PART IB Phenylethanol Glycosides Tiv Thaiv Myocardial Hypertrophy Induced Los ntawm Abdominal Aortic Constriction Ntawm ECE-1 Demethylation Inhibition Thiab PI3K/PKB/eNOS Pathway Enhancement
Mar 06, 2022
Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com
Qiong-Ling Fan, 1 Jia-Wei Wang, 1 Shi-Lei Zhang, et al.
Phenylethanoild glycosides (CPhGs)yog cov khoom tseem ceeb hauv paus ntawm pharmacological kev ua nyob rau hauvCistanche tubulosathiab muaj ntau yam teebmeem pharmacological. Txawm li cas los xij, nws tsis paub meej tias CPhGs(Phenylethanoild glycosides los ntawm cistanche) muaj cov nyhuv ameliorative ntawm lub siab tshaj qhov ua rau myocardial hypertrophy. Hauv txoj kev tshawb no, txiv neej SD nas hnyav (200 ± 20) g tau tsim cov qauv plawv hypertrophy los ntawm plab aortic coarctation (AAC). Tom qab ua haujlwm, cov nas tau muab tshuaj nrog cov tshuaj sib xyaw rau 6 lub lis piam (CPhGs(Phenylethanoild glycosides los ntawm cistanche) 125, 250, thiab 500 mg / kg / d thiab valsartan 8.3 mg / kg / d). Echocardiography, lub plawv qhov hnyav Performance index (HWI), cross-sectional area of cardiomyocytes (CSCA), fibrosis area, plasma endothelin 1(ET-1), and proinflammatory factor levels are detected. Peb cov txiaj ntsig tau pom tias CPhGs sib txawv(Phenylethanoild glycosides) ntau npaum li cas txo qis sab laug ventricular posterior phab ntsa thickness (LVPWT), sab laug ventricular end-diastolic txoj kab uas hla (LVED), HWI, CSCA, fibrosis cheeb tsam, ET-1, proinflammatory yam, arterial natriuretic peptide (ANP), hlwb natriuretic peptide (BNP ), endothelin converting enzyme 1(ECE-1) mRNA theem, cyclooxygenase 2 (COX-2), siab mobility pawg box 1 (HMGB-1) protein ntau, thiab ECE{{8} } qib demethylation thaum nce sab laug ventricular ejection fractions (LVEF), sab laug ventricular fractional shortening (LVFS), phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-PKB), thiab phosphoricated nitrate. synthetase (p-eNOS). -e indexes ntawm CPhGs(Phenylethanoild glycosides los ntawm cistanche) 250 thiab 500 mg / kg pawg tau txawv ntawm AAC pawg; piv nrog valsartan pawg (AV), qhov ntsuas ntawm CPhGs 500 mg / kg pawg tsis sib txawv. Hauv kev xaus, CPhGs(Phenylethanoild glycosides los ntawm cistanche) ameliorated myocardial hypertrophy nas los ntawm AAC, uas tej zaum yuav muaj feem xyuam rau ECE- 1 demethylation inhibition thiab PI3K/PKB/eNOS li.
2. Cov ntaub ntawv thiab cov txheej txheem
2.1. Tsiaj.
70 txiv neej SPF SD nas hnyav 200 ± 20 g (10 ib pab pawg) tau muab los ntawm Kev Tshawb Fawb Tsiaj Center ntawm Xinjiang Medical University (Urumqi, Suav). Tus nab npawb ntawv tso cai tsim tsiaj yog SYXK(XIN)2016-0003 thiab daim ntawv pov thawj tus lej yog SCXK(XIN)2016-002. -e txoj kev tshawb fawb tau pom zoo los ntawm Pawg Saib Xyuas Kev Ncaj Ncees Tsiaj ntawm Thawj Tsev Kho Mob ntawm Xinjiang Medical University (kev pom zoo naj npawb 20180223-183). -ey tau khaws cia tsis pub nkag mus rau dej thiab zaub mov hauv cov tawb yas thiab khaws cia rau hauv 12 teev lub teeb / tsaus ntuj ntawm qhov kub ntawm 22 ± 2 degree thiab av noo ntawm 45-55 feem pua hauv peb Lub Chaw Tshawb Fawb Tsiaj.
2.2. Khoom siv.
CPhGs(Phenylethanoild glycosides los ntawm cistanche) , uas nws cov ntsiab lus ntawm echinacea yog ntau tshaj 40 feem pua thiab acteoside yog ntau tshaj 16 feem pua, tau yuav los ntawm Hetian Di Chen Medical Biotechnology Co., Ltd. Valsartan tau yuav los ntawm Beijing Novartis Pharma Co., Ltd. Carboxymethylcellulose sodium (CMC-Na) yog muab los ntawm Xian Zhengtian Pharmaceutical Accessories Co., Ltd. Thiab CPhGs tau yaj hauv 0.5 feem pua CMC-Na.
Phenylethanoild glycosides los ntawm cistanche
2.3. Abdominal Aortic Coarctation (AAC).
Tom qab tshuaj loog los ntawm 2.5 feem pua pentobarbital sodium (2.5 ml / kg) (Merck, Darmstadt City, Hessen, Lub teb chaws Yelemees), cov nas tau shaved thiab khaws cia rau hauv cov cua sov. -en lub plab kab noj hniav ntawm nas tau qhib rau 2 cm nyob rau hauv nruab nrab ntawm lub plab mog nyob rau hauv ib tug tsis muaj menyuam, lub plab viscera raug thawb mus rau sab xis, thiab lub plab aorta yog tag nrho nthuav tawm ntawm 0.5 cm. saum lub raum txoj hlab ntsha. Number 8 koob tau ligated nrog tus xov tooj 2 hlau nyob rau hauv parallel mus rau lub plab kab noj hniav raug sutured [18]. Lub plab aortas ntawm nas nyob rau hauv pawg sham tau sib cais yam tsis muaj ligation thiab tsis muaj pab pawg tswj kev phais. -e hnub tom qab ua haujlwm, AC 125 mg / kg, AC 250 mg / kg, thiab AC 500 mg / kg tau noj nrog CPhGs(Phenylethanoild glycosides los ntawm cistanche)los ntawm gavage; AV pab pawg tau noj nrog valsartan los ntawm gavage; thiab pawg tswj hwm, pab pawg AAC, thiab pab pawg neeg txaj muag tsis tau noj nrog CPhGs lossis valsartan, ib hnub ib zaug rau 42 hnub sib law liag. Cov koob tshuaj tau teem tseg raws li cov lus qhia yav dhau los thiab kev sim ua ntej [8].
2.4. Echocardiography.
Rau lub lis piam tom qab AAC, cov nas tsuag tau tshuaj loog nrog 2.5 feem pua pentobarbital sodium thiab ua echocardiography (Philips HD11 XE, Amsterdam, Netherlands). LVPWT, LVED, LVEF, thiab LVFS tau ntsuas plaub zaug los xam qhov nruab nrab raws li qhov ntsuas kawg.
2.5. HeartWeight Index (HWI).
Tom qab echocardiography, cov nas raug tua los ntawm kev sau cov ntshav los ntawm lub plab aorta. -e lub siab ntawm nas raug tshem tawm ntawm lub hauv siab thiab hnyav tom qab ntxuav los ntawm ib txwm saline (4 degree) 3 zaug thiab ntws nrog cov ntawv lim huv. -e HWI raug xam los ntawm cov qauv hauv qab no: HWI � lub plawv hnyav lub cev hnyav. (1)
2.6. Hematoxylin-Eosin thiab Masson Staining.
-e myocardium ntawm sab laug ventricular tau kho los ntawm 4 feem pua paraformaldehyde embedded nyob rau hauv parafin, crosscut nrog ib seem thickness ntawm 4 μm, qhuav nyob rau hauv ib lub qhov cub ntawm 60 degree rau 2 teev, thiab ces stained nrog hematoxylin-eosin los yog Masson (Solaibio, Beijing, Tuam Tshoj). -e qauv ntawm myocardium tau pom nrog ib qho kev hloov pauv theem-qhov sib txawv ntawm lub tshuab ntsuas (Leica, Frankfurt, Lub teb chaws Yelemees). Image J software tau siv los xam CSAC thiab thaj tsam fibrosis.
2.7. Enzyme-Linked Immune Sorbent Assay (ELISA).
Lub plab aortic ntshav tau muab rho tawm los ntawm nas thiab muab tso rau hauv lub raj tshuaj tiv thaiv rau 2 h ntawm chav tsev kub thiab ces centrifuged ntawm 3000 r / min ceev. -e supernatant tau nqus nrog pipettes (Eppendorf, Hamburg, Lub teb chaws Yelemees) thiab khaws cia ntawm 80 degree rau kev sim. Plasma ET-1, interleukin 1 (IL-1 ), interleukin 6 (IL-6), qog necrosis factor Evidence-Based Complementary and Alternative Medicine
(TNF- ) (Bioss, Beijing, Tuam Tshoj), thiab cyclooxygenase 1 (COX-1) (Cusabio, Wuhan, Tuam Tshoj) qib nas raug kuaj pom raws li ELISA cov lus qhia.
2.8. Methylation-Specific PCR.
DNA tau muab rho tawm los ntawm sab laug ventricular myocardial cov ntaub so ntswg ntawm nas los ntawm DNA extraction kit (Tiangen Biotech, Beijing, Suav). -e tus nqi nqus (A tus nqi) yog sib npaug rau OD260/OD280 (1.8∼1.9). 20 ul genomic DNA (500 pg-1 ug) tau hloov kho nrog sulfurous acid los ntawm Methylation-Gold Kit (ZYMO, Orange County, CA, USA). -e MSP cov tshuaj tiv thaiv yog 25 μL, suav nrog 3.0 μL ntawm sulfite hloov pauv DNA qauv, 2.5 μL ntawm 10 × tsis, 2.5 μL ntawm 2.5 hli dNTPs, 1.0 μL ntawm 10 μm primer, 0.1 μL ntawm Hotstar KARA Biotechnology (NTP) , thiab 14.9 μL ntawm ddH2O. -ere yog 45 cycles nyob rau hauv tag nrho: denaturation ntawm 95 degree, annealing ntawm 55 degree, thiab extension ntawm 72 degree rau 30 s. Tom qab PCR, 5 μL cov khoom raug cais los ntawm 2 feem pua agarose gel electrophoresis. -e DNA tau kho los ntawm M. Sssl methyltransferase (New England Biolabs, Ipswich, MA, USA) tau siv los ua kev tswj hwm zoo ntawm cov tshuaj methylation, thiab DNA ntawm pawg tswj hwm ib txwm siv los tswj qhov zoo ntawm demethylation. Thaum kawg, agarose gel tau yees duab thiab tshuaj xyuas los ntawm Bio-Rad gel imaging system. -e demethylation qib tau xam los ntawm cov qauv hauv qab no:
Tag nrho cov primers tsim nyob rau hauv Methprimer (Applied Biosystems, MA, USA) thiab synthesized los ntawm Sangon Biotech (Shanghai, Tuam Tshoj) yog teev nyob rau hauv Table 1.
2.10. Western Blot.
Proteins tau muab rho tawm los ntawm sab laug ventricular myocardium nrog Ripa Buffffer (Scientific) thiab protease inhibitor ntxiv. Protein concentration tau txiav txim los ntawm BCA txoj kev (Solarbio, Beijing, Suav). Cov protein tau noj ntawm 50 ug thiab denatured ntawm 100 ° C rau 5 feeb. Proteins raug cais los ntawm sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, Tuam Tshoj) thiab tom qab ntawd xa mus rau polyvinylidene fluoride (PVDF) daim nyias nyias los ntawm cov khoom siv electrophoretic (Bio-Rad, CA, USA). Tom qab raug thaiv nrog 3 feem pua BSA rau 2 teev nyob rau hauv chav tsev kub, cov membranes tau incubated nrog anti-PI3K antibody, antiphospho-PI3K antibody, anti eNOS antibody, anti-phospho-eNOS (1: 1000, Bioss, Beijing, Tuam Tshoj), anti-COX{17}} antibody, anti-HMGB-1 antibody (1: 1000, Proteintech, Chicago, IL, USA), thiab anti-ECE-1 antibody (1: 2000, Signalway Antibody , College Park, MD, USA), thawj cov tshuaj tiv thaiv thaum hmo ntuj ntawm 4 degree . Hnub tom qab, tom qab ntxuav nrog TBST 3 zaug, cov membranes tau incubated nrog HRP-labeled tshis tiv thaiv luav IgG (1: 25000, 1 h, Zhongshanjinqiao, Shanghai, Tuam Tshoj) ntawm chav tsev kub. Tom qab tau ntxuav nrog TBST 3 zaug, cov daim nyias nyias tau pom los ntawm Pierce™ ECL Western Blotting Substrate thiab ua duab siv FluorChem E Imaging System (Protein Simple, USA), uas tau ua rau -actin raws li kev tswj hwm sab hauv.
Cov txiaj ntsig ntawm Phenylethanoild glycosides los ntawm cistanche
cov txiaj ntsig cistanche
3. Cov txiaj ntsig
3.1. CPhGs(Phenylethanoild glycosides los ntawm cistanche) Txhim kho plawv muaj nuj nqi hauv nas tom qab AAC.
-e nas tau txais gavage los ntawm hnub tom qab AAC. -e zoo tswj pab pawg (AV) tau muab gavage nrog valsartan 8.3 mg / kg / d, thiab CPhGs pawg (AC) tau muab gavage nrog CPhGs 125, 250, thiab 500 mg / kg / d, raws li. Tom qab rau lub lis piam 'nruam koob tshuaj, cov nas tau ua echocardiography nyob rau hauv 2.5 feem pua sodium pentobarbital tshuaj loog. Ob tus nas tuag tom qab AAC: ib tus hauv pawg qauv tuag rau hnub peb tom qab AAC los ntawm pulmonary edema; Lwm tus hauv AC 500 mg / kg tuag hnub tom qab kev phais xav tias yog tshwm sim los ntawm kev phais mob. Raws li pom nyob rau hauv daim duab 2, piv nrog rau pawg sham, LVED, LVEF, thiab LVFS nyob rau hauv AAC pab pawg tau txo qis heev tab sis LVPWT hauv pab pawg AAC tau nce ntau. Piv nrog rau pawg AAC, LVED, LVEF, thiab LVFS tau nce thiab LVPWT tau txo qis rau qhov sib txawv ntawm txhua pawg AC koob tshuaj; rau LVPWT, qhov kev hloov pauv tseem ceeb tsis pom nyob hauv AC 125 mg / kg pawg tab sis tau pom hauv AC 250 thiab 500 mg / kg pawg. Piv nrog rau pawg AV, LVPWT thiab LVED hauv AC 125 mg / kg pawg tau pom qhov sib txawv tseem ceeb, thaum LVPWT, LVED, LVFS, thiab LVFS hauv AC 250 thiab 500 mg / kg pawg tsis yog.
3.2. CPhGs(Phenylethanoild glycosides los ntawm cistanche) Txo HWI, CSAC, thiab Fibrosis Area hauv nas tom qab AAC.
-en peb ntsuas lub plawv ntawm txhua tus nas los xam HWI thiab muab cov ntaub so ntswg myocardial rau HE thiab Masson staining los soj ntsuam cov kev hloov pauv ntawm cheeb tsam cross-sectional (CSAC) thiab thaj chaw fibrosis hauv cardiomyocytes. Raws li pom nyob rau hauv daim duab 3(a), 3(d), thiab 3(e), piv nrog rau pawg sham, lub plawv qhov hnyav (HW) thiab HWI hauv pawg AAC tau txo qis heev; piv nrog pawg AAC, HW thiab HWI hauv AC250 thiab 500 mg / kg pawg tau txo qis; piv nrog AV pawg, HW thiab HWI tau nce ntau hauv AC 125 mg / kg pawg tab sis tsis nyob hauv AC 250 thiab 500 mg / kg pawg. Nyob rau hauv daim duab 3(b) thiab 3(f), piv nrog rau pawg sham, Masson staining cov txiaj ntsig tau pom tias thaj chaw fibrosis hauv pawg AAC tau nce ntau. Rau thaj tsam fibrosis, qhov kev txo qis tseem ceeb tau pom hauv AC 125, 250, thiab 500 mg / kg pawg thaum piv rau pawg AAC, thaum muaj kev nce ntxiv hauv AC 125 thiab 250 mg / kg pawg thiab qhov txo qis hauv AC 500 mg / kg pawg tau pom thaum piv rau AV pawg. Nyob rau hauv daim duab 3(c) thiab 3(g), HE staining cov txiaj ntsig tau pom tias CSAC hauv pab pawg AAC tau nce ntau ntxiv nyob rau hauv kev sib piv nrog rau hauv pawg sham; piv nrog pawg AAC, CSAC hauv AC 250 thiab 500 mg / kg pawg tau txo qis; piv nrog rau hauv AV pawg, txawm li cas los xij, CSAC tau nce ntau hauv AC 125 thiab 250 mg / kg pawg, thiab tsis muaj qhov sib txawv tseem ceeb hauv AC 500 mg / kg pawg.
3.3. CPhGs(Phenylethanoild glycosides los ntawm cistanche) Txo mRNA Qib ntawm Myocardial Hypertrophy Genes hauv Myocardial Ntsws ntawm nas tom qab AAC.
ANP, BNP, thiab myosin hnyav saw (-MHC) yog ib qho tseem ceeb ntawm lub plawv hypertrophy yam [19, 20]. Peb tau kuaj pom qhov sib txheeb ntawm cov theem ntawm peb lub plawv hypertrophy noob hauv cov ntaub so ntswg myocardial. Raws li pom nyob rau hauv daim duab 4, piv nrog cov sham pawg, cov txheeb ze qhia theem ntawm ANP, BNP, thiab -MHC mRNA nyob rau hauv cov pab pawg neeg AAC tau nce ho; Txawm li cas los xij, piv nrog pab pawg AAC, qhov txheeb ze qhia theem ntawm peb lub myocardial hypertrophy genes hauv AC 250 Cov Ntawv Pov Thawj-raws li kev sib txuas thiab lwm yam tshuaj 5.
