李俊峰, 舒建昌, 楊欽河, 馬 民, 張玉佩, 顏顯欣
(1暨南大學(xué)附屬第一醫(yī)院消化科, 廣東 廣州 510630; 2暨南大學(xué)附屬第四醫(yī)院消化科, 廣東 廣州 510175; 3暨南大學(xué)中醫(yī)學(xué)院, 廣東 廣州 510632)
大葉茜草素抑制大鼠CFSC-2G細(xì)胞活化和膠原合成*
李俊峰1△, 舒建昌2▲, 楊欽河3, 馬 民3, 張玉佩3, 顏顯欣3
(1暨南大學(xué)附屬第一醫(yī)院消化科, 廣東 廣州 510630;2暨南大學(xué)附屬第四醫(yī)院消化科, 廣東 廣州 510175;3暨南大學(xué)中醫(yī)學(xué)院, 廣東 廣州 510632)
目的觀察大葉茜草素(mollugin)對大鼠肝星狀細(xì)胞系CFSC-2G活化和膠原合成的影響并探討其分子機(jī)制。方法小劑量(10 μmol/L)過氧化氫(H2O2)誘導(dǎo)CFSC-2G細(xì)胞30 min后,再加入不同濃度(0、20、40、60和120 μmol/L)的mollugin處理。MTT法檢測細(xì)胞活力,real-time PCR和Western blot法分別檢測核因子E2相關(guān)因子2(Nrf2)、血紅素加氧酶-1(HO-1)、核因子κB(NF-κB) p65、Bcl-2、Bcl-xL、Bax以及肝星狀細(xì)胞活化標(biāo)志物α-平滑肌肌動蛋白和Ⅰ型膠原蛋白的mRNA和蛋白表達(dá),并用Western blot法檢測p38絲裂原活化蛋白激酶(p38 MAPK)的磷酸化水平。結(jié)果低劑量H2O2可以誘導(dǎo)CFSC-2G細(xì)胞活化,mollugin明顯促進(jìn)p38 MAPK磷酸化,上調(diào)Nrf2和HO-1的mRNA和蛋白表達(dá),下調(diào)NF-κB p65、Bcl-2和Bcl-xL的mRNA和蛋白表達(dá),抑制H2O2誘導(dǎo)活化的CFSC-2G細(xì)胞活力和膠原合成(P<0.05)。結(jié)論Mollugin 可能通過上調(diào)Nrf2和HO-1并下調(diào)NF-κB p65和Bcl-2表達(dá),抑制CFSC-2G細(xì)胞活化和膠原合成。
大葉茜草素; CFSC-2G細(xì)胞; 細(xì)胞活力; 膠原合成
肝纖維化是慢性肝損傷-修復(fù)、細(xì)胞外基質(zhì)過度沉積和疤痕形成的病理過程,最終導(dǎo)致肝硬化或肝癌形成,是全世界肝病死亡的主要原因[1]。氧化應(yīng)激通過核因子κB (nuclear factor-κB, NF-κB)和B細(xì)胞白血病/淋巴瘤2(B-cell leukemia/lymphoma-2, Bcl-2)等信號途徑誘導(dǎo)靜止?fàn)顟B(tài)肝星狀細(xì)胞(hepatic stellate cells, HSC)活化并轉(zhuǎn)分化為表達(dá)α-平滑肌肌動蛋白(α-smooth muscle actin, α-SMA)和分泌Ⅰ型膠原蛋白(collagen type I, ColⅠ)的肌成纖維細(xì)胞樣細(xì)胞(myofibroblast-like cells, MF),是肝纖維化形成的重要機(jī)制之一[2]。核因子E2相關(guān)因子2/血紅素加氧酶1(nuclear factor E2-related factor 2/heme oxygenase-1, Nrf2/HO-1)是細(xì)胞內(nèi)抗氧化反應(yīng)的主要調(diào)控子,上調(diào)Nrf2和HO-1表達(dá)可以抑制氧化應(yīng)激和親電子物質(zhì)、保護(hù)肝組織和細(xì)胞并抑制HSC活化和膠原的合成[3-4]。大葉茜草素(mollugin)是從中藥茜草中提取的萘醌類化合物,具有抗氧化和抗炎癥等藥理作用[5],它是否可通過抗氧化應(yīng)激抑制肝星狀細(xì)胞的活化及膠原合成未見報道,本研究旨在探討mullogin對肝星狀細(xì)胞活化的作用及其機(jī)制。
1.1細(xì)胞系 大鼠肝星狀細(xì)胞系CFSC-2G購自中科院上海細(xì)胞生物研究所。
1.2試劑 大葉茜草素(廣州來普達(dá)克生物技術(shù)有限公司);MTT(Sigma);抗磷酸化p38絲裂原活化蛋白激酶(phosphorylated p38 mitogen-activated protein kinase,p-p38 MAPK)兔單克隆抗體(Cell Signaling Technology);抗Nrf2和HO-1兔多克隆抗體,抗NF-κB p65、Bcl-2、Bcl-xL、Bax、α-SMA、ColⅠ和β-actin抗體(Santa Cruz);Trizol和Taq DNA polymerase (Invitrogen);SYBR PrimeScript PCR 試劑盒(TaKaRa)。
2.1細(xì)胞系CFSC-2G的培養(yǎng) 將CFSC-2G細(xì)胞置于含10%FBS和1%青-鏈霉素的 DMEM/F12培養(yǎng)基中,37 ℃、5%CO2、飽和濕度培養(yǎng)箱中常規(guī)傳代培養(yǎng),取對數(shù)生長期細(xì)胞用于實驗。
2.2MTT法檢測mollugin對CFSC-2G細(xì)胞生長的影響 取對數(shù)生長期的CFSC-2G細(xì)胞, 以每孔1×104個細(xì)胞的濃度接種于96孔板,每組設(shè)4個復(fù)孔,孵育24 h后,棄上清液,加入10 μmol/L H2O2預(yù)處理30 min,后再加入濃度分別為0、20、40、60和120 μmol/L的mollugin處理細(xì)胞,設(shè)不加H2O2及mollugin組為空白對照組,DMSO+H2O2為陰性對照組,繼續(xù)培養(yǎng)24 h,每孔加入MTT (5 g/L) 20 μL,避光孵育4 h;棄上清液,每孔加入150 μL DMSO,應(yīng)用酶標(biāo)儀檢測各孔在570 nm波長下的吸光度(A)值,計算細(xì)胞抑制率。
抑制率(%)=(A空白對照組-A實驗組)/A空白對照組。
2.3Real-time PCR檢測mRNA表達(dá) 根據(jù)GenBank編號查到相應(yīng)蛋白質(zhì),根據(jù)編碼序列設(shè)計引物。Nrf2上游引物5′-TACTCCCAGGTTGCCCACA-3′,下游引物5′-CATCTACAAACGGGAATGTCTGC-3′;HO-1上游引物5′-AGATTGCCCAGAAAGCCCTGGAC-3′,下游引物 5′-AACTGTCGCCACCAGAAAGCTGAG-3′;Bcl-2上游引物5′-CATTTCCACGTCAACAGAATTG-3′,下游引物5′-AGCACAGGATTGGATATTCCAT-3′;Bcl-xL 上游引物5′-GAACGGCGGCTGGGATACTTTT-3′,下游引物5′-GAGAAGGGGGTGGGAGGGTAGA-3′;Bax上游引物5′-TGGCAGCTGACATGTTTTCTGAC-3′,下游引物5′-TCACCCAACCACCCTGGTCTT-3′;α-SMA上游引物5′-TGAAGAGCATCCCACCCT-3′,下游引物5′-ACGAAGGAATAGCCACGC-3′;Collagen I上游引物5′-CAGCCGCTTCACCTACAGC-3′, 下游引物5′-TTTTGTATTCAATCACTGTCTTGCC-3′;β-actin上游引物5′-GCGCGGCTACAGCTTCA-3′,下游引物5′-TCTCCTTAATGTCACGCACGAT-3′,由上海捷瑞生物工程有限公司合成。按照Trizol試劑盒說明提取細(xì)胞總RNA,采用Oligo(dT)引物和隨機(jī)引物進(jìn)行逆轉(zhuǎn)錄,以cDNA為模板進(jìn)行熒光定量PCR。反應(yīng)條件為:預(yù)變性 95 ℃ 30 s; 95 ℃ 5 s, 60 ℃ 34 s, 共40個循環(huán),用2-ΔΔCt方法進(jìn)行相對定量分析。
2.4Western blot檢測蛋白表達(dá) 收集裂解細(xì)胞BCA法定量蛋白,蛋白經(jīng)SDS-PAGE分離后,將凝膠中的蛋白轉(zhuǎn)移至PVDF膜,用封閉液封閉2 h后,加入稀釋的Ⅰ抗(p38 MAPK單克隆抗體1∶500;Nrf2和HO-1多克隆抗體1∶800;Bcl-2、Bcl-xL和Bax單克隆抗體1∶1 000;β-actin抗體1∶1 000),4 ℃下孵育過夜,次日TBS洗3遍,每次10 min,加入1∶1 000稀釋HRP標(biāo)記的Ⅱ抗,室溫下孵育2 h。加ECL顯色劑經(jīng)凝膠成像分析系統(tǒng)檢測,ImageJ軟件進(jìn)行灰度分析,以目標(biāo)蛋白與β-actin的灰度值比值作為目標(biāo)蛋白的相對量,進(jìn)行定量分析。
采用SPSS 13.0統(tǒng)計軟件進(jìn)行分析,數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示,兩組間比較采用t檢驗,多組間比較采用單因素方差分析,以P<0.05為差異有統(tǒng)計學(xué)意義。
小劑量H2O2增加了CFSC-2G細(xì)胞活力。mollugin抑制了H2O2預(yù)處理的CFSC-2G活力,差異具有統(tǒng)計學(xué)意義(P<0.05),其中120 μmol/L的mollugin抑制作用最明顯,因此選擇120 μmol/L 作為后續(xù)實驗的基礎(chǔ)濃度,見圖1。
Figure 1. The effects of mollugin at different concentrations on the viability of CFSC-2G cells treated with H2O2. Mean±SD.n=4.▲P<0.05vsblank control group;*P<0.05vsH2O2group.
圖1Mollugin抑制經(jīng)H2O2活化的CFSC-2G細(xì)胞活性
H2O2預(yù)處理組與空白對照組比較,肝星狀細(xì)胞活化標(biāo)志物α-SMA和ColⅠ、抗氧化調(diào)控因子Nrf2和HO-1以及促細(xì)胞增殖蛋白Bcl-2、Bcl-xL和Bax的mRNA表達(dá)明顯增加,表明H2O2誘導(dǎo)CFSC-2G細(xì)胞活化和增殖。 Mollugin上調(diào)Nrf2和HO-1 mRNA表達(dá),抑制Bcl-2、Bcl-xL、Bax、α-SMA和ColⅠ mRNA的表達(dá),表明mollugin 明顯抑制H2O2預(yù)處理的CFSC-2G細(xì)胞活化和膠原合成,差異有統(tǒng)計學(xué)意義(P<0.05),見圖 2。
H2O2上調(diào)Nrf2、HO-1、NF-κB p65、Bcl-2、Bcl-xL、Bax、α-SMA和ColⅠ蛋白表達(dá)。Mollugin促進(jìn)CFSC-2G細(xì)胞p38 MAPK磷酸化和Nrf2、HO-1蛋白的表達(dá),抑制NF-κB p65、Bcl-2、Bcl-xL、Bax、α-SMA和ColⅠ蛋白的表達(dá),差異具有統(tǒng)計學(xué)意義(P<0.05),見圖3~5。
Figure 2. Mollugin promoted the mRNA expression of Nrf2 and HO-1, and inhibited Bcl-2, Bcl-xL, α-SMA and ColⅠmRNA expression in H2O2-treated CFSC-2G cells. Mean±SD.n=4.*P<0.05vsblank control group;▲P<0.05vsH2O2group.
圖2Mollugin對Nrf2、HO-1、Bcl-2、Bcl-xL、Bax、α-SMA和ColⅠmRNA表達(dá)的影響
Figure 3. Mollugin promoted p-p38 MAPK, Nrf2 and HO-1 protein expression in H2O2-treated CFSC-2G cells. Mean±SD.n=4.*P<0.05vsblank control group;▲P<0.05vsH2O2group.
圖3CFSC-2G細(xì)胞p-p38MAPK、Nrf2和HO-1的蛋白表達(dá)情況
Figure 4. Mollugin inhibited NF-κB p65, Bcl-2 and Bcl-xL protein expression in H2O2-treated CFSC-2G cells. Mean±SD.n=4.*P<0.05vsblank control group;▲P<0.05vsH2O2group.
圖4大葉茜草素抑制NF-κBp65、Bcl-2和Bcl-xL蛋白表達(dá)
Figure 5. Mollugin inhibited α-SMA and ColⅠprotein expression in H2O2-treated CFSC-2G cells. Mean±SD.n=4.*P<0.05vsblank control group;▲P<0.05vsH2O2group.
圖5Mollugin抑制CFSC-2G細(xì)胞α-SMA和ColⅠ蛋白表達(dá)
Nrf2/HO-1是細(xì)胞內(nèi)抗氧化反應(yīng)的主要調(diào)控子,氧化應(yīng)激誘導(dǎo)p38 MAPK磷酸化,導(dǎo)致細(xì)胞質(zhì)內(nèi)Nrf2抑制劑Kelch樣ECH相關(guān)蛋白1(Kelch-like ECH-associated protein 1,Keap1)降解,促使Nrf2移位至細(xì)胞核內(nèi)并結(jié)合抗氧化反應(yīng)元件(anti-oxidant response element,ARE),從而介導(dǎo)HO-1表達(dá)[9]。NF-κB是一種重要的氧化還原敏感核轉(zhuǎn)錄因子,與肝星狀細(xì)胞活化和肝纖維化形成密切相關(guān)。H2O2可磷酸化NF-κB抑制蛋白(nuclear factor-κB inhibitor protein, IκB),促使NF-κB p65從NF-κB/IκBα復(fù)合體解離出來,移位至細(xì)胞核內(nèi),上調(diào) Bcl-2和Bcl-xL等表達(dá),促進(jìn)HSC增殖,抑制其凋亡[7,10]。 HO-1抑制NF-κB p65表達(dá),抑制肝纖維化形成[11]。
大葉茜草素通過激活p38 MAPK磷酸化,上調(diào)Nrf2/HO-1,下調(diào)NF-κB p65、Bcl-2和Bcl-xL表達(dá),保護(hù)神經(jīng)細(xì)胞、結(jié)腸上皮細(xì)胞免受炎性損傷、抑制腫瘤細(xì)胞增殖及誘導(dǎo)腫瘤細(xì)胞凋亡[12-14]。高濃度的ROS誘導(dǎo)HSC死亡,而低劑量的ROS誘導(dǎo)HSC活化、增殖和膠原的形成[15]。H2O2具有較長的半衰期,良好的膜通透性和細(xì)胞內(nèi)濃度高,常被作為第二信使物質(zhì)模擬細(xì)胞內(nèi)氧化應(yīng)激系統(tǒng)誘導(dǎo)HSC活化[16]。本實驗采用小劑量(10 μmol/L)H2O2預(yù)處理具有原代細(xì)胞特性的大鼠肝星狀細(xì)胞CFSC-2G,構(gòu)建氧化應(yīng)激誘導(dǎo)肝星狀細(xì)胞活化模型,觀察大葉茜草素對活化的CFSC-2G細(xì)胞生長、抗氧化調(diào)控因子Nrf2和HO-1及促細(xì)胞炎癥和增殖蛋白NF-κB p65、Bcl-2、Bcl-xL和Bax表達(dá)的影響,結(jié)果顯示小劑量H2O2可以誘導(dǎo)CFSC-2G細(xì)胞活化和增殖;大葉茜草素促進(jìn)p38 MAPK磷酸化,上調(diào)抗氧化調(diào)控因子Nrf2/HO-1,下調(diào)促細(xì)胞炎癥和增殖蛋白NF-κB p65、Bcl-2和Bcl-xL及肝星狀細(xì)胞活化標(biāo)志物α-SMA和ColⅠ mRNA和蛋白的表達(dá),抑制了H2O2預(yù)處理的CFSC-2G細(xì)胞活化和膠原的合成。
綜上所述,大葉茜草素可能通過上調(diào)Nrf2/HO-1和下調(diào)NF-κB p65、Bcl-2、Bcl-xL、Bax、α-SMA和ColⅠ,促進(jìn)p38 MAPK磷酸化表達(dá),抑制肝星狀細(xì)胞的活化和膠原合成,為預(yù)防和治療肝纖維化提供新的靶點和藥物。
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Mollugin inhibits viability and collagen synthesis of rat CFSC-2G cells
LI Jun-feng1, SHU Jian-chang2, YANG Qin-he3, MA Min3, ZHANG Yu-pei3, YAN Xian-xin3
(1DepartmentofGastroenterology,TheFirstAffiliatedHospitalofJinanUniversity,Guangzhou510630,China;2DepartmentofGastroenterology,TheFourthAffiliatedHospitalofJinanUniversity,Guangzhou510175,China;3CollegeofTraditionalChineseMedicine,JinanUniversity,Guangzhou510632,China.E-mail:ljfmed@163.com)
AIM: To investigate the effects of mollugin on the viability and collagen synthesis of rat hepatic stellate cell line CFSC-2G.METHODSThe activation of CFSC-2G cells was induced with low concentration (10 μmol/L) of hydrogen peroxide (H2O2) for 30 min in the experiment. The viability of the CFSC-2G cells after exposed to mollugin at different concentrations (0, 20, 40, 60 and 120 μmol/L) was detected by MTT assay. The mRNA and protein expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), nuclear factor-κB (NF-κB) p65, Bcl-2, Bcl-xL, Bax, and hepatic stellate cell activation markers α-smooth muscle actin (α-SMA) and collagen type I (Col Ⅰ) were detected by real-time PCR and Western blot. The phosphorylation level of p38 mitogen-activated protein kinase (p38 MAPK) was determined by Western blot.RESULTSMollugin significantly inhibited the viability and collagen synthesis of activated CSFC-2G cells induced by H2O2. The expression of Nrf2, HO-1 and Bax at mRNA and protein levels, and the phosphorylation level of p38 MAPK were promoted, while the levels of NF-κB p65, Bcl-2, Bcl-xL, α-SMA and ColⅠwere inhibited by mollugin (P<0.05).CONCLUSIONMollugin may inhibit H2O2-induced viability and collagen synthesis of the CSFC-2G cells by activating Nrf2 and HO-1, and blocking the NF-κB p65 and Bcl-2 expression.
Mollugin; CFSC-2G cells; Cell viability; Collagen systhesis
1000- 4718(2017)12- 2259- 05
2017- 05- 02
2017- 08- 08
廣東省中醫(yī)藥局資助項目(No.20141069)
▲共同第一作者
△通訊作者 Tel: 020-38688918; E-mail: ljfmed@163.com
R285.5; R575
A
10.3969/j.issn.1000- 4718.2017.12.023
(責(zé)任編輯: 林白霜, 羅 森)