楊明美 馬月紅 李鎖 王仕忠 郭盛華

安石榴苷對(duì)中波紫外線(xiàn)誘導(dǎo)HaCaT細(xì)胞光損傷的預(yù)防作用研究

楊明美 馬月紅 李鎖 王仕忠 郭盛華

目的探討安石榴苷對(duì)中波紫外線(xiàn)(UVB)誘導(dǎo)角質(zhì)形成細(xì)胞損傷的保護(hù)機(jī)制。方法培養(yǎng)的HaCaT細(xì)胞分為空白對(duì)照組、安石榴苷組、UVB組、安石榴苷+UVB組。噻唑藍(lán)(MTT)法檢測(cè)細(xì)胞增殖能力,Hoechst/碘化丙錠(PI)染色和流式細(xì)胞儀檢測(cè)細(xì)胞凋亡,RT-PCR法測(cè)定金屬基質(zhì)蛋白酶1(MMP1)及其組織抑制因子1(TIMP1)mRNA表達(dá)水平,Western印跡檢測(cè)絲裂原活化蛋白激酶(MAPK)通路相關(guān)蛋白P38、JNK、ERK的磷酸化水平變化。結(jié)果MTT試驗(yàn)示,10～40 μmol/L安石榴苷對(duì)UVB誘導(dǎo)的HaCaT細(xì)胞損傷有較佳的預(yù)保護(hù)作用。UVB組HaCaT細(xì)胞強(qiáng)Hoechst和強(qiáng)PI雙染細(xì)胞較空白對(duì)照組增多,安石榴苷+UVB組較UVB組減少。流式細(xì)胞儀分析,UVB組凋亡細(xì)胞百分率(9.82%±0.11%)高于空白對(duì)照(1.24%±0.91%,P＜0.01),而安石榴苷(10、20、40 μmol/L)+UVB組凋亡細(xì)胞百分率(分別為6.38%±0.14%、5.24%±0.17%、3.77%±0.11%)較UVB組低,差異有統(tǒng)計(jì)學(xué)意義(均P＜0.01)。UVB組MMP1 mRNA相對(duì)表達(dá)量(12.376±0.602)高于空白對(duì)照組(1.007±0.147,P＜0.01),而TIMP1 mRNA相對(duì)表達(dá)量(0.103±0.006)低于空白對(duì)照組(1.006±0.139,P＜0.01),安石榴苷組MMP1及TIMP1 mRNA與空白對(duì)照組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(均P＞0.05)。安石榴苷預(yù)處理的HaCaT細(xì)胞經(jīng)30 mJ/cm2UVB照射后MMP1 mRNA相對(duì)表達(dá)量較UVB組降低(均P＜0.01),而TIMP1 mRNA較UVB組升高(均P＜0.01)。Western印跡示,經(jīng)UVB照射后,HaCaT細(xì)胞p-ERK、p-JNK及p-p38表達(dá)升高(均P＜0.01)。安石榴苷組HaCaT細(xì)胞p-ERK、p-JNK及p-p38表達(dá)沒(méi)有明顯改變(P＞0.05),而安石榴苷+UVB組有不同程度下降(均P＜0.01)。結(jié)論安石榴苷對(duì)UVB引起HaCaT細(xì)胞損傷有一定的預(yù)防作用。

角蛋白細(xì)胞;紫外線(xiàn);基質(zhì)金屬蛋白酶1;細(xì)胞外信號(hào)調(diào)節(jié)MAP激酶類(lèi);安石榴苷;基質(zhì)金屬蛋白酶抑制因子

石榴提取物中的活性成分有著廣泛的生物學(xué)效應(yīng),其對(duì)人體的藥理作用主要體現(xiàn)在抗氧化、抗癌、抗菌、抗病毒、抗紫外線(xiàn)損傷以及改善代謝綜合征等方面[1-3],這些作用主要與石榴中含有的安石榴苷、沒(méi)食子酸等酚類(lèi)、鞣花單寧以及尿石素等成分有關(guān)[2,4],而安石榴苷是石榴提取物中主要活性成分。我們檢測(cè)了安石榴苷對(duì)中波紫外線(xiàn)(UVB)照射后HaCaT細(xì)胞凋亡的影響,觀察安石榴苷單獨(dú)或聯(lián)合UVB照射對(duì)金屬基質(zhì)蛋白酶1(MMP1)、組織抑制因子1(TIMP1)以及絲裂原活化蛋白激酶(MAPK)通路相關(guān)分子磷酸化的影響,探討安石榴苷預(yù)防UVB誘導(dǎo)的角質(zhì)形成細(xì)胞凋亡的作用。

材料與方法

一、材料

UVB輻照儀為德國(guó)Waldmann Medizintechnik公司產(chǎn)品,波長(zhǎng)280～320 nm,照射強(qiáng)度1.35 mW/cm2。安石榴苷為美國(guó)Sigma公司產(chǎn)品,純度≥98%。HaCaT細(xì)胞株購(gòu)于中國(guó)科學(xué)院細(xì)胞庫(kù)。噻唑藍(lán)(MTT)、RPMI 1640培養(yǎng)基、0.25%胰酶、胎牛血清均為美國(guó)Gibco公司產(chǎn)品。細(xì)胞凋亡壞死試劑盒(Hoechst和PI染色)為上海碧云天生物技術(shù)有限公司產(chǎn)品。Annexin V/PI凋亡檢測(cè)試劑盒為美國(guó)BD公司產(chǎn)品。Trizol為美國(guó)Invitrogen公司產(chǎn)品。cDNA第一鏈合成試劑盒、Taq酶為美國(guó)Thermo Fisher公司產(chǎn)品。SYBR Green試劑盒為日本Toyobo公司產(chǎn)品。p-JNK(T183/Y185)、p-P38(T180/Y182)、p-ERK(T202/Y204)、Akt、JNK、P38MAPK 抗 體 均 為 美 國(guó)CST公司產(chǎn)品。β肌動(dòng)蛋白、羊抗兔二抗、全蛋白抽提試劑盒,ECL檢測(cè)試劑盒為南京凱基生物科技發(fā)展有限公司產(chǎn)品。

二、方法

1.實(shí)驗(yàn)分組及設(shè)置:安石榴苷溶于二甲基亞砜溶液(DMSO)中制成1 000 mmol/L儲(chǔ)存液,-20℃保存,臨用時(shí)用新鮮培養(yǎng)基調(diào)至終濃度為5、10、20、40、80 μmol/L,DMSO終濃度0.1%(v/v),不加安石榴苷組加入含同等濃度DMSO的培養(yǎng)基,實(shí)驗(yàn)分為空白對(duì)照組(不加入安石榴苷,不照射UVB)、安石榴苷組、UVB組、安石榴苷 +UVB組。加藥組細(xì)胞經(jīng)安石榴苷處理24 h后行UVB照射。

2.細(xì)胞培養(yǎng)及UVB照射:HaCaT細(xì)胞用含10%小牛血清的RPMI 1640培養(yǎng)基培養(yǎng)24 h后,吸去培養(yǎng)皿內(nèi)的培養(yǎng)液,磷酸鹽緩沖液(PBS)洗滌1次,加入0.5 ml或2 ml PBS,置于UVB輻照儀下,照射劑量為30 mJ/cm2,照射結(jié)束后吸去PBS,加入無(wú)血清培養(yǎng)基繼續(xù)培養(yǎng)。

3.MTT法檢測(cè)HaCaT細(xì)胞活性:將傳代細(xì)胞調(diào)整至1×104～2×104/ml后接種入96孔培養(yǎng)板,每孔150 μl,每一處理因素設(shè)立3個(gè)復(fù)孔。長(zhǎng)至60%細(xì)胞融合后對(duì)各組細(xì)胞進(jìn)行處理,再置37℃5%CO2條件下繼續(xù)培養(yǎng)24 h。每孔加入10 μl MTT溶液(5 g/L),在37℃、5%CO2下孵育2 h后棄上清液,加入DMSO 100 μl/孔溶解,置酶標(biāo)儀于490 nm波長(zhǎng)測(cè)吸光度(A值)。

4.Hoechst和PI染色:經(jīng)過(guò)步驟2處理,將細(xì)胞繼續(xù)培養(yǎng)12 h后,吸盡培養(yǎng)液,PBS漂洗3次,加1 ml細(xì)胞染色緩沖液,依次加入5 μl Hoechst染液和5 μl PI染液,混勻,4℃孵育30 min,置熒光顯微鏡下觀察。正常細(xì)胞呈現(xiàn)弱藍(lán)色熒光或伴有弱紅色熒光,凋亡或壞死細(xì)胞呈現(xiàn)強(qiáng)藍(lán)色和強(qiáng)紅色熒光。

5.Annexin V-FITC和PI標(biāo)記細(xì)胞后流式細(xì)胞儀檢測(cè):經(jīng)過(guò)步驟2處理后,細(xì)胞繼續(xù)培養(yǎng)12 h,用0.25%胰酶消化液(不含EDTA)消化后收集細(xì)胞懸液,離心,棄上清,預(yù)冷PBS漂洗3次,加100 μl緩沖液混勻后吸入流式細(xì)胞儀上樣管,依次加入5 μl Annexin和5 μl PI,搖勻,室溫避光孵育15 min,每管加入400 μl緩沖液,上機(jī)。單標(biāo)記Annexin VFITC細(xì)胞亞群處于早期凋亡,雙標(biāo)記Annexin VFITC和PI為中晚期凋亡,單標(biāo)記PI為壞死。

6.實(shí)時(shí)PCR檢測(cè):步驟2處理后,將細(xì)胞在培養(yǎng)基中繼續(xù)培養(yǎng)6 h,使用Trizol法提取細(xì)胞總RNA,逆轉(zhuǎn)錄試劑盒合成第一鏈cDNA。按SYBR Green熒光定量檢測(cè)試劑盒說(shuō)明書(shū)進(jìn)行定量檢測(cè),終體系為20 μl,引物序列及產(chǎn)物大小見(jiàn)表1。反應(yīng)條件為95℃5 min,95℃15 s,60℃30 s,72℃30 s,40個(gè)循環(huán)。所有樣品的目的基因和GAPDH進(jìn)行熒光定量 PCR 反應(yīng),分別取得 Ct值,采用 2-ΔΔCt方法分析樣品各基因的相對(duì)表達(dá)差異。

7.Western印跡:步驟2處理后,將細(xì)胞在培養(yǎng)基中繼續(xù)培養(yǎng)1 h,收集細(xì)胞前徹底吸除培養(yǎng)液,冷PBS洗1次后,加入150 μl含有蛋白酶抑制劑的細(xì)胞裂解液,在冰上操作收獲細(xì)胞,Bradford法測(cè)定蛋白濃度后進(jìn)行12%聚丙烯酰胺凝膠電泳(SDSPAGE)和轉(zhuǎn)膜(PVDF)。PVDF膜常溫下5%牛血清白蛋白(BSA)封閉1 h后,加入一抗(1∶1 000),4℃搖床過(guò)夜,磷酸鹽吐溫緩沖液(PBST)洗滌3次,每次5 min。然后加入二抗(1∶2 000),常溫下作用1 h,PBST洗滌3次,每次5 min。然后ECL發(fā)光、壓片。應(yīng)用生物圖像分析軟件Gel-Pro32分析各樣本的蛋白水平,以β肌動(dòng)蛋白作為內(nèi)參照。

表1 定量PCR檢測(cè)的引物序列及產(chǎn)物大小

結(jié) 果

一、安石榴苷對(duì)HaCaT細(xì)胞活性的影響以及對(duì)UVB的預(yù)防作用

MTT法檢測(cè)各組細(xì)胞活性,空白對(duì)照組A值為0.824±0.014,UVB 組為 0.410±0.023,5、10、20、40、80 μmol/L 安石榴苷組分別為 0.824±0.008、0.827±0.016、0.824±0.009、0.825±0.010、0.820±0.008。 5、10、20、40、80 μmol/L 安石榴苷 +UVB 組分別為0.449±0.007、0.497±0.012、0.591±0.020、0.720±0.046、0.457±0.015。與空白對(duì)照組比較,UVB組細(xì)胞活性顯著降低(P＜0.01),而安石榴苷組與空白對(duì)照組細(xì)胞活性差異無(wú)統(tǒng)計(jì)學(xué)意義;與UVB組比較,5～80 μmol/L安石榴苷 +UVB組的細(xì)胞活力有不同程度上升(均P＜0.05),其中10～40 μmol/L安石榴苷有較佳的預(yù)保護(hù)作用(均P＜0.01),故后續(xù)試驗(yàn)采用了 10、20、40 μmol/L 作為預(yù)處理濃度。

二、凋亡相關(guān)檢測(cè)

1.Hoechst和PI染色:UVB組HaCaT細(xì)胞凋亡水平較空白對(duì)照組增加。安石榴苷+UVB組凋亡細(xì)胞較UVB組減少。見(jiàn)圖1。

圖1 Hoechst和PI染色觀察HaCaT細(xì)胞凋亡 空白對(duì)照組(1a)和安石榴苷組(1b～1d)細(xì)胞呈淡藍(lán)色伴有淡紅色熒光,UVB組(1e)出現(xiàn)部分強(qiáng)藍(lán)色強(qiáng)紅色雙染細(xì)胞,安石榴苷+UVB組(1f～1h)強(qiáng)藍(lán)色強(qiáng)紅色細(xì)胞減少,凋亡水平減低。1a:空白對(duì)照;1b:10 μmol/L安石榴苷;1c:20 μmol/L 安石榴苷;1d:40 μmol/L 安石榴苷;1e:UVB;1f:10 μmol/L 安石榴苷 +UVB;1g:20 μmol/L 安石榴苷 +UVB;1h:40 μmol/L 安石榴苷+UVB

2.Annexin V-FITC和PI標(biāo)記細(xì)胞后流式細(xì)胞儀分析:凋亡細(xì)胞百分率為早期凋亡+晚期凋亡細(xì)胞群比例(即Q2+Q4群細(xì)胞百分比)。安石榴苷(10、20、40 μmol/L)組與空白對(duì)照組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(均P＞0.05),UVB組凋亡細(xì)胞百分率明顯高于空白對(duì)照組(P＜0.01),而安石榴苷(10、20、40 μmol/L)+UVB組凋亡細(xì)胞百分率與UVB組比較有不同程度降低,差異有統(tǒng)計(jì)學(xué)意義(均P＜0.01),見(jiàn)表 2。

三、RT-PCR測(cè)定 MMP1 mRNA及 TIMP1 mRNA的表達(dá)

UVB組MMP1 mRNA相對(duì)表達(dá)量明顯高于空白對(duì)照組(P＜ 0.01), 空白對(duì)照組與 10、20、40 μmol/L安石榴苷組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(F=0.42,P＞0.05)。UVB 組與 10、20、40 μmol/L安石榴苷 +UVB組MMP1 mRNA相對(duì)表達(dá)量組間比較差異有統(tǒng)計(jì)學(xué)意義(F=270.71,P＜0.01),分別與UVB組比較(Dunnett-t檢驗(yàn)),均P＜0.01,見(jiàn)表2。

UVB組TIMP1 mRNA相對(duì)表達(dá)量明顯低于空白對(duì)照組(P＜ 0.01),空白對(duì)照組與 10、20、40 μmol/L安石榴苷組組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(F=1.35,P＞ 0.05)。UVB組與10、20、40 μmol/L安石榴苷 +UVB組間比較差異有統(tǒng)計(jì)學(xué)意義(F=1053.21,P＜0.01),各組間經(jīng)Dunnett-t檢驗(yàn),均P＜0.01,見(jiàn)表2。

四、HaCaT細(xì)胞MAPK信號(hào)通路在安石榴苷保護(hù)下經(jīng)UVB的激活和變化

UVB組與空白對(duì)照組比較,HaCaT細(xì)胞總ERK、JNK及p38未見(jiàn)明顯改變,磷酸化ERK、JNK及p38表達(dá)均升高。 10、20、40 μmol/L 安石榴苷組HaCaT 細(xì)胞磷酸化ERK、JNK及p38表達(dá)無(wú)明顯改變,而10、20、40 μmol/L安石榴苷+UVB組細(xì)胞磷酸化ERK、JNK及p38表達(dá)均有不同程度下降,見(jiàn)圖2。

圖2 HaCaT細(xì)胞MAPK信號(hào)家族在安石榴苷和UVB作用下表達(dá)的變化

表2 安石榴苷及UVB照射對(duì)HaCaT細(xì)胞凋亡率、MMP1和TIMP1 mRNA表達(dá)的影響(±s)

表2 安石榴苷及UVB照射對(duì)HaCaT細(xì)胞凋亡率、MMP1和TIMP1 mRNA表達(dá)的影響(±s)

注:n=3;a:與空白對(duì)照組比較,P＞0.05;b:與空白對(duì)照組比較,P＜0.01;c:與UVB組比較,P＜0.01

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討 論

石榴提取物具有明確的光保護(hù)作用[5],安石榴苷是石榴提取物中的主要有效單體成分[6]。

本實(shí)驗(yàn)結(jié)果顯示,5～80 μmol/L安石榴苷單獨(dú)處理對(duì)細(xì)胞活性無(wú)影響,而同等濃度的安石榴苷在一定程度上可減輕UVB照射對(duì)HaCaT細(xì)胞的損傷,其中10～40 μmol/L安石榴苷有較佳的預(yù)防UVB光損傷的能力,5～40 μmol/L安石榴苷的防光能力隨濃度增高而增強(qiáng),而80 μmol/L安石榴苷防光能力較弱,推測(cè)可能安石榴苷濃度過(guò)高,影響了細(xì)胞增殖。30 mJ/cm2UVB照射可誘使HaCaT細(xì)胞發(fā)生凋亡,安石榴苷預(yù)處理可明顯降低UVB照射所致的HaCaT細(xì)胞凋亡,表明安石榴苷可減輕UVB照射對(duì)HaCaT細(xì)胞的刺激損傷,但對(duì)細(xì)胞增殖作用的影響不明顯。

TIMP與MMP的平衡在細(xì)胞外基質(zhì)的合成和降解中發(fā)揮作用,MMP1及TIMP1的表達(dá)失去平衡,可致膠原降解增加,引起光損傷的發(fā)生[7]。目前認(rèn)為,UVB可誘導(dǎo)三個(gè)MAPK亞族激活,包括ERK、JNK和P38,誘導(dǎo)角質(zhì)形成細(xì)胞凋亡[8]。本實(shí)驗(yàn)中我們發(fā)現(xiàn),30 mJ/cm2UVB照射可以顯著增加MMP1 mRNA的水平,抑制TIMP1 mRNA的產(chǎn)生,導(dǎo)致了二者間動(dòng)態(tài)平衡的改變,與Zaid等[9]的研究結(jié)果一致。30 mJ/cm2UVB組HaCaT細(xì)胞對(duì)MAPK信號(hào)家族有著不同程度的激活作用,與Rattan等[8]研究相符。安石榴苷預(yù)處理再照UVB的HaCaT細(xì)胞 MMP1、TIMP1表達(dá)量變化以及MAPK信號(hào)通路標(biāo)記物較UVB組明顯有差異,而安石榴苷組對(duì)這些指標(biāo)沒(méi)有影響。Kaur和Saraf[10]研究發(fā)現(xiàn),許多草本提取物具有很強(qiáng)的紫外線(xiàn)吸收能力;Kapoor和Saraf[11]研究證實(shí),石榴皮提取物是強(qiáng)效遮光劑,因此我們推測(cè),安石榴苷可能是通過(guò)對(duì)紫外線(xiàn)的吸收作用減少紫外線(xiàn)對(duì)細(xì)胞的損害,但是UVB致細(xì)胞凋亡的信號(hào)傳導(dǎo)通路是一個(gè)復(fù)雜的過(guò)程,TIMP、MMP和MAPK雖然起著重要作用,但并不代表全部,安石榴苷是否還有其他的途徑影響細(xì)胞的凋亡進(jìn)程還有待進(jìn)一步探討。

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2013-10-11)

(本文編輯:吳曉初)

Preventive effects of punicalagin against ultraviolet B-induced damage to human HaCaT keratinocytes

Yang Mingmei*,Ma Yuehong,Li Suo,Wang Shizhong,Guo Shenghua.*Department of Dermatology,Changzhou No.2 People's Hospital Affiliated to Nanjing Medical University,Changzhou 213003,China

Guo Shenghua,Email:czeygsh@163.com

ObjectiveTo investigate the mechanisms underlying the protection by punicalagin against ultraviolet B(UVB)-induced damage to keratinocytes.MethodsCultured human HaCaT keratinocytes were divided into several groups:blank control group receiving no treatment,punicalagin groups treated with various concentrations of punicalagin,UVB group irradiated with UVB at 30 mJ/cm2,combination groups pretreated with different concentrations of punicalagin followed by UVB radiation at 30 mJ/cm2.The concentrations of punicalagin were 5,10,20,40 and 80 μmol/L in the cell proliferation assay,10,20 and 40 μmol/L in the other assays.After additional culture for different durations,methyl thiazolyl tetrazolium(MTT)assay was performed to evaluate the proliferation of HaCaT cells,Hoechst and propidium iodide(PI)staining as well as flow cytometry to detect the apoptosis in cells,reverse transcription-PCR to quantify the mRNA expressions of matrix metalloproteinase-1(MMP1)and tissue inhibitor of metalloproteinase-1(TIMP1)in HaCaT cells,Western blot to determine the phosphorylation levels of the mitogen-activated protein kinase(MAPK)pathway-related proteins including P38,JNK and ERK.Statistical analysis was carried out byttest,one-way analysis of variance,and Dunnett'st-test.ResultsAs the MTT assay showed,punicalagin at 10-40 μmol/L showed stronger pre-protective effects against UVB-induced damage to HaCaT cells compared with punicalagin at the other concentrations.The number of cells highly positive for both Hoechst and PI staining was larger in the UVB group than that in the blank control group,but smaller in the combination groups than in the UVB group.The percentage of apoptotic cells increased significantly in the UVB group compared with the blank control group(9.82%±0.11%vs.1.24%±0.91%,P＜0.01),but decreased significantly in the three combination groups(punicalagin(10,20 and 40 μmol/L)+UVB)compared with the UVB group(6.38%±0.14%,5.24%±0.17%and 3.77%±0.11%vs.9.82%±0.11%,allP＜0.01).The expression of MMP1 mRNA was significantly higher,but that of TIMP1 mRNA was significantly lower in the UVB group than in the blank control group(bothP＜0.01),whereas no statistically significant difference was observed in the expression of MMP1 or TIMP1 mRNA between the punicalagin groups and blank control group(allP＞0.05).The pretreatment with punicalagin significantly reduced the expression level of MMP1 mRNA(P＜0.01),but elevated that of TIMP1 mRNA(P＜0.01)in the combination groups compared with the UVB group.As Western blot showed,the phosphorylation levels of P38,JNK and ERK were markedly increased in the UVB group(allP＜0.01),but experienced no significant changes in the punicalagin groups(allP＞0.05)compared with the blank control group,and decreased to different degrees in the combination groups compared with the UVB group(allP＜0.01).ConclusionPunicalagin has a pre-protective effect on UVB-induced damage to HaCaT cells.

Keratinocytes;Ultraviolet rays;Matrix metalloproteinase 1;Extracellular signal-regulated MAP kinases;Punicalagin;Matrix metalloproteinase inhibitory factor

10.3760/cma.j.issn.0412-4030.2014.07.008

213003江蘇常州,南京醫(yī)科大學(xué)附屬常州第二人民醫(yī)院皮膚科(楊明美、馬月紅、李鎖、郭盛華),中心實(shí)驗(yàn)室(王仕忠)

郭盛華,Email:czeygsh@163.com