韓宗萍 卓飛霞 李芳 夏瑾瑜 黃明星
【摘要】目的 探索何首烏對(duì)肝細(xì)胞脂質(zhì)沉積的影響及其機(jī)制。方法 利用永生化肝細(xì)胞系LO2,采用1 mmol/L游離脂肪酸(FFA)處理,建立脂肪肝沉積細(xì)胞模型。同時(shí)加入不同濃度的何首烏提取物處理(0、2.5、5、10 μg/mL),采用油紅O染色以觀察何首烏對(duì)LO2細(xì)胞脂肪沉積的作用;采用實(shí)時(shí)熒光定量PCR進(jìn)行實(shí)驗(yàn)組LO2細(xì)胞內(nèi)法尼酯X受體(Fxr)基因及其下游通路膽鹽輸出泵(Bsep)、小分子異源二聚體伴侶(Shp)、膽固醇7α-羥化酶1(Cyp7a1)關(guān)鍵基因的檢測(cè);并采用蛋白免疫印跡法檢測(cè)FXR及其下游通路關(guān)鍵蛋白的表達(dá)水平。結(jié)果 何首烏處理后的LO2細(xì)胞中,油紅O的染色量會(huì)隨著藥物濃度增加而增加;Fxr的mRNA表達(dá)下降,而FXR蛋白誘導(dǎo)促進(jìn)的下游基因Bsep、Shp的表達(dá)相應(yīng)下降,而其下游抑制基因Cyp7a1的表達(dá)則上升(P均<0.05)。何首烏處理的LO2肝細(xì)胞中,F(xiàn)XR的蛋白水平明顯降低,蛋白激酶C (PKC)蛋白磷酸化水平明顯降低,而PKC總蛋白表達(dá)水平無(wú)明顯改變,SHP的蛋白水平也明顯下降。結(jié)論 何首烏可通過抑制FXR-PKC/SHP信號(hào)轉(zhuǎn)導(dǎo)通路,從而促進(jìn)LO2肝細(xì)胞內(nèi)的脂質(zhì)沉積。為何首烏導(dǎo)致膽汁淤積所引起的肝損傷的治療提供新的理論依據(jù)。
【關(guān)鍵詞】何首烏;藥物性肝損傷;脂質(zhì)沉積;法尼酯X受體信號(hào)通路
The effect and mechanism of FXR signaling pathway on lipid accumulation induced by Polygonum multiflorum thumb Han Zongping, Zhuo Feixia, Li Fang, Xia Jinyu, Huang Mingxing. Department of Clinical Nutrition, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, China
Corresponding author,Huang Mingxing, E-mail: huangmx5@mail.sysu.edu.cn
【Abstract】Objective To investigate the effect and mechanism of Polygonum multiflorum thumb (PMT) on lipid accumulation in hepatic cells. Methods Immortalized hepatocyte LO2 cell line was treated with? non free fatty acids (FFA) to establish the lipid accumulation cell model. Following treatment with 0, 2.5, 5 and 10 μg/mL PMT for 24 h, the LO2 cells were assessed for lipid accumulation by Oil red O staining. The expression levels of Fxr, Bsep, Shp and Cyp7a1 were quantitatively measured by real-time quantitative fluorescence PCR. The expression levels of FXR and key proteins in the downstream signaling pathway were detected by Western blot. Results In LO2 cells treated with PMT, the intensity of Oil red O staining was increased with the increasing concentration of PMT. The expression level of Fxr mRNA was significantly down-regulated, those of Bsep and Shp mRNA in the downstream signaling pathway were down-regulated accordingly, whereas that of Cyp7a1 mRNA in LO2 cells was significantly up-regulated (all P < 0.05). In PMT-treated LO2 cells, the expression level of FXR protein was significantly down-regulated, the phosphorylated level of PKC was considerably decreased, the expression level of total PKC was not significantly changed and that of SHP protein was significantly down-regulated. Conclusions PMT may aggravate lipid accumulation via suppressing the FXR-PKC/SHP signaling pathway. These findings provide novel theoretical evidence for the treatment of liver injury caused by PMT induced-intrahepatic cholestasis.
【Key words】Polygonum multiflorum thumb; Drug-induced liver injury; Lipid accumulation;
FXR signaling pathway
近年來(lái),藥物性肝損傷的發(fā)生率隨著人均藥物處方的增加而大幅升高,引起國(guó)內(nèi)外的廣泛關(guān)注,特別是保健食品添加劑和中草藥導(dǎo)致的肝損傷占藥物性肝損傷中的比例高達(dá)16.1%[1-3]。何首烏是國(guó)內(nèi)中醫(yī)常用的藥物,為蓼科植物何首烏的干燥塊根[4-7]。目前常用于補(bǔ)肝腎中藥類保健品和護(hù)發(fā)制劑,以及大眾日常餐飲、食療保健等。但英國(guó)藥品與健康產(chǎn)品管理局曾于2006年通報(bào)過何首烏制劑的肝損傷案例,其后國(guó)內(nèi)陸續(xù)出現(xiàn)了大量關(guān)于何首烏導(dǎo)致肝損傷報(bào)道,甚至致死或肝移植案例[4-7]。歐洲和美國(guó)等藥品監(jiān)管部門均出臺(tái)了對(duì)何首烏及含何首烏的制劑進(jìn)行監(jiān)管的相關(guān)政策[3]。目前認(rèn)為何首烏引起的肝損傷中36.0%為膽汁淤積的表現(xiàn)和癥狀[4-7]。
然而,目前何首烏導(dǎo)致的肝內(nèi)膽汁淤積的確切機(jī)制尚不清楚。目前藥物性肝內(nèi)膽汁淤積的主要機(jī)制是膽汁合成和轉(zhuǎn)運(yùn)受損,這個(gè)過程受膜受體、核受體及其轉(zhuǎn)錄因子轉(zhuǎn)錄等一系列調(diào)控,其中法尼酯X受體(FXR)是常見的膽汁酸合成的生物感受器和受體[8-10]。FXR調(diào)節(jié)膽汁酸代謝的膽鹽輸出泵(Bsep)基因介導(dǎo)肝內(nèi)一價(jià)膽汁酸向膽管內(nèi)轉(zhuǎn)運(yùn),F(xiàn)XR也調(diào)控肝臟Mrp2、Ntcp等膽汁相關(guān)基因表達(dá),調(diào)控肝細(xì)胞和膽小管的膽汁酸鹽及其有機(jī)陰離子的攝取和排泄。我們推測(cè),F(xiàn)XR信號(hào)通路的抑制和功能下調(diào)導(dǎo)致膽汁淤積的發(fā)生[8-10]。
本研究通過LO2肝細(xì)胞脂質(zhì)沉積模型,初步發(fā)現(xiàn)了何首烏可通過調(diào)節(jié)FXR介導(dǎo)的信號(hào)通路而引發(fā)脂質(zhì)沉積。從而為明確何首烏藥物性膽汁淤積肝病的發(fā)病機(jī)制提供了重要的研究基礎(chǔ),為治療何首烏導(dǎo)致藥物膽汁淤積的作用靶點(diǎn)提供重要的理論參考價(jià)值。
材料與方法
一、細(xì)胞系及實(shí)驗(yàn)試劑
人永生化肝細(xì)胞系LO2購(gòu)自中國(guó)醫(yī)學(xué)科學(xué)院細(xì)胞庫(kù),凍存于實(shí)驗(yàn)室液氮罐。何首烏水提取物粉末購(gòu)自南京澤朗醫(yī)藥有限公司。試驗(yàn)開展前將何首烏粉末均勻混懸于0.5%羧甲基纖維素鈉鹽(CMC-Na)溶液。胎牛血清(FBS)、RPMI-1640培養(yǎng)基、0.25%胰蛋白酶、磷酸鹽緩沖液(PBS)均購(gòu)自Gibco公司;油紅O及游離脂肪酸(FFA)購(gòu)自Sigma公司;抗FXR抗體、抗PKC抗體、抗磷酸化PKC抗體、抗小分子異源二聚體伴侶(SHP)抗體、Actin抗體購(gòu)自Cell Signaling Technology公司,抗SHP抗體購(gòu)自Abcam公司。總RNA提取試劑TRIzol Reagent購(gòu)自Life公司。
二、實(shí)時(shí)熒光定量PCR(RT-PCR)
采用TRIzol法提取各組細(xì)胞總RNA,使用HiScript Ⅱ qRT SuperMix Ⅱ(Vazyme)逆轉(zhuǎn)錄獲取cDNA。按實(shí)時(shí)熒光定量TapMan 探針法試劑盒LightCycler 480 Probe Master (Roche, Indianapolis, IN)說明書,依次加入試劑:Probe Master,cDNA,Primer Forward/Reverse,Probe,ddH2O,混勻。95℃反應(yīng)10 min,95℃反應(yīng)15 s,60℃反應(yīng)60 s,40個(gè)循環(huán),PCR儀器自動(dòng)收集熒光信息,獲取相關(guān)基因的Ct值,計(jì)算得出目的基因的相對(duì)含量。
三、蛋白免疫印跡法
根據(jù)何首烏不同濃度的處理設(shè)為實(shí)驗(yàn)組,未加何首烏處理為對(duì)照組。收集各實(shí)驗(yàn)組及對(duì)照組細(xì)胞,采用裂解液消化細(xì)胞獲得總蛋白裂解液樣品,各組蛋白BCA法定量后,進(jìn)行SDS-PAGE;電泳完成后,將3張3M濾紙和1張PVDF膜,浸泡在甲醇去離子水中5 min后,與專用濾紙和纖維墊浸泡于1×轉(zhuǎn)膜緩沖液中;剝下凝膠,去掉濃縮膠部分,并把濾紙和PVDF膜裁成凝膠大小;按照三明治夾心法進(jìn)行轉(zhuǎn)膜,300 mA轉(zhuǎn)膜2 h;立刻取出PVDF膜,然后用蛋白封閉液封閉1 h;采用待測(cè)蛋白的一抗室溫孵育PVDF膜3 h后,TBST洗膜3次,每次15 min;采用相對(duì)應(yīng)的二抗孵育PVDF膜1 h后,TBST洗膜3次,每次15 min;于暗房,避光采用ECL顯色液顯色底片,并采用X線片壓片發(fā)光顯影定影而獲得數(shù)據(jù)片。
四、統(tǒng)計(jì)學(xué)處理
采用SPSS 25.0進(jìn)行統(tǒng)計(jì)學(xué)分析,正態(tài)分布計(jì)量資料以? 表示,比較采用成組t檢驗(yàn)(雙側(cè)檢驗(yàn)),P < 0.05為差異有統(tǒng)計(jì)學(xué)意義。
結(jié)果
一、何首烏對(duì)LO2肝細(xì)胞脂肪沉積的促進(jìn)作用
利用永生化肝細(xì)胞系LO2,采用FFA(1 mmol/L)處理24 h,建立脂肪肝沉積細(xì)胞模型,同時(shí)加入不同濃度的何首烏提取物進(jìn)行處理(0、2.5、5、10 μg/mL),24 h后用油紅O染色以觀察何首烏對(duì)LO2細(xì)胞脂肪沉積的作用。結(jié)果顯示,與對(duì)照組相比,何首烏處理后的LO2細(xì)胞中,油紅O的染色量會(huì)隨著藥物濃度增加而增加(圖1),提示何首烏可促進(jìn)LO2細(xì)胞中脂肪的沉積。
二、何首烏導(dǎo)致脂肪肝沉積細(xì)胞模型中Fxr及Fxr下游基因的mRNA表達(dá)情況
研究中收集2組何首烏提取物(0、5 μg/ml)
處理過的脂肪肝沉積LO2細(xì)胞模型(1 mmol/L FFA處理24 h)進(jìn)行了mRNA芯片檢測(cè),發(fā)現(xiàn)FXR通路的相關(guān)基因變化比較明顯,進(jìn)而采用RT-PCR進(jìn)行Fxr及其下游基因的驗(yàn)證檢測(cè)。結(jié)果顯示,F(xiàn)xr的mRNA表達(dá)明顯下降,而FXR蛋白誘導(dǎo)促進(jìn)的下游基因Bsep、Shp的表達(dá)相應(yīng)下降,而其下游抑制基因膽固醇7α-羥化酶1(Cyp7a1)的表達(dá)則上升(圖2)。由此可見LO2細(xì)胞中何首烏誘導(dǎo)的脂質(zhì)積累,可能是通過抑制FXR信號(hào)通路相關(guān)基因的表達(dá)所致。
三、何首烏通過FXR通路的介導(dǎo)促進(jìn)了LO2肝細(xì)胞的脂肪沉積
為了探索FXR通路在何首烏介導(dǎo)的LO2肝細(xì)胞的脂肪沉積作用,使用不同濃度何首烏提取物(2.5、5、10 μg/ml)與FFA共同處理LO2細(xì)胞24 h后,采用蛋白免疫印跡法檢測(cè)FXR及其下游通路相關(guān)蛋白的表達(dá)水平。結(jié)果顯示,經(jīng)何首烏處理后,F(xiàn)XR的蛋白水平明顯降低,蛋白激酶C(PKC)蛋白磷酸化水平明顯降低,而PKC的總蛋白表達(dá)水平無(wú)明顯改變,同時(shí)SHP的蛋白水平也明顯降低(圖3)。此結(jié)果提示何首烏能通過抑制FXR-PKC/SHP信號(hào)轉(zhuǎn)導(dǎo)通路,促進(jìn)LO2肝細(xì)胞內(nèi)脂肪的沉積。
討論
近年來(lái)關(guān)于何首烏肝毒性報(bào)道頻出,給臨床安全性和合理用藥帶來(lái)挑戰(zhàn)。國(guó)家食品藥品監(jiān)督管理局(SFDA)不良反應(yīng)監(jiān)測(cè)中心報(bào)告提示何首烏及其相關(guān)制劑的不良反應(yīng)報(bào)告超過2萬(wàn)份,主要為肝損害的不良反應(yīng)。SFDA不良反應(yīng)監(jiān)測(cè)中心監(jiān)測(cè)到的數(shù)據(jù)只反映了中藥肝毒性的一部分,在中醫(yī)處方、老百姓自行服用的情況下發(fā)生肝損傷更嚴(yán)重[11-15]。國(guó)外非常重視何首烏引起肝毒性,2012年9月美國(guó)國(guó)家醫(yī)學(xué)圖書館發(fā)布的LiverTox(Clinical and Research Information on Drug-induced Liver Injury)數(shù)據(jù)庫(kù)中收錄約600種具有肝損傷的西藥和中草藥,其中何首烏作為一個(gè)專題被收錄,數(shù)據(jù)庫(kù)專門收錄了何首烏及其制劑肝損傷的報(bào)道,其中何首烏導(dǎo)致肝內(nèi)膽汁淤積的病例數(shù)據(jù)最多見,其治療效果欠佳,是目前臨床上亟需解決的問題之一[11-14]。
FXR是核受體超家族成員[16-18]。FXR通過調(diào)控一系列膽汁酸相關(guān)基因的表達(dá),在膽汁酸合成、轉(zhuǎn)運(yùn)和代謝中發(fā)揮了重要作用。FXR主要通過2條通路激活,其中第一條即在肝細(xì)胞內(nèi)激活,第二條通路在小腸上皮細(xì)胞激活[16-18]。膽汁酸本身可激活腸上皮細(xì)胞的FXR,F(xiàn)XR是成纖維細(xì)胞生長(zhǎng)因子(FGF)19(在小鼠為FGF15)的上游轉(zhuǎn)錄因子,可啟動(dòng)腸上皮細(xì)胞大量表達(dá)FGF19。腸腔內(nèi)FGF19釋放到循環(huán)血液中,通過門脈循環(huán)到達(dá)肝臟后,F(xiàn)GF19結(jié)合和激活肝FGF受體4并與b-Klotho組成受體復(fù)合物后進(jìn)一步激活c-jun氨基末端激酶與PKC/SHP等信號(hào)轉(zhuǎn)導(dǎo)通路,來(lái)抑制CYP7A1等因子的合成,從而抑制膽汁酸合成,由此也構(gòu)成了膽汁酸的負(fù)反饋調(diào)節(jié)通路[16-18]。
本研究團(tuán)隊(duì)長(zhǎng)期致力于FGF15/19信號(hào)軸介導(dǎo)藥物性肝損傷的臨床和基礎(chǔ)研究,發(fā)現(xiàn)FGF15具有促進(jìn)小鼠肝細(xì)胞再生的作用,但其作用不依賴于膽汁酸。此外,本團(tuán)隊(duì)也通過研究揭示了酒精性肝損傷的主要原因是腸道Fgf15缺失,并證明了Fgf15缺失后腸道的通透性增加,從而加重肝損傷[19-21]。
本研究從體外細(xì)胞試驗(yàn)發(fā)現(xiàn)何首烏對(duì)LO2肝細(xì)胞脂肪沉積的促進(jìn)作用,LO2細(xì)胞中何首烏誘導(dǎo)的脂質(zhì)積累,其機(jī)制可能通過抑制FXR信號(hào)通路相關(guān)基因的表達(dá)所致。何首烏能通過抑制FXR-PKC/SHP信號(hào)轉(zhuǎn)導(dǎo)通路,促進(jìn)LO2肝細(xì)胞內(nèi)脂肪的沉積。因此,本研究提示何首烏可導(dǎo)致肝細(xì)胞內(nèi)FXR基因表達(dá)下調(diào),F(xiàn)XR基因膽汁轉(zhuǎn)運(yùn)信號(hào)通路受到抑制,進(jìn)一步介導(dǎo)何首烏抑制膽汁轉(zhuǎn)運(yùn)信號(hào)通路,促進(jìn)肝細(xì)胞內(nèi)脂質(zhì)沉積。由此,揭示了何首烏導(dǎo)致藥物性膽汁淤積致病分子機(jī)制,為臨床上治療膽汁淤積所導(dǎo)致的肝損傷提供新的理論依據(jù)。
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(收稿日期:2021-06-06)
(本文編輯:楊江瑜)