Obacunone Attenuates Liver Fibrosis Nrog Txhim Kho Cov nyhuv Anti-Oxidant Ntawm GPx-4 Thiab Inhibition Ntawm EMT

Thov hu rauoscar.xiao@wecistanche.comyog xav paub ntxiv

Abstract:Obacunone, limonin triterpenoid muab rho tawm los ntawm Phellodendronchinense Schneid los yog Dic-tamnus dasycarpusb Turcz tsob nroj, elicits ntau yam pharmacological teebmeem xws li anti-inflammatory, anti-neoplastic, anti-oxidation, thiab anti- ntsws-fibrosis sawv daws yuav. Txawm li cas los xij, cov nyhuv anti-fibrotic ntawm oba-cone thiab cov ncauj lus kom ntxaws hauv lub cev hauv daim siab fibrosis tseem tsis meej. Lub siab fibrosis yog ib yam kab mob debilitating hem tib neeg kev noj qab haus huv. Hloov pauv kev loj hlob zoo (TGF)- / P-Smad yog txoj hauv kev tseem ceeb ntawm fibrosis featured nrog epithelial-mesenchymal transformations (EMT) thiab collagen depositions, nrog rau ntau cov pa-dawb radicals. Nrf-2 ua raws li qhov tseem ceeb tiv thaiv oxidative regulator tsav cov kab lus ntawm ntau yam tshuaj tiv thaiv kab mob ntsig txog cov noob. Glutathione peroxidase-4(GPx-4) yog ib tug tswv cuab ntawm tsev neeg glutathione peroxidase uas ncaj qha inhibits phospholipid oxidation kom txo oxidative kev nyuaj siab. Hauv txoj kev tshawb fawb tam sim no, peb tsom mus tshawb nrhiav lub luag haujlwm ntawm tus txha caj qaum hauv nas lub siab fibrosis qauv raug ntxias los ntawm carbon tetrachloride (CCl4) thiab hauv cov kab mob hepatic stellate (LX2 cell line) sib tw nrog TGF- . Obacunone tau pom tias muaj zog ameliorative cuam tshuam rau daim siab fibrosis ob qho tib si nyob rau hauv activated LX2 thiab nyob rau hauv nas daim siab cov ntaub so ntswg nrog txo qib ntawm -SMA, collagen1, thiab vimentin. Obacunone kuj zoo kawg nkaus suppressed TGF- / P-Smad cov cim thiab cov txheej txheem EMT. Lub caij no, obacunone tau siv lub zog los tiv thaiv oxidation los ntawm kev txo cov qib ntawm cov pa oxygen reactive (ROS) hauv ob qho tib si qauv. Cov nyhuv antioxidant ntawm cov pob txha caj qaum tau raug ntaus nqi los ntawm kev ua kom GPx-4 thiab Nrf-2. Tsis tas li ntawd, qhov kev kho mob ntawm pob txha caj qaum ntawm LX2 hlwb tau raug tshem tawm hauv vitro ntxiv nrog GPx-4 antagonist RSL3, nyob rau tib lub sijhawm nrog cov qib siab ntawm ROS.puritans vitamin cYog li, peb pom tau hais tias cov pob txha muaj peev xwm ua rau lub siab mob plab los ntawm kev txhim kho GPx-4 teeb liab thiab inhibition ntawm TGF- /P-Smad txoj kev thiab EMT txheej txheem.

Ntsiab lus:pob txha; daim siab fibrosis; EMT; kab mob siab stellate hlwb; GPx-4;TGF- ; oxidative stress

Thov nias ntawm no kom paub ntxiv

1. Taw qhia

Cov kab mob siab ntev feem ntau yog tshwm sim los ntawm tus kab mob siab kab mob, tsis muaj cawv steatosis, cawv kab mob siab, thiab kab mob siab autoimmune. Kev raug mob siab tas mus li yuav xaus rau hauv daim siab fibrosis, lossis txawm tias mob qog noj ntshav, uas cuam tshuam rau tib neeg kev noj qab haus huv [1]. Lub siab fibrosis yog ib hom kab mob uas tsis kho cov lus teb nyob rau hauv qhov kev raug mob tsis tu ncua uas tshwm sim los ntawm exaggerated tsub zuj zuj ntawm extracellular matrix (ECM) thiab caws pliav tsim [2]. Hepatic stellate cells (HSCs) feem ntau yog nyob rau hauv ib qho chaw so nyob rau hauv ib txwm muaj mob, lawv tau los ua aberrantly activated nyob rau hauv lub siab mob ntev thiab featured nrog siab proliferation thiab secretions ntawm ECM [3], denoted li myofibroblasts [4]. Nkag siab txog cov txheej txheem ntawm HSC ua kom muaj txiaj ntsig zoo rau kev txhim kho cov tshuaj kho tshiab rau kev kho mob siab fibrosis.

Kev hloov pauv kev loj hlob (TGF-) txhawb cov lus teb inflammatory los ntawm kev txhawb nqa kev qhia ntawm Interleukin-1 (IL-1) thiab Interleukin-6(IL-6) hauv monocytes [5]. Ntxiv mus, nws ua rau mob qog noj ntshav los ntawm kev hloov pauv ntawm epithelial-mesenchymal (EMT) thiab qog lub cev tiv thaiv kab mob [6-8]. EMT hais txog cov txheej txheem physiological dynamic uas nyob rau hauv epithelial hlwb poob lawv polarity thiab hloov mus rau hauv mesenchymal hlwb xws li fibroblasts thiab myofibroblasts, yog li ua rau kev loj hlob ntawm fibrosis [9]. Qhov tshwm sim ntawm EMT yog ib txwm cuam tshuam nrog kev txo qis ntawm E-cadherin thiab kev txhim kho ntawm mesenchymal cell markers, xws li N-cadherin thiab qwj [10]. TGF- -Smad activation yog ib txoj hauv kev tseem ceeb hauv lub cev hauv EMT nrog rau kev tsim myofibroblast [11].

Oxidative stress yog suav hais tias yog ib qho ntawm thawj qhov ua rau fibrosis nrog overproduction ntawm reactive oxygen hom (ROS) [12]. Cov qib siab ntawm ROS ua rau cov qauv kev puas tsuaj rau biomolecules, xws li cov protein, lipids, thiab DNA, thaum kawg ua rau kev raug mob ntawm tes thiab txhawb nqa fibrosis [13]. Nyob rau hauv oxidative kev nyuaj siab, lub dimer ntawm Nrf-2-Keep-1 yog dissociated, nuclear respiratory yam tseem ceeb-2(Nrf-2) yog thauj mus rau hauv lub nucleus, thiab induced qhia ntau yam antioxidant enzymes los ntawm kev khi nrog cov tshuaj tiv thaiv antioxidant (ARE) txo cov qib ROS [14]. Glutathione peroxidase-4 (GPx-4) ​​ua raws li cov tshuaj tiv thaiv antioxidant nrog glutathione peroxidase kev ua haujlwm, feem ntau yog lub luag haujlwm rau inhibition ntawm phospholipid oxidation thiab ROS ntau lawm hauv cov txheej txheem ferroptosis [15].sibCov kev tshawb fawb nas tau pom tias fibrosis hnyav dua tom qab poob ntawm Nrf-2 lossis GPx-4 [16,17].

Cistanche tuaj yeem tiv thaiv kev laus

Tam sim no, muaj ntau yam tshuaj muaj rau kev kho mob ntawm daim siab fibrosis, nrog rau cov uas muaj cov khoom ntawm kev tiv thaiv kab mob [18], antioxidant [19], thiab peroxisome proliferator-activated receptors (PPAR) agonist [20]. Nyob rau hauv ib txoj kev tshawb no tsis ntev los no, Prof. Yu pom tias cov neeg mob uas muaj daim siab cirrhosis muaj ntau dua lipid oxidation theem nyob rau hauv kev sib piv rau cov kev noj qab haus huv tswj nyob rau hauv cov ntaub ntawv kuaj mob [21]. Ntau cov tshuaj antioxidant xws li resveratrol [19] thiab ursolic acid|13] tau pom tias muaj txiaj ntsig zoo ntawm fibrosis. Cov ntaub ntawv pov thawj no qhia tau hais tias kev tiv thaiv oxidation yog ib lub tswv yim zoo rau kev kho cov kab mob fibrosis.

Pirfenidone (PFD) yog ib qho tshuaj pyridine rau idiopathic pulmonary fibrosis (IPF), thaum nws tseem ua rau muaj txiaj ntsig zoo ntawm daim siab fibrosis hauv ntau qhov kev tshawb fawb [22,23]. Yog li, PFD raug xaiv los ua cov tshuaj tswj tau zoo hauv txoj kev tshawb no. Obacunone (Oba) yog lub ntuj limonoid compound muab rho tawm los ntawm cov nroj tsuag rutaceous xws li Phellodendri thiab Dictamnus-dasycarpus [24], uas muaj ntau yam khoom lom xws li tshuaj tiv thaiv kab mob |25, antioxidation [26], thiab anti-tumor sawv daws yuav [27]. Cov ntawv ceeb toom tsis ntev los no tau tshaj tawm tias Oba yog tus muaj zog Nrf-2 agonist, uas ua rau lub ntsws fibrosis zoo los ntawm CCL4 induction thiab oxidative puas rau MDA-MB-231 hlwb tshwm sim los ntawm H, O2. Qhov tseem ceeb tshaj, Oba tsis muaj tshuaj lom rau nas [28]. Txawm li cas los xij, tsis muaj lus ceeb toom txog qhov cuam tshuam ntawm Oba hauv daim siab fibrosis tau tshaj tawm, tshwj xeeb tshaj yog ntawm GPx-4 kho kom haum rau cov tshuaj tiv thaiv phospholipid oxidation, TGF- -Smad signals, thiab cov txheej txheem EMT. Ntawm no, peb npaj siab los tshawb txog cov teebmeem ntawm Oba ntawm daim siab fibrosis thiab txhais cov txheej txheem molecular hauv qab.

2. Cov txiaj ntsig

2.1. Oba Inhibited TGF- -Induced Lx-2 Activation

Since the activated HSCs (aHSCs)played a critical role in liver fibrosis [3], we first observed the effects of Oba on liver fibrosis in aHSCs. In this experiment, we used the Cell Counting Kit-8(CCK8) to detect the toxicity of Oba on LX-2 cells, a type of human HSCs line. We found that LX-2 cells survival was not significantly affected by Oba at the concentration range of 10 to 160 uM during 24/48/72 h treatment (Figure 1A). Then, the LX-2 cells were stimulated with 10 ng/mL TGF-β for 24 h and then treated with Oba or not. the protein expression of α-Smooth muscle actin (α-SMA) was effectively inhibited at concentrations of >20 awm. Interestingly, nrog kev nce concentration ntawm Oba, a-SMA inhibition nyhuv tsis tau nce ntxiv (Daim duab 1B).cistanche yog dab tsiYog li, peb xaiv 20 μM concentration rau cov kev sim hauv qab no, nrog PFDserving raws li kev tswj hwm zoo. Oba tau zoo inhibited cov protein ntau ntawm -SMA, collagen-1, vimentin, thiab cov ntaub so ntswg loj hlob (CTGF) (Daim duab 1C), hauv aHSCs[3,4]. Tsis tas li ntawd, Oba txo qhov mRNA qhia theem ntawm -SMA, collagen-1, thiab vimentin (Daim duab 1D). Cov txiaj ntsig ntawm immunofluorescence tau tshawb xyuas ntxiv tias qhov zoo staining cov cim ntawm -SMA tau pom tseeb txo qis los ntawm Oba (xws li hauv TGF- pawg, raws li cov xov tooj liab suav txo tom qab kho nrog Oba) hauv Lx-2(Daim duab 1E). Cov txiaj ntsig no qhia tias Oba ua rau muaj kev cuam tshuam zoo ntawm Lx-2 ua kom.

2.2.Oba Inhibited TGF- /Smad Signals, EMT txheej txheem, thiab Exerted Anti-Inflammatory Effect

Txhawm rau txheeb xyuas cov txheej txheem molecular hauv qab cov teebmeem saum toj no los tiv thaiv fibrosis, cov qib protein qhia ntawm TGF-Receptor, P-Smad, thiab -SMA tau pom, uas tau cuam tshuam loj heev los ntawm Oba hauv aHSCs (Daim duab 2A). Tsis tas li ntawd, cov protein. qhia theem ntawm N-cadherin thiab qwj tau txo qis, thaum cov protein qhia theem ntawm E-cadherin tau nce qib (Daim duab 2B), qhia tias txoj hauv kev EMT raug thaiv ib yam nkaus [3]. Ntxiv rau, peb pom tias Oba ua rau txo qis ntawm IL-6(Daim duab 2C).Anti aging cistancheTxhawm rau soj ntsuam ntxiv cov txiaj ntsig ntawm Oba ntawm qhov mob, peb thawj zaug ua rau HepG-2 hlwb (ib hom tib neeg hepatocellular carcinoma cell kab) nrog 20 nmol CCl4, ua raws li kev kho mob nrog 20 uM Oba. Cov txiaj ntsig no tau pom tias Oba tuaj yeem txo cov mRNA thiab cov protein qhia theem ntawm IL-6 hauv CCl4-stimulated HepG-2(Daim duab 2D).Ntawm tag nrho, oba tau nthuav tawm ntau yam kev tiv thaiv kab mob. los ntawm inhibiting TGF- / Smad signals, thiab EMT txheej txheem, nrog rau cov tshuaj tiv thaiv.

2.3. Cov teebmeem Anti-Fibrosis ntawm Oba yog nyob ntawm kev txhim kho Antioxidant muaj peev xwm los ntawm kev ua kom GPXx-4

In order to further define the underlying molecular mechanisms of Oba in aHSCs, we discovered that Oba activated the Nrf-2/Keap-1 pathway and reduced the protein expressions of Nox-2 (Figure 3A).In addition, we found that Oba increased the protein expression level of GPx-4(Figure 3B). To validate the importance of the GPx-4-mediated signal, RSL3, which is an inhibitor of GPx-4 [29], was selected to deal with Lx-2. Examination by using the CCK8 （Figure 3C）revealed that at concentrations of RSL3>{{0}}.75 μM, cell viability ntawm Lx-2 tau poob qis; Yog li ntawd, 0.75-μM RLS raug xaiv los cuam tshuam rau hauv kev qhia ntawm GPx-4.Cov txiaj ntsig ntawm Western blot pom tau tias thaum RSL3 tau ntxiv rau hauv aHSCs, GPx-4 cov lus qhia protein raug txo thiab cov cim profibrotic suav nrog -SMA thiab vimentin tau thim rov qab tag nrho, qhia qhov tseem ceeb ntawm GPx-4 hauv kev kho cov teebmeem kev tiv thaiv fibrosis ntawm Oba (Daim duab 3D). Lub caij no, Oba pom tseeb txo qis ROS qib ntawm aHSCs los ntawm ROS fluorescent detectors, thiab cov nyhuv no tau pom meej thaiv los ntawm RSL3 (Daim duab).Cov ntaub ntawv no tau qhia tias qis phospholipid ROS theem kho los ntawm GPx-4 yog lub ntsiab tiv thaiv fibrosis. signal of Ob.

2.4. Oba Attenuates CCl4-Induced Mouse Liver Fibrosis

CCl4-induced siab fibrosis yog ib qho ntawm cov qauv nas uas nquag siv los kawm txog daim siab fibrosis [3]. Nyob rau theem histological, Oba txo qis hauv daim siab pathology, ua pov thawj los ntawm ameliorated collagen depositions qhia nrog Masson staining thiab anti-SMA staining. (Daim duab 4A-D). Tom qab ntawd, qib ntshav ntawm alanine transaminase (ALT) / aspartate transaminase (AST) tau txiav txim siab los ntsuas qhov kev puas tsuaj ntawm daim siab, piv rau cov nas CCL4, cov koob tshuaj tsawg Oba pawg thiab PFD pawg tau txo qis ALT qib (p.<0.05)(figure 4e).="" expectedly,="" oba="" inhibited="" the="" protein="" expression="" levels="" of="" collagen-1,="" ctgf,="" and="" α-sma="" in="" mouse="" fibrotic="" livers,="" which="" were="" consistent="" with="" the="" immunohistologic="" results="" (figure="" 4f).="" these="" in-vivo="" results="" illustrated="" that="" oba="" significantly="" improved="" the="" ccl4-induced="" mouse="" liver="">

2.5. Oba txhim kho qhov kev nthuav qhia ntawm GPx-4, Txo qhov mob, EMT-Related Protein Expressions, thiab Txo cov Lipid Oxidation Levels hauv CCl4-Induced Liver Fibrosis

Raws li pom nyob rau hauv daim duab 5A, Oba inhibited cov protein qhia ntawm IL-6 thiab qhov tshwm sim ntawm EMT nyob rau hauv nas fibrotic siab nrog txo qis cov qwj thiab N-cadherin tab sis ntau dua E-cadherin. Tsis tas li ntawd, cov lus qhia protein ntawm Nrf-2 thiab GPx-4 tau pom tseeb tau nce ntxiv tom qab kev kho mob nrog cov koob tshuaj nruab nrab thiab siab ntawm Oba (Daim duab 5B).cistanche cov txiaj ntsigImmunohistochemical Staining (IHC) cov txiaj ntsig tau pom tias piv nrog cov qauv pab pawg, cov lej ntawm GPx-4 cov hlwb zoo (xim av) hauv cov pab pawg siab- thiab nruab nrab-dose Oba tau nce ntau (Daim duab 5C, E). Peb tom ntej no tau sim cov theem ntawm oxidative kev nyuaj siab nyob rau hauv nas lub siab thiab pom tias, piv rau cov tswj, cov qauv pab pawg neeg lub ROS fluorescence siv tau pom meej dua. Ntawm qhov tod tes, theem ntawm ROS tau txo qis tom qab kev kho mob nrog Oba (Daim duab 5D, F).

3. Kev sib tham

Fibrosis yog ib hom kab mob sib kis thoob ntiaj teb. Txawm li cas los xij, suav nrog kev kis tus kab mob, haus dej cawv lossis tsis muaj cawv kab mob siab, thiab cov lus teb autoimmune, txhua yam xaus nrog fibrosis vim mob siab mob siab [1]. Kev ua kom HSCs yog qhov tshwm sim hauv nruab nrab ntawm daim siab fibrosis. HSCs tau qhib nrog kev tsim tawm tsis zoo ntawm ntau yam ntawm fibrogenic yam thiab ECM depositions [2,3]. TGF- -txog canonical Smad activation signaling, EMT txheej txheem nrog rau IL-6- ntsig txog kev mob rhiab tau raug tshaj tawm tias koom nrog cov txheej txheem saum toj no [3,4]. Txhawm rau ntsuas qhov cuam tshuam los tiv thaiv fibrosis ntawm Oba ntawm daim siab fibrosis, ua ntej peb tsom mus rau kev txheeb xyuas nws cov teebmeem ntawm Lx-2 kev ua kom raug ntxias los ntawm TGF-, thiab tom qab ntawd txheeb xyuas nws cov teebmeem hauv vivo los tiv thaiv fibrosis los ntawm kev siv CCl4-induced siab fibrosis nas qauv nyob rau hauv kawg.

Oba yog lub ntuj limonoid compound [24] thiab nws cov teebmeem lom neeg feem ntau suav nrog kev tiv thaiv kab mob [25], antioxidant [26], thiab tiv thaiv qog [27] ua haujlwm. Tsis ntev los no, nws tau pom tias Oba yog ib qho muaj zog thiab muaj txiaj ntsig Nrf-2 agonist thiab tawm dag zog los tiv thaiv pulmonary fibrosis hauv cov nas [28]. Txawm li cas los xij, cov pov thawj tseem tsis muaj nyob rau hauv nws cov tshuaj tiv thaiv kab mob siab fibrosis thiab cuam tshuam txog cov txheej txheem molecular. Ntawm no, cov lus tshaj tawm ntawm -SMA, collagen 1, thiab CTGF txhua tus tau cuam tshuam los ntawm Oba hauv Lx-2 qauv txhawb nqa nrog TGF- . Kev txhim kho EMT teeb liab kuj tseem muaj txiaj ntsig zoo los ntawm Oba.

Nws tau raug lees paub tias oxidative kev nyuaj siab yog ib qho tseem ceeb pab rau fibrosis [12]. Reactive oxygen hom (ROS) yog ib lub kaus rau cov oxidized molecules los yog ions nrog lom lom thiab overproductions ntawm ntau yam ROS nyob rau hauv hepatocytes yuav ua rau nws tuag thiab elicit daim siab fibrosis [13]. Ntawm qhov tsis sib xws, so HSCs tau qhib ua haujlwm tom qab lub siab raug mob ntawm tes thiab ROS ntau dhau ua rau muaj kev ua kom HSCs ntau dua thiab xaus nrog daim siab fibrosis [12]. Yog li, dampening lub exaggerated oxidative kev nyuaj siab dhau los ua ib qho kev kho mob zoo rau fibrosis.

Nrf-2 yog ib qho tseem ceeb regulator ntawm anti-oxidative kev nyuaj siab, lub activated phosphorylated Nrf-2 yog translocated rau hauv lub nucleus los tsav lub up-regulation ntawm downstream antioxidant proteins, GST, HO1[15]. Ntau lub ntuj tshuaj activators ntawm Nrf2 tau tshaj tawm tias muaj cov teebmeem los tiv thaiv fibrosis, xws li tert-butylhydroquinone thiab curcumin. Ntawm tag nrho cov anti-oxidant proteins, glutathione peroxidase -4(GPx{11}}) yog ib tug tswv cuab ntawm glutathione peroxidase tsev neeg (GPx1-8) thiab nws pab txhawb lub redox tshuav nyiaj li cas los ntawm kev txo cov glutathione [ 30-32]. Nws tau raug pov thawj tias qhov kev qhia ntau dua ntawm glutathione peroxidase -7 inhibited tsis-alcoholic fatty siab kab mob los ntawm kev txo qis ntawm ROS [33]. GPx-4 txo phospholipid peroxide ntawm lipid membrane los tiv thaiv cov hlwb los ntawm oxidative raug mobJ15]. Kazuya Tsubouchi et al. pom tias qhov kev qhia ntawm GPx-4 txo qis hauv nas lub ntsws fibrosis cov ntaub so ntswg tshwm sim los ntawm bleomycin, thiab fibrosis hnyav dua thaum GPx-4 noob raug tshem tawm. Tsis tas li ntawd, lipid peroxidation thiab TGF- / P-Smad txoj hauv kev tau txhim kho zoo hauv lub ntsws fibroblast thaum GPx -4 gene depleted [17]. Tsis ntev los no, ntau cov pov thawj txhawb nqa tias phospholipid-derived ROS tau koom nrog hauv kev tuag ntawm tes tshwj xeeb, hu ua ferroptosis. GPx-4 inhibitor, RSL3, yog siv los ua ferroptosis inducer ib yam nkaus [21,34].

Hauv peb qhov kev sim, peb pom tias Oba tau siv lub peev xwm antioxidant los ntawm kev tswj hwm Nrf-2 thiab GPx-4 thiab txo qis- tswj Nox-2. Qhov tseem ceeb, cov nyhuv anti-fibrosis ntawm Oba tau ua rau tsis muaj zog, thaum GPx-4 noob tau inhibited los ntawm RSL3, GPx-4 inhibitor. Raws li txoj cai, qhov txo qis ROS qib los ntawm Oba kev kho mob tau pom zoo ib yam nkaus. Cov kev soj ntsuam hauv vitro no tau txhawb siab tias GPx-4-kev kho mob los tiv thaiv phospholipid ROS yog qhov tseem ceeb rau kev tiv thaiv kev tiv thaiv ntawm Oba.

Txhawm rau kom ua tiav cov txiaj ntsig kev kho mob los tiv thaiv fibrosis saum toj no ntawm Oba hauv LX2 hlwb, CCl4-induced nas mob siab fibrosis tau kuaj thiab. CCl4 yog ib hom tshuaj lom hepatotropic, microsomal enzyme P450 kev sib haum xeeb degradation uas ua rau cov dawb radicals reactive heev los rhuav tshem kev ncaj ncees ntawm cov cell membrane, tig mus, ua rau lub siab ua haujlwm tsis zoo thiab raug mob, nws thiaj li hloov mus rau hauv daim siab fibrosis [35]. Hauv peb qhov kev sim, Oba tau nthuav tawm cov txiaj ntsig zoo ntawm CCl-induced siab fibrosis. Nws alleviated lub siab puas tsuaj nrog qis qis ntawm AST, thaj tsam ntawm collagen fiber ntau ua me me, thiab cov protein qhia ntawm a-SMA, CTGF, Collagen-1, qwj, N-cadherin, thiab IL{{10} }} tau txo tag nrho. Thaum lub teeb liab los tiv thaiv oxidant, cov lus qhia protein ntawm Nrf-2, thiab GPx{13}} tau nce siab dhau lawm, qhov siab dua ntawm ROS tsis kam. Cov txiaj ntsig no tau lees paub ntxiv txog qhov cuam tshuam hauv vitro los tiv thaiv fibrosis los ntawm Oba thiab lwm yam teeb liab, tshwj xeeb tshaj yog inhibition ntawm EMT thiab anti-oxidant los ntawm Nrf-2 thiab GPx{17}}.

Hauv cov ntsiab lus, peb xaus lus tias Oba txo TGF- -induced HSCs activation thiab CCl4-induced hepatic fibrosis los ntawm ntau yam txheej txheem. Ua ntej, Oba inhibited qhov classical TGF- /Smad txoj kev, EMT, thiab IL-6 ntau lawm. Qhov thib ob, nws tau nthuav tawm lub peev xwm antioxidant zoo los ntawm kev txhim kho cov lus ntawm Nrf-2 thiab GPx-4. GPx-4kev kho mob los tiv thaiv phospholipid ROS feem ntau ua rau nws cov teebmeem los tiv thaiv fibrosis. Yog li ntawd, Oba tuaj yeem ua tus neeg sib tw tshuaj tua kab mob rau kev tiv thaiv thiab kho fibrosis.

Oba tau qhia qee qhov kev kho mob ntawm CCl 4-induced siab fibrosis hauv C57 nas. Oba tuaj yeem txo qis-tswj cov kev qhia ntawm fibrosis protein biomarkers, xws li: -SMA, CTGF, thiab lwm yam. Meanwhile, Oba txo cov protein qhia ntawm I-6, E-cadherin, thiab lwm yam. Nws yog nthuav tias Mid-Oba qhia pom cov txiaj ntsig zoo dua rau qis-Oba thiab siab-Oba raws li kev txheeb ze ntawm cov proteins no, txawm hais tias, nws tsis yog qhov tseem ceeb. Txawm li cas los xij, nws tau qhia tias qhov kev siv tshuaj zoo tshaj plaws tuaj yeem yog qhov tseem ceeb hauv cov txheej txheem no, thiab peb yuav tshawb txog qhov tshwm sim no hauv kev sim tom ntej. Tsis tas li ntawd, peb pom tias qis ROS thiab qib siab ntawm daim siab fibrosis nrog kev tswj hwm kev qhia ntawm Nrf{10}} thiab GPx{11}}. Yog li, peb ntseeg tias Oba tuaj yeem txo lub siab fibrosis los ntawm kev tswj cov Nrf-2 thiab GPx-4. Txawm li cas los xij, PFD tej zaum yuav muaj kev kho mob zoo dua li Oba hauv vivo thiab vitro. Txawm li cas los xij, Oba tau zoo dua PFD ntawm cov tshuaj tiv thaiv oxidant, nws yog qhov tsim nyog los ua kom muaj peev xwm nrhiav tau cov tshuaj tshiab los tiv thaiv daim siab-fibrogenic nyhuv los ntawm cov tshuaj tiv thaiv oxidant, thiab ua rau me ntsis pab rau tib neeg noj qab haus huv.

4. Cov ntaub ntawv thiab cov txheej txheem

4.1. Tshuaj thiab Reagents

Oba (no.B20553, purity Ntau dua lossis sib npaug li 98 feem pua) thiab RSL3 (no. S81241, purity Ntau dua lossis sib npaug li 98 feem pua) tau yuav los ntawm YUANYE Bio-Technology Co., Ltd.(Shanghai, Tuam Tshoj) thiab yaj. nyob rau hauv dimethyl sulfoxide (DMSO) (no.R21950; YUANYE) mus rau ib tug concentration ntawm 72.64 mg / mL thiab 6 mmol, raws li zoo raws li. Tom qab lim kom tsis muaj menyuam, cov txiaj ntsig tau raug khaws cia ntawm -20 degree C. Cov tshuaj tau ntxiv rau hauv kab lis kev cai nruab nrab kom ua tiav qhov kawg ntawm DMSO concentration ntawm<1%. tgf-β="" was="" purchased="" from="" peprotech="" china="" (suzhou,="" china).="" ccl4="" was="" purchased="" from="" jiangsu="" qiangsheng="" chemical="" (jiangsu,="" china).="" a="" ros="" fluorescence="" probe="" kit="" was="" purchased="" from="" applygen(beijing,="" china).="" trizol,="" sybr="" premix="" ex="" taq="" reagents,="" revertaid="" first="" strand="" cdna="" synthesis="" kit,="" ripa,="" bca="" protein="" assay="" kit,="" and="" sds-page="" kit="" were="" purchased="" from="" thermo="" fisher="" scientific(waltham,="" ma,="" usa).="" the="" cell="" counting="" kit-8="" (cck8)was="" purchased="" from="" saint-bio="" (shanghai="" china).="" the="" primers="" were="" designed="" and="" synthesized="" by="" tsingke="" (xi'an,="" china).dapi,="" goat="" anti-mouse="" igg="" h&l(alexa="" fluor="" 594)(ab150120),="" anti-alpha="" smooth="" muscle="" actin="" antibody(ab7817),="" anti-vimentin="" antibody="" (ab20346),="" anti-il-6="" antibody="" (ab233706),anti-collagen-1="" antibody="" (ab34710),anti-smad3="" (phospho)="" antibody="" (ab52903),="" and="" anti-nox2/gp91phox="" antibody(ab80508)="" were="" purchased="" from="" abcam(cambridge,="" ma,="" usa).="" e-cadherin="" polyclonal="" antibody="" (20874-1-ap),="" n-cadherin="" polyclonal="" antibody="" (22018-1-ap),="" gpx4="" antibody="" (14432-1-ap),β-actin="" polyclonal="" antibody(20536-1-ap),="" nrf2="" polyclonal="" antibody="" (16396-1-ap),="" and="" keap1="" polyclonal="" antibody="" (10503-2-ap)="" were="" purchased="" from="" proteintech="" (rosemount,="" min,="" usa).ctgf="" polyclonal="" antibody(wl02602)="" and="" snail="" polyclonal="" antibody="" (wlo1863)were="" purchased="" from="" wanleibio="" (shenyang,="">

4.2. Cell Cultures thiab Experimental Design

Tib neeg kab mob siab stellate cell kab Lx-2 thiab tib neeg lub siab mob qog noj ntshav kab mob Hepg-2 (txhua yam tau los ntawm Lub Tsev Haujlwm Saib Xyuas Tshuaj, Lub Tsev Kawm Ntawv Qib Siab Fourth Military Medical University, Tuam Tshoj) tau coj mus rau hauv Dulbecco's Modified Eagle Medium ( DMEM) ntxiv nrog 10 feem pua ​​​​fetal bovine serum thiab 1 feem pua ​​​​penicillin (Thermo Fisher Scientific) thiab streptomycin (Thermo Fisher Scientific), thiab tom qab ntawd khaws cia ntawm 37C hauv qhov chaw noo ntawm 5 feem pua ​​​​CO2 thiab 95 feem pua ​​​​ntawm huab cua. Txhawm rau ntsuas qhov cuam tshuam ntawm Oba ntawm Lx-2 cell viability, Lx-2 tau planked nrog 1 × 10 * cells/zoo hauv 96-zoo phaj thiab incubated rau 24 teev ua ntej ntxiv ntawm Oba. Tom qab ntawd, Lx-2 raug kho nrog ntau qhov sib txawv ntawm Oba (10,20, 40,80, thiab 160 wM thiab kab lis kev cai rau 24,48, lossis 72 h. Cell viability raug kuaj los ntawm CCK8. Txhawm rau ntsuas qhov Cov nyhuv ntawm Oba ntawm Lx-2 ua kom, Lx-2 tau ua ntej kev coj noj coj ua nrog qhov nruab nrab tsis muaj ntshav rau 12 h, ua raws li kev txhawb nqa nrog TGF- (10ng / mL, 24 h) thiab kho nrog Oba los yog PFD. Txhawm rau ntsuas qhov cuam tshuam ntawm Oba ntawm qhov mob, Hepg-2 tau txhawb nqa los ntawm CCl4 (20 nm, 8 h) thiab kho nrog 20 μM Oba. Thaum kawg ntawm kab lis kev cai, cov hlwb tau sau thiab raug rau Western blotting thiab real-time qPCR tsom xam.

4.3. Tsiaj txhu thiab kev sim tsim

Txiv neej C57 nas uas hnyav 20± 0.5 g (Lub Laboratory Tsiaj Center ntawm Lub Tsev Kawm Ntawv Qib Siab Fourth Military Medical University) tau nyob hauv qhov tsim nyog ntawm 25 degree ± 2 degree qhov kub thiab txias 12-h lub teeb / tsaus voj voog nrog pub dawb rau dej thiab zaub mov. Txhua qhov kev sim hauv qhov kev tshawb fawb no tau ua tiav raws li cov txheej txheem pom zoo los ntawm Pawg Neeg Saib Xyuas Tub Rog Hauv Tsev Kawm Qib Siab Fourth Military Medical Care. Cov nas c57 tau muab faib ua 6 pawg (n=6 hauv txhua pab pawg) raws li hauv qab no: i) sham tswj pawg, i) pawg qauv, ii) pab pawg siab Oba (CCl4 ntxiv rau 6mg / kg Oba), io Cov pab pawg nruab nrab-Oba (CCl4 ntxiv rau 3 mg / kg Oba), ø) qis-Oba pawg (CCl4 ntxiv rau 1.5 mg / kg Oba), thiab vi) PFD pawg (CCl4＋ PFD). feem pua ​​CCl4/olive oil, 5 mL/kg intraperitoneal txhaj) peb zaug hauv ib lub lis piam rau ib lub sij hawm 6-lub lim tiam kom induce daim siab fibrosis. Lwm pab pawg tau noj nrog sib txawv koob tshuaj Oba los yog PFD thiab sib npaug ntim ntawm CCil4 intraperitoneal kab mob rau ib lub sij hawm 6-lub lim tiam. Ib qho sib npaug ntawm cov roj txiv roj hloov CCl4 tau muab rau C57 los ntawm ipas tswj sham. Txhua qhov kev sim hauv kab lus no tau ua tiav raws li cov txheej txheem pom zoo los ntawm Pawg Neeg Saib Xyuas Tub Rog Tub Rog Hauv Tsev Kawm Ntawv Qib Siab Fourth ntawm Tsiaj. Saib xyuas.

4.4.Western Blotting

C57 daim siab los yog kab mob hlwb tau homogenized los yog lysed nyob rau hauv RIPA tsis los ntawm kev siv cov ntaub so ntswg lapping apparatus los yog cell scraping (Service, KZ-2). Protein concentration tau ntsuas siv BCA Protein Assay Kit. Lub lysate tau centrifuged (4 degree, 12, 000 rpm, 5 min), thiab cov supernatant tau sau. Cov kauj ruam ua haujlwm tau ua raws li tau tshaj tawm yav dhau los [14]. Cov qauv no tau raug soj ntsuam raws li Chemiluminescent Imaging thiab Analysis system (Beijing Sage Creation Science Co, MiniChemi 610).

4.5. Real-Time qPCR Kev Ntsuam Xyuas

C57 daim siab los yog kab lis kev cai tau homogenized los yog lysed hauv Trizol reagent nrog kev pab los ntawm cov ntaub so ntswg lapping apparatus kom rho tag nrho RNA. Tag nrho RNA tau thim rov qab rau hauv cDNA los ntawm kev siv RevertAid First Strand cDNA Synthesis Kit. -actin tau siv rau kev siv sab hauv. RT-qPCR tau ua tiav ntawm 7500 Real-time qPCR System (Applied Biosystems) nrog SYBR Premix Ex Taq reagents. Cov primers ntawm tib neeg cov noob siv tau teev nyob rau hauv Table 1.

Kab lus no yog muab rho tawm los ntawm Molecules 2021, 26, 318. https://doi.org/10.3390/molecules26020318 https://www.mdpi.com/journal/molecules