npauj npaim xiav pea paj tshuaj yej, piv txwv li, mob plab, xeev siab, thiab kev tsis haum tshuaj, tsis tshua muaj qhia. Sappan ntoo kuj nquag siv los ua zaub mov xim, thiab hauv cov zej zog (xws li cov zej zog hauv Thaib teb) feem ntau yog siv los ua dej haus. Txawm li cas los xij, kev tiv thaiv kev ua haujlwm (tiv thaiv kev xeeb tub hauv cov txiv neej) ntawm sappan ntoo extract yog pom hauv cov qauv nas, vim nws cuam tshuam cov spermatogenesis60. Ntawm qhov tod tes, lub plawv ntawm Caesalpinia sappan L. yog siv los ua tshuaj kho mob rau cov tsos mob gynecological, suav nrog oligomenorrhea thiab amenorrhea61. Yog li ntawd, kev nyab xeeb ntawm kev siv mus sij hawm ntev ntawm HydroZitLa hauv tib neeg tseem yuav tsum tau paub tseeb hauv kev sim tshuaj.

Txoj kev tshawb no muaj qee qhov kev txwv. Cov ntsiab lus tseem ceeb ntawm lub luag haujlwm rau CaOx pob zeb inhibitory kev ua haujlwm tsis raug txheeb xyuas thiab tus yam ntxwv. Cov txheej txheem tseeb ntawm HydroZitLa los tiv thaiv CaOx pob zeb tsim hauv nas tsis tau tshawb xyuas ntau. Cov tshuaj tiv thaiv kab mob ntawm cov tshuaj HydroZitLa tsis tau tshawb xyuas. Qhov koob tshuaj ntawm HydroZitLa uas tuaj yeem ua rau cov zis citrate ntau ntxiv hauv cov nas nephrolithic tsis tau tshawb pom. Cov phenolic profile ntawm HydroZitLa yuav tsum tau tshawb xyuas ntxiv. Cov txheej txheem ntawm kev txhawb nqa lub neej ntev los ntawm HydroZitLa hauv C. elegans tsis tau tshawb nrhiav ntau. Thaum kawg, cov ntsiab lus tshwj xeeb hauv HydroZitLa lub luag haujlwm rau lub neej ntev thiab kev tiv thaiv kev laus tsis raug txheeb xyuas thiab tus yam ntxwv.

Hauv kev xaus, HydroZitLa tau tsim los rau tib lub sijhawm ua rau muaj kev pheej hmoo ntawm CaOx pob zeb tsim los ntawm kev muab cov kua dej, citrate, thiab ntuj antioxidants. Kev sim, HydroZitLa inhibited intrarenal CaOx deposit nyob rau hauv nephrolithic nas. Lub CaOx pob zeb inhibitory kev ua tau zoo ntawm HydroZitLa yog sib npaug rau tam sim no siv potassium citrate (Uralyt-U). HydroZitLa ua tau zoo attenuated oxidative stress ob leeg hauv vitro thiab hauv vivo. Remarkably, HydroZitLa ncua lub neej thiab ncua qhov pib ntawm kev laus hauv C. elegans. Tsis tas li ntawd, HydroZitLa cuam tshuam telomere shortening, p16 qhia, thiab ntxov ntxov senescence nyob rau hauv tib neeg lub raum hlwb. Cov kev tshawb pom ua ntej no tau qhia tias HydroZitLa yog qhov chaw kho mob tau txais txiaj ntsig zoo rau CaOx nephrolithiasis kev kho mob nrog rau kev txhawb nqa mus ntev thiab kev tiv thaiv kev laus. Kev sim tshuaj yog tsim nyog los lav qhov kev kho mob ntawm HydroZitLa hauv tib neeg.

Cov ntaub ntawv thiab cov txheej txheem

HydroZitLa ntau lawm, cov ntsiab lus antioxidant, thiab khoom noj khoom haus tseeb. HydroZitLa yog cov tshuaj cog qoob loo los ntawm cov dej haus concentrate hauv zos muaj nyob hauv Tailand32. Cov dej haus yog npaj los ntawm diluting HydroZitLa concentrate (55 mL nyob rau hauv ib lub hnab kaw) nrog dej haus (mus txog rau lub ntim kawg ntawm 500 mL). Nws muaj pinkish-ntsuab xim nrog ib tug qab qaub-thiab-qab zib saj. Cov khoom xyaw tseem ceeb ntawm HydroZitLa yog txiv tsawb qia (Musa sapientum L.) dej extract, thiab citric acid (16 mEq / hnab). Tsob ntoo Tai tshuaj ntsuab suav nrog Clitoria ternatea L. (Butterfly blue pea paj hmoov) thiab Caesalpinia sappan L. (Sappan ntoo hmoov) tau siv los ua cov xim ntuj. Stevia thiab sucralose yog sweeteners. Cov khoom no tau pom zoo los ntawm Tai FDA thaum lub Plaub Hlis 10, 2019, nyob rau hauv qeb ntawm cov dej haus hauv lub thawv ntim khoom ntim (Food serial no. 10–{11}},244–{13}}).

Tag nrho cov antioxidant muaj peev xwm (TAC) ntawm HydroZitLa yog 21.87 ± 7.96 mg vitamin C sib npaug antioxidant muaj peev xwm rau ib lub hnab (Ntxiv daim duab 8). Tag nrho cov ntsiab lus phenolic (TPC) yog 19.99 ±0.83 mg gallic acid sib npaug / 100 g. Tag nrho cov ntsiab lus favonoid (TFC) yog 4.59 ± 0.31 mg catechin sib npaug / 100 g. Rau kev tswj kom zoo, TAC, TPC, thiab TFC tau siv los ua kev tswj xyuas tsis zoo. Cov theem ntawm TAC, TPC, thiab TFC nyob rau hauv lub txiv tsawb qia, npauj npaim xiav pea paj, thiab sappan ntoo rho tawm ntawm ntau ntau thiab cia lub sij hawm ntsiab lus tsis sib txawv heev. Hauv kev tsim cov HydroZitLa, TAC, TPC, TFC, thiab brix tus nqi (17 ± 2) tau siv los ua cov kev tswj xyuas zoo kom tswj tau qhov sib xws ntawm cov khoom siv.

Tag nrho lub zog ntawm HydroZitLa ib lub hnab (kev pabcuam) yog 25 kcal. Tsis pom muaj cov protein, rog, thiab cov roj cholesterol. Tag nrho cov carbohydrates yog 6 g, qab zib yog<1 g, and sodium was 10 mg. The potassium content was 240 mg/100 g. Soluble and insoluble dietary fibers were 0.74 and 0.43 g/100 g, respectively. Te pH of  HydroZitLa was 3.4.

Kev soj ntsuam toxicity.Lub xub ntiag ntawm Escherichia coli, Clostridium spp., Salmonella spp., thiab Staphylococcus aureus hauv HydroZitLa tau txheeb xyuas los ntawm Bureau of Quality and Safety of Food, Ministry of Public Health, Thaib teb. Cov hlau profile ntawm HydroZitLa tau txheeb xyuas los ntawm ICP-OES.

Cov tshuaj toxicity hauv vivo ntawm HydroZitLa hauv cov nas tau kuaj los ntawm Lub Tsev Haujlwm Saib Xyuas Kev Noj Qab Haus Huv, Ministry of Public Health, Thaib teb. HydroZitLa lossis dej (tswj) tau hais lus rau cov nas ntawm koob tshuaj 20 mL / kg (n=10 ib pab). Cov nas tau soj ntsuam rau 14 hnub ua ntej necropsy. Tag nrho cov kab mob hauv cov kab mob visceral tau kuaj thiab muab piv ntawm HydroZitLa thiab pawg tswj hwm.

CaOx aggregation assay.Noob COM crystals tau npaj 62 thiab diluted rau 50 mg/mL hauv 0.05 M Tris/0.15 NaCl, pH 6.5. HydroZitLa, los yog BSA, los yog cov dej distilled (dawb) (200 µL) tau ntxiv rau lub tshiab npaj ua hauj lwm noob COM ncua kev kawm ntawv (2 mL). Tom qab sib tov, absorbance ntawm 620 nm yog ntsuas raws li hauv paus (AT0). Tom qab incubation ntawm 37 degree rau 10 min, qhov absorbance ntawm 620 nm tau ntsuas dua (AT10). Aggregation coefficient (AC) raug xam raws li nram no: AC=((AT0− AT10)/10) × 1000. Tus nqi AC siab dua qhia tias muaj COM aggregation ntau dua.

Cell kab, cytotoxicity, thiab intracellular ROS kev txiav txim.HK{{0}} hlwb (ATCC, VA, USA) tau khaws cia hauv DMEM ntxiv nrog 10 feem pua ​​​​fetal bovine serum thiab 1 feem pua ​​Pen-Strep hauv qab 37 degree, 5 feem pua ​​CO2, thiab 95 feem pua ​​​​cov av noo. Lub cytotoxicity ntawm HydroZitLa hauv HK-2 hlwb raug soj ntsuam los ntawm MTT assay63. HK-2 hlwb raug noob (200 hlwb/zoo), loj hlob ib hmos nyob rau hauv ib tug 96- zoo phaj, thiab kho nrog ntau yam concentrations ntawm HydroZitLa (0 raws li kev tswj, 1.25 feem pua ​​, 2.5 feem pua ​​, 5 feem pua ​​, 10 feem pua, 20 feem pua, 40 feem pua, 50 feem pua, 80 feem pua, thiab 100 feem pua ​​v/v) rau 24 teev. Tom qab ntxuav, MTT tov tau ntxiv, incubated rau 1 h, thiab ces muab pov tseg. Dimethyl sulfoxide tau ntxiv rau solubilize formazan crystals. Absorption ntawm 570 nm tau ntsuas. Cell viability (feem pua ​​​​) tau suav nrog siv cov hlwb tsis kho raws li kev tswj hwm (100 feem pua ​​​​ntawm kev muaj sia nyob).

Qhov inhibition ntawm intracellular ROS ntau lawm los ntawm HydroZitLa yog txiav txim los ntawm dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay nyob rau hauv ib tug 96- zoo phaj 64,65. HK-2 hlwb tau ua ke nrog 0.1 M DCFH-DA kua thiab incubated ntawm 37 degree rau 30 min. Tom qab ntxuav, hlwb raug sib tw nrog H2O2 lossis COM (nrog thiab tsis muaj 10 feem pua ​​v/v HydroZitLa). Fluorescent siv (zoo siab ntawm 485 nm thiab tawm ntawm 535 nm) tau ntsuas thaum pib (T0) thiab 60 min (T60). Arbitrary fluorescent unit (AFU), uas qhia txog theem ntawm ROS tiam, raug xam raws li hauv qab no: AFU= T60/T0.

Protein carbonylation ntsuas.Protein carbonyl cov ntsiab lus, raws li qhov taw qhia ntawm cov protein oxidation, tau ntsuas raws li peb cov ntaub ntawv ua ntej 64-66. Te kho HK-2 hlwb (hauv 100- hli phaj) tau lysed los ntawm radioimmunoprecipitation assay tsis muaj cov tshuaj tiv thaiv kab mob protease. Tag nrho cov protein concentration tau ntsuas los ntawm Bradford kev soj ntsuam. Lub cell lysate yog incubated nrog 10 mM dinitrophenylhydrazine (DNPH) tov los yog 2 N HCl rau 1 h nyob rau hauv qhov tsaus ntuj nti. Txias 20 feem pua ​​​​ntawm trichloroacetic acid tau ntxiv thiab incubated rau 10 feeb ntawm dej khov. Cov pellets tau sau los ntawm centrifugation, ntxuav nrog ethyl acetate: ethanol (1: 1), thiab rov ua dua nrog 6 M guanidine HCl. Absorbance (A) ntawm 375 nm tau ntsuas. Cov protein carbonyl cov ntsiab lus normalized los ntawm tag nrho cov protein concentration yog xam raws li nram no: ((ADNPH - AHCl) × 45.45) / protein concentration.

Tus qauv nas rau CaOx nephrolithiasis.EG (1 feem pua ​​v/v) ntxiv rau hauv cov dej haus (dawb nkag mus rau ad libitum, rau 35 hnub) yog siv rau induction ntawm CaOx nephrolithiasis nyob rau hauv nas. Txiv neej Wistar nas (6-8 lub lis piam, 180-300 g) tau muab faib ua plaub pawg, xws li, EG (n=6), EG plus HydroZitLa (n=6), EG plus Uralyt-U (n=6), thiab ib txwm tswj (n{10}}) pawg. Tag nrho cov nas tau nyob hauv lub tawb hlau stainless ntawm 25 degree hauv qab 12: 12 h lub voj voog tsaus ntuj. Ob tug nas tswj tau lub hom phiaj tsuas yog siv rau kev sib piv ntawm CaOx deposits nyob rau hauv nas raum thiab immunohistochemistry, tsis yog rau cov zis chemistry. HydroZitLa thiab Uralyt-U tau raug quab yuam noj ob zaug ib hnub (sab sawv ntxov thiab yav tsaus ntuj) ntawm tag nrho cov tshuaj citrate ntawm 2 mEq ib hnub. Qhov kev txhaj tshuaj ntawm citrate no sib npaug rau 60.8 mEq citrate hauv tib neeg suav nrog raws li kev hloov pauv koob tshuaj uas tau npaj los ntawm Reagan-Shaw li al.67. 24-h cov zis tau sau thaum kawg ntawm kev cuam tshuam (hnub 35) siv thymol ua cov tshuaj tiv thaiv. Cov nas tsuag tau tshuaj loog nrog isoflurane thiab txi. Ob lub raum raug tshem tawm, txiav, thiab kho nyob rau hauv 10 feem pua ​​​​ntawm formalin tsis nyob rau hauv kev tshawb fawb histology.

Txhua tus nas tau yuav los ntawm National Laboratory Animal Center, Salaya Campus Mahidol University, Nakhonpathom, Thaib teb. Cov txheej txheem kev sim tau ua los ntawm cov txheej txheem rau cov tsiaj sim los ntawm National Research Council of Thailand thiab tau pom zoo los ntawm Institutional Animal Care & Use Committee, Kws Qhia Ntawv ntawm Tshuaj, Chulalongkorn University (No. 022/2560). Peb txoj kev tshawb fawb tau ua raws li ARRIVE cov lus qhia.

CaOx deposition nyob rau hauv lub raum seem los ntawm polarized microscopy.Formalin-taw paraffin-embedded nas raum seem tau npaj los ntawm cov txheej txheem siv cov ntaub so ntswg. H&E staining tau ua raws li tus txheej txheem txheej txheem. Birefringent CaOx crystal deposits nyob rau hauv H&E-stained seem tau pom tau siv lub teeb pom kev zoo polarized (OLYMPUS BX50, Nyiv).

Yasue staining rau CaOx histochemistry.Lub raum cov ntaub so ntswg tau deparaffinized, rehydrated, submerged nyob rau hauv 5 feem pua ​​​​acetic acid rau 30 min kom tshem tawm calcium phosphate thiab calcium carbonate, thiab ntxuav nrog distilled dej 68. Sections tau incubated nrog 5 feem pua ​​AgNO3 rau 12 min, ntxuav, thiab incubated nrog saturated rubeanic. acid nyob rau hauv 10 feem pua ​​ammonium rau 1 min. Cov stained seem tau muab yaug nrog 50 feem pua ​​​​ethanol, ntxuav nrog dej distilled, lub cev qhuav dej, tshem tawm, thiab mounted. Black CaOx precipitates tau pom nyob rau hauv lub teeb tsom iav69.

Immunohistochemical staining.Tom qab deparaffinization thiab rehydration, antigen retrieval yog ua los ntawm boiling nyob rau hauv sodium citrate tsis siv lub microwave. Endogenous H2O2 yog inactivated los ntawm incubating nrog 0.3 feem pua ​​H2O2 rau 30 min. Nonspecific binding yog thaiv los ntawm incubating nrog ib txwm nees serum rau 20 min. Tom qab ntawd cov seem tau tsim nrog 1: 1,000 4-HNE (ab46545, Abcam, Cambridge, UK) lossis cleaved caspase-3 (5A1E, Cell Signaling Technology, MA, USA) thawj cov tshuaj tiv thaiv ntawm 4 degree thaum hmo ntuj, ua raws li incubation nrog cov tshuaj tiv thaiv theem nrab ntawm 37 degree rau 30 min. Tom qab ntxuav, ntu tau incubated nrog ABC reagent (VECTASTAIN® ABC cov khoom siv) rau 30 min, soaked nyob rau hauv 1 feem pua ​​​​diaminobenzidine staining reagent rau 5 min, thiab counterstained nrog hematoxylin rau 5 min. Thaum kawg, ntu tau lub cev qhuav dej, tshem tawm, mounted, thiab pom nyob rau hauv lub teeb lub teeb.

Kev ntsuas cov zis citrate, oxalate, uric acid, thiab iCOCI. Cov zis theem ntawm citrate tau txiav txim siab los ntawm kev ua haujlwm siab ua kua chromatography (Varian Medical Systems, CA, USA) siv ROAorganic Acid H ntxiv rau kab (30}0 × 7.8 mm) (Phenomenex, CA, USA), thiab eluted los ntawm 5 mM H2SO4 ntawm tus nqi ntws ntawm 0.5 mL / min. Cov qib urinary oxalate thiab uric acid tau ntsuas los ntawm capillary electrophoresis (Beckman Coulter, CA, USA) sib cais ntawm 25 degree nrog qhov hluav taws xob ntawm -20 kV. Kev kuaj zis iCOCI tau ua raws li peb daim ntawv tshaj tawm dhau los 38.

C. elegans lim thiab tu.C. elegans hom tsiaj qus Bristol N2 thiab nws qhov chaw kuaj zaub mov E. coli OP50 tau txais los ntawm Caenorhabditis Genetics Center, University of Minnesota, USA. Lawv tau cog thiab khaws cia rau hauv nematode kev loj hlob nruab nrab agar daim hlau nrog cov nyom ntawm E. coli OP50 ntawm 20 degree 70. Txhua qhov kev sim tau ua nyob rau hauv cov hnub nyoog-synchronized cov neeg laus worms.

Kev ntsuas lub neej.Kev soj ntsuam Lifespan tau ua nyob rau hauv kua media71. Luv luv, 10 hnub nyoog-synchronized cov neeg laus nematodes tau raug xa mus rau hauv ib qho 24-zoo phaj nrog M9 tsis nrog E. coli OP50 thiab 5-fuoro-2'- deoxyuridine rau kev tiv thaiv ntawm progeny zus tau tej cov. Ntau qhov ntau ntawm HydroZitLa tau ntxiv siv cov dej distilled los tswj lub tsheb. Alive nematodes raug suav txhua txhua 24 teev. Nematodes raug suav hais tias tuag thaum lawv tsis teb rau maj mam prodding siv platinum voj.

Pharyngeal pumping assay.Pharyngeal pumping (qhov qhia txog kev noj zaub mov muaj peev xwm) tau ntsuas hauv cov neeg laus nematodes (~10) piv ntawm kev tswj hwm thiab HydroZitLa supplementation, los ntawm kev saib xyuas cov pharyngeal contraction rau 30 s hauv txhua 24 teev nyob rau hauv lub stereomicroscope (SMZ{ {3}}, Motic, Suav) nyob rau hnub 0 (ua ntej ntxiv), 5, 10, thiab 1572.

Kev ntsuas lipofuscin.Kev sib sau ntawm lipofuscin nyob rau hauv cov tsiaj qus-hom nematodes tom qab ntxiv nrog ntau qhov ntau ntawm HydroZitLa (20 feem pua ​​, 30 feem pua ​​, 40 feem pua ​​​​v / v) tau saib xyuas rau 5 hnub. E. coli OP50-cov kab mob noj tau siv los tswj. Tom qab qhov kev cuam tshuam, cov cab tau ntxuav siv M9 tsis muaj ob peb zaug thiab muab tso rau ntawm lub khob swb rau hauv ib qho kev poob ntawm sodium azide. Fluorescent imaging tau ua los ntawm ZEISS LSM 700 confocal microscope. Cov duab tau raug tshuaj xyuas ntxiv siv Image J software, thiab cov fluorescence siv tau nthuav tawm raws li arbitrary units (AU)72.

Kev ntsuas qhov txheeb ze telomere ntev. Qhov txheeb ze telomere ntev (RTL) tau ntsuas hauv HK-2 hlwb kho nrog H2O2 (25 µM), sodium oxalate (NaOx, 900 µM), thiab COM (25 µg / cm2) nrog lossis tsis muaj HydroZitLa (10 feem pua ​​v /v). RTL tau txiav txim siab los ntawm lub sij hawm ntawm lub sij hawm muaj nuj nqis polymerase saw cov tshuaj tiv thaiv (qPCR)73. Nws yog xam raws li qhov piv ntawm cov ntawv luam ntawm telomeric rov qab mus rau daim ntawv luam ntawm ib tug tib-copy noob (36B4 noob) uas yog proportional rau qhov nruab nrab telomere ntev. Cov primers siv tau raws li hauv qab no: Telomere (rau pem hauv ntej) 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3}′, Telomere (rov qab) 5′-GGC TTGCCTTACTTACTTACTTACTTACTTACGTTACCT{{18}GATAGTTACCT{{18}GATAGGATGTGWward) {23 }}′, and 36B4 (reverse) 5′-CCCTTTATCATCAACGGGTACAA-3′. PCR tau nthuav dav ntawm 95 degree rau 10 min, ua raws li 40 lub voj voog ntawm 95 degree rau 15 s thiab 54 degree rau 1 min.

Ob chav staining ntawm SA-gal thiab p16.HK{{0}} cov hlwb loj hlob ntawm daim npog npog tau kho nrog H2O2, NaOx, thiab COM nrog thiab tsis muaj 10 feem pua ​​v/v HydroZitLa rau 72 h rau induction ntawm SIPS. Tom qab kev kho mob, cov hlwb raug kho thiab stained nrog cov tshuaj tshiab tshiab X-gal (Vivantis, Malaysia) rau 12–16 h, ntxuav, permeabilized nrog 0.1 feem pua ​​Triton X-100 (Amresco, TX, USA) rau 3 min , thiab thaiv kev sib khi tsis sib xws nrog 1 feem pua ​​​​cov tshuaj nees ib txwm muaj (Gibco, MA, USA) ntawm 37 degree rau 1 h. Cells raug incubated nrog 1:10,000 p16 thawj antibody (ab108349, Abcam) ntawm 4 degree thaum hmo ntuj, ua raws li 1:10,000 Alexa Fluor® 488-conjugated secondary antibody ( Cell Signaling Technology) ntawm 37 degree rau 30 min. Cov stained npog yog mounted nrog Fluoroshield mounting nruab nrab nrog 4′, 6-diamidino-2-phenylindole (Abcam). SA- -gal-positive senescent hlwb (xiav) thiab p16- qhia cov hlwb (ntsuab) tau pom thiab ua duab siv EVOS FL Auto 2 cov duab duab (Thermo Fisher Scientific, MA, USA).

Kev txheeb cais.Tag nrho cov ntaub ntawv tau nthuav tawm raws li qhov txhais tau tias ± tus qauv sib txawv lossis nruab nrab (interquartile ntau), raws li qhov tsim nyog. Ob-tus qauv t-test los yog Mann-Whitney xeem tau ua los ntsuas qhov sib txawv ntawm ob pawg. Ib txoj kev tsom xam ntawm qhov sib txawv lossis Kruskal-Wallis xeem, ua raws li kev sib piv ntau yam, tau siv los ntsuas qhov sib txawv ntawm peb lossis ntau pawg. GraphPad Prism Sofware version 9 (GraphPad Sofware Inc., CA, USA) tau ua haujlwm rau kev suav thiab cov duab. P<0.05 was considered significant.

Cov ntaub ntawv

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73. Cawthon, RM Telomere ntsuas los ntawm quantitative PCR. Nucleic Acids Res. 30, e47 (2002).

Kev lees paub

Txoj kev tshawb fawb no tau txais kev txhawb nqa nyiaj txiag los ntawm Research Gap Fund los ntawm Ministry of Science thiab Technology, National Science thiab Technology Development Agency (NSTDA) thiab Chulalongkorn University (GB-rgf_60_2905_0003 rau CB), thiab los ntawm Thaib Kev Tshawb Fawb Kev Tshawb Fawb thiab Innovation, Industry Division (RDG6150088 to CB), thiab Kws Qhia Ntawv ntawm Tshuaj, Chulalongkorn University (Matching Fund). NL tau txais 90th Ib Xyoo ntawm Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund). Cov kws sau ntawv ua tsaug lees paub txog kev tshawb fawb thiab kev txhawb nqa tshiab los ntawm Chulalongkorn University Technology Center thiab Thailand Science Research thiab Innovation Fund (TSRI) (CU_FRB640001_01_2). Ua tsaug rau Miss Natcha Madared rau lawv cov kev pab zoo heev. Ua tsaug rau cov neeg ua haujlwm nyob rau hauv cov tsiaj chaw ntawm Kws Kho Mob, Chulalongkorn University. Ua tsaug rau Assistant Prof. Dr. Sitthichai Ukritchon thiab Prof. Dr. Manathip Osiri rau lawv txoj kev txhawb nqa ntawm kev siv polarized microscopy.

Sau cov nyiaj pab

NL: Ua kev sim ntawm tes thiab nas, thiab sau cov ntaub ntawv. NC: Sau cov ntaub ntawv, thiab pab tsim lub tswv yim tshawb fawb thiab kev txheeb xyuas cov ntaub ntawv. MP: Conceived thiab ua C. elegans sim, thiab ua C. elegans cov ntaub ntawv tsom xam. DJ: Pab hauv immunohistochemistry thwmsim thiab duab. CM: Pab hauv kev faib cov ntaub so ntswg. KC: Ua telomere shortening thiab senescence thwmsim. AL: Tswj kom nkag mus rau tsiaj chaw rau kev sim nas. TT: Pab kom xeeb tub thiab tsim C. elegans sim. CB: Tsim thiab tsim cov kev tshawb fawb, ua cov ntaub ntawv txheeb xyuas, thiab sau cov ntawv sau. Txhua tus kws sau ntawv tau tshuaj xyuas cov ntawv sau.

Kev nyiam sib tw

CB, NC thiab NL yog cov neeg tsim khoom ntawm HydroZitLa. Chulalongkorn University thiab cov neeg tsim khoom muaj cov cuab yeej txawj ntse rau HydroZitLa. Tsis muaj lwm tus kws sau ntawv muaj kev tsis sib haum xeeb los tshaj tawm txog txoj haujlwm tam sim no.

Cov ntaub ntawv ntxiv

Cov ntaub ntawv ntxivCov ntawv online muaj cov khoom siv ntxiv muaj nyob rau ntawm lub vev xaib.

Kev sau ntawvthiab thov cov ntaub ntawv yuav tsum tau hais rau CB

Reprints thiab tso cai cov ntaub ntawvmuaj nyob rau ntawm lub vev xaib.

Publisher daim ntawvSpringer Nature tseem nyob nruab nrab ntawm kev txiav txim plaub ntug hauv daim duab qhia kev tshaj tawm thiab cov koom haum koom nrog.

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