Abstract
Tumor necrosis factor-α (TNF-α)-driven inflammatory reaction plays a crucial role in the initiation of liver fibrosis. We herein attempted to design genetically engineered adipose-derived stem cells (ASCs) producing etanercept (a potent TNF-α inhibitor), and to determine the anti-fibrotic potential of the secretome released from the etanercept-synthesizing ASCs (etanercept-secretome). First, we generated the etanercept-synthesizing ASCs by transfecting the ASCs with mini-circle plasmids containing the gene insert encoding for etanercept. We subsequently collected the secretory material released from the etanercept-synthesizing ASCs and determined its anti-fibrotic effects both in vitro (in thioacetamide [TAA]-treated AML12 and LX2 cells) and in vivo (in TAA-treated mice) models of liver fibrosis. We observed that while etanercept-secretome increased the viability of the TAA-treated AML12 hepatocytes (p = 0.021), it significantly decreased the viability of the TAA-treated LX2 HSCs (p = 0.021). In the liver of mice with liver fibrosis, intravenous administration of the etanercept-secretome induced significant reduction in the expression of both fibrosis-related and inflammation-related markers compared to the control group (all Ps < 0.05). The etanercept-secretome group also showed significantly lower serum levels of liver enzymes as well as pro-inflammatory cytokines, such as TNF-α (p = 0.020) and IL-6 (p = 0.021). Histological examination of the liver showed the highest reduction in the degree of fibrosis in the entanercept-secretome group (p = 0.006). Our results suggest that the administration of etanercept-secretome improves liver fibrosis by inhibiting TNF-α-driven inflammation in the mice with liver fibrosis. Thus, blocking TNF-α-driven inflammation at the appropriate stage of liver fibrosis could be an efficient strategy to prevent fibrosis.
Highlights
Liver fibrosis is characterized by cell death of hepatocytes, hepatic inflammation, and activation of hepatic stellate cells (HSCs)
We obtained the secretome from these etanercept-adipose-derived stem cells (ASCs) after a series of processes, including centrifugation and filtering, all of which are mentioned in the methods section
Control secretome refers to the secretome obtained from empty vector-transfected ASCs, and etanercept-secretome refers to the secretome obtained from etanercept-synthesizing ASCs
Summary
Liver fibrosis is characterized by cell death of hepatocytes, hepatic inflammation, and activation of hepatic stellate cells (HSCs). Tumor necrosis factor-α (TNF-α) is one of the predominant cytokines involved in all these steps leading to liver fibrosis It is produced by the activated macrophages and other liver cells in response to liver injuries. Numerous studies have shown that the stem-cell secretome has similar therapeutic potential as stem cells because the main working mechanism of stem cells is secretome-mediated [23,24,25,26,27,28,29,30] In this experiment, we expected that etanercept-synthesizing ASCs would release a variety of anti-inflammatory and immunomodulatory materials along with etanercept, the sum of which could exert the strengthened anti-fibrotic potential during the process of liver fibrosis. Aprlaasbminiodseentcreoadtimngenfot rtoettahneeprcaerpent.taAlrpalbaisnmosied trreesautmltseinnt tthoethreelepaasreenotfaml pinlai-scmiricdlerepsluasltms iidnsthenecroedleiansge foofr etanemrcienpi-tc. ir(cSloeurce frpolmasmhtitdpss://wwewn.bcoiodciantg.com/gefonromics/celtoannienrgce-pkitt.s/mini(cSiroculerc-edna-vecfrtoomrthetcthpnso:/l/owgwy-wno.bnio-icnatte.gcoramti/vgee-nsoumstiacisn/ecdlo-nexinpgr-eksistiso/nm).in(iDci)rcElffe-edctnsao-vf ethcteoer-ttaencehrnceoplotg-sye-cnroetno-mineteognratthiveec-eslul vsitaabinileitdy-eoxfpArMesLsi1o2nh).ep(aDto) cyEtfefse.ctTsheoifndthiveideutaalnseerccreeptot-mseectrreetaotmmeenotsn, esthpeecicaelllyl evtaianbeirlciteypt-osfecrAeMtomL1e2, ihnedpuacteodcystiegsn.ifiTchanetlyinhdiigvhideur aclellsveciarbetiolimtyeoftAreMatmL1e2nhtse,paetsopceyctieasll2y4 hetaanfteerrcterpeta-tsmecernett.om(Ee),Effinedctuscoedf estiagnneifricceapntt-lsyecrheitgohmere ocneltlhevicaebllilvitiyaboilfityAoMf LLX122 HheSpCast.oEctyatneserc2e4pth-seacfrteetromtreeasitgmneifinct.an(tEly) dEefcferectasseodf tehteanceerllcevpiat-bsielcitryetoofmTeAoAn-ttrheeacteedll vLiXa2biclietlylso,feLspXe2cHiaSllCys4. 8Ethanaeftrecreptrte-saetmcreetnotm. eVasligunesifiacraenptlryesdeenctreedasaesd mtheeance±ll svtaianbdilairtdy dofevTiAatAio-ntroefattehdreLeXin2dceeplelsn,deesnpteecixaplleyri4m8ehntsa.ftAerbbtrreeavtimateionnt.s:VCaltu, ceosnatrreolp; rCeSs,ecnotendtroals smeceraento±mseta. nEdSa,redtadneevricaetpiotn-seocfrtehtroeme ein; dTeApAen, tdheinotaceextpaemriimdee;nHts.SAC,bhberepvaitaictiostnesl:laCtte, cceolnl;trmolc;ECtaSn, ceorcnetprot,l esteacnreertcoempte-.eEncSo, deitnagnemrcienpicti-rscelcerDetNomAe; ;mTcAMAo,ckth, imoainciectiarmcleidDeN; HASwCi,thhoeputateinccsotdelilnagteetcaenlle;rmcecpEt;tapnpeMrcoecpkt,, peatarennetrecreapltp-elanscmodidinwg itmhoinuitceirnccleodDinNg Aet;anmerccMepotc;kT,NmF-iαni,ctiurcmleorDnNecAroswisiftahcotuotr-αe.ncoding etanercept; ppMock, parenteral plasmid without encoding etanercept; TNF-α, tumor necrosis factor- α
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