food & nutrition reserch ORIGINAL ARTICLE Protective effect of oil from Cornus wilsonin fruits ginst crbon tetrchloride-induced heptic fibrosis in mice Qing Liu 1, Qing Liu 2,3, Xiohu Lei 1, Zhenyu Co 1, Ju Zhng 1, To Kung 1, Guoxing Liu 1, Yu Fng 1, Ke Qin 1, Jie Fu 1, Huihui Du 1, Likun Yn 1, Zhihong Xio 3, Chngzhu Li 3, Xundi Xu 1 * 1 Hunn Provincil Key Lbortory of Heptobiliry Disese Reserch & Division of Hepto-Biliry-Pncretic Surgery, Deprtment of Surgery, The Second Xingy Hospitl, Centrl South University, Chngsh, Chin; 2 College of Life Sciences nd Technology, Centrl South University of Forestry nd Technology, Chngsh, Chin; 3 Hunn Provincil key Lbortory of Oils & Fts Moleculr Structure nd Function, Hunn Acdemy of Forestry, Chngsh, Chin Populr scientific summry We evluted the heptoprotective effect of oil from Cornus Wilsonin Fruits. CWO protected the liver from dmge cused by vi ntioxidnt nd nti-inflmmtory effects. We found tht CWO cn ttenute TGF-β1/Smd3 signling pthwy in liver, thereby reducing -induced heptic fibrosis in mice. Our work is expected to promote CWO s n edible oil for liver protection. Abstrct Bckground: Cornus wilsonin Wnger is widely distributed woody oil plnt in south Chin; oil extrcted from its fruits hs been the min source of edible oil for locl residents for hundreds of yers. Previous studies hve demonstrted tht Cornus wilsonin oil (CWO) hs hypolipidemic ctivity in rts. However, the heptoprotective effects of CWO nd their underlying mechnisms re not cler. Objective: The purpose of this study ws to explore the protective effects nd mechnisms of the CWO ginst crbon tetrchloride ( )-induced heptic fibrosis in mice. Methods: Heptic fibrosis mouse model ws induced by intrperitonel injection with 1 ml/kg (mixed 1:4 in olive oil) twice week for 6 weeks. In the mentime, the mice were orlly dministrted with CWO (0.5, 2 ml/kg) once dily for 6 weeks. Serologicl chnges s well s oxidtive stress, inflmmtory, nd histologicl ltertion in the liver were determined. Results: The results showed tht CWO significntly ttenuted -induced serologicl chnges in mice, s ssessed by serum mrkers, including lnine minotrnsferse (ALT), sprtte minotrnsferse (AST), procollgen III, collgen type IV, hyluronic cid, nd lminin. At the sme time, CWO significntly improved -induced liver histologicl chnges, s detected by hemtoxylin nd eosin (H&E), Sirius red, nd Msson s trichrome stining. In ddition, tretment with CWO reduced oxidtive stress nd inflmmtion in the liver. Furthermore, CWO lso reduced the expression of extrcellulr mtrix (ECM) in liver induced by, nd TGF-β1/Smd3 signling my be involved in the process. Conclusions: CWO meliortes -induced heptic fibrosis by ttenuting heptic oxidtive stress, reducing heptic inflmmtion nd inhibiting TGF-β1/Smd3 signling pthwy in liver. CWO my be potentilly beneficil edible oil for the djuvnt tretment of heptic fibrosis. Keywords: Cornus wilsonin oil; heptic fibrosis; oxidtive stress; inflmmtion; TGF-b1 To ccess the supplementry mteril, plese visit the rticle lnding pge Received: 15 December 2019; Revised: 4 Mrch 2020; Accepted; 23 Mrch 2020; Published: 8 My 2020 Food & Nutrition Reserch 2020. 2020 Qing Liu et l. This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License (http:// cretivecommons.org/licenses/by/4.0/), llowing third prties to copy nd redistribute the mteril in ny medium or formt nd to remix, trnsform, nd build upon the mteril for ny purpose, even commercilly, provided the originl work is properly cited nd sttes its license. 1
Qing Liu et l. Heptic fibrosis is chronic disese cused by liver injury, nd its occurrence nd development re complex pthologicl processes involving multiple fctors (1 4). Long-term chronic liver injury stimultes heptocyte deth nd regenertion, ggrvtes heptic oxidtive stress nd chronic inflmmtion, ctivtes heptic stellte cells (HSCs), nd ultimtely leds to the deposition of extrcellulr mtrix (ECM), including α-smooth muscle ctin (α-sma) nd type I collgen (COL1A1) (1, 5 6). Activtion of HSCs is key event in the development of heptic fibrosis. Due to chronic liver dmge, HSCs cn be ctivted, thereby ggrvting heptic fibrosis (7). In ddition, berrnt ctivity of TGF-β1/Smd3 signling pthwy is one of the most prominent drivers for the ctivtion nd trnsformtion of HSCs into myofibroblsts (8). Recently, mny studies hve shown tht mny nturl substnces hve ntifibrotic nd liver protective effects (9 11). Cornus wilsonin Wnger is widely distributed woody oil plnt in south Chin, which hs high fruit yield with high oil content. Becuse Cornus wilsonin oil (CWO) contins mny beneficil chemicls, such s unsturted ftty cids (UFAs), phytosterol, lph tocopherol, nd squlene, it hs been the min source of edible oil for locl residents for hundreds of yers. Previous studies hve lso demonstrted tht CWO hs hypolipidemic ctivity in rts due to its high content of UFAs (12). However, it is still not cler whether CWO cn llevite heptic fibrosis. Here, we first investigted the role of CWO in heptic fibrosis nd explored its possible mechnisms. In the present study, we estblished crbon tetrchloride ( )-induced heptic fibrosis model. To study the role of CWO in heptic fibrosis, we treted model mice with different doses of CWO. We found tht CWO cn significntly improve liver histopthology nd serologicl mrker levels, ultimtely ttenuting the degree of heptic fibrosis induced by. The mechnism my be tht CWO cn improve heptic oxidtive stress, improve heptic inflmmtion, nd inhibit TGF-β1/Smd3 signling pthwy in the liver. Tken together, our studies demonstrted tht CWO plys n importnt role in countercting the development of -induced heptic fibrosis, suggesting tht CWO might hve the potentil to llevite heptic fibrosis. Mterils nd methods Cornus wilsonin oil CWO, supplied by the Hunn Acdemy of Forestry (Chngsh, Chin), ws obtined by subcriticl extrction. The ftty cid composition of CWO ws determined by gs chromtogrphy mss spectrometer (GC/ MS). The phytosterol content of CWO ws determined by spectrophotometry. The vitmin E nd squlene content of CWO were determined by high-performnce liquid chromtogrphy. Regents (C112040) nd olive oil (O108686) were purchsed from Alddin (Alddin, Shnghi, Chin). Hemtoxylin nd eosin (H&E) stining kit (G1120), Sirius red stining kit (G1471), nd Msson s trichrome stining kit (G1340) were ll purchsed from Solrbio (Solrbio, Beijing, Chin). TRIzol regent ws purchsed from Thermo (Thermo Scientific, MA, USA). Antibodies ginst β-ctin (60008-1-AP) nd F4/80 (28463-1-AP) were purchsed from Proteintech (Proteintech, Rosemont, IL, USA). Antibodies ginst α-sma (b32575), TGF-β1 (b215715), nd p-smd3 (b52903) were purchsed from Abcm (Abcm, Cmbridge, UK). Antibodies ginst COL1A1 (BA0325) were purchsed from Boster (Boster, Beijing, Chin). A Universl Two-Step Test Kit (PV-9000) ws purchsed from Zsbio (Zsbio, Beijing, Chin). Totl superoxide dismutse (SOD) (A001-1), mlondildehyde (MDA) (A003-1), nd reduced glutthione (GSH) (A006-1) test kits were obtined from the Nnjing Jincheng Biotechnology Institute (Nnjing, Chin). Animls nd tretment C57BL/6J mice were obtined from the Jckson Lbortory (JAX, ME, USA). All mice were housed in well-ventilted sterile cges nd provided with food nd wter d libitum. After 1 week of dpttion, 24 mice were rndomly divided into four groups: norml control (NC) group tht received olive oil, model group treted with, low-dose group (LDG) treted with + CWO (0.5 ml/kg, IG), nd high-dose group (HDG) treted with +CWO (2 ml/kg, IG). When the mice were 6 weeks old, ws dissolved in olive oil to form 20% solution nd injected intrperitonelly into mice twice week t dose of 5 ml/kg; the NC group ws injected with the sme mount of olive oil. The use of CWO ws orlly dministered dily ccording to the corresponding dose. After 6 weeks of continuous dministrtion, the mice were scrificed 48 h fter the lst injection without overnight fsting. At scrifice, mice were nesthetized with diethyl ether; blood nd liver smples were collected. Blood smples were centrifuged t 4,000 rpm for 20 min t 4 C, nd the superntnt ws tken for biochemicl nlysis. The liver ws dissected nd wshed with ice-cold sline. After weighing, the left outer lobe ws immersed in 4% prformldehyde, nd the remining liver tissue ws immeditely frozen in liquid nitrogen nd finlly trnsferred to -80 C for long-term storge. The niml experiments were pproved by the Animl Cre nd Use Committee of Centrl South University. 2
Protective effect of oil from Cornus wilsonin fruits Biochemicl nlysis Serum ws collected s mentioned bove. We mesured the serum concentrtions of lnine minotrnsferse (ALT) nd sprtte minotrnsferse (AST) using utomtic biochemicl nlyzers (Abbott, Chicgo, IL, USA) nd then detected the serum levels of procollgen III (PC III), collgen type IV (IV-C), hyluronic cid (HA), nd lminin (LN) with rdioimmunossy kits (Yunde Bio-Medicine, Beijing, Chin). To evlute oxidtive stress, we used commercil kits to detect SOD, GSH, nd MDA levels in liver homogentes ccording to the mnufcturer s protocols. Histopthology Liver tissues fixed in 4% prformldehyde were embedded in prffin nd then cut into 4-µm-thick sections. To quntify the heptic fibrosis re, these sections were prepred for stining with H&E, Sirius red, nd Msson s trichrome. For Sirius red collgen stining, thin sections were deprffinized ccording to stndrd procedures nd stined with Sirius red stining solution for 1 h t room temperture. After wshing with wter, the sections were grdully dehydrted in ethnol nd then seled with neutrl blsm. For Msson s trichrome stining, the sections were routinely deprffinized nd hydrted nd then stined in steps using Msson s trichrome kit. The severity of heptic fibrosis ws ssessed under light microscopy (Olympus, Hmburg, Germny). Imges were obtined of rndom fields, nd representtive views of liver sections re shown. Immunohistochemistry The expression of α-sma, COL1A1, nd F4/80 in the liver ws observed by immunohistochemicl stining, s described below. Briefly, the tissue sections were deprffinized nd hydrted, incubted with 0.3% hydrogen peroxide to block endogenous peroxidse, subjected to ntigen repir with citrte t high pressure, nd finlly incubted t 37 C with α-sma (1:500 dilution), COL1A1 (1:500 dilution), nd F4/80 (1:1000 dilution) ntibodies for 1 h. After the sections were incubted with the primry ntibodies, they were processed using Universl Two-Step Test Kit. Therefter, the sections were counterstined with hemtoxylin nd seled with neutrl blsm. Rel-Time Quntittive Polymerse Chin Rection (RT-qPCR) Approximtely 20 mg of liver tissue ws homogenized in 1 ml of TRIzol regent. Totl RNA ws extrcted ccording to the mnufcturer s instructions. The entire experimentl procedure ws crried out ccording to the instructions; mrna expression fold chnges were nlysis by the reltive quntifiction method (2 ΔΔCt ). The following primer pirs were used: IL-6, (forwrd) 5 -GACTTCCATCCAGTTGCCTT-3 nd (reverse) 5 -ATGTGTAATTAAGCCTCCGACT-3 ; IL-1β, (forwrd) 5 -TGAAATGCCACCTTTTGACAGT-3 nd (reverse) 5 -TTCTCCACAGCCACAATGAGT-3 ; TNFα, (forwrd) 5 -AGCACAGAAAGCATGATCCG-3 nd (reverse) 5 -CACCCCGAAGTTCAGTAGACA-3 ; MCP-1, (forwrd) 5 -AGCAGCAGGTGTCCCAAA-3 nd (reverse) 5 -CTGAAGACCTTAGGGCAGAT-3 ; β-ctin, (forwrd) 5 -ACATCCGTAAAGACCTCTATGCC-3, nd (reverse) 5 -TACTCCTGCTTGCTGATCCAC-3. Western blotting Liver tissue totl protein ws extrcted with rdioimmunoprecipittion ssy (RIPA) buffer, nd the concentrtion ws determined by the Bicinchoninic Acid (BCA) method. Thirty microgrms of liver protein lyste ws electrophoresed on 10% sodium dodecyl sulphte polycrylmide gel electrophoresis (SDS-PAGE). The seprted proteins were trnsferred onto polyvinylidene fluoride (PVDF) membrnes, which were blocked with 5% skim milk powder for 1 h t room temperture. Then, the membrnes were incubted overnight t 4 C with α-sma (1:1,000 dilutions), COL1A1 (1:500 dilutions), TGF-β1 (1:1,000 dilutions), nd p-smd3 (1:2,000 dilutions) primry ntibodies. β-actin (1:5,000 dilution) ws used s control. After wshing with TBST, the membrnes were incubted with secondry ntibodies (1:5,000 dilution) for 1 h t room temperture, then detected with enhnced chemiluminescence system. Imges were cptured, nd Imge J softwre ws used to nlyze the grey vlues of the trget protein bnds. Sttisticl nlyses The dt re expressed s the men ± stndrd devition (SD) nd were nlyzed using SPSS 19.0 softwre. Except pthologicl dt, ll other experimentl dt were nlyzed by one-wy nlysis of vrince (ANOVA) followed by Tukey multiple comprison test. The pthologicl dt of the liver were nlyzed by the Kruskl Wllis nonprmetric test, followed by Mnn Whitney U-test. P < 0.05 ws tken to indicte tht difference ws sttisticlly significnt. Histogrms were generted using GrphPd Prism 7 softwre (Sn Diego, CA, USA). Results Chemicl composition nd physicochemicl properties of CWO CWO ws qulittively nd quntittively chrcterized by GC/MS, reveling totl of six mjor chemicl constituents; the results of GC/MS nlysis of ftty cid methyl esters re shown in Fig. 1 nd Tble 1. In ddition to ftty cid components, we lso found some other 3
Qing Liu et l. Fig. 1. Ftty cid composition of CWO. The ftty cid composition of CWO ws detected by gs chromtogrphy-mss spectrometer. Tble 1. Reltive content of ftty cids in Cornus wilsonin oil (CWO) No. Ftty cid Reltive % 1 C16:0 (Plmitic) 15.48 2 C16:1 (Plmitoleic) 1.10 3 C18:0 (Steric) 1.89 4 C18:1 (Oleic) 35.71 5 C18:2 (Linoleic) 44.49 6 C18:3 (Linolenic) 1.32 Sturted ftty cids 17.37 Monounsturted ftty cids 36.81 Polyunsturted ftty cids 45.81 Tble 2. Other beneficil fctors in Cornus wilsonin oil (CWO) No. Compound Content 1 Phytosterol 1.98 mg/g 2 Vitmin E (lph tocopherol) 0.60556 mg/g 3 Squlene 0.0324 g/kg beneficil fctors in CWO, including phytosterols, vitmin E (lph tocopherol), nd squlene (Tble 2). The physicochemicl properties of CWO (cid vlue, 5.31 mg KOH/g; iodine vlue, 104.2 g/100 g; sponifiction vlue, 200.89 mg KOH/g) re similr with other edible oils, such s penut oil nd soyben oil. CWO ttenuted -induced liver injury To test the effect of CWO on heptic fibrosis, we used -induced heptic fibrosis model in this study. The -induced mouse heptic fibrosis model lrgely mimicked humn heptic fibrosis. Gross exmintion showed tht pthologicl chnges, such s smll nodules on the surfce of the liver, occurred in the livers of mice injected with compred with those of mice injected with olive oil, but CWO tretment ttenuted pthologicl chnges in the liver. As shown in Fig. 2, H&E stining showed tht the model group hd pseudolobule formtion in the liver nd inflmmtory cell infiltrtion in the portl re, while CWO reduced inflmmtory cell infiltrtion nd pseudolobule formtion, especilly in the HDG. The liver/body weight rtio of the model group ws significntly (P < 0.01) higher thn tht of the NC group; however, CWO could significntly (P < 0.05) reduce the rtio in HDG (Fig. 2b nd Supplementry Tble S1). ALT nd AST re serologicl mrkers of liver dmge, nd tretment resulted in significnt (P < 0.01) increses in ALT nd AST, wheres CWO effectively (P < 0.01) reduced the levels of these mrkers (Fig. 2c). Other serum biochemicl prmeters, including PC III, IV-C, HA, nd LN, re importnt mrkers of the severity of heptic fibrosis. In the model group, the levels of serum PC III were significntly (P < 0.01) incresed compred with those in the NC group. Compred with tretment lone, tretment with CWO long with significntly (P < 0.05) decresed the serum levels of precollgen type III in dose-dependent mnner (Fig. 2d). Similr results were observed for other heptic fibrosis serologicl mrkers, such s IV-C, HA, nd LN (Fig. 2d). CWO improved -induced histopthologicl ltertions To ssess the effect of CWO on -induced heptic fibrosis, Sirius red stining nd Msson s trichrome were used to quntify the re of heptic fibrosis. In the NC group, the liver hd norml lobulr structure with centrl vein nd rdil heptic cord, wheres tretment resulted in severe vcuolr degenertion of heptocytes, inflmmtory cell infiltrtion, impirment of lobulr structure, lrge fibrous septum formtion, pseudolobule formtion, nd collgen fiber deposition. However, CWO 4
Protective effect of oil from Cornus wilsonin fruits b c d Fig. 2. CWO ttenuted -induced liver injury. () The morphology of whole livers nd H&E-stined liver sections (100 ) from the four groups (norml control group: olive oil tretment; model group: tretment; low-dose group: + CWO (0.5 ml/kg) tretment; high-dose group: + CWO (2 ml/kg) tretment). Representtive photogrphs re shown. (b) The liver-to-body weight rtios of the indicted four groups. (c) ALT nd AST levels in serum derived from the mice in the indicted four groups were determined with stndrd enzymtic ssy kits. (d) Serum levels of PC III, IV-C, HA, nd LN were detected using commercilly vilble kits. The dt re expressed s the men ± SD (n = 6/group); **P < 0.01, #P < 0.05, ##P < 0.01. significntly ttenuted the series of liver histopthologicl chnges cused by, especilly in the HDG (Fig. 3 nd Tble 3). CWO inhibited oxidtive stress cused by Oxidtive stress is n importnt fctor in the process of heptic fibrosis, nd excessive oxidtive stress cn excerbte heptic fibrosis. Liver oxidtive stress cn be evluted by mesuring liver tissue metrics, including SOD, GSH, nd MDA levels. In this study, we hypothesized tht CWO might ttenute -induced liver injury by inhibiting oxidtive stress. Compred with olive oil tretment lone, tretment significntly (P < 0.01) reduced SOD nd GSH levels in liver tissue. However, tretment with high-dose CWO significntly (P < 0.05) reversed -induced SOD nd GSH depletion (Fig. 4 nd b). In contrst, compred with olive oil tretment lone, tretment significntly (P < 0.01) incresed the levels of MDA, while high-dose CWO tretment mrkedly (P < 0.05) reduced MDA levels (Fig. 4c). CWO ttenuted CCl4-induced inflmmtory response Inflmmtory response is n importnt process in the development of heptic fibrosis. tretment cn ggrvte the inflmmtory response in mouse liver. In this study, we ssumed tht CWO tretment could reduce -induced liver inflmmtion. As shown in Fig. 5, compred with the NC group, tretment significntly incresed the number of F4/80 positive cells in the liver. However, concurrent dministrtion of CWO mrkedly reduced the number of F4/80 positive cells. Similrly, tretment significntly (P < 0.01) incresed the trnscript levels of IL-6, IL-1β, TNFα, nd monocyte chemottrctnt protein-1 (MCP-1) in the liver; concurrent dministrtion of CWO mrkedly (P < 0.05) reduced their trnscript levels in the liver compred with dministrtion lone in dose-dependent mnner. 5
Qing Liu et l. b Fig. 3. CWO suppressed heptic fibrogenesis in -induced mouse model. () Representtive histology of Sirius red, Msson s trichrome re shown (100 ). (b) Quntifiction of positive stining res ws mesured by Imge J softwre. The dt re expressed s the men ± SD (n = 6/group); ****P < 0.0001, ##P < 0.01, ####P < 0.0001. Tble 3. Degree of heptic fibrosis in ech group Group n Pthologicl grding of heptic fibrosis P 0 I II III IV V VI Norml control (NC) 6 6 0 0 0 0 0 0 Model 6 0 0 0 2 3 1 0 Low-dose group (LDG) 6 0 1 3 2 0 0 0 b High-dose group (HDG) 6 0 3 2 1 0 0 0 c Dt re presented s the men of 10 fields. n, Number of mice. Significnt difference versus NC group (P < 0.01). b Significnt difference versus model group (P < 0.01). c Significnt difference versus model group (P < 0.01). b c Fig. 4. CWO ttenuted oxidtive stress in -induced mouse model. ( c) The content of SOD, GSH, nd MDA in the liver ws detected using commercilly vilble kits. The dt re presented s the men ± SD (n = 6/group); **P < 0.01, #P < 0.05. 6
Protective effect of oil from Cornus wilsonin fruits b c d e Fig. 5. Effect of CWO on proinflmmtory cytokines nd chemokines. () The expression of F4/80 in the liver tissues ws determined using immunohistochemistry ssys (200 ). (b) The trnscript levels of IL-6, IL-1β, TNFα, nd MCP-1 in the liver were determined using RT-qPCR ssys. The dt re expressed s the men ± SD (n = 6/group); **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01, ###P < 0.001. b c Fig. 6. CWO reduced the expression of ECM nd inhibited TGF-β1/Smd3 signling pthwy. () The expression of α-sma nd COL1A1 in the liver tissues ws determined using immunohistochemistry ssys (200 ). Quntifiction of positive stining res ws mesured by Imge J softwre. (b) The protein levels of α-sma nd COL1A1 in the liver tissues of the four groups of mice were determined using Western blot ssys. (c) The protein levels of TGF-β1 nd p-smd3 in the liver tissues of the four groups of mice were determined using Western blot ssys. The dt re presented s the men ± SD of three independent experiments; **P < 0.01, #P < 0.05, ##P < 0.01. 7
Qing Liu et l. Effect of CWO on ECM nd TGF-β1/Smd3 signling pthwy α-sma nd COL1A1 re the min ECM proteins nd secreted by ctivted HSCs, nd their expression increses the production of chemokines tht cn recruit vrious inflmmtory cells, thereby further ggrvting heptic fibrosis. Immunohistochemicl stining of α-sma nd COL1A1 ws used to evlute ECM deposition. As shown in Fig. 6, the expression of α-sma nd COL1A1 ws observed in smooth muscle cells of blood vessels in the NC group. In the CCl4-induced model group, α-sma nd COL1A1 expression ws significntly elevted in the portl res nd collgen fiber deposition res. In contrst, the expression of α-sma nd COL1A1 protein in the CWO tretment group ws significntly lower thn tht in the CCl4-induced model group. Western blot ssys ws lso used to detect the expression of α-sma nd COL1A1. The expression of α-sma nd COL1A1 ws gretly (P < 0.001) enhnced by tretment. However, concurrent dministrtion of CWO mrkedly (P < 0.05) decresed α-sma nd COL1A1 expression compred with dministrtion lone in dose-dependent mnner (Fig. 6b). TGF-β1/Smd3 signling pthwy is one of the most importnt pthwys to ctivte HSCs nd promote heptic fibrosis. In the Western blot ssys, we found tht expression of Trnsforming Growth Fctor-β1 (TGFβ1) nd p-smd3 ws mrkedly (P < 0.001) enhnced by tretment compred with NC group, while concurrent tretment with CWO lrgely (P < 0.05) restored the levels of TGF-β1 nd p-smd3, especilly in the HDG (Fig. 6c). Discussion In the present study, we investigted the efficcy of orl CWO for tretment of -induced heptic fibrosis in mice. Through 6-week in vivo experiment, our reserch demonstrted tht supplementry CWO ws effective in ttenuting liver injury nd heptic fibrosis, suggesting tht CWO hs potentil vlue for the prevention nd tretment of heptic fibrosis. Our experiments further suggested tht the effectiveness of this tretment my be ttributble to its ntioxidnt cpcity, nti-inflmmtory effect nd its effects on inhibiting TGF-β1/Smd3 signling pthwy. Serologicl mrkers re importnt indictors for ssessing liver injury nd heptic fibrosis. Plsm ALT nd AST re sensitive indictors of liver dmge. HA, PC III, IV-C, nd LN re mrkers tht reflect the severity of heptic fibrosis (13 15). To confirm the heptoprotective effect of CWO, we detected these serologicl mrkers to ssess liver function nd the degree of heptic fibrosis. We found tht CWO reduced ALT nd AST levels in dose-dependent mnner nd lso improved the levels of serum mrkers of heptic fibrosis, including HA, PC III, IV-C, nd LN. These results indicted tht CWO hs heptoprotective effect nd cn llevite liver injury nd heptic fibrosis. Histopthologicl nlysis is used s direct mens of evluting the protective effects of CWO. Heptic fibrosis histopthology is chrcterized by heptocyte structurl disorder, extensive heptic stetosis, blloon-like chnges, inflmmtory necrosis, collgen deposition, nd diffuse fibrous septum formtion (16 17). In the present study, the H&E, Sirius red, nd Msson s trichrome results showed tht CWO cn reduce liver inflmmtion, inhibit collgen fiber deposition, nd improve liver tissue structure. The results of immunohistochemicl stining of α-sma nd COL1A1 further confirmed tht CWO cn llevite the deposition of collgen fibers nd the formtion of pseudolobules, indicting tht CWO cn meliorte liver histopthologicl chnges. Regrding the mechnism of heptic fibrosis, growing number of studies hve shown tht oxidtive stress contributes to the formtion of heptic fibrosis nd tht bnorml oxidtive stress conditions fcilitte the ctivtion of vrious signling pthwys, thereby cusing lipid peroxidtion, ctivting HSCs, nd ccelerting the development of heptic fibrosis (18 20). SOD nd GSH re importnt ntioxidnts in orgnisms; they cn inhibit free rdicl-initited lipid peroxidtion nd protect ginst free rdicl dmge to body tissues (21 22). Their content in liver tissue cn reflect the ntioxidnt cpcity of the liver. MDA is one of the most importnt products of lipid peroxidtion; it cuses cross-linking of mcromolecules such s proteins nd nucleic cids, nd it cn denture biofilms nd produce cytotoxicity (21 22). Its liver content cn lso reflect the oxidtive stress stte of the liver. In the present study, the liver content of SOD nd GSH ws decresed in the -treted model group compred with the NC group, wheres the liver content of MDA ws incresed. However, CWO tretment could reverse this trend nd reduce heptic oxidtive stress cused by. The mechnism my be relted to the fct tht CWO is rich in UFAs nd other ntioxidnt components, including phytosterol, lph tocopherol, nd squlene. These dt suggest tht CWO exerts protective effect ginst heptic fibrosis by inhibiting oxidtive stress. Liver inflmmtion is nother importnt mechnism for the development of heptic fibrosis (23 24). Chronic liver inflmmtion is often ccompnied by the upregultion of proinflmmtory cytokines nd chemokines, which cn ggrvte heptic fibrosis (23 24). IL-6, IL-1β, nd TNFα re importnt proinflmmtory cytokines; MCP-1 is mjor chemokine; nd F4/80 is mrker of mouse mcrophges. IL-6 nd IL-1β cn induce heptic inflmmtion nd ECM synthesis in liver (25). TNFα ggrvtes the development of heptic fibrosis cused by vrious fctors (26 28). MCP-1 hs 8
Protective effect of oil from Cornus wilsonin fruits chemotctic ctivity for monocytes nd cn ctivte monocytes nd mcrophges (29). In this study, dministrtion of CWO could reduce the number of F4/80 positive cells in liver, nd inhibit the trnscript levels of IL-6, IL-1β, TNFα, nd MCP-1 in the liver. These results indicte tht CWO cn llevite heptic fibrosis by inhibiting liver inflmmtion. In ddition to the bove mechnisms leding to heptic fibrosis, ctivtion of HSCs is nother one of the most widely ccepted mechnisms. Activtion of HSCs is n importnt event in the process of heptic fibrosis, nd ctivted HSCs trnsform into myofibroblst-like phenotype, fter which they secrete ECM proteins such s α-sma nd COL1A1 (30 32). Therefore, the presence of α-sma nd COL1A1 is often considered s HSC ctivity mrkers during the progression of heptic fibrosis. TGF-β1, pleiotropic cytokine, plys key role in the development of heptic fibrosis nd cn ctivte Smd3 to ccelerte fibrosis progression. Mny studies hve indicted tht TGF-β1/Smd3 signling pthwy is n importnt pthwy to promote the ctivtion of HSCs nd the production of ECM (33 35). In the present study, our results showed tht tretment could ctivte TGF-β1/ Smd3 signling pthwy nd increse ECM expression in liver tissue, while concurrent dministrtion of CWO could inhibit the TGF-β1/Smd3 signling pthwy nd reduce ECM expression in dose-dependent mnner. Therefore, these dt suggest tht the key protective effect of CWO ginst heptic fibrosis my be medited by inhibition the ctivtion of TGF-β1/Smd3 signling pthwy. Conclusion In summry, we hve demonstrted for the first time tht CWO cn meliorte -induced heptic fibrosis by ttenuting heptic oxidtive stress, reducing heptic inflmmtion, nd inhibiting TGF-β1/Smd3 signling pthwy in liver. Our findings indicte tht CWO my be potentilly beneficil edible oil for the djuvnt tretment of heptic fibrosis. 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