Tshooj 1 Kev Tiv Thaiv Los Ntawm Phenylethanol Glycosides Los Ntawm Cistanche Tubulosa Ntawm Bovine Serum Albumin-induced Hepatic Fibrosis hauv nas

Hu rau: Audrey Hu Whatsapp / hp: 0086 13880143964 Email:audrey.hu@wecistanche.com

Abstract

Keeb kwm:Cistanche tubulosa yog ib hom tshuaj suav tshuaj ntsuab uas tau siv dav los tswj kev tiv thaiv kab mob.Phenyl ethanol glycosides los ntawm cistanche(CPhGs) los ntawm cov nroj tsuag no yog cov khoom siv tau zoo. Lub hom phiaj ntawm txoj kev tshawb no yog los soj ntsuam kev tiv thaiv thiab kho cov teebmeem ntawm CPhGs ntawm BSA-induced hepatic fibrosis nyob rau hauv nas thiab lwm yam molecular mechanisms nrog hepatic stellate hlwb. Biejiarangan (BJRG), lwm cov tshuaj suav tshuaj ntsuab, tau siv los tswj qhov zoo. Cov hau kev: Hauv kev sim vivo, 75 SD nas tau muab faib ua 6 pawg: ib txwm (kho dej-kho), qauv (BSA-kho), tshuaj zoo (BSA-kho ntxiv rau BJRG 60{{29} } mg/kg/day), thiab BSA-treated plus CPhGs (125, 250, thiab 500 mg/kg/day) pawg. Lub siab thiab tus po, cov qib ntshav ntawm aspartate aminotransferase (AST), alanine aminotransferase (ALT), hexadecenoic acid (HA), laminin (LN), hom III procollagen (PCIII), hom IV collagen (IV-C), hydroxyproline ( Hyp), thiab kev hloov pauv kev loj hlob 1 (TGF- 1) tau ntsuas hauv nas lub siab. Cov qib histopathological rau daim siab fibrosis raug soj ntsuam rau txhua pab pawg siv H&E thiab Masson's trichrome staining. Kev qhia ntawm TGF- 1, collagen I (Col-I), thiab collagen III (Col-III) tau txiav txim siab los ntawm txoj kev tiv thaiv kab mob immunohistochemical. Cov teebmeem no tau raug soj ntsuam ntxiv hauv vitro los ntawm kev txiav txim siab qhia theem ntawm NF-κB p65 thiab Col-I los ntawm kev txheeb xyuas PCR ntawm lub sijhawm. Col-I protein qhia kuj tau tshuaj xyuas los ntawm western blotting. Cov txiaj ntsig: Txhua pawg koob tshuaj (125, 250, thiab 500 mg / kg / hnub) ntawm CPhGs ua rau txo qis lub siab thiab tus po, txo qis ALT, AST, HA, LN, PCIII, IV-C qib ntshav, TGF- 1 cov ntsiab lus (P < 0.01,="" p="">< 0.01,="" thiab="" p="">< 0.01),="" thiab="" hyp="" cov="" ntsiab="" lus.="" cphgs="" kuj="" tau="" txo="" qis="" qhov="" o="" ntawm="" daim="" siab="" hlwb="" thiab="" tiv="" thaiv="" hepatocyte="" necrosis="" thiab="" inflammatory="" cell="" infiltration.="" immunohistochemical="" cov="" txiaj="" ntsig="" tau="" pom="" tias="" cphgs="" txo="" qis="" kev="" qhia="" ntawm="" tgf-="" 1="" (p="">< 0.01,="" p="">< 0.01,="" thiab="" p="">< 0.01),="" col-="" i,="" thiab="" col-iii.="" cov="" teebmeem="" hauv="" vitro="" ntawm="" cphgs="" (100,="" 75,="" 50,="" thiab="" 25="" ug="" ml)="" ntawm="" hsc-t6="" tau="" pom="" tias="" cphgs="" txo="" qis="" mrna="" qhia="" ntawm="" nf-κb="" p65="" thiab="" col-i,="" thiab="" cphgs="" kuj="" txo="" qis="" col-i="" protein="" qhia.="" cov="" ntsiab="" lus:="" cphgs="" muaj="" cov="" txiaj="" ntsig="" zoo="" los="" tiv="" thaiv="" kab="" mob="" siab,="" thiab="" tuaj="" yeem="" siv="" los="" ua="" cov="" tshuaj="" hepatoprotective="" rau="" kev="" kho="" mob="" siab="">

Ntsiab lus: Hepatic fibrosis, Cistanche tubulosa, Phenylethanol glycoside, Chemokine BSA, Kev Tiv Thaiv thiab Kho.

Keeb kwm

Hepatic fibrosis yog ib lub qhov txhab kho mob rau lub siab mob hnyav uas tshwm sim hauv cov kab mob ntawm cov kab mob siab ntev uas tshwm sim los ntawm ntau yam. Cov yam tseem ceeb no yog kis kab mob, haus cawv, cholestasis, thiab kab mob metabolic thiab autoimmune [1–3]. Progressive tsub zuj zuj ntawm extracellular matrix (ECM) thiab txo qis remodeling cuam tshuam lub cev architecture ntawm lub siab, ua rau mob siab fibrosis [4]. Hepatic fibrosis yog qhov tseem ceeb hauv cov kab mob siab ntev thiab feem ntau txhim kho mus rau qhov tsis tuaj yeem ua rau cirrhosis thiab carcinogenesis. Tam sim no, tsis muaj txoj hauv kev lossis cov tshuaj siv tau zoo rau kev kho mob hepatic fibrosis. Yog li ntawd, nws yog ib qho tseem ceeb kom nrhiav tau cov tshuaj tiv thaiv kab mob siab fibrosis uas yuav ua rau txo qis kev mob siab rau fibrosis thiab mob qog noj ntshav.Cistanche tubulosaW (ntawm tsev neeg Orobanchaceae) yog cov kab mob cab uas loj hlob nyob rau sab qab teb ntawm Xinjiang hauv Suav teb [5]. Cov neeg feem ntau siv nws los txhawb lub raum, txhawb cov ntshav, so lub plab, thiab ncua kev laus. Nws yog officially teev nyob rau hauv Suav Pharmacopoeia [6]. C. tubulosa muaj ntau yam active Cheebtsam. Cov no suav nrogPhenyl ethanol glycosides los ntawm cistanche(CPhGs), iridoids, thiab polysaccharides. Raws li ib tug ntawm ntau yam active Cheebtsam nyob rau hauvC. tubulosa, CPhGs tau nthuav tawm kev ntseeg siab antioxidant, tiv thaiv kev qaug zog, neuroprotective, thiab tiv thaiv cov teebmeem hauv vivo thiab hauv vitro kev tshawb fawb [7]. Nyob rau hauv xyoo tas los no, nws tau tshaj tawm tias CPhGs (Phenyl ethanol glycosides los ntawm cistanche) muaj cov teebmeem hepatoprotective. Cov txheej txheem muaj peev xwm hauv qab cov teebmeem no yog tshem tawm cov dawb radicals, kev tiv thaiv kab mob siab, kev tiv thaiv kab mob, inhibition of apoptosis, inhibition ntawm HBsAg thiab HBeAg, thiab inhibition ntawm HBV DNA replication thiab lwm yam [8-11]. Txawm li cas los xij, qee qhov kev tshawb fawb hauv cov ntaub ntawv hais txog kev tiv thaiv kab mob siab fibrosis ntawm cov duab. Yog li, txoj kev tshawb no tsom mus tshawb xyuas cov tshuaj tiv thaiv kab mob siab ntawm GPhCs los ntawm kev siv tus qauv ntawm bovine serum albumin (BSA) induced hepatic fibrosis hauv nas. Cov txheej txheem molecular cuam tshuam tau tshawb xyuas hauv HSC-T6 hlwb.

Cov txheej txheem

Tshuaj thiab reagents

Cov khoom siv Hydroxyproline (Alkaline hydrolysis) (Ntau: 20140616) tau yuav los ntawm Nanjing Jiancheng Bioengineering Institute (Tuam Tshoj). Rat TGF- 1 Sandwich ELISA cov khoom siv (Ntau: 238240615) tau yuav los ntawm Lianke Biotech Co., Ltd. (Tuam Tshoj). Luav Anti-Collagen I antibody (Ntau: 140619), Luav Anti-Collagen III antibody (Ntau: 980788 W), thiab Rabbit Anti-TGF- 1 antibody (Ntau: 140619) yog muab los ntawm Beijing Biosynthesis Biotechnology Co., LTD (Tuam Tshoj). Npog nrog lub cev yaj cov ntshav (cov kua dej ua haujlwm) (Ntau: WP141214), PV-6000 (Lot: WK141225), thiab DAB cov khoom siv (Lot: K136621D) tau yuav los ntawm Zhongshan Golden Choj Biotech Co., Ltd. (Suav teb) . Kev kuaj xyuas cov tshuaj tiv thaiv thawj zaug tau ua los ntawm kev siv 2. Antibody tov (Alk-Phos. Conjugated, Anti-Lavbit) thiab 2. Antibody tov (Alk-Phos. Conjugated, Anti-nas) (ntau: 272387) muas los ntawm Invitrogen Company (USA ). Bovine serum albumin (BSA) (Ntau: SLBG8239V) tau yuav los ntawm Sigma (USA). Ua ntej siv, BSA tau npaj ntawm 18 g / L hauv ib txwm saline, cov kab mob tshem tawm los ntawm kev pom, thiab BSA khaws cia ntawm 4 degree. Freund's incomplete adjuvant muaj 1 g ntawm lipid los ntawm yaj cov plaub hau (Lot: AF0220LA14, Shanghai yuan ye Bio-Technology Co., Ltd. (Tuam Tshoj,) yog tov nrog 2 g kua paraffin (Lot: 20130815, Tianjin Fuyu Fine Chemical Co., Ltd. Ltd. (Tuam Tshoj,), sterilized nyob rau hauv ib chav autoclave, thiab khaws cia ntawm 4 degree . Cov tshuaj zoo BJRG tau txais los ntawm Biejiarangan ntsiav tshuaj los ntawm Inner Mongolia Furui Medical Science Co., Ltd. (Suav teb). BJRG ntsiav tshuaj hauv dej distilled ntawm qhov concentration ntawm 600 mg / kg.

Cov ntaub ntawv cog

C. tubulosa(Orobanchaceae tsev neeg) tau yuav los ntawm thaj tsam Minfeng ntawm Xinjiang ntawm Tuam Tshoj. Cov ntaub ntawv tau lees paub los ntawm tus kws tshawb fawb Jun Zhao, Lub Chaw Haujlwm Tseem Ceeb rau Uighur Tshuaj, Lub Tsev Haujlwm ntawm Materia Medica ntawm Xinjiang. Daim ntawv pov thawj tau muab tso rau hauv lub koom haum ntawm Materia Medica ntawm Xinjiang.

Kev npaj ntawm CPhGs(Phenyl ethanol glycosides los ntawm cistanche)

Qhuav thiab sliced ​​​​rhizomes ntawmC. tubulosa(6. Ethanol extracts raug ntxuav los ntawm kev siv AB -8 cob kom tau phenyl ethanol glycosides (CPhGs). Cov kev daws teeb meem ntawm CPhGs siv rau ntau pawg hauv kev tshawb fawb tsiaj (500 mg / kg, 250 mg / kg, thiab 125 mg / kg, raws li) tau yaj hauv 0.5 feem pua ​​(5 g / L) carboxymethyl cellulose (sodium ntsev).

Quantification ntawm CPhGs(Phenyl ethanol glycosides los ntawm cistanche)

Cov ntsiab lus ntawm ob yam khoom (echinacoside thiab acteoside) hauv CPhGs tau txiav txim siab los ntawm HPLC siv txoj kev qhia yav dhau los (Zhang li al. 2004) [12]. HPLC tau ua los ntawm kev siv Shimadzu LC-10HPLC nruab nrog lub ntsuas UV. HPLC kem yog Phenomenex Gemini ODS kem (250 × 4.6 mm, 5 μm). Cov theem isocratic mobile muaj xws li methanol-acetonitrile-1 feem pua ​​​​acetic acid (15:10:75, v/v/v). Elution yog rau 40 min thiab tus nqi ntws tau khaws cia ntawm 0.6 mL / min. Qhov ntsuas kub ntawm sab hauv tau khaws cia tsis tu ncua ntawm 30 degree. UV nrhiav tau ntawm 334 nm.

Tsiaj txhu thiab HSC-T6 cell kab

[Qib SPF] noj qab nyob zoo txiv neej Sprague–Dawley (SD) nas (180–220 g) tau muas los ntawm Xinjiang Medical University Animal Center, Daim ntawv tso cai No.: SCXK (Tshiab) 2011–0004. Cov nas tsuag tau noj tshwj xeeb-tsis muaj kab mob (SPF) chow. Tag nrho cov txheej txheem cuam tshuam txog kev sim tsiaj tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Thawj Tsev Kho Mob ntawm Xinjiang Medical University. Cov nas tau nyob hauv tawb nyob rau hauv kev tswj hwm ib puag ncig (25 degree thiab 12 h lub teeb / tsaus voj voog) thiab muaj kev nkag tau dawb rau cov qauv nas pellet zaub mov thiab dej kais. Cov nas tau acclimate ua ntej kho. Ib qho immortalized nas hepatic stellate cell kab, HSC-T6, tau txais los ntawm Wuhan Procell Gene Biotechnology Co., LTD. (Wuhan, Suav teb). HSC-T6 hlwb raug coj los ntawm Dulbecco's Modified Eagle's Medium (High Glucose) (DMEM, Beijing, Tuam Tshoj) ntxiv nrog 10 feem pua ​​​​fetal bovine serum (Gibco, South America), 100 IU/ml penicillin, thiab 100 ug/ml streptomycin (Beijing). , Tuam Tshoj) nyob rau hauv ib tug humidified incubator ntawm 37 degree nrog 5 feem pua ​​CO2.

BSA-induced daim siab raug mob thiab kev kho mob

Xya caum-tsib SD nas tau muab faib ua rau pawg: Ib txwm (kho cov dej tsis huv), qauv (BSAtreated), tshuaj zoo (BSA-kho ntxiv rau BJRG 600 mg / kg / hnub), thiab BSA-kho ntxiv CPhGs(Phenyl ethanol glycosides los ntawm cistanche)(125, 25{11}}, 5{{20}}0 mg/kg/day) pawg. BSA-treated plus CPhGs (125, 250, 500 mg/kg/day) pab pawg muaj 13 nas nyob rau hauv ib pab, thiab lwm pab pawg muaj 12 nas nyob rau hauv ib pab. Tus qauv BSA-induced daim siab raug mob yog muab faib ua thawj rhiab heev ua raws li kev tiv thaiv kab mob) [13]. Tsuas yog cov pab pawg ib txwm muaj, lwm pab pawg tau muab ntau qhov kev txhaj tshuaj subcutaneous nrog 0.5 ml (9 mg / ml) BSA Freund's incomplete adjuvant nyob rau hnub 1, hnub 15, hnub 22, hnub 29, thiab hnub 36 rau thawj rhiab heev. Xya hnub tom qab txhaj koob thib tsib, cov ntshav tau txais los ntawm cov nas retinal leeg plexus thiab kuaj cov ntshav albumin los tiv thaiv lub cev. BSA antibody nyob rau hauv nas ntshav tau kuaj pom los ntawm ob txoj kev agar diffusion. Kev txhaj tshuaj tiv thaiv tau ua los ntawm kev tswj hwm 0.4 ml ntawm BSA hauv cov ntsev ib txwm yog los ntawm cov hlab ntsha caudal hauv BSA cov tshuaj tiv thaiv kab mob zoo nas ob zaug hauv ib lub lis piam rau kaum zaug. Cov concentrations thiab lub sij hawm txhaj tshuaj yog 5.00, 5.50, 6.00, 6.50, 7.00, 7.50, 8.50, 9.00, 9.50 thiab 10.00 g/L hnub 46, hnub 50, hnub 53, hnub 57, hnub 60, hnub 64, hnub 67, hnub 71, hnub 74 thiab hnub 78, raws li. Hauv cov pab pawg ib txwm muaj, cov kua qaub ib txwm siv rau kev txhaj tshuaj tiv thaiv kab mob thawj zaug (sensitization) thiab kev txhaj tshuaj thib ob (tshem tawm) tsis yog BSA, thiab lwm yam mob tau zoo ib yam li cov hauv pawg qauv. Cov pab pawg ib txwm tau muab cov dej distilled hauv qhov ncauj nrog koob tshuaj 10 ml / kg / hnub. Cov pab pawg qauv tau hais lus 10 ml / kg / hnub 0.5 feem pua ​​CMC-Na tov. Cov pab pawg tshuaj zoo tau noj 600 mg / kg / hnub BJRG. BSA-kho ntxiv rau CPhGs (125,

250, thiab 500 mg / kg / hnub) pab pawg tau hais lus 125, 250, thiab 500 mg / kg / hnub CPhGs(Phenyl ethanol glycosides los ntawm cistanche), raws. Txhua hnub noj nas txuas ntxiv rau ob lub lis piam tom qab txhaj tshuaj kawg. Tom qab lub sijhawm sim, nas tau yoo mov rau 12 teev ua ntej 10 feem pua ​​​​chloral hydrate thiab tom qab ntawd euthanized tam sim ntawd. Cov qauv ntshav tau sau los ntawm txhua tus nas thiab siv tam sim ntawd. Lub siab tau sau rau ob lub hom phiaj: (1) khaws cia hauv cov kua nitrogen rau cov khoom siv Hyp thiab (2) kho hauv 10 feem pua ​​​​ formaldehyde rau kev kuaj histological thiab immunohistochemical. Tag nrho lub sijhawm ntawm kev tshawb fawb tsiaj yog 93 hnub.

Daim siab thiab tus po

Lub siab thiab tus po tau txiav tawm los ntawm laparotomy thiab ntxuav nrog 4 degree ib txwm salines. Tom qab nqus dej ntau dhau nrog cov ntawv lim, lub siab thiab tus po tau raug ntsuas los ntsuas cov ntsuas sib txuas: Cov txheeb ze lub cev hnyav=lub cev hnyav (g) / tus kheej lub cev hnyav (g) × 100 feem pua ​​[14].

Kev sim ntawm tes

HSC-T6 hlwb tau plated hauv 96-zoo phaj. Thaum xub thawj, cov hlwb raug coj los ntawm DMEM uas muaj 10 feem pua ​​FBS rau 48 teev. Qhov nruab nrab tau hloov nrog DMEM yam tsis muaj FBS kom tshaib plab rau 12 teev. Cov hlwb tom qab ntawd tau coj mus kuaj nrog DMEM uas muaj 5.0 ng/mL TGF- 1 (tsis muaj FBS) rau 24 teev. Thaum kawg, qhov sib txawv ntawm CPhGs(Phenyl ethanol glycosides los ntawm cistanche)(100ug / ml, 50ug / ml, thiab 25ug / ml), acteoside (6 ug / ml, 3 ug / ml, thiab 1.5 ug / ml), thiab echinacoside (500ug / ml, 250ug / ml, thiab 125ug / ml) tau nqa tawm nyob rau hauv lub phaj nyob rau hauv quadruplicate qhov dej thiab incubated rau 48 h.

Kev txheeb xyuas PCR tiag tiag

MRNA qhia theem ntawm NF-κB, p65, thiab collagen kuv tau txiav txim siab los ntawm PCR lub sijhawm. Txhawm rau txiav txim siab mRNA kab lus hauv HSC-T6 hlwb, cov hlwb (4 × 105 hlwb) tau cog rau hauv 6-zoo daim hlau nrog 3 mL DMEM nrog 10 feem pua ​​FBS thiab incubated hmo ntuj ntawm 37 degree thiab 5 feem pua ​​​​CO2, tom qab ntawd cov xov tooj ntawm tes kab lis kev cai xov xwm tau hloov mus rau DMEM tsis muaj ntshav qab zib. Tom ntej no, CPhGs(Phenyl ethanol glycosides los ntawm cistanche)(100 ug/ml, 50 ug/ml, thiab 25 ug/ml), acteoside (6 ug/ml, 3 ug/ml, thiab 1.5 ug/ml), thiab echinacoside (500 ug/ml) , 250 ug / ml, thiab 125 ug / ml) tau ntxiv rau hauv qhov dej. Tom qab 48 teev ntawm incubation nrog CPhGs los yog monomeric compositions, tag nrho RNA tau muab rho tawm siv TRIzol reagent (Invitrogen, USA) thiab agitated vigorously nrog chloroform rau 15 s. Tom qab zaum ntawm chav sov li 3 min, lysate tau centrifuged ntawm 12, 000 × g rau 15 min ntawm 4 degree . RNA nyob rau hauv aqueous theem yog precipitated nrog isopropanol, thiab lub Upper aqueous theem tau pauv mus rau ib tug tshiab microcentrifuge raj. RNA tau precipitated los ntawm kev ntxiv 0.75 feem pua ​​​​ethanol, tom qab ntawd lub microcentrifuge raj thiab centrifuged ntawm 12, 000 × g ntawm 4 degree tsis pub ntau tshaj 5 min. Cov supernatant raug tshem tawm thiab RNA tau qhuav ntawm chav sov li 5-10 min. Cov txheej txheem tshwj xeeb ntawm primers (Sangon, Shanghai, Suav teb) uas tau siv rau kev nthuav dav ntawm nas -actin [GenBank: NM_031144.3], Collagen I [GenBank: NM_ 053304.1], thiab NF -κB p65 [GenBank: NM_199267.2] noob tau tsim los siv Batch Primer 3. Lub rau pem hauv ntej (fw) thiab thim rov qab (rv) primers tau raws li hauv qab no: Collagen I (fw: GGA GAG AGC ATG ACC GAT GG , rv: GGG ACT TCT TGA GGT TGC CA), NF-κB p65 (fw: CAT ACG CTG ACC CTA GCC TG, rv: TTT CTT CAA TCC GGT GGC GA), -actin (fw: TAA GGC CAA CCG TGA AAA GAT G, rv: AGA GGC ATA CAG GGA CAA CAC A). Cov txiaj ntsig tau zoo li qub rau mRNA ntawm lub tsev tu noob -actin raws li kev tswj hwm sab hauv thiab tau nthuav tawm raws li qib mRNA txheeb ze. Cov tshuaj tiv thaiv tau ua nrog 8 μL iQ SYBR Green Supermix, 1 μL 10 pM primer khub, 8.5 μL dej distilled, thiab 2.5 μL cDNA. Txhua cov tshuaj tiv thaiv polymerase tau ua raws li cov hauv qab no: 95 degree rau 3 min, tom qab ntawd 40 lub voj voog ntawm 10 s ntawm 95 degree, 30 s ntawm 55 degree, thiab 10 s ntawm 55 degree - 95 degree rau kev txuas ntxiv, ua raws li kev ntsuas fluorescence ib zaug. . Cov txiaj ntsig kawg tau piav qhia nrog cov txiaj ntsig txheeb ze (2-ΔΔCt). Kev suav thiab kev tshuaj xyuas tau ua los ntawm iQ5 Real-Time PCR Detection System.

Western blot tsom xam

Collagen I (Abcam, Cambridge, UK, Art No: ab34710) protein qhia theem tau txiav txim siab los ntawm Western blotting [nrog -actin (Lot: 60008-1-}lg; Proteintech, Tuam Tshoj)) raws li kev tswj xyuas hauv tsev. Tag nrho cov cell extracts tau npaj siv Radioimmunoprecipitation assay (RIPA) (Thermo Scientific, USA) tsis muaj 1 feem pua ​​Halt protease inhibitor cocktail (Thermo Scientific, USA) thiab 1 feem pua ​​Halt phosphatase inhibitor cocktails (Thermo Scientific, USA). Cov protein concentration tau ntsuas thiab ntsuas los ntawm Bradford txoj kev [18]. Protein (10-50 ug) tau muab cais rau ntawm 10 feem pua ​​​​SDS-PAGE gel thiab xa mus rau PVDF daim nyias nyias (Millipore, USA). Membranes raug thaiv rau 1 h nyob rau hauv chav tsev kub nrog 5 feem pua ​​BSA, thiab cov thawj cov tshuaj tiv thaiv (Anti-Colla gen I antibody, 1:200 dilution los yog nas mAb ntawm -actin, 1: 5000 dilution) tau incubated ntawm 4 degree thaum hmo ntuj. Qhov sib thooj Alk-Phos. conjugated secondary antibodies tau incubated nyob rau hauv chav tsev kub. Thaum kawg, cov ntaub so ntswg tau ntxuav peb zaug nrog 1 × Tris–HCl saline nrog 0.1 feem pua ​​​​Tween 20, thiab cov cim tau raug kuaj thiab pom los ntawm GEL DOC XR Imaging System (Bio-Rad). Kev tsom xam Densitometric tau ua tiav ntawm cov proteins txaus siab thiab normalized rau -actin los ntawm GEL DOC Image Studio software (Bio-Rad). -actin tau siv los ua kev tswj hwm sab hauv.

Kev txheeb cais

Shapiro-Wilk normality test thiab Levene's variance homogeneity test tau siv los txheeb xyuas qhov qub thiab Koj li al. DARU Journal of Pharmaceutical Sciences (2015) 23:52 Nplooj 4 ntawm 13 homogeneity ntawm variance. Kev soj ntsuam ntawm qhov sib txawv (ANOVA) ua raws li Tukey's post hoc test tau siv los txheeb xyuas qhov sib txawv ntawm cov ntaub ntawv tsis sib xws, feem ntau faib cov ntaub ntawv. Kruskal-Wallis non-parametric test tau siv los txheeb xyuas cov ntaub ntawv tsis nquag faib lossis homogeneous. Cov txiaj ntsig tau qhia tias txhais tau tias ± SD. Qhov tseem ceeb tau teeb tsa ntawm P < 0.05.="" tag="" nrho="" cov="" ntaub="" ntawv="" tau="" txheeb="" xyuas="" los="" ntawm="" spss="" 16.0="" software="" (xinjiang="" medical="">

Cov txiaj ntsig

Kev txiav txim siab ntawm CPhGs

CPhGs hauv C. tubulosa muaj ob phenylethyl cawv glycosides, echinacoside, thiab acteoside, thiab lawv cov ntsiab lus hauv CPhGs tau txiav txim siab los ntawm HPLC tsom xam (Fig. 1) kom yog 42.71 ± 0.42 feem pua ​​thiab 14.27 ± {{7 }}.18 feem pua, feem.

Daim siab thiab tus po

Raws li daim duab 1 tau pom, daim siab thiab tus po qhov ntsuas ntawm pawg qauv tau nce siab [P < 0="" 01,="" p="">< 0.05].="" lub="" siab="" thiab="" tus="" po="" qhov="" ntsuas="" ntawm="" cov="" tshuaj="" zoo="" bjrg="" thiab="" cphgs="" ntawm="" ntau="" pawg="" koob="" tshuaj="" tau="" txo="" qis="" dua="" piv="" nrog="" cov="" qauv="" pawg="" [pliver="0.004," pliver="0.003," pliver="0." 004,="" pliver="0.005;" pspleen="0.017," pspleen="0.027," pspleen="0.024," pspleen="0.070," ntsig="" txog].="" lub="" siab="" thiab="" tus="" kab="" mob="" spleen="" indices="" tau="" qis="" dua="" li="" cov="" qauv="" ntawm="">

Cov teebmeem ntawm CPhGs ntawm ALT, AST cov dej num, thiab daim siab fibrosis cim

Hauv kev tshawb fawb tam sim no, qib ntshav ntawm cov enzymes hepatic AST thiab ALT tau nce ntxiv hauv pawg qauv, qhia txog kev puas tsuaj rau hepatocellular hauv BSAinduced daim siab fibrosis nas. Txawm li cas los xij, cov kev sim.

qhia tias kev kho mob nrog BJRG (600 mg/kg) thiab CPhGs (125, 250, thiab 5{{10}}0 mg/kg) txo qis AST [PAST < 0="" .="" ,="" re="" feem]="" thiab="" alt="" [palt="0}.117," palt="0.139," palt="0.189," palt="0.255," ntsig="" txog]="" qib="" hauv="" cov="" kab="" mob="" siab="" fibrosis="" nas="" .="" (table="" 2).="" cov="" theem="" ntawm="" ha,="" ln,="" pc="" thiab="" iv-c="" hauv="" cov="" qauv="" nas="" tau="" nce="" ntau="" [pha="">< 0.001,="" pln="">< 0.001,="" ppciii="0.002," piv-c="">< 0.001,="" ntsig="" txog].="" piv="" nrog="" rau="" pawg="" qauv,="" theem="" ntawm="" ha="" [pha="0.009," pha="0.007," pha="0.009," pha="0.023," ntsig="" txog],="" ln="" [pln="0.011," p="" ln="0.004," p="" ln="0.026," p="" ln="0.069," ntsig="" txog],="" pc="" iii="" [p="" pciii="" {="" {47}}.006,="" p="" pciii="0.067," p="" pciii="0.136," p="" pciii="0.296," ntsig="" txog],="" thiab="" iv-c="" [p="" iv-c="">< 0.001,="" p="" iv-c="">< 0.001,="" p="" iv-c="">< 0.001,="" p="" iv-c="">< 0.001,="" ntsig="" txog]="" hauv="" cov="" nas="" tau="" poob="" qis="" los="" ntawm="" bjrg="" (600="" mg="" kg)="" thiab="" cphgs="" ntawm="" ntau="" pawg="" tshuaj="" (125,="" 250,="" thiab="" 500="" mg/kg)="" (table="" 3).hyp="" cov="" ntsiab="" lus="" thiab="" tgf-="" 1="" cov="" ntsiab="" lus="" collagen="" kuj="" tau="" kuaj="" pom="" los="" ntawm="" kev="" ntsuas="" hyp="" qib="" hauv="" daim="" siab.="" raws="" li="" qhia="" hauv="" table="" 4,="" qhov="" txhais="" tau="" tias="">qib hauv pawg qauv tau nce siab dua li cov pab pawg ib txwm muaj, tab sis nws tau txo qis hauv pawg BJRG thiab cov pab pawg sib txawv CPhGs koob tshuaj. Lub siab siab ntawm TGF{{0}}} ntawm pawg qauv hauv nas tau nce ntau [P < 0="" 001].="" piv="" nrog="" rau="" pawg="" qauv,="" tgf-="" 1="" qib="" ntawm="" cov="" tshuaj="" zoo="" thiab="" cov="" pab="" pawg="" sib="" txawv="" ntawm="" cphgs="" tau="" poob="" qis="" [p="" tgf-="" 1="">< 0.001,="" p="" tgf-="" 1="">< 0.001,="" p="" tgf-="" 1="">< 0.001,="" ptgf-="" 1="">< 0.001,="" ntsig="" txog].="" cov="" txiaj="" ntsig="" tau="" muab="" sau="" tseg="" hauv="" table="">

Kev kuaj mob histopathological

Kev soj ntsuam ntawm cov kab mob siab ib txwm stained nrog H&E thiab Masson's trichrome pom cov kab mob siab sib txawv thiab kab mob siab sinusoids. Lub siab cov ntaub so ntswg nyob rau hauv cov qauv nas muaj teeb meem, thiab daim siab cov ntaub so ntswg thiab hepatic sinusoids tau hloov los ntawm ib tug loj npaum li cas ntawm cov ntaub so ntswg. Txawm li cas los xij, ntau dua li qub cytoarchitecture thiab cov ntaub so ntswg tsawg dua tau kuaj pom hauv pawg kho mob dua li cov hauv pawg qauv.

H&E staining

Nyob rau hauv ib txwm pab pawg neeg, hepatic lobule yam ntxwv kev ncaj ncees yam tsis muaj qhov txawv txav chaw portal thiab hepatic sinusoids tau pom. Cov kab mob hepatic tau muab tso rau hauv ib qho kev txiav txim siab, nrog lub hauv paus puag ncig thiab meej. Lub nuclei yog nyob rau hauv nruab nrab ntawm lub cell, nrog ntau cytoplasm. Tsuas yog thaj chaw portal muaj cov ntaub so ntswg me me (Fig. 2a). Hauv pawg qauv, cov qauv lobular tau raug puas tsuaj loj heev. Cov hlwb hauv siab pom cov dej me me degeneration, feem ntau ballooning degeneration thiab / lossis fatty degeneration. Kev tsim ntawm inflammatory cell infiltration, dav fibrous cov ntaub so ntswg hyperplasia, tsim ntawm ib tug loj tus naj npawb ntawm cov fibrous septum, phua lobules, thiab tseem ceeb daim siab hlwb proliferation kuj tau pom. Cov kev soj ntsuam no tau lees paub qhov ua tiav ntawm kev tsim cov nas tiv thaiv kab mob raug mob tsiaj tus qauv ntawm kab mob siab fibrosis (Fig. 2b). Piv nrog rau pawg qauv, muaj kev txo qis hauv cov cell infiltration hauv CPhGs sib txawv. Tsis tas li ntawd pom muaj tsawg necrosis thiab fatty degeneration ntawm daim siab hlwb nrog rau alleviation ntawm fibrosis. CPhGs txo qis cov kab mob ntawm BSA-vim kab mob siab fibrosis hauv nas, txo qhov o ntawm daim siab hlwb, thiab tiv thaiv kab mob hepatocyte necrosis thiab inflammatory cells infiltration, qhia tias CPhGs ua rau muaj kev tiv thaiv ntawm BSA-induced nas hepatic fibrosis. (Fig. 2c–f). Masson's trichrome staining Nyob rau hauv ib txwm pab pawg neeg, daim siab cov ntaub so ntswg yog ib txwm. Lub hepatic portal cheeb tsam tau pom ib tug me me ntawm xiav collagen fibers, thiab daim siab cov ntaub so ntswg yog ib txwm tsim (Fig. 3a). Piv nrog rau daim siab cov ntaub so ntswg ntawm ib txwm pab pawg neeg, cov ntaub so ntswg fibrous proliferated los ntawm lub hauv paus nplooj ntawv thiab nthuav mus rau hauv lub siab parenchyma. Collagen fibers txuas ntxiv thiab txuas thiab

enveloped tag nrho lobule thiab ncig lub hauv paus hlab ntsha. Cov teebmeem no, nrog rau hepatocyte fibrosis, lobular structural puas, periportal fibrosis, thiab pseudolobule tsim (Fig. 3b) muab pov thawj tias tus qauv tsim. Piv nrog rau cov qauv pab pawg, cov collagen fibers nyob rau hauv lub zoo tshuaj tswj pab pawg neeg tau maj mam ncua sab nraud ntawm lub peripheral portal cheeb tsam (Fig. 3c). Com pared nrog cov qauv pab pawg, cov collagen fibers nyob rau hauv sib txawv CPhGs koob tshuaj pawg tau txo qis, fiber ntau proliferation yog inhibited, thiab proliferation ntawm fibrous cov ntaub so ntswg nyob rau hauv lub siab parenchyma ho txo. Cov txiaj ntsig no tau qhia tias CPhGs tiv thaiv nas los ntawm BSA-induced hepatic fibrosis (Fig. 3d–f ).

Immunohistochemical staining

Qhov tseem ceeb, kev qhia ntawm collagen hom I thiab collagen hom III ua lub luag haujlwm tseem ceeb hauv kev txhim kho cov kab mob siab fibrosis, thiab lawv cov tiam thiab tso tawm hauv daim siab cov ntaub so ntswg tuaj yeem ua qhov tseem ceeb ntawm kev tiv thaiv kab mob siab. Cov txiaj ntsig tau muab sau tseg hauv Table 5

Collagen type I

Cov pab pawg ib txwm qhia txog collagen hom kuv feem ntau hauv cov hlab ntsha thiab thaj chaw portal. Cov qauv pab pawg tau nthuav tawm cov collagen hom I vascular fibrosis hauv cov chaw portal. Nyob rau hauv qhov chaw ntawm Disse, collagen hom kuv staining tau pom streaks los yog raws li ib tug patchy tis. Collagen encased fibrous septa los ua pseudolobule. Hauv cov kev sim no, tsawg dua fibrous septa tau tsim rau BJRJ (600 mg / kg) thiab ntau pab pawg ntawm CPhGs (125, 250, thiab 500 mg / kg) [PCol I=0.002, PCol I {{ 6}}.001, PCol I=0.023, thiab P Col I=0.044, ntsig txog]. Cov txiaj ntsig semiquantitative tau qhia tias qhov kev qhia ntawm lawv cov collagen hom kuv tau qis dua piv nrog cov qauv pawg (Fig. 4).

Collagen type III

Collagen hom III tau qhia tsis muaj zog hauv cov pab pawg ib txwm muaj, thiab thaj chaw peripheral nyob ib puag ncig lub portal thiab hepatic veins tso tawm me me ntawm cov xim daj zoo nkauj tsis tas yuav fibers (Fig. 5a). Nyob rau hauv cov qauv pab pawg neeg, collagen fibers nthuav dav thiab tuab cords, qhia tias muaj zog qhia, feem ntau nyob rau hauv lub portal thiab fibrous cov ntaub so ntswg (Fig. 5b). Collagen fibers ntawm BJRJ (600 mg / kg) thiab ntau pawg ntawm CPhGs (125, 250, thiab 500 mg / kg) tau filamentous thiab faib nyob ib ncig ntawm cov hlab ntsha hauv nruab nrab thiab cov chaw portal. Piv nrog rau pawg qauv, cov collagen fibers tau txo qis, staining yog daj ntseg thiab nyias, thiab immunohistochemical staining yog weakly zoo (Fig. 5c–f ). Cov txiaj ntsig ib nrab ntawm cov txiaj ntsig no qhia tau hais tias cov hlwb qhia tau zoo hauv pawg koob tshuaj zoo thiab sib txawv [PCol III=0.015, PCol III=0.001, PCol III=0.010, thiab P Col III=0.037, ntsig txog] txawv ntawm cov hauv pawg qauv.

TGF- 1

Tsis muaj kev qhia me me ntawm TGIF{{0}} nyob rau hauv cov kab mob siab nas. Kev nthuav qhia tau txwv rau qee cov hlwb sib txuas (Fig. 6a). Hauv pawg qauv, TGF- 1 qhia tau dav dav hauv thaj chaw portal, fibrous qhov chaw, lub siab lub hlwb, cov hlwb inflammatory, cov phab ntsa sinusoidal thiab cytoplasm. Ib qho chaw portal tshwj xeeb tau nthuav tawm cov lus pom zoo nrog cov xim daj-daj (Daim duab 6b). Muaj qee qhov kev qhia me me hauv thaj chaw portal thiab fibrous septa ntawm BJRJ (600 mg / kg) thiab ntau pawg ntawm CPhGs (125, 250, thiab 500 mg / kg). Qhov loj ntawm qhov zoo staining hauv cov pab pawg no tau txo qis dua piv nrog cov qauv ntawm pawg. Staining nyob rau hauv lub interstitial hlwb nyob rau hauv lub fibrous septa thiab cytoplasm ntawm inflammatory cells raug txo (Fig. 6c–f ). Cov txiaj ntsig ib nrab tau qhia tias BJRJ thiab ntau pawg koob tshuaj ntawm CPhGs [P TGF- 1=0.001, P TGF- 1 < 0.001,="" p="" tgf-="" 1="0.009," ptgf-="" 1="0" .004,="" ntsig="" txog]="" yog="" qhov="" tseem="" ceeb="" piv="" nrog="" rau="" pawg="" qauv.="" raws="" li="" tau="" hais="" ua="" ntej="" hauv="" tsab="" xov="" xwm,="" tgf-="" 1="" yog="" ib="" qho="" tseem="" ceeb="" cytokine="" hauv="" pathophysiology="" ntawm="" daim="">

fibrosis, stimulating zus tau tej cov extracellular matrix [19]. Peb tau pom tias theem ntawm TGF- 1 tau nce hauv cov qauv hauv pawg raws li qhov hnyav ntawm daim siab fibrosis. Kev qhia theem ntawm TGF- 1 hauv daim siab tau zoo ib yam nrog cov ntshav qab zib TGF- 1 qib. Qhov no qhia tau hais tias CPhGs tuaj yeem txo lub siab fibrosis vim TGF- 1 qhia los ntawm kev koom tes hauv kev sib txuas thiab degradation ntawm ECM. Kev qhia ntawm collagen hom I, collagen hom III, thiab TGF- 1 tuaj yeem kuaj xyuas cov txheej txheem pathological ntawm hepatic fibrosis. Lawv cov qib qhia hauv pawg kho mob tau txo qis thiab qhia tau tias CPhGs tuaj yeem txhim kho collagenase kev ua haujlwm, tswj kev sib npaug ntawm daim siab ECM synthesis thiab degradation, yog li ncua thiab tiv thaiv kev tsim ntawm daim siab fibrosis.