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Antioxidant Effect of Filipendula glaberrima Nakai Extract in HepG2 Cells
Biomed Sci Letters 2022;28:25-33
Published online March 31, 2022;  https://doi.org/10.15616/BSL.2022.28.1.25
© 2022 The Korean Society For Biomedical Laboratory Sciences.

Mijin Hong* and Dahyun Hwang†,**

Department of Biomedical Laboratory Science, Hoseo University, Asan-si, Chungcheongnam-do 31499, Korea
Correspondence to: Dahyun Hwang. Department of Biomedical Laboratory Science, Hoseo University, 20, Hoseo-ro 79 beon-gil, Baebang-eup, Asan-si, Chungcheongnam-do 31499, Korea.
Tel: +82-41-540-9628, Fax: +82-41-540-9997, e-mail: hdh@hoseo.edu
*Undergraduate student, **Professor.
Received March 2, 2022; Revised March 27, 2022; Accepted March 28, 2022.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
The imbalance of oxidative stress due to the excessive production of reactive oxygen species (ROS) leads to the pathogenesis of liver disease. To prevent this, the role of antioxidant mechanisms is important. Antioxidant studies have been reported on the Filipendula glaberrima Nakai. However, studies applied to HepG2 cells, which are human liver cells, have not yet been conducted. In this study, 70% ethanol extract of Filipendula glaberrima Nakai (FGE) was prepared and antioxidant activity was investigated. It was confirmed whether FGE pretreatment could reduce hydrogen peroxideinduced oxidative stress in HepG2 cells. The increase in gene expression of antioxidant biomarkers and the scavenging ability of ROS were measured, and Hoechst 33342 staining was used to know the inhibitory effect of the apoptosis. As a result, FGE significantly increased SOD (2.6-fold), CAT (4.4-fold), MT-1A (3.1-fold), GPx (4-fold), and G6PD (2.4)-fold compared to the H2O2-treated group. FGE directly inhibited ROS production from 13.4 to 3.6 (the fluorescence mean of DCF-DA) and also reduced apoptotic cells from 45% to 10% (Hoechst 33342 staining) at 2.5 μg/mL. These results demonstrate the excellent antioxidant activity of FGE and show that it can be used as a functional food to prevent liver disease.
Keywords : Anti-oxidant, Filipendula glaberrima Nakai, HepG2, Functional foods
꽌 濡

솢꽦궛냼醫(reactive oxygen species, ROS)쓽 怨쇰룄븳 깮꽦 떖삁愿怨 吏덊솚, 떦눊蹂, 怨좏삁븬, 媛꾩쭏솚怨 媛숈 留뚯꽦吏덊솚怨 옄媛硫댁뿭吏덊솚 벑쓽 떎뼇븳 吏덈퀝쓣 쑀諛쒗븳떎(Valko et al., 2007). ROS뒗 superoxide anion (O2), hydroxy radical (-OH), hydrogen peroxide (H2O2) 벑쓣 留먰븯硫 誘명넗肄섎뱶由ъ븘 샇씉쓽 遺궛臾쇰줈 깮꽦맂떎. 씤泥댁쓽 쟻떦븳 ROS뒗 꽭룷 떊샇 쟾떖, 떊寃 쟾떖뿉 以묒슂븳 뿭븷쓣 븯硫 삉븳 젙긽 議곗쭅뿉꽌 빆긽꽦 쑀吏뿉 愿뿬븳떎(Kwak and Lee, 2014). 洹몃윭굹 씤泥닿 怨쇰룄븯寃 궛솕쟻 뒪듃젅뒪瑜 諛쏆쓣 寃쎌슦 怨쇱엵쓽 ROS媛 깮꽦릺怨 씠뒗 떒諛깆쭏, 吏吏, 빑궛(DNA, RNA)怨 媛숈 꽭룷 援ъ꽦 슂냼瑜 넀긽 諛 뙆愿댁떆궓떎(Zhang et al., 2016). 듅엳 H2O2뒗 떎瑜 솢꽦궛냼醫낅낫떎 諛섍컧湲곌 湲멸퀬 뙆愿댁쟻씤 free radical 以 븯굹씤 hydroxy radical (-OH)濡쒖쓽 蹂삎씠 돺湲 븣臾몄뿉 궛솕 넀긽쓽 二쇱슂 썝씤씠 맂떎(Clarkson and Thompson, 2000). 뵲씪꽌 솢꽦궛냼醫낆쓽 젣嫄곕 넻븳 吏덈퀝쓽 삁諛 諛 愿由ш 븘슂븳 떎젙씠硫 留뚯꽦吏덊솚쓽 삁諛⑷낵 끂솕 뼲젣 벑 깮泥 議곗젅 湲곕뒫씠굹 깮泥 諛⑹뼱뒫젰쓣 吏땶 씪遺 꽦遺꾨뱾쓣 씠슜븳 湲곕뒫꽦 떇뭹뿉 븳 뿰援ш 솢諛쒗엳 吏꾪뻾릺怨 엳떎(Chang et al., 2013; Cho et al., 2014).

媛꾩 쑀빐臾쇱쭏쓣 泥섎━븯湲 쐞븳 슚냼뱾쓣 깮궛븯뿬 쑀빐臾쇱쭏쓣 泥섎━븯뒗 씪李⑥쟻씤 빐룆 옉슜쓣 븯뒗 옣湲곕떎(Kim et al., 2019). ROS 怨쇱엵 깮꽦쑝濡 씤븳 궛솕쟻 뒪듃젅뒪쓽 遺덇퇏삎 媛 泥대궡쓽 二쇱쐞 媛꾩꽭룷, 궡뵾꽭룷 洹몃━怨 媛꾩꽦긽 꽭룷(Hepatic stellate cell)뿉 쁺뼢쓣 二쇨퀬 媛꾩꽟쑀솕瑜 珥됱쭊떆궓떎(Gandhi, 2012; Cichoz-Lach and Michalak, 2014). 씠뒗 媛 뿼利, 媛꾧꼍蹂利, 媛꾩꽭룷븫怨 媛숈 媛꾩쭏솚쑝濡 씠뼱吏 닔 엳쑝硫 씠瑜 삁諛⑺빐 以 닔 엳뒗 빆궛솕젣(antioxidant)媛 깮由ы솢꽦 臾쇱쭏濡쒖꽌 겕寃 媛곴킅諛쏄퀬 엳떎(Leem et al., 2011; Marí et al., 2013).

쁽옱 슦由 二쇰뿉꽌 젒븷 닔 엳뒗 泥쒖뿰 빆궛솕젣濡쒕뒗 鍮꾪誘 C, 뵆씪蹂대끂씠뱶瑜, 移대줈떚끂씠뱶瑜, 깂땶, 븞넗떆븘땶, 넗肄뷀럹 벑씠 엳쑝硫 깮泥 궡 끂솕瑜 뼲젣븯怨 吏덈퀝쓽 삁諛⑹뿉 슚怨쇱쟻씤 寃껋쑝濡 븣젮졇 엳떎(Atoui et al., 2005; Suh et al., 2019). 빆궛솕젣 以묒뿉꽌룄 BHT (butylated hydroxytoluene)怨 BHA (butylated hydroxyanisole) 媛숈 빀꽦 빆궛솕젣뒗 졃븯怨 슦닔븳 솢꽦 슚怨쇰 媛吏吏留 諛쒖븫꽦 諛 븞쟾꽦뿉 븳 臾몄젣媛 젣湲곕릺硫댁꽌 洹쇰옒뿉뒗 빀꽦 빆궛솕젣쓽 떒젏쓣 蹂댁셿븳 泥쒖뿰쑀옒 빆궛솕젣 뿰援ш 袁몄븯寃 吏꾪뻾릺怨 엳떎(Kim et al., 2009; Na et al., 2016; Cha et al., 2017). 洹몃윭굹 泥쒖뿰 빆궛솕젣쓽 寃쎌슦, 빀꽦 빆궛솕젣뿉 鍮꾪빐 깮泥 씠슜瑜좎씠 궙湲 븣臾몄뿉 鍮꾧탳쟻 궙 빆궛솕 솢꽦쓣 蹂댁씤떎(Jeong et al., 2006). 빆궛솕젣쓽 씠윴 떒젏뱾쓣 蹂댁셿븯湲 쐞빐, 씤泥댁뿉 臾댄빐븯硫댁꽌 깮泥 씠슜瑜좎씠 넂븘 빆궛솕 슚뒫씠 슦닔븳 泥쒖뿰 빆궛솕젣뿉 븳 吏냽쟻씤 媛쒕컻씠 슂援щ릺뼱吏꾨떎.

꽣由ы(Filipendula glaberrima Nakai, FG) 源딆 궛냽뿉꽌 옄씪뒗 옣誘멸낵 냽쓽 뿬윭빐궡씠 떇臾쇱씠떎. 蹂 냽 떇臾쇱쓽 遺遺꾩쓽 醫낆씠 븳援, 씪蹂, 留뚯< 벑 룞遺곸븘떆븘뿉 옄깮븯뒗뜲, 듅엳 FG뒗 븳諛섎룄뿉留 遺꾪룷븯뒗 듅궛醫낆쑝濡 븣젮졇 엳떎(Yeo et al., 1992). 삉븳 꽣由ы씠 냼뿼, 吏꾪넻, 넻뭾쓽 移섎즺 紐⑹쟻쑝濡 궗슜릺뿀떎뒗 蹂닿퀬 슦닔븳 빆궛솕 솢꽦뒫쓣 媛吏 뵆씪蹂대끂씠뱶 怨꾩뿴 以 븯굹씤 移댄뀒궓 꽦遺꾩쓣 룷븿븳떎뒗 蹂닿퀬媛 엳떎(Yeo et al., 1992b; Lee et al., 2020). 吏湲덇퉴吏 꽣由ы쓣 냼옱濡 븳 빆궛솕 뿰援ш 吏꾪뻾릺뼱 솕쑝굹 씤泥 媛꾩꽭룷뿉 쟻슜븳 뿰援щ뒗 븘吏 吏꾪뻾맂 諛붽 뾾떎. 뵲씪꽌 씤泥 媛꾩꽭룷뿉 꽣由ы 異붿텧臾쇱쓣 씠슜븳 빆궛솕 솢꽦뿰援щ뒗 泥쒖뿰 빆궛솕젣濡쒖꽌쓽 怨좊媛移섍 엳쓣 寃껋쑝濡 궗猷뚮맂떎.

蹂 뿰援ъ뿉꽌뒗 泥쒖뿰臾 냼옱씤 꽣由ы 뿉깂삱 異붿텧臾(ethanol extract of Filipendula glaberrima Nakai, FGE)쓽 빆궛솕 슚怨쇰 룊媛븯湲 쐞빐 씤媛 媛꾩븫꽭룷二쇱씤 HepG2뿉 H2O2濡 쑀諛쒕맂 궛솕쟻 뒪듃젅뒪뿉 빆븯뿬 씠뱾 異붿텧臾쇱씠 꽭룷瑜 蹂댄샇븷 닔 엳뒗吏 븣븘蹂댁븯떎. 蹂 떆猷뚯쓽 빆궛솕 솢꽦쓣 痢≪젙븿쑝濡쒖뜥 媛꾩쭏솚쓣 삁諛⑺븷 닔 엳뒗 嫄닿컯湲곕뒫떇뭹씠굹 泥쒖뿰깮臾쇱옄썝쓽 냼옱濡쒖쓽 솢슜媛뒫꽦쓣 蹂닿퀬옄 븯떎.

옱猷 諛 諛⑸쾿

떆猷뚯쓽 異붿텧諛⑸쾿

FGE 寃쎄린룄 寃쎌젣怨쇳븰吏꾪씎썝 諛붿씠삤꽱꽣(Suwon, Korea)뿉꽌 援ъ엯뻽떎. FG쓽 吏긽遺 遺遺(55.5 g)쓣 떎삩뿉꽌 븯猷 룞븞 70% 닔꽦 뿉깂삱(1 L)濡 異붿텧븯떎. 씠썑 異붿텧臾쇱쓣 뿬怨쇳븯怨(Advantec No.2) 40℃쓽 삩룄濡 媛먯븬냽異뺥븳 뮘(EYELA rotary evaporator, Tokyo, Japan) 룞寃 嫄댁“븯뿬 옍瑜섎Ъ 7.55 g쓣 뼸뿀떎. 嫄댁“맂 異붿텧臾쇱쓣 DMSO뿉 100 mg/mL 냽룄濡 끃씠怨 利앸쪟닔濡 쟻젅엳 씗꽍븯뿬 떎뿕뿉 궗슜븯떎.

꽭룷諛곗뼇

蹂 떎뿕뿉 궗슜맂 꽭룷뒗 씤媛 媛꾩븫꽭룷二쇱씤 HepG2濡 븳援 꽭룷二 뻾(Korean Cell Line Bank, Seoul, Korea)뿉꽌 遺꾩뼇 諛쏆븯떎. 꽭룷뒗 10% fetal bovine serum (FBS; Life Technologies, Grand Island, NY, USA)怨 1% penicillinstreptomycin (P/S; GenDEPOT, Katy, TX, USA)쓣 븿쑀븳 Dulbeccós modified Eaglés medium (DMEM; HyClone, San Angelo, TX, USA)쓣 궗슜븯뿬 37℃, 5% CO2 議곌굔쓽 諛곗뼇湲곗뿉꽌 諛곗뼇븯떎.

HepG2 꽭룷쓽 꽭룷룆꽦 痢≪젙

FGE쓽 HepG2 꽭룷뿉 븳 븞쟾꽦쓣 痢≪젙븯湲 쐞빐 HepG2 cell (5×105 cell/mL)瑜 96 well plate뿉 200 μL뵫 遺꾩<븯怨 16~20떆媛 젙룄 5% CO2 incubator (37℃)뿉꽌 븞젙솕븯떎. FGE (0.625~2.5 μg/mL)濡 24떆媛 泥섎━븯怨 꽭룷뿉 궛솕쟻 뒪듃젅뒪瑜 쑀諛쒗븯湲 쐞빐 400 μM쓽 H2O2瑜 9떆媛 泥섎━븯떎.

꽭룷룆꽦쓣 痢≪젙븯湲 쐞빐 긽痢듭븸쓣 젣嫄고븯怨 SFM (Serum Free Media)뿉 끃씤 10% EZ cytox (DoGenBio, Seoul, Korea) 100 μL瑜 泥④븯떎. 30遺 썑 450 nm뿉꽌 씉愿묐룄瑜 痢≪젙븯떎. 긽 꽭룷 깮議댁쑉(%) 쓬꽦 議곌뎔뿉 븳 諛깅텇쑉濡 몴떆릺뿀떎. 꽭룷 삎깭뒗 룄由쏀쁽誘멸꼍(CKX53, OYMPUS, Tokyo, Japan)쓣 궗슜븯뿬 愿李고븯떎.

젙웾쟻 떎떆媛 以묓빀슚냼 뿰뇙 諛섏쓳(qRT-PCR)

HepG2 꽭룷 (5×105 cell/mL)瑜 24 well plate뿉 500 μL뵫 遺꾩<븯怨 16~20떆媛 젙룄 5% CO2 incubator (37℃)뿉꽌 븞젙솕븯떎. 洹몃━怨 FGE (0.625~2.5 μg/mL)濡 24떆媛 泥섎━븯怨 꽭룷뿉 궛솕쟻 뒪듃젅뒪瑜 쑀諛쒗븯湲 쐞빐 400 μM쓽 H2O2瑜 9떆媛 泥섎━븯떎. mRNA 닔以뿉꽌 쑀쟾옄쓽 諛쒗쁽 젙룄瑜 솗씤븯湲 쐞빐 Total RNA Extraction Kit (iNtRON BIOTECHNOLOGY, Daejeon, Korea)瑜 궗슜븯뿬 RNA瑜 遺꾨━븯떎. 遺꾨━맂 RNA뒗 ReverTra AceTM qPCR RT Master Mix (TOYOBO, Osaka, Japan)瑜 궗슜븯뿬 cDNA濡 뿭쟾궗 떆궓 썑 qPCR Magnetic Induction Cycler PCR Machine (biomolecular systems, Coomera, Australia)쓣 궗슜븯뿬 SYBR® Green Realtime PCR Master Mix 떆빟(TOYOBO, Osaka, Japan)뿉 쓽빐 吏꾪뻾븯떎. 蹂 떎뿕뿉 궗슜맂 primer쓽 뿼湲곗꽌뿴 Table 1뿉 굹궡뿀쑝硫, mRNA쓽 諛쒗쁽 蹂솕뒗 2-ΔΔCt method 諛⑸쾿쓣 궗슜븯뿬 GAPDH 議곌뎔쓣 1濡 꽕젙븯뿬 몴쁽븯떎(Livak and Schmittgen, 2001).

Primer list

Gene Forward Reverse
SOD1 5'-AAG GCC GTG TGC GTG CTG AA-3' 5'-CAG GTC TCC AAC ATG CCT CT-3'
CAT 5'-AAG GTT TGG CCT CAC AAG G-3' 5'-CGG CAA TGT TCT CAC ACA G-3'
MT-1A 5'-CTC GAA ATG GAC CCC AACT-3' 5'-ATA TCT TCG AGC AGG GCT GTC-3'
GPx 5'-GTG TAT GCC TTC TCG GCG CG-3' 5'-CGT TGC GAC ACA CCG GAG AC-3'
G6PD 5'-CCG GAT CGA CCA CTA CCT GGG CAAG-3' 5'-GTT CCC CAC GTA CTG GCC CAG GAC CA-3'
GAPDH 5'-ATG GTG AAG GTC GGT GTG AAC-3' 5'-TTG ATG TTA GTG GGG TCT CGC-3'

*Abbreviations: superoxide dismutase (SOD), catalase (CAT), metallothioneins (MT-1A), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PD), glyceraldehyde 3-phosphate dehydrogenase (GAPDH)



솢꽦궛냼醫(Reactive oxygen species, ROS) 냼嫄곕뒫 痢≪젙

HepG2 꽭룷뿉 FGE瑜 泥섎━븯쓣 븣 ROS쓽 냼嫄곕뒫쓣 蹂닿린 쐞븯뿬 2,7-dichlorofluorescein diacetate (DCF-DA; Sigma-Aldrich Co. St. Louis, U.S.A)瑜 궗슜븯뿬 Fluorescence Activated Cell Sorter (FACS; BD FACSAriaTM II Cell Sorter)濡 痢≪젙븯떎. HepG2 꽭룷瑜 6 well plate뿉 5×105 cell/mL쓽 理쒖쥌 냽룄媛 릺룄濡 꽭룷 슜븸쓣 遺꾩<븳 썑 37℃, 5% CO2 諛곗뼇湲곗뿉꽌 24떆媛 룞븞 諛곗뼇븳 뮘, FGE (0.625~2.5 μg/mL)濡 24떆媛 泥섎━븯怨 400 μM쓽 H2O2瑜 12떆媛 泥섎━븯뿬 궛솕쟻 뒪듃젅뒪瑜 쑀諛쒗븯떎. 諛곗뼇 썑 phosphate-buffered saline (PBS)쑝濡 1쉶 꽭泥숉븯怨 20 μM쓽 DCF-DA瑜 泥섎━븯떎. 20遺 뮘 PBS濡 꽭泥숉븳 떎쓬 trypsin-EDTA瑜 씠슜븯뿬 꽭룷瑜 쉶닔븯怨 excitation 485 nm, emission 535 nm쓽 議곌굔뿉꽌 FACS濡 痢≪젙븯떎.

Hoechst 33342 뿼깋

HepG2 꽭룷瑜 cell culture slide뿉 5×104 cell/mL쓽 理쒖쥌 냽룄媛 릺룄濡 꽭룷 슜븸쓣 遺꾩<븳 썑 諛곗뼇湲곗뿉꽌 24떆媛 룞븞 諛곗뼇븳 뮘, FGE (0.625~2.5 μg/mL)濡 24떆媛 泥섎━븯怨 400 μM쓽 H2O2瑜 12떆媛 諛섏쓳떆耳곕떎. PBS濡 2쉶 꽭泥 썑, 10% formalin쓣 泥섎━븯뿬 1떆媛 怨좎젙떆궓 뮘 PBS濡 꽭泥숉븯怨 Hoechst 33342 (Sigma-Aldrich Co. St. Louis, U.S.A)濡 30遺 룞븞 뿼깋떆궎怨, 뿼깋 썑 꽭泥숉븯뿬 삎愿 쁽誘멸꼍 븯뿉꽌 400諛곕줈 愿李고븯떎.

넻怨 泥섎━

Windows슜 SPSS 踰꾩쟾 12.0 (SPSS, Chicago, IL, USA)쓣 궗슜븯뿬 넻怨 泥섎━瑜 吏꾪뻾븯떎. 紐⑤뱺 떎뿕 寃곌낵뒗 떎뿕쓽 룊洹 ± SD濡 몴떆븯떎. 媛 洹몃9媛 李⑥씠쓽 넻怨꾩쟻 쑀쓽꽦 씪썝諛곗튂 遺꾩궛遺꾩꽍(one-way ANOVA)쓣 궗슜븯怨 P < 0.05 닔以뿉꽌 寃利앺븯떎.

寃 怨

FGE쓽 HepG2 꽭룷 꽭룷룆꽦

蹂 떎뿕뿉꽌뒗 FGE씠 HepG2 꽭룷쓽 利앹떇뿉 誘몄튂뒗 쁺뼢쓣 븣븘蹂닿린 쐞빐 HepG2 꽭룷뿉 븳 룆꽦쓣 솗씤븯떎(Fig. 1). 쓬꽦 議곌뎔쓣 젣쇅븳 紐⑤뱺 援곗뿉 400 μM쓽 H2O2瑜 泥섎━븯뿬 궛솕쟻 뒪듃젅뒪瑜 쑀룄븯怨, 400 μM쓽 H2O2 떒룆 泥섎━援곗뿉꽌뒗 쓬꽦 議곌뎔 鍮 쑀쓽쟻씤 꽭룷 깮議댁쑉 媛먯냼(빟 20%)瑜 굹깉떎. 諛섎㈃뿉 FGE쓽 0.625~2.5 μg/mL쓽 냽룄뿉꽌뒗 냽룄 쑀쓽쟻쑝濡 利앷맂 꽭룷 깮議댁쑉쓣 蹂댁쑝硫 紐⑤뱺 냽룄뿉꽌 꽭룷룆꽦 굹굹吏 븡븯떎. 뵲씪꽌 HepG2 꽭룷뿉 FGE씠 꽭룷 깮議댁쑉뿉 쁺뼢쓣 겕寃 誘몄튂吏 븡뒗 냽룄 踰붿쐞뿉꽌 썑냽 떎뿕쓣 吏꾪뻾븯떎.

Fig. 1. Cytotoxic effects of FGE against H2O2-induced oxidative stress in HepG2 cells. HepG2 cells were treated with FGE (0.625, 1.25 and 2.5 μg/mL) for 24 h and then 400 μM H2O2 induced oxidative stress for a further 9 h. The cell viability was measured using EZ cytox. The present data were expressed mean ± SD. Different superscripts mean a significant difference among groups (P<0.05) based on one-way ANOVA and Duncan's multiple range test. FGE, ethanol extract of Filipendula glaberrima Nakai; NC, Negative control; Asc, Ascorbic acid.

FGE쓽 빆궛솕 솢꽦 슚怨

蹂 뿰援ъ뿉꽌뒗 RT-PCR쓣 씠슜븯뿬 FGE쓽 빆궛솕 諛⑹뼱 떆뒪뀥씤 3媛쒖쓽 二쇱슂 슚냼 諛 떒諛깆쭏(SOD, CAT, MT-1A)怨 glutathione 궗뿉 愿뿬븯뒗 슚냼뱾(GPx, G6PD)쓽 mRNA 諛쒗쁽뼇쓣 솗씤븯떎(Fig. 2, Fig. 3). H2O2濡 궛솕쟻 뒪듃젅뒪媛 쑀룄맂 HepG2 꽭룷뿉꽌 FGE뒗 紐⑤몢 넻怨꾩쟻쑝濡 쑀쓽븳 빆궛솕 슚怨쇰 굹궡뿀떎.

Fig. 2. Effect of FGE on the expression of (A) SOD, (B) CAT and (C) MT-1A genes in H2O2-induced HepG2 cells. HepG2 cells were treated with FGE (0.625, 1.25 and 2.5 μg/mL) for 24 h and then 400 μM H2O2 induced oxidative stress for a further 9 h. The mRNA expressions were analyzed by qRT-PCR. The cells were collected and (A) SOD, (B) CAT and (C) MT-1A mRNA level were estimated NC was treated with only the SFM (Serum Free Media). The present data were expressed mean ± SD. Different superscripts mean a significant difference among groups (P<0.05) based on one-way ANOVA and Duncan's multiple range test. FGE, ethanol extract of Filipendula glaberrima Nakai; NC, Negative control; Asc, Ascorbic acid.

Fig. 3. Effect of FGE on the expression of (A) GPx, (B) G6PD genes in H2O2-induced HepG2 cells. HepG2 cells were treated with FGE (0.625, 1.25 and 2.5 μg/mL) for 24 h and then 400 μM H2O2 induced oxidative stress for a further 9 h. The mRNA expressions were analyzed by qRT-PCR. The cells were collected and (A) GPx, (B) G6PD mRNA level were estimated NC was treated with only the SFM (Serum Free Media). The present data were expressed mean ± SD. Different superscripts mean a significant difference among groups (P<0.05) based on one-way ANOVA and Duncan's multiple range test. FGE, ethanol extract of Filipendula glaberrima Nakai; NC, Negative control; Asc, Ascorbic acid.

FGE쓽 理쒓퀬 냽룄씤 2.5 μg/mL뿉꽌 H2O2 떒룆 泥섎━援 鍮 SOD (2.6諛), CAT (4.4諛), MT-1A (3.1諛), GPx (4諛), G6PD (2.4)諛곕줈 mRNA 닔以씠 紐⑤몢 쑀쓽쟻쑝濡 利앷븯떎. 뵲씪꽌 FGE뒗 mRNA 닔以뿉꽌 紐⑤몢 냽룄 쓽議댁쟻쑝濡 빆궛솕 솢꽦 슚怨쇰 蹂댁씤 寃껋쓣 븣 닔 엳떎.

꽭룷 궡 ROS 냼嫄 슚怨

HepG2 꽭룷뿉 H2O2뿉 쓽빐 쑀룄맂 꽭룷 궡 ROS 깮꽦뿉 븳 FGE쓽 쁺뼢쓣 솗씤븯怨좎옄 FACS瑜 씠슜빐 DCF-DA쓣 寃異쒗븯뿬 ROS 깮꽦웾쓣 痢≪젙븯떎(Fig. 4). 씠瑜 젙웾遺꾩꽍 븳 寃곌낵, H2O2 떒룆 泥섎━援곌낵 鍮꾧탳뻽쓣 븣 룊洹 DCF-DA 삎愿 媛믪씠 FGE쓽 理쒓퀬 냽룄씤 2.5 μg/mL뿉꽌 13.4뿉꽌 3.6쑝濡 3諛 씠긽 媛먯냼 븳 寃껋쓣 蹂 닔 엳떎(Fig. 4B). 씠寃껋 HepG2 꽭룷뿉 H2O2濡 쑀諛쒕맂 ROS瑜 FGE媛 냼嫄곗떆耳곗쓬쓣 굹궡硫 蹂 寃곌낵뿉 뵲씪 FGE媛 쑀쓽븳 ROS 냼嫄곕뒫씠 엳쓬쓣 솗씤븯떎.

Fig. 4. Effect of FGE on the contents of ROS in H2O2-induced HepG2 cells. The cells were treated with FGE and then stimulated with 400 μM H2O2 for 12 h. (A) ROS was determined by FACS and (B) analysis of statistical results of FACS detection. The present data were expressed mean ± SD. Different superscripts mean a significant difference among groups (P<0.05) based on one-way ANOVA and Duncan's multiple range test. FGE, ethanol extract of Filipendula glaberrima Nakai; NC, Negative control; Asc, Ascorbic acid.

Apoptosis 뼲젣 슚怨(Hoechst 33342)

H2O2 쑀룄뿉 븳 FGE쓽 꽭룷 蹂댄샇 슚怨쇰 룊媛븯湲 쐞빐 Hoechst 33342 뿼깋쓣 닔뻾븯떎(Fig. 5). H2O2 떒룆 泥섎━援곗뿉꽌뒗 깮議 꽭룷 닔媛 쟻쑝硫댁꽌 꽭룷 궗硫몄씠 吏꾪뻾릺뼱 빑쓽 쓳異 諛 遺꾩뿴 삎깭瑜 蹂댁씠뒗 쟾삎쟻씤 apoptosis 듅吏뺤쓣 굹궡뿀떎(Fig. 5A). 諛섎㈃뿉, FGE瑜 냽룄 蹂꾨줈 泥섎━븳 援곗뿉꽌뒗 냽룄 쓽議댁쟻쑝濡 젙긽쟻쑝濡 삩쟾븳 빑 삎깭瑜 蹂댁씠뒗 꽭룷媛 利앷븯떎. 씠瑜 젙웾遺꾩꽍 븳 寃곌낵, apoptotic cell 議곌뎔(5%) 鍮꾧탳뻽쓣 븣 H2O2 떒룆 泥섎━援곗뿉꽌 45%濡 겕寃 利앷븯吏留 FGE瑜 븿猿 泥섎━뻽쓣 븣, apoptotic cell씠 30.0%, 17.5%, 10.0%濡 냽룄 쓽議댁쟻쑝濡 媛먯냼븯떎(Fig. 5B). 뵲씪꽌 蹂 寃곌낵뿉 뵲瑜대㈃, FGE뒗 HepG2 꽭룷뿉 옉슜븯뿬 빆궛솕 솢꽦쓣 利앹쭊떆궡쑝濡쒖뜥 H2O2뿉 쓽븳 궛솕쟻 넀긽쓣 蹂댄샇븷 닔 엳쓬쓣 삎깭븰쟻쑝濡 솗씤븯떎.

Fig. 5. Protective effect of FGE against H2O2-induced apoptosis in HepG2 cells. The cells were treated with FGE and then stimulated with 400 μM H2O2 for 12 h. Fixed cells were stained with Hoechst 33342 and examined by fluorescence microscope (Magnification×400). Apoptotic HepG2 cells are indicated by arrows. (A) The morphology of cell nucleus and (B) analysis of statistical results of apoptotic cells. FGE, ethanol extract of Filipendula glaberrima Nakai; NC, Negative control; Asc, Ascorbic acid.
怨 李

誘명넗肄섎뱶由ъ븘쓽 젙긽쟻씤 샇씉 궗怨쇱젙뿉꽌 솢꽦궛냼뒗 냼웾뵫 깮꽦릺怨 꽭룷쓽 湲곕뒫 쑀吏뿉 븘닔쟻씤 臾쇱쭏씠떎(Kwak and Lee, 2014). 洹몃윭굹 솢꽦궛냼醫 깮꽦쓽 遺덇퇏삎쑝濡 씤븳 怨쇰룄븳 궛솕쟻 뒪듃젅뒪뒗 끂솕 諛 媛곸쥌 吏덊솚뱾쓽 諛쒕퀝湲곗쟾怨 誘명넗肄섎뱶由ъ븘쓽 湲곕뒫 옣븷瑜 珥됱쭊떆궓떎(Maxwell, 1995). 궛솕쟻 뒪듃젅뒪뒗 媛 뿼利, 媛꾧꼍솕瑜 鍮꾨’븳 떎뼇븳 媛꾩쭏솚쓽 諛쒕퀝쓣 쑀諛쒗븯뒗 二쇱슂 썝씤씠 릺硫 媛꾩뿉 留ㅼ슦 痍⑥빟븯떎怨 븣젮졇 엳떎(Sanchez-Valle et al., 2012).

蹂 뿰援щ뒗 in vitro뿉꽌 H2O2뿉 쓽빐 쑀諛쒕맂 궛솕 뒪듃젅뒪濡쒕꽣 FGE쓽 HepG2 cell 蹂댄샇 슚怨쇰 솗씤븯湲 쐞빐 닔뻾릺뿀떎. 媛꾩 깮泥 궡뿉꽌 빐룆 諛 깮솕븰쟻 궗 媛숈 以묒슂븳 湲곕뒫쓣 닔뻾븯뒗 以묒떖쟻씤 湲곌쑝濡 媛꾩꽭룷媛 궛솕뒪듃젅뒪뿉 빆븯뿬 蹂댄샇 슚怨쇰 愿李고븯湲 쐞빐 쟻빀븯떎怨 뙋떒뻽怨, 蹂 뿰援ъ뿉꽌뒗 씤媛 媛꾩븫꽭룷二쇱씤 HepG2 꽭룷二쇰 궗슜븯떎(Gao et al., 2016). 씪諛섏쟻쑝濡 HepG2 꽭룷뿉 H2O2濡 궛솕쟻 뒪듃젅뒪瑜 쑀諛쒖떆궎湲 쐞빐 100~1,000 μM쓽 냽룄媛 꽑깮릺뼱吏뒗뜲, 蹂 뿰援ъ뿉꽌뒗 꽭룷뿉 빟 20% 젙룄쓽 쑀쓽쟻씤 룆꽦쓣 쑀諛쒗븯뒗 냽룄씤 400 μM쓣 꽑깮븯뿬 떎뿕쓣 吏꾪뻾븯떎. FGE뒗 0.625~2.5 μg/mL쓽 냽룄뿉꽌 꽭룷뿉 룆꽦쓣 굹궡吏 븡뒗 냽룄 踰붿쐞뿉꽌 떎뿕쓣 吏꾪뻾뻽떎.

ROS뒗 깮泥 궡 떊泥댁쓽 援ъ꽦臾쇱쭏怨 媛뺥븳 諛섏쓳꽦쓣 蹂댁씠硫 궛솕쟻 뒪듃젅뒪瑜 쑀諛쒗븯뒗 臾쇱쭏濡 꽭룷쓽 궛솕怨쇱젙쓣 씪쑝궎뒗 二쇱썝씤씠떎(Choi et al., 2012). 洹몃윭굹 씤泥 궡뿉꽌 ROS쓽 吏냽쟻씤 깮꽦 遺덇뵾븯硫, 洹몃쭔겮 ROS쓽 냼嫄곕 넻븳 씤泥 궡 궗쓽 洹좏삎씠 以묒슂븯떎. FGE쓽 吏곸젒쟻씤 ROS 냼嫄곕뒫쓣 蹂닿퀬옄 H2O2瑜 泥섎━븯뿬 깮꽦맂 怨쇰룄븳 ROS 깮꽦쓣 쑀諛쒗븯뿬 DCF-DA瑜 泥섎━빐 FACS 遺꾩꽍쓣 떎떆븯떎. 蹂 뿰援ъ뿉꽌 FGE媛 H2O2뿉 쓽빐 怨쇰룄븯寃 깮꽦맂 ROS瑜 슚怨쇱쟻쑝濡 媛먯냼떆궎뒗 寃껋쓣 솗씤븯떎. 뵲씪꽌 FGE쓽 ROS 냼嫄곕뒫쓣 痢≪젙빐 궛솕쟻 넀긽쓣 뼲젣븯뒗 슚怨쇰 寃利앺븯떎.

H2O2뒗 꽭룷留 넻怨쇨 슜씠븯硫 궛솕쟻 뒪듃젅뒪瑜 利앷떆궡뿉 뵲씪 誘명넗肄섎뱶由ъ븘 留됱쟾쐞瑜 媛먯냼떆耳 寃곌뎅 apoptosis瑜 쑀諛쒖떆궎뒗 寃껋쑝濡 븣젮졇 엳떎(Youn et al., 2015). Apoptosis媛 吏꾪뻾릺硫 꽭룷뒗 硫댁뿭 諛섏쓳쓣 씪쑝耳, 빑쓽 쓳異 諛 DNA媛 議곌컖굹뒗 삎깭븰쟻 듅吏뺤쓣 蹂댁씤떎. 뵲씪꽌 빑 궡 DNA뿉 듅씠쟻쑝濡 寃고빀븯뒗 삎愿 뿼깋泥댁씤 Hoechst 33342瑜 궗슜븯뿬 빑쓽 삎깭븰쟻씤 듅吏뺤쓣 愿李고뻽떎(Kim et al., 2010). 洹 寃곌낵 HepG2 꽭룷二쇱뿉 H2O2 泥섎━援곗쓽 꽭룷빑뿉꽌 빑 쓳異뺢낵 遺꾩뿴濡 씤븳 apoptotic body媛 愿李곕맂 諛섎㈃, FGE瑜 냽룄 쓽議댁쟻쑝濡 泥섎━븳 援곗뿉꽌뒗 젙긽 꽭룷援곗쓽 삎깭 쑀궗븳 빑쓽 삎깭瑜 愿李고븷 닔 엳뿀떎. 씠뒗 HepG2 꽭룷뿉 FGE쓽 쟾泥섎━媛 H2O2濡 쑀諛쒕맂 궛솕쟻 넀긽쑝濡쒕꽣 蹂댄샇븯뒗 슚怨쇨 엳쓬쓣 삁痢≫빐 蹂 닔 엳떎. 빑쓽 삎깭븰쟻씤 愿李곗쓣 넻빐 FGE쓽 궛솕쟻 뒪듃젅뒪濡쒕꽣쓽 꽭룷 蹂댄샇 슚怨쇰 媛떆쟻쑝濡 솗씤뻽떎硫 FGE쓽 슦닔븳 빆궛솕 솢꽦쓣 寃利앺븯湲 쐞빐 빆궛솕쓽 몴쟻씤 諛붿씠삤 留덉빱뱾쓣 痢≪젙븯떎. 蹂 뿰援ъ뿉꽌뒗 二쇱슂 빆궛솕 슚냼뱾쓽 쑀쟾옄 諛쒗쁽뼇쓣 蹂닿린 쐞빐 RT-PCR쓣 씠슜븯떎. 洹 寃곌낵 SOD, CAT, GPx, G6PD, MT-1A怨 媛숈 빆궛솕 諛붿씠삤 留덉빱뿉꽌 H2O2 떒룆 泥섎━븳 議곌뎔뿉 鍮꾪빐 mRNA 諛쒗쁽씠 쑀쓽쟻쑝濡 利앷븯떎.

슦由 紐몄 젙긽쟻쑝濡 깮泥 湲곕뒫쓣 쑀吏븯湲 쐞빐 떎뼇븳 궡 • 쇅遺쟻 옄洹뱀뿉 븳 옄泥댁쟻씤 諛⑹뼱 硫붿빱땲利섏쓣 媛吏怨 엳떎. 몴쟻쑝濡 SOD 諛 CAT 媛숈 빆궛솕怨 슚냼, glutathione 궗怨꾩뿉 냽븯뒗 GPx, G6PD 洹몃━怨 떎紐⑹쟻 떒諛깆쭏濡 媛꾩<릺뒗 MT-1A怨 媛숈 빆궛솕 諛⑹뼱떆뒪뀥씠 엳떎(Salla et al., 2016). 빆궛솕怨 슚냼씤 SOD CAT뒗 꽭룷 궡 怨쇱엵쑝濡 깮꽦릺뒗 씪李⑥쟻씤 radical쓽 怨쇰룄븳 삎꽦쓣 젣嫄고븯뒗뜲 湲곗뿬븯怨 GSH뒗 솚썝 삎깭씤 GSH 궛솕 삎깭씤 GSSG濡 議댁옱븯硫 GPx뒗 궛솕, 솚썝 諛섏쓳쓣 넻빐 궛솕 뒪듃젅뒪瑜 젣嫄고븯뒗 뒫젰쓣 蹂댁쑀븯怨 엳떎(Clarkson and Thompson, 2000b; Zhang et al., 2009). 삉븳 G6PD쓽 솢꽦씠 媛먯냼븷 寃쎌슦 NADPH 媛숈 glutathione쓣 솚썝삎쑝濡 쑀吏떆耳쒖<뒗 蹂댁“씤옄쓽 깮꽦쓣 븯떆耳 꽭룷 궡쓽 옄쑀씪뵒移쇱씠굹 怨쇱궛솕臾쇱쓣 슚怨쇱쟻쑝濡 빐룆븯吏 紐삵븯寃 맂떎(Kim et al., 2008). MT-1A 以묎툑냽 씠삩(Cu, Cd, Hg, Pb, Pt)怨 넂 移쒗솕젰쓣 蹂댁뿬 SOD 媛숈 빆궛솕 슚냼뿉 븳 湲덉냽 蹂댁“씤옄瑜 젣怨듯븿쑝濡쒖뜥 媛꾩젒쟻씤 빆궛솕젣濡쒖꽌 湲곕뒫쓣 븷 닔 엳떎(Álvarez-Barrios et al., 2021). 利, 씤泥댁쓽 젙긽쟻씤 諛⑹뼱 硫붿빱땲利섏뿉 뵲瑜몃떎硫 씤泥댁쓽 꽭룷 궡뿉꽌 궛솕 솚썝 긽깭瑜 議곗젅븯뿬 ROS 깮꽦 뼲젣뿉 愿뿬븯吏留, 씠 떆뒪뀥쓽 遺덇퇏삎 怨쇰룄븳 ROS瑜 깮꽦떆耳 궛솕쟻 뒪듃젅뒪瑜 珥덈옒븯怨 寃곌뎅 留뚯꽦吏덊솚쓽 諛쒕퀝 湲곗쟾쑝濡쒖꽌 쁺뼢쓣 誘몄튇떎(Kim et al., 2012). 뵲씪꽌, 蹂 寃곌낵뒗 FGE媛 빆궛솕 諛붿씠삤 留덉빱뱾쓽 쑀쟾옄 諛쒗쁽쓣 利앷떆궡쑝濡쒖뜥 궛솕쟻 뒪듃젅뒪濡쒕꽣 꽭룷뿉 蹂댄샇 슚怨쇰 蹂댁떎怨 蹂 닔 엳떎. 利, FGE媛 H2O2濡 씤빐 쑀諛쒕맂 怨쇰룄븳 ROS쓽 깮꽦쑝濡 씤빐 HepG2 꽭룷쓽 빑 쓳異 諛 遺꾩뿴怨 媛숈 珥덇린 apoptotic body뿉 븳 蹂댄샇 슚怨쇰 蹂댁씤 寃껋씠 FGE쓽 빆궛솕 諛⑹뼱 硫붿빱땲利 湲곗옉뿉 뵲瑜 寃껋엫쓣 굹궡怨 엳떎.

紐⑤뱺 寃곌낵瑜 醫낇빀븯뿬 蹂 븣, FGE뒗 HepG2 媛꾩꽭룷뿉 H2O2濡 씤빐 쑀룄맂 궛솕쟻 뒪듃젅뒪濡쒕꽣 蹂댄샇 슚怨쇰 蹂댁떎. 씠뒗 FGE쓽 슦닔븳 빆궛솕 슚뒫쓣 利앸챸븯硫 뼢썑 媛 넀긽 蹂댄샇 諛 媛 湲곕뒫 媛쒖꽑 슚怨쇰 媛뽯뒗 媛 嫄닿컯쓽 湲곕뒫꽦 냼옱濡쒖뜥 솢슜맆 닔 엳쓣 寃껋씠씪 궗猷뚮맂떎. 諛섎㈃, 蹂 뿰援ъ뿉꽌뒗 꽭룷뿉 FGE쓽 떒룆 泥섎━뿉 쓽븳 빆궛솕 솢꽦뿉 븳 쁺뼢쓣 議곗궗븯吏 紐삵뻽뒗뜲, 異뷀썑 FGE媛 꽭룷 利앹떇 삉뒗 빆궛솕 쑀쟾옄 솢꽦솕뿉 湲곗뿬뻽뒗吏 異붽쟻씤 寃利앹씠 븘슂븷 寃껋쑝濡 궗猷뚮맂떎. 삉븳, FGE뿉 쓽븳 빆궛솕 슚怨쇨 誘명넗肄섎뱶由ъ븘쓽 궡씤꽦 떊샇 쟾떖 寃쎈줈뿉 뼱뼸寃 쁺뼢쓣 誘몄튂뒗吏뿉 븳 異붽쟻씤 湲곗쟾 뿰援ш 뜑 븘슂븷 寃껋쑝濡 깮媛곷맂떎.

ACKNOWLEDGEMENT

This research was supported by the Academic Research Fund of Hoseo University in (2021-0482).

CONFLICT OF INTEREST

The authors have declared no conflict of interest.

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