茵陳蒿湯對肝纖維化大鼠肝組織內(nèi)質(zhì)網(wǎng)應(yīng)激caspase-12通路的影響

李木松　張貴賢　魏媛媛　劉震霞

(保定市傳染病醫(yī)院肝五A科,保定,071000)

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茵陳蒿湯對肝纖維化大鼠肝組織內(nèi)質(zhì)網(wǎng)應(yīng)激caspase-12通路的影響

李木松張貴賢魏媛媛劉震霞

(保定市傳染病醫(yī)院肝五A科,保定,071000)

目的:觀察茵陳蒿湯對膽汁瘀積性肝纖維化大鼠肝組織內(nèi)質(zhì)網(wǎng)應(yīng)激caspase-12通路的影響,探討茵陳蒿湯抗膽汁瘀積性肝纖維的作用機(jī)制。方法:將35只SD大鼠分為假手術(shù)組和造模組,采用膽總管結(jié)扎法復(fù)制膽汁瘀積性肝纖維化大鼠模型,1周后將造模組動(dòng)物分為模型組和中藥組,中藥組予以3.5 g/kg茵陳蒿湯灌胃,4周后處死動(dòng)物,HE、Masson染色觀察肝臟組織病理學(xué)變化;Western blot法檢測各組大鼠肝臟GRP78、eIF2α和Caspase-12蛋白表達(dá)變化。結(jié)果:肝臟組織病理學(xué)變化顯示模型組大鼠肝纖維化程度較假手術(shù)組明顯增加(P<0.05),中藥組大鼠肝纖維化程度較模型組減輕(P<0.05),但仍重于假手術(shù)組。Western blot結(jié)果顯示,模型組肝臟GRP78、eIF2α和Caspase-12蛋白的表達(dá)與假手術(shù)組相比顯著升高(P<0.01),中藥組較模型組eIF2α和Caspase-12明顯降低(P<0.01)。結(jié)論:抑制eIF2α和Caspase-12的活化,從而減少肝細(xì)胞凋亡,進(jìn)而抑制內(nèi)質(zhì)網(wǎng)應(yīng)激在膽汁瘀積性肝纖維化中的促進(jìn)作用,是茵陳蒿湯抗膽汁瘀積性肝纖維化的作用機(jī)制之一。

膽汁瘀積性肝纖維化;茵陳蒿湯;內(nèi)質(zhì)網(wǎng)應(yīng)激;細(xì)胞凋亡;Caspase-12

肝纖維化的發(fā)生發(fā)展與肝細(xì)胞的凋亡密切相關(guān),抑制肝細(xì)胞凋亡可有效地防止肝纖維化的進(jìn)展[1-2]。內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic Reticulum Stress,ERS)與細(xì)胞凋亡及器官纖維化的發(fā)生發(fā)展關(guān)系密切[3-4]。Caspase-12是內(nèi)質(zhì)網(wǎng)特有的Caspase,Caspase-12的活化可誘發(fā)凋亡蛋白酶級聯(lián)反應(yīng)。茵陳蒿湯有較好的保護(hù)肝細(xì)胞、抗肝纖維化作用[5-6],本研究通過觀察茵陳蒿湯對肝組織內(nèi)質(zhì)網(wǎng)應(yīng)激Caspase-12通路的影響,進(jìn)一步闡明茵陳蒿湯防治肝纖維化的作用機(jī)制。

1　材料與方法

1.1動(dòng)物及試劑35只SPF級雄性SD大鼠,體質(zhì)量(180±20) g,由河北醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心提供。HE染液和Masson染液購自福州邁新生物技術(shù)公司;細(xì)胞組織裂解液RIPA和蛋白酶抑制劑PMSF購自上海碧云天公司;丙烯酰胺和甲叉雙丙烯酰胺購自美國Sigma公司;GRP78、eIF2α和Caspase-12和β-actin抗體購自美國Santa Cruz公司。

1.2藥品制備茵陳蒿湯《傷寒論》由茵陳18 g,梔子12 g,大黃(去皮)6 g,根據(jù)《中藥藥理研究方法學(xué)》大鼠劑量=人劑量×35(人轉(zhuǎn)換因子)/6(大鼠轉(zhuǎn)換因子)計(jì)算出人鼠等效劑量為3.5 g/kg。按傳統(tǒng)工藝制備成水煎劑,減壓濃縮至0.35 g/mL備用。

1.3實(shí)驗(yàn)方法將實(shí)驗(yàn)動(dòng)物隨機(jī)分為造模組(25只)和假手術(shù)組(10只),采用膽管結(jié)扎法復(fù)制膽汁瘀積性肝纖維化模型。術(shù)前禁食不禁水12 h,用鹽酸氯胺酮肌肉注射進(jìn)行麻醉(100 mg/kg),常規(guī)皮膚消毒后,沿腹部正中線切開皮膚以充分暴露膽總管,于膽總管遠(yuǎn)近端分別結(jié)扎后從中間離斷,關(guān)腹;假手術(shù)組僅開腹暴露并游離膽總管但不結(jié)扎,關(guān)腹。于造模1周后,造模組隨機(jī)分為模型組(11只),中藥組(各12只)。于4周末處死動(dòng)物。按造模時(shí)方法鹽酸氯胺酮麻醉,腹主動(dòng)脈采血,分離血清,-80 ℃冰箱保存。取全肝臟稱重后,于肝右葉固定位置切取1.0 cm×1.0 cm大小2塊肝組織,中性甲醛溶液固定,石蠟包埋后行HE、Masson染色;其余肝組織于液氮中速凍后轉(zhuǎn)移至-80 ℃冰箱保存?zhèn)溆谩?/p>

1.4病理學(xué)檢查將甲醛固定后的肝組織行石蠟包埋,蘇木精-伊紅(HE)染色和Masson三色膠原纖維染色,400倍光鏡下觀察肝組織病理變化。肝纖維化程度參照文獻(xiàn)[7],每個(gè)“+”記1分,總積分越高,表明肝臟纖維化程度及病理損害越重。

1.5Western blot法檢測肝組織GRP78、eIF2α和Caspase-12蛋白表達(dá)在緩沖液樣品中將細(xì)胞裂解進(jìn)行SDS-PAGE檢測。聚丙烯酰胺凝膠電泳后,電轉(zhuǎn)移法將蛋白轉(zhuǎn)移至PVDF濾膜上。用5%的脫脂牛奶封閉3 h,將膜用兔抗大鼠單克隆培養(yǎng)。免疫反應(yīng)性蛋白用增強(qiáng)的化學(xué)發(fā)光試劑顯影。ECL顯色,Quantity One軟件測定分析結(jié)果。采用β-actin作為內(nèi)參照,以光密度比值表示該蛋白相對表達(dá)水平。

2　結(jié)果

2.1一般情況模型組大鼠體質(zhì)量較假手術(shù)組增長緩慢,2 d后開始出現(xiàn)小便發(fā)黃,7 d后在尾巴、耳朵、皮毛出現(xiàn)黃染,膽總管漸進(jìn)性擴(kuò)張,肝臟體積增大,色澤黃染,第3～4周肝臟質(zhì)地逐漸變硬,4周表面呈桔皮樣,彈性明顯降低。有4只動(dòng)物在造模后第1周和第4周因消化道出血和腹水死亡。

2.2肝組織病理學(xué)變化HE和Masson染色顯示,假手術(shù)組大鼠肝細(xì)胞索以肝小葉中央靜脈為中心呈放射狀排列,肝小葉結(jié)構(gòu)清晰,有少量炎性細(xì)胞浸潤,無明顯壞死,無膠原纖維增生;模型組肝索排列紊亂,匯管區(qū)纖維結(jié)締組織大量增生,增生的膽管上皮細(xì)胞分化為小膽管,與纖維組織形成間隔,肝組織結(jié)構(gòu)破壞嚴(yán)重,大量膠原組織沉積形成纖維間隔,可見假小葉形成;中藥組可見增生的膽管上皮細(xì)胞周圍有纖維組織增生,匯管區(qū)面積擴(kuò)大,較模型組炎性細(xì)胞浸潤和肝細(xì)胞壞死減少(P<0.05)。見圖1,圖2,表1。

圖1　HE染色檢測各組大鼠肝組織病理改變

圖2　Masson三色染色檢測各組大鼠肝組織病理改變

組別只數(shù)肝纖維化程度-++++++假手術(shù)組1010000模型組110344中藥組101711

圖3　茵陳蒿湯對膽汁瘀積性肝纖維化大鼠內(nèi)質(zhì)網(wǎng)應(yīng)激Caspase-12通路的影響

2.3Western blot法檢測茵陳蒿湯對膽汁瘀積性肝纖維化大鼠內(nèi)質(zhì)網(wǎng)應(yīng)激Caspase-12通路的影響模型組大鼠與假手術(shù)組相比肝臟GRP78、eIF2α和Caspase-12蛋白表達(dá)明顯增多差異有統(tǒng)計(jì)學(xué)意義(P<0.01),中藥組較模型組eIF2α和Caspase-12明顯降低(P<0.01)。見圖3,表2。

表2　茵陳蒿湯對膽汁瘀積性肝纖維化大鼠內(nèi)質(zhì)網(wǎng)應(yīng)激Caspase-12通路的影響

注:**P<0.01,與假手術(shù)組比較;△△P<0.01,與模型組比較。

3　討論

內(nèi)質(zhì)網(wǎng)參與新生蛋白質(zhì)的合成、儲存、加工修飾和運(yùn)輸。錯(cuò)誤折疊蛋白在內(nèi)質(zhì)網(wǎng)腔過多聚積從而引發(fā)ERS[7]。ESR有利于恢復(fù)細(xì)胞內(nèi)環(huán)境穩(wěn)態(tài),是一種重要的細(xì)胞自我防御機(jī)制,但高強(qiáng)度或持續(xù)的ERS則可通過激活下游Caspase-12的活化,繼而誘發(fā)凋亡蛋白酶級聯(lián)反應(yīng)[8]。研究表明,ERS誘導(dǎo)的肝細(xì)胞凋亡參與了肝纖維化的發(fā)生機(jī)制[9]。葡萄糖調(diào)節(jié)蛋白-78(Glucose-regulated Protein,GRP78)是內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)的感受器,應(yīng)激狀態(tài)下GRP78大量表達(dá),與未折疊蛋白結(jié)合,恢復(fù)蛋白質(zhì)的正確構(gòu)象。因此,GRP78被認(rèn)為是ERS的標(biāo)志性分子。GRP78也通過磷酸化真核細(xì)胞起始因子2α(Eukaryotic Initiationfactor2α,eIF2α)抑制細(xì)胞蛋白質(zhì)合成,并通過活化Caspase-12,發(fā)生凋亡蛋白酶級聯(lián)反應(yīng)[10]。Caspase-12位于內(nèi)質(zhì)網(wǎng)腔內(nèi),是內(nèi)質(zhì)網(wǎng)特有的Caspase,也是ERS介導(dǎo)細(xì)胞凋亡的一個(gè)主要調(diào)節(jié)因子[11],其活化是不依賴線粒體凋亡途徑和細(xì)胞色素C來完成。ERS與細(xì)胞凋亡及器官纖維化密切相關(guān)[12-13]。

本研究發(fā)現(xiàn),通過膽管結(jié)扎誘導(dǎo)的膽汁瘀積性肝纖維化模型大鼠肝組織結(jié)構(gòu)紊亂,大量纖維結(jié)締組織增生,膠原纖維形成纖維間隔,可見假小葉形成,纖維化程度積分明顯增高,證明模型復(fù)制成功[14-15]。內(nèi)質(zhì)網(wǎng)應(yīng)激的標(biāo)志性蛋白GRP78表達(dá)增強(qiáng),以及eIF2α和Caspase-12表達(dá)明顯增加,提示在膽汁瘀積性肝纖維過程中發(fā)生了內(nèi)質(zhì)網(wǎng)應(yīng)激,從而導(dǎo)致肝細(xì)胞凋亡的發(fā)生。茵陳蒿湯由茵陳蒿、梔子和大黃組成,首載于《傷寒論》,有保肝利膽退黃等功效[16],為臨床上治療濕熱內(nèi)蘊(yùn)型肝膽疾病的首選方劑,本實(shí)驗(yàn)研究提示,茵陳蒿湯可明顯改善膽汁瘀積性肝纖維化大鼠肝纖維化程度,提示該方具有抗膽汁瘀積所造成的肝纖維化作用。膽汁瘀積性肝纖維化大鼠肝臟GRP78、eIF2α和Caspase-12蛋白表達(dá)明顯增加,用藥后eIF2α和Caspase-12明顯降低,提示抑制eIF2α的表達(dá)和Caspase-12的活化,從而減少肝細(xì)胞凋亡,進(jìn)而抑制ERS在膽汁瘀積性肝纖維化中的促進(jìn)作用,是茵陳蒿湯抗膽汁瘀積性肝纖維化的作用機(jī)制之一。

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(2015-09-21收稿責(zé)任編輯：王明)

Effects of Yinchenhao Decoction on Endoplasmic Reticulum Stress Pathway of Caspase-12 in Hepatic Fibrosis Rats

Li Musong, Zhang Guixian, Wei Yuanyuan, Liu Zhenxia

(Liver diseases five A department of Baoding Infectious Disease Hospital, Baoding 071000, China)

Objective:To observe the effects of Yinchenhao decoction on endoplasmic reticulum stress caspase-12 pathway of cholestasis liver fibrosis rat and its action mechanism on anti bile siltation liver fiber.Methods:Thirty five SD rats were divided into sham operation group and the duplicate model group. The common bile duct ligation method was used to replicate cholestasis liver fibrosis in rats. After 1 week of building, the rats were divided into model group and TCM group. TCM group was given3.5 g/kg Yinchenhao decoction by intragastric administration. The rats were killed after four weeks. HE and Masson staining method were used to observe the changes of the liver tissue pathology, and Western blot method was used to detect GRP78, eIF2 alpha and Caspase-12 protein expression changes in liver tissue of each group.Results:The liver histopathology examination showed that the liver fibrosis rat model was duplicated successfully. The degree of liver fibrosis in the TCM group was lower than that of the model group (P<0.05), but it was more severe than that of the Sham operation group. Blot Western showed that the expression of Caspase-12, eIF2α and GRP78 in the model group was significantly higher than those of the Sham operation group (P<0.01). Besides, eIF2α and Caspase-12 significantly decreased in the TCM group (P<0.01).Conclusion:Inhibiting eIF2α and caspase-12 activation, reducing apoptosis of liver cells, thereby inhibiting the promoting effect of ERS in the liver fibrosis of bile stasis is one possible action mechanism of Yinchenhao decoction in removing bile stasis liver fibrosis.

Cholestatic liver fibrosis; Yinchenhao decoction; Endoplasmic reticulum stress; Apoptosis; Caspase-12

李木松(1976.12—),男,本科,主治醫(yī)師,肝五科主任,研究方向:中西醫(yī)結(jié)合防治肝病,E-mail:602366035@qq.com

R285.5

A doi：10.3969/j.issn.1673-7202.2016.09.050