劉珍財,趙海霞,陳 茜,劉光耀,袁 丁,張長城
·論著·
·中醫(yī)·中西醫(yī)結(jié)合研究·
五子衍宗方對環(huán)磷酰胺致肝損傷的保護(hù)作用及其機(jī)制研究
劉珍財,趙海霞,陳 茜,劉光耀,袁 丁,張長城*
目的研究五子衍宗方(WP)對環(huán)磷酰胺(CTX)致肝損傷的保護(hù)作用及其機(jī)制,以期為臨床治療提供借鑒。方法2015年9月—2016年6月,將8周齡SPF級BALB/c雄性小鼠40只隨機(jī)分為正常對照組、模型對照組、WP低劑量組、WP高劑量組,每組10只。應(yīng)用WP提取黃酮類化合物并將其溶于1%羧甲基纖維素鈉(CMC-Na),WP低劑量組和WP高劑量組提前3 d分別給予黃酮類化合物140、280 mg/kg,灌胃給藥30 d,1次/d;從第3天開始,除正常對照組腹腔注射等量0.9%氯化鈉溶液外,其余各組均腹腔注射CTX 50 mg/kg造模,每隔2 d注射1次,共10次。模型對照組血清丙氨酸氨基轉(zhuǎn)移酶(ALT)、天冬氨酸氨基轉(zhuǎn)移酶(AST)水平與正常對照組比較有統(tǒng)計學(xué)差異提示造模成功。最后一次注射CTX 12 h后眼球取血、脫臼處死小鼠。檢測血清ALT、AST水平;取肝臟,計算肝臟系數(shù),HE染色法觀察肝組織形態(tài)學(xué)表現(xiàn),檢測肝組織中超氧化物歧化酶(SOD)、丙二醛(MDA)水平。結(jié)果模型對照組小鼠血清ALT、 AST水平高于正常對照組(P<0.01);WP低劑量組、WP高劑量組小鼠血清ALT、 AST水平低于模型對照組(P<0.05)。模型對照組肝臟系數(shù)高于正常對照組、WP低劑量組、WP高劑量組(P<0.01)。模型對照組小鼠肝小葉和肝索排列混亂,肝細(xì)胞腫脹明顯并伴有壞死,胞質(zhì)疏松。WP低劑量組、WP高劑量組肝小葉排列較整齊,肝細(xì)胞腫脹和胞質(zhì)疏松程度較模型對照組明顯減輕,肝細(xì)胞形態(tài)均勻,排列規(guī)整。模型對照組肝組織SOD水平低于正常對照組、WP低劑量組、WP高劑量組,MDA水平高于正常對照組、WP低劑量組、WP高劑量組(P<0.05)。結(jié)論WP對CTX所致肝損傷具有良好的保護(hù)作用,其機(jī)制可能與提高機(jī)體的抗氧化能力,清除體內(nèi)自由基有關(guān)。
藥物性肝損傷;環(huán)磷酰胺;藥理作用分子作用機(jī)制;五子衍宗方
劉珍財,趙海霞,陳茜,等.五子衍宗方對環(huán)磷酰胺致肝損傷的保護(hù)作用及其作用機(jī)制研究[J].中國全科醫(yī)學(xué),2017,20(27):3426-3430.[www.chinagp.net]
LIU Z C,ZHAO H X,CHEN Q,et al.Protective effect and mechanism of Wuzi-Yanzong prescription against the liver injury induced by cyclophosphamid[J].Chinese General Practice,2017,20(27):3426-3430.
環(huán)磷酰胺(CTX)是目前臨床上廣泛應(yīng)用的烷化劑類抗癌藥物,也是臨床上常用的免疫抑制劑,常用于治療白血病、淋巴瘤和乳腺癌等多種惡性腫瘤與非惡性腫瘤。因CTX是細(xì)胞周期非特異性藥物,在對腫瘤細(xì)胞產(chǎn)生殺傷作用的同時,也會對正常細(xì)胞和組織產(chǎn)生毒性作用。CTX的代謝產(chǎn)物能夠和體內(nèi)生物大分子如蛋白質(zhì)、脂質(zhì)、DNA結(jié)合,導(dǎo)致DNA損傷,從而引起肝損傷、骨髓抑制、生殖毒性等毒副作用。CTX的臨床應(yīng)用也因此受到限制,故減少CTX的毒副作用尤為重要。已有研究結(jié)果表明,CTX可致肝損傷,并且可利用CTX來建立實驗性肝損傷模型[1-2]。
五子衍宗方(WP)由枸杞子、五味子、菟絲子、覆盆子、車前子組成?,F(xiàn)代藥理學(xué)研究顯示,WP具有抗氧化、抗衰老、增強(qiáng)免疫功能等功效,如WP可明顯提高小鼠睪丸的抗氧化損傷能力,改善精子質(zhì)量[3-4],增強(qiáng)免疫低下小鼠脾臟功能,提高機(jī)體免疫功能[5]。有研究顯示,WP對乙醇性肝損傷、脂肪肝等有一定的保護(hù)作用[6]。本課題組前期研究也發(fā)現(xiàn),WP對CTX所致肝損傷具有保護(hù)作用[7-8],但其有效成分及其作用機(jī)制目前尚不清楚。黃酮類化合物是WP的主要活性成分之一,易溶于乙酸乙酯中。多項研究表明,天然黃酮類化合物對肝臟有保護(hù)作用[9-10]。因此,本課題組利用CTX建立肝損傷模型,進(jìn)一步探討WP對CTX所致肝損傷的保護(hù)作用及其機(jī)制。
1.1 材料
1.1.1 實驗動物 2015年9月—2016年6月,選取8周齡SPF級Balb/C雄性小鼠40只,體質(zhì)量22~25 g,由湖北省實驗動物中心提供〔動物許可證號:SCXK(鄂)2008-0005〕,分籠飼養(yǎng),環(huán)境溫度(20±2)℃,相對濕度55%~65%,12 h光照,嚙齒類動物飼料喂養(yǎng),自由飲水,適應(yīng)性飼養(yǎng)1個月。
1.1.2 藥品與試劑 枸杞子、五味子、菟絲子、覆盆子、車前子,均購自本草堂大藥房;CTX,江蘇恒瑞醫(yī)藥股份有限公司;丙氨酸氨基轉(zhuǎn)移酶(ALT)、天冬氨酸氨基轉(zhuǎn)移酶(AST)、超氧化物歧化酶(SOD)、丙二醛(MDA)測定試劑盒購自南京建成生物工程研究所。
1.2 方法
1.2.1 黃酮類化合物的提取 稱取4副藥(1副藥:枸杞子40 g,五味子5 g,菟絲子40 g,覆盆子20 g,車前子10 g),總重460 g,加4 000 ml 75%乙醇浸泡過夜,第2天回流提取3次,第1次1.5 h,第2次1.5 h,第3次1.0 h;合并提取液,旋轉(zhuǎn)蒸發(fā)儀(75 ℃)濃縮至1 000 ml;石油醚脫脂3次,第1次提取液與石油醚體積比1∶1,根據(jù)顏色調(diào)整比例,第2次提取液與石油醚體積比1∶1,第3次提取液與石油醚體積比2∶1。出現(xiàn)乳化層,超聲4 min后再分液,棄石油醚層,之后與乙酸乙酯以1∶1比例萃取3次;取上層溶液,蒸餾回收乙酸乙酯得到黃酮類化合物,干燥后計算提取率為0.729%。
1.2.2 肝損傷模型 將小鼠隨機(jī)分為4組:正常對照組、模型對照組、WP低劑量組和WP高劑量組,每組10只。WP低劑量組和WP高劑量組提前3 d分別給予黃酮類化合物140、280 mg/kg〔黃酮類化合物溶于1%羧甲基纖維素鈉(CMC-Na)〕,灌胃給藥30 d,1次/d;從第3天開始,除正常對照組腹腔注射等量0.9%氯化鈉溶液外,其余各組均腹腔注射CTX 50 mg/kg造模,每隔2 d注射1次,共10次。模型對照組血清ALT、AST水平與正常對照組有統(tǒng)計學(xué)差異提示造模成功。
1.2.3 ALT、AST水平及小鼠肝臟系數(shù)測定 小鼠末次給藥12 h后(期間禁食不禁水),稱重,眼球取血后靜置30 min,4 000 r/min低溫離心10 min(離心半徑10 cm),取上清液,按ALT、AST測定試劑盒要求測定ALT、AST水平。眼球取血后脫臼處死小鼠,取出肝臟,0.9%氯化鈉溶液洗凈后用濾紙擦去表面水分并稱重,計算肝臟系數(shù)〔肝臟系數(shù)=肝臟質(zhì)量(mg)/體質(zhì)量(g)〕。
1.2.4 肝組織形態(tài)學(xué)表現(xiàn) 于每只小鼠肝左葉同一部位取相同大小的肝組織,10%甲醛固定,石蠟包埋,切片,HE染色,光學(xué)顯微鏡(×200)觀察小鼠肝組織形態(tài)變化。
1.2.5 SOD、MDA水平測定 稱取一定量的肝組織,加4 ℃ 0.9%氯化鈉溶液制成10%的組織勻漿,以3 000 r/min低溫離心3次,5 min/次,離心半徑10 cm,取上清液。按SOD、MDA測定試劑盒要求測定SOD、MDA水平。
2.1 4組小鼠血清ALT、AST水平比較 4組小鼠血清ALT、AST水平比較,差異有統(tǒng)計學(xué)意義(P<0.01)。模型對照組小鼠血清ALT、AST水平高于正常對照組,差異有統(tǒng)計學(xué)意義(P<0.01),說明肝損傷模型建立成功;WP低劑量組、WP高劑量組小鼠血清ALT、AST水平低于模型對照組,差異有統(tǒng)計學(xué)意義(P<0.01,見表1)。
2.2 4組小鼠肝臟系數(shù)比較 4組小鼠肝臟系數(shù)比較,差異有統(tǒng)計學(xué)意義(P<0.05)。模型對照組肝臟系數(shù)高于正常對照組、WP低劑量組、WP高劑量組,差異有統(tǒng)計學(xué)意義(P<0.01,見表2)。
表1 4組小鼠血清ALT、AST水平比較
注:與正常對照組比較,aP<0.05;與模型對照組比較,bP<0.05;ALT=丙氨酸氨基轉(zhuǎn)移酶,AST=天冬氨酸氨基轉(zhuǎn)移酶,WP=五子衍宗方
表2 4組小鼠肝臟系數(shù)比較
注:與模型對照組比較,aP<0.05
2.3 4組小鼠肝組織形態(tài)學(xué)表現(xiàn) 正常對照組小鼠肝小葉結(jié)構(gòu)完整、清晰可辨,肝細(xì)胞以中央靜脈為中心,呈放射狀排列,且大小形態(tài)無異常;中央靜脈及肝血竇未見出血、充血和水腫;肝匯管區(qū)未見纖維組織增生及炎性細(xì)胞浸潤,未見明顯肝細(xì)胞變性(見圖1A,本文圖1彩圖見本刊官網(wǎng)www.chinagp.net電子期刊相應(yīng)文章)。模型對照組小鼠肝小葉和肝索排列混亂,難以分辨;肝細(xì)胞腫脹明顯、胞質(zhì)很疏松、細(xì)胞結(jié)構(gòu)辨識不清,有肝細(xì)胞壞死(見圖1B)。WP低劑量組小鼠肝小葉有損傷,但仍有一部分肝小葉排列整齊,肝細(xì)胞有炎性細(xì)胞浸潤,肝細(xì)胞腫脹、胞質(zhì)疏松(見圖1C)。WP高劑量組肝小葉排列較整齊,肝細(xì)胞腫脹和胞質(zhì)疏松程度較模型對照組明顯減輕,肝細(xì)胞形態(tài)均勻,排列規(guī)整,與正常對照組無明顯差異(見圖1D)。
2.4 4組小鼠肝組織SOD、MDA水平比較 4組小鼠肝組織SOD、MDA水平比較,差異有統(tǒng)計學(xué)意義(P<0.05)。模型對照組肝組織SOD水平低于正常對照組、WP低劑量組、WP高劑量組,MDA水平高于正常對照組、WP低劑量組、WP高劑量組,差異有統(tǒng)計學(xué)意義(P<0.05,見表3)。
CTX是一種很強(qiáng)的氮芥類烷化劑,具有廣泛的抗腫瘤活性,在體外無烷化活性,其進(jìn)入機(jī)體后先經(jīng)肝臟中的微粒體功能氧化酶分解成酰胺氮芥、丙烯醛和大量的活性氧自由基,然而酰胺氮芥和活性氧自由基能夠與DNA結(jié)合,引起DNA鏈斷裂,從而抑制細(xì)胞增殖。臨床研究發(fā)現(xiàn),CTX在臨床使用過程中出現(xiàn)了嚴(yán)重的肝損傷[11],并且可利用CTX來建立肝損傷模型[12]。CTX能夠引起血清ALT、AST水平升高,肝組織中谷胱甘肽(GSH)和SOD活性降低、MDA水平升高,肝組織形態(tài)發(fā)生變化,膽汁分泌發(fā)生異常[13]。因此,研究各種可以降低或拮抗CTX毒副作用的藥物,具有十分重要的意義。李育浩等[14]通過建立乙醇性肝損傷大鼠模型發(fā)現(xiàn),WP通過降低血、肝過氧化脂質(zhì)(LPO)水平,糾正肝內(nèi)脂質(zhì)代謝異常繼而減少組織細(xì)胞的損傷來保護(hù)肝臟。WP及其組成成分可以減少自由基,提高機(jī)體的抗氧化能力,減少外周血白細(xì)胞線粒體DNA缺失,保護(hù)氧化應(yīng)激對肝組織造成的損傷。有研究顯示,枸杞中所含的甜菜堿可抑制脂肪在肝細(xì)胞內(nèi)沉淀,促進(jìn)肝細(xì)胞新生,同時可防止四氯化碳引起的肝功能紊亂,這可能是其可修復(fù)受損內(nèi)質(zhì)網(wǎng),促進(jìn)抗氧化酶的活化或合成,從而消除自由基,保護(hù)肝細(xì)胞[15]。菟絲子黃酮是一種天然有效的自由基清除劑,可減少脂質(zhì)過氧化,對慢性肝損傷同樣具有良好的保護(hù)作用[16]。文獻(xiàn)報道,五味子制劑聯(lián)合黃芪飲片治療慢性乙型肝炎,能夠增強(qiáng)肝臟的解毒功能,有利于肝細(xì)胞功能的恢復(fù)[17]。研究也發(fā)現(xiàn),厚葉南五味子可顯著降低急性肝損傷小鼠血清ALT、AST水平,有效改善小鼠肝組織的病理損傷[18]?,F(xiàn)代藥理學(xué)研究表明,五味子提取物對多種原因引起的肝損傷具有保護(hù)作用[19]。本課題組前期研究也發(fā)現(xiàn)WP對CTX所致肝損傷具有保護(hù)作用[7-8],但其具體機(jī)制尚不清楚,因此進(jìn)一步研究WP對肝損傷的保護(hù)作用及其機(jī)制,將對臨床治療肝疾病提供新思路。
注: A為正常對照組,B為模型對照組,C為五子衍宗方(WP)低劑量組,D為WP高劑量組
圖1 4組小鼠肝組織損傷(HE染色,×200)
Figure1 Injury in the liver tissue specimens of mice in the 4 groups identified by pathological examination
Table3ComparisonofSODandMDAlevelsinlivertissuespecimensofmiceinthe4groups
組別只數(shù)SOD(U/mgprot)MDA(nmol/mgprot)正常對照組1037.98±2.2213.63±2.79模型對照組1030.97±1.91a27.51±1.99aWP低劑量組1034.56±0.98b18.41±4.30bWP高劑量組1037.58±1.65b14.96±2.92bF值35.8041.90P值<0.01<0.01
注:與正常對照組比較,aP<0.05;與模型對照組比較,bP<0.05;SOD=超氧化物歧化酶,MDA=丙二醛
ALT、AST水平在肝臟疾病的診斷中具有重要意義[20]。當(dāng)肝臟有實質(zhì)性損害時,如肝細(xì)胞變性壞死,細(xì)胞膜通透性增加,分布在胞質(zhì)中的ALT、AST釋放入血,使得血清中酶含量增高。因此,血清ALT、AST水平可以反映肝臟的受損傷程度。
研究表明,WP及其各組分均具有良好的清除自由基、抗氧化作用,而氧化應(yīng)激是CTX誘導(dǎo)肝損傷的主要原因之一[21]。SOD是細(xì)胞內(nèi)天然的氧自由基清除劑,而MDA則是自由基與細(xì)胞表面不飽和酸的反應(yīng)(脂質(zhì)過氧化反應(yīng))產(chǎn)物。SOD活力的高低間接反映了機(jī)體清除自由基的能力,而MDA則反映了機(jī)體細(xì)胞受自由基攻擊的嚴(yán)重程度。在生理狀態(tài)下,機(jī)體產(chǎn)生的自由基與抗氧化防御系統(tǒng)處于動態(tài)平衡,而CTX代謝產(chǎn)物丙烯醛還會干擾體內(nèi)的抗氧化系統(tǒng),導(dǎo)致某些功能酶活性改變甚至喪失,使細(xì)胞內(nèi)環(huán)境穩(wěn)態(tài)遭到破壞,引起氧化應(yīng)激性肝損傷。另外,當(dāng)血清中抗氧化防御系統(tǒng)防御能力降低時,肝組織中MDA水平升高,SOD活性降低,引起氧化應(yīng)激反應(yīng),導(dǎo)致細(xì)胞損傷甚至細(xì)胞死亡。而WP可提高CTX小鼠機(jī)體抗氧化能力,降低小鼠血漿、肝組織MDA水平,增強(qiáng)SOD活性,拮抗CTX所產(chǎn)生的肝毒性,保護(hù)肝臟。
本實驗結(jié)果表明,模型對照組小鼠注射CTX后,肝臟系數(shù)顯著升高,肝損傷嚴(yán)重,并伴有肝細(xì)胞壞死,血清ALT、AST水平和肝組織MDA水平顯著升高,SOD活性顯著降低,表明造模成功。WP灌胃后,小鼠血清ALT、AST水平明顯降低,肝組織MDA水平降低,受損肝組織SOD活性明顯增強(qiáng),表明WP能夠拮抗CTX誘導(dǎo)所引起的肝損傷。
綜上所述,WP對CTX所致肝損傷具有良好的保護(hù)作用,其機(jī)制可能與其提高機(jī)體的抗氧化能力,清除體內(nèi)自由基有關(guān)。
作者貢獻(xiàn):劉珍財進(jìn)行研究設(shè)計與實施、資料收集整理、撰寫論文并對文章負(fù)責(zé);趙海霞、陳茜、劉光耀、袁丁進(jìn)行研究實施與評估、資料收集;張長城進(jìn)行質(zhì)量控制及審校。
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[1]王曉丹,杜軍,劉克,等.黃傘脂溶性成分對環(huán)磷酰胺致小鼠肝損傷的保護(hù)作用[J].中成藥,2013,35(9):2013-2016.DOI:10.3969/j.ssn.1001-1528.2013.09.045. WANG X D,DU J,LIU K,et al.Protective effect of liposoluble component of Pholiota adipose on liver injury induced by cyclophosphamide in mice[J].Chinese Traditional Patent Medicine,2013,35(9):2013-2016.DOI:10.3969/j.ssn.1001-1528.2013.09.045.
[2]梁園,龍騰云,陳紅霞,等.玉郎傘多糖對環(huán)磷酰胺致小鼠肝損傷的保護(hù)作用[J].中國藥師,2014,17(11):1800-1803.DOI:10.3969/j.issn.1008-049X.2014.11.003. LIANG Y,LONG T Y,CHEN H X,et al.Protective effect of Yulangsan polysaccharide on liver injury induced by Cyclophosphamide in mice[J].China Pharmacist,2014,17(11):1800-1803.DOI:10.3969/j.issn.1008-049X.2014.11.003.
[3]XU Y P,LIU B X,ZHANG X P,et al.A Chinese Herbal Formula,Wuzi Yanzong Pill,improves spermatogenesis by modulating the secretory function of Sertoli cells[J].Chin J Integr Med,2014,20(3):194-199.DOI:1007/s11655-014-1743-4.
[4]張長城,賈亮亮,許佳,等.五子衍宗湯對環(huán)磷酰胺致小鼠生精障礙保護(hù)作用的研究[J].中國中醫(yī)基礎(chǔ)醫(yī)學(xué)雜志,2010,16(7):570-571. ZHANG C C,JIA L L,XU J,et al.Protective effect of Wuzi yanzong decoction on dyszoospermia induced by cycoxan in mice[J].Chinese Journal of Basic Medicine in Traditional Chinese Medicine,2010,16(7):570-571.
[5]王洪武,賈亮亮,徐媛青,等.五子衍宗湯對環(huán)磷酰胺致免疫低下小鼠的免疫調(diào)節(jié)作用[J].中國實驗方劑學(xué)雜志,2011,17(4):144-146.DOI:10.3969/j.issn.1005-9903.2011.04.044. WANG H W,JIA L L,XU Y Q,et al.Immuno-regulation effects of Wuzi Yanzong decoction on immunosuppression mice[J].Chinese Journal of Experimental Traditional Medical Formula,2011,17(4):144-146.DOI:10.3969/j.issn.1005-9903.2011.04.044.
[6]歐琴,何堅.加味五子衍宗湯治療脂肪肝82例[J].陜西中醫(yī),2003,24(7):587.DOI:10.3969/j.issn.1000-7369.2003.07.007. OU Q,HE J.Treatment of 82 cases of fatty liver with flavored Wuzi Yanzong decoction[J].Shaanxi Journal of Traditional Chinese Medicine,2003,24(7):587.DOI:10.3969/j.issn.1000-7369.2003.07.007.
[7]黃威峰,劉苗苗,袁丁,等.五子衍宗方對環(huán)磷酰胺致小鼠肝臟細(xì)胞DNA損傷的保護(hù)作用[J].中成藥,2015,37(5):1093-1096.DOI:3969/j.ssn.1001-1528.2015.05.038. HUANG W F,LIU M M,YUAN D,et al.Protective effect of Wuzi Yanzong recipe on DNA damage induced by cyclophosphamide in liver cells of mice[J].Chinese Traditional Patent Medicine,2015,37(5):1093-1096.DOI:3969/j.ssn.1001-1528.2015.05.038.
[8]劉苗苗,袁丁,黃威峰,等.五子衍宗方對環(huán)磷酰胺致小鼠DNA損傷的影響[J].中國中醫(yī)藥信息雜志,2014,21(6):38-40.DOI:10.3969/j.issn.1005-5304.2014.06.014. LIU M M,YUAN D,HUANG W F,et al.Effect of Wuzi Yanzong Fang against cyclophosphamide induced DNA damage in mice[J].Chinese Journal of Information on TCM,2014,21(6):38-40.DOI:10.3969/j.issn.1005-5304.2014.06.014.
[9]黃秀曼,杜鋼軍,孫婷,等.過山蕨總黃酮對肝損傷的保護(hù)作用[J].中草藥,2012,43(12):2458-2463. HUANG X M,DU G J,SUN T,et al.Protection of total flavonoids from Camptosorus sibiricus on hepatic injury[J].Chinese Traditional and Herbal Drugs,2012,43(12):2458-2463.
[10]王志旺,王瑞瓊,郭玫,等.甘肅產(chǎn)藏藥五脈綠絨蒿總黃酮對小鼠實驗性肝損傷的保護(hù)作用[J].中國實驗方劑學(xué)雜志,2013,19(2):206-209.DOI:10.13422/j.cnki.syfjx.2013.02.006. WANG Z W,WANG R Q,GUO M,et al.Study on liver protection of total flavones of meconopsis quintuplinervia from Gansu Province in mice[J].Chinese Journal of Experimental Traditional Medical Formulae,2013,19(2):206-209.DOI:10.13422/j.cnki.syfjx.2013.02.006.
[11]張曉峰.環(huán)磷酰胺口服致老年人急性肝損傷[J].藥物不良反應(yīng)雜志,2005,7(6):436.DOI:10.3969/j.issn.1008-5734.2005.06.014. ZHANG X F.Oral cyclophosphamide-associated acute liver impairment[J].Adverse Drug Reactions Journal,2005,7(6):436.DOI:10.3969/j.issn.1008-5734.2005.06.014.
[12]張善玉,樸娟玉,宛瑩,等.不同給藥次數(shù)環(huán)磷酰胺誘導(dǎo)小鼠肝損傷的對比研究[J].延邊大學(xué)醫(yī)學(xué)學(xué)報,2009,32(1):22-25.DOI:10.16068/j.1000-1824.2009.01.032. ZHANG S Y,PIAO J Y,WAN Y,et al.Comparative study of the liver injury induced with different medication times of cyclophosphamide in mice[J].Journal of Medical Science Yanbian University,2009,32(1):22-25.DOI:10.16068/j.1000-1824.2009.01.032.
[13]施暢,廖明陽.環(huán)磷酰胺與異環(huán)磷酰胺致肝毒性機(jī)制的比較[J].衛(wèi)生毒理學(xué)雜志,2000,14(2):99-102.DOI:10.3969/j.issn.1002-3127.2000.02.010. SHI C,LIAO M Y.A comparative study on the mechanism(s)of the hepatotoxicity induced by Cyclophasphamide and Ifosfamide in rats[J].Journal of Health Toxicology,2000,14(2):99-102.DOI:10.3969/j.issn.1002-3127.2000.02.010.
[14]李育浩,鄧響嘲,吳清和,等.五子衍宗丸對乙醇性肝損傷大鼠脂質(zhì)代謝的影響[J].中國中藥雜志,1994,19(5):300-302. LI Y H,DENG X C,WU Q H,et al.Effects of Wuzi Yanzong pills on lipid in rats with alcohol-induced liver injury[J].China Journal of Chinese Materia Medica,1994,19(5):300-302.
[15]劉英娟,杜金梁,賈睿,等.枸杞多糖對四氯化碳致建鯉急性肝損傷的保護(hù)作用[J].浙江農(nóng)業(yè)學(xué)報,2015,27(1):37-43.DOI:10.3969/j.issn:1004-1524.2015.01.08. LIU Y J,DU J L,JIA R,et al.Protective effects of Lycium barbarum polysaccharides against carbon tertrachloride(CCl4)induced liver accurate injury in Carprinus carpoivar Jian[J].Acta Agriculturae Zhejiangensis,2015,27(1):37-43.DOI:10.3969/j.issn:1004-1524.2015.01.08.
[16]宋敏,柴連琴,左百樂.菟絲子黃酮對小鼠慢性肝損傷的影響[J].上海畜牧獸醫(yī)通訊,2009(3):11.DOI:10.3969/j.issn.1000-7725.2009.03.006. SONG M,CHAI L Q,ZUO B L.Effects of flavonoids from semen cuscutae on chronic liver injury in mice[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2009(3):11.DOI:10.3969/j.issn.1000-7725.2009.03.006.
[17]燕菲,張巧艷,張宏,等.五味子與黃芪多糖協(xié)同保護(hù)對乙酰氨基酚致小鼠急性肝損傷[J].藥學(xué)實踐雜志,2009,27(5):340-344.DOI:10.3969/j.issn.1006-0111.2009.05.007. YAN F,ZHANG Q Y,ZHANG H,et al.Synergistic protective effect of Fructus Schisandrae chinensis with Astragalus polysaccharides on paracetamol-induced acute liver injury in mice[J].Journal of Pharmaceutical Practice,2009,27(5):340-344.DOI:10.3969/j.issn.1006-0111.2009.05.007.
[18]劉瑛,劉元,王麗,等.厚葉南五味子對小鼠急性肝損傷的保護(hù)作用[J].中國熱帶醫(yī)學(xué),2015,15(12):1424-1427.DOI:10.13604/j.cnki.46-1064/r.2015.12.03. LIU Y,LIU Y,WANG L,et al.Protective effects of Kadsura coccinea on acute liver injury of rats[J].China Tropical Medicine,2015,15(12):1424-1427.DOI:10.13604/j.cnki.46-1064/r.2015.12.03.
[19]李宜軒,陳建光,李鳳,等.北五味子提取物對實驗性肝纖維化大鼠肝損傷的保護(hù)作用[J].吉林大學(xué)學(xué)報(醫(yī)學(xué)版),2014,40(2):285-288.DOI:10.13481/j.1671-587x.20140215. LI Y X,CHEN J G,LI F,et al.Protective effects of extract fructus Schisandrae Chinensis on hepatic injury in experimental hepatic fibrosis rats[J].Journal of Jilin University(Medicine Edition),2014,40(2):285-288.DOI:10.13481/j.1671-587x.20140215.
[20]LIU W,GAO F F,LI Q,et al.Protective effect of astragalus polysaccharides on liver injury induced by several different chemotherapeutics in mice[J].Asian Pac J Cancer Prev,2014,15(23):10413-10420.DOI:10.7314/APJCP.2014.15.23.10413.
[21]KORKMAZ A,TOPAL T,OTER S.Pathophysiological aspects of cyclophosphamide and ifosfamide induced hemorrhagic cystitis;implication of reactive oxygen and nitrogen species as well as PARP activation[J].Cell Biol Toxicol,2007,23(5):303-312.DOI:10.1007/s10565-006-0078-0.
(本文編輯:崔莎)
ProtectiveEffectandMechanismofWuzi-YanzongPrescriptionAgainsttheLiverInjuryInducedbyCyclophosphamide
LIUZhen-cai,ZHAOHai-xia,CHENQian,LIUGuang-yao,YUANDing,ZHANGChang-cheng*
MedicalCollegeofChinaThreeGorgesUniversity,Yichang443002,China
*Correspondingauthor:ZHANGChang-cheng,Professor;E-mail:greatwall@ctgu.edu.cn
ObjectiveTo study the protective effect and mechanism of Wuzi-Yanzong prescription(WP) against the liver injury induced by cyclophosphamide(CTX),so as to provide a reference for clinical treatment of the disease.MethodsFrom September 2015 to June 2016,40 8-week-old SPF grade male BALB/c mice were randomly divided into normal control group,model control group,WP low-dose group,and WP high-dose group,with 10 mice in each.Flavonoids solution was made by dissolving the flavonoids extracted from WP in 1% CMC-Na.WP low-dose and high-dose groups were respectively intragastrically administered with flavonoids solution 140 mg/kg,280 mg/kg,once a day for 30 days from the 1st day of intervention.For modeling,from the 3rd day of intervention,mice in the control group were given intraperitoneal injection of 0.9% sodium chloride solution,while those in the other groups
intraperitoneal injection of CTX 50 mg/kg,once three days for 10 times.Successful modeling was considered when the levels of serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in the model control group were statistically different from those in the normal control group.Twelve hours after the last time of injection,all the mice were sacrificed by cervical dislocation after taking blood from the eyeball.The serum levels of ALT and AST were detected,the liver was obtained to calculate the liver index,to observe the tissue morphology changes after HE staining and to determine the levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in liver tissues.ResultsThe serum ALT and AST levels in the model control mice were higher than those in the normal control group(P<0.01).The serum ALT and AST levels in the WP low-dose and high-dose groups were lower than those in the model control group(P<0.05).The liver index in the model control group was higher than that in the other 3 groups(P<0.01).In the model control group,the hepatic lobules and hepatic cords were disordered,and the hepatocytes were swollen and accompanied by necrosis and cytoplasm loose.However,in the WP low-dose and high-dose groups,liver lobules arranged neatly,liver cell swelling and cytoplasmic extent were significantly reduced compared with those in the model control group,liver cell morphology was uniform,and arranged in regular.The SOD level of liver tissue in the model control group was lower while the MDA level was higher than in the normal control group(P<0.01).WP low-dose group,WP high-dose group had higher SOD level but lower MDA level than the model control group(P<0.05).ConclusionWP has good protective effects on liver injury caused by CTX and its mechanism is probably related to the scavenging of free radical and increasing of cell anti-oxidative function.
Drug-induced liver injury;Cyclophosphamide;Molecular mechanisms of pharmacological action;Wuzi-yanzong prescription
國家自然科學(xué)基金資助項目(81573931,81373881,81503334);三峽大學(xué)人才科研啟動基金(KJ2014B067)
R 575.1
A
10.3969/j.issn.1007-9572.2017.27.020
2017-04-18;
2017-07-18)
443002 湖北省宜昌市,三峽大學(xué)醫(yī)學(xué)院
*通信作者:張長城,教授; E-mail:greatwall@ctgu.edu.cn