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WHO《西太平洋地区医学索引》来源期刊  
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医药导报, 2021, 40(5): 616-621
doi: 10.3870/j.issn.1004-0781.2021.05.008
沙棘熊果酸对酒精性肝病模型大鼠的保护作用*
Protective Effect of Ursolic Acid Extracted from Hippophae rhamnoides L. on Alcohol-Induced Liver Disease in Rats
李可欣1,, 张男男1, 侯瑞丽1, 张文龙2, 高龙1, 苗晓涵1, 戈娜1,

摘要:

目的 探讨沙棘熊果酸对酒精性肝病模型大鼠的保护作用及其机制。方法SPF级雄性Wistar大鼠50只,随机分为5组:正常对照组、模型对照组和熊果酸小、中、大剂量(50,100,150 mg·kg-1·d-1)组,每组10只。采用灌胃方式给药,持续8周,末次给药12 h后,腹主动脉取血,留取肝脏组织。测定大鼠血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平及肝匀浆中三酰甘油(TG)含量;苏木精-伊红 ( HE )染色观察大鼠肝脏形态结构改变;TUNEL法测定大鼠肝细胞凋亡率;免疫印迹法(Western blotting)检测大鼠肝组织Bcl-2、Bax及cleaved caspase-3的蛋白表达变化。结果HE染色显示,模型对照组大鼠肝索排列紊乱,且细胞浆内散在空泡,肝组织内炎症细胞聚集,肝细胞呈现片状坏死。但熊果酸各剂量组较模型对照组肝脏脂肪变性明显改善,炎症细胞浸润及坏死区域明显减少,组织结构趋向正常,且熊果酸中、大剂量组血清ALT、AST水平及肝组织TG含量明显降低(P<0.05)。TUNEL结果显示,熊果酸大剂量组染色阳性细胞数及肝细胞凋亡指数较模型对照组明显降低(P<0.05);免疫印迹法检测结果显示,与模型对照组比较,熊果酸各剂量组肝组织Bcl-2蛋白表达均明显升高,Bax蛋白表达均明显下降,且cleaved caspase-3蛋白表达量均增多。结论 沙棘熊果酸对大鼠酒精性肝病有改善作用,其机制可能与调控肝细胞凋亡关键蛋白Bcl-2、Bax、cleaved caspase-3的表达水平,进而抑制肝细胞的异常凋亡有关。

关键词: 熊果酸 ; 沙棘 ; 肝病 ; 酒精性 ; 细胞凋亡

Abstract:

Objective To explore the effect and mechanism of ursolic acid (UA) extracted from Hippophae rhamnoides L.on alcohol-induced liver disease in rats. Methods Fifty male Wistar rats (SPF)were randomly divided into five groups:normal control group,model control group,low-,medium- and high-dose UA group,10 rats in each group.The rats were administered by intragastric administration for eight consecutive weeks.After 12 hours of last administration,blood was collected by abdominal aorta and the liver was obtained for histopathological and biochemical assays.The levels of ALT and AST in serum was detected.The content of TG in liver homogenate was determined.The morphological changes of hepatic tissue in rats were observed by HE staining.The apoptotic rate of rat liver cells was detected by TUNEL method.The protein expressions of Bcl-2,Bax and cleaved caspase-3 in liver tissue were detected by Western blotting. Results In model control group,HE staining showed that the hepatic cord was arranged disorderly,and there were lots of vacuoles in the cytoplasm of hepatocytes.The inflammatory cells in the liver tissue gathered,and the hepatocytes showed flake necrosis in model control group. Compared with model control group,ursolic acid significantly improved the steatosis of the liver.In the UA group,infiltration and necrosis of inflammatory cells were significantly decreased,the tissue structure tended to be normal,and the activity of serum ALT,AST,and the content of TG in the medium- and high-dose UA group liver tissue were significantly decreased (P<0.05).TUNEL results showed that the number of positive cells and the apoptosis index of hepatocytes in the high dose of UA group were significantly lower than in model control group (P<0.05).Western blotting results showed that the protein expression of Bcl-2 in liver tissue of UA groups was significantly increased,the protein expression of Bax was significantly decreased,and the protein expression of cleaved caspase-3 was increased,compared with model control group. Conclusion Ursolic acid extracted from Hippophae rhamnoides L.could improve the liver injury induced by alcohol.The mechanism might be related to the regulation the protein expressions of Bcl-2,Bax and cleaved caspase-3,key proteins of apoptosis,thereby inhibiting the abnormal apoptosis of hepatocytes.

Key words: Ursolic acid ; L. ; Liver disease ; alcohol ; Cell apoptosis

开放科学(资源服务)标识码(OSID)

酒精性肝病(alcoholic liver disease,ALD)是指长期和(或)大量酒精摄入所导致的肝脏损伤,肝细胞凋亡、脂肪变性、炎症、坏死等是它的主要表现形式[1,2]。世界卫生组织(WHO)在2018年《全球酒精与健康报告》中指出,全世界每年因饮酒而死亡的人数占全部死亡人数的5.3%,约300万人,其中2013年北京302医院接诊的ALD患者人数是2002年的2倍[3]。因此积极寻找一种预防及辅助治疗ALD的天然活性物质具有重要意义。沙棘(Hippophae rhamnoides L.)蒙名其察日嘎纳,含多种生物活性成分,是珍贵的药食两用植物[4]。本课题组前期从沙棘中提取的熊果酸是一种天然植物化合物,近年来已被广泛应用于食品、药品及化妆品中[5]。熊果酸具有保肝、抗炎、抗氧化、抗肿瘤等多种作用[6]。因此,本实验以沙棘熊果酸为研究对象,通过酒精诱导大鼠肝损伤模型,探讨沙棘熊果酸对酒精诱导性肝损伤的改善效果及可能的作用机制,为酒精诱导肝损伤的防治提供参考。

1 材料与方法
1.1 材料

沙棘果由内蒙古农业大学于呼和浩特市和林县境内野生沙棘林采集、提供,经内蒙古农业大学生命科学学院王玉珍教授鉴定(Shaji-2012-1215),分离纯化由内蒙古科技大学进行,并应用红外光谱、氢核磁共振谱(1H-NMR)和碳核磁共振谱(13C-NMR)鉴定为五环三萜类化合物[7]。熊果酸分子式为C30H48O3,相对分子质量为456.68,含量93.8%。

1.2 主要试剂和仪器

无水乙醇购自西陇科学股份有限公司;丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、三酰甘油(TG)试剂盒均购自南京建成生物工程研究所;TUNEL细胞凋亡检测试剂盒购自Roche公司(批号:11684817910);鼠抗GAPDH多克隆抗体购自美国Santa Cruz Biotechnology公司(批号:sc-47724);BCA蛋白浓度试剂盒(批号:P0012)、超敏电化学发光试剂盒(批号:P0018FS)、RIPA裂解液(批号:P0013B)、苯甲基磺酰氟(批号:ST506)、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)蛋白上样缓冲液(批号:P0015)均购自江苏碧云天生物技术研究所;羊抗兔Bcl-2(批号:MABC573)、Bax(批号:AB2915)、cleaved caspase-3(批号:ABC495)多克隆抗体购自美国Millipore公司;辣根酶标记山羊抗兔IgG(批号:ZB-2301)、辣根酶标记山羊抗小鼠IgG(批号:ZB-2305)均购自北京中杉金桥生物技术有限公司。

RM2135型石蜡切片机(德国LEICA公司);CX41照相显微镜(日本Olympus公司);BAYGENE电泳仪(北京百晶生物技术有限公司);550型全自动多功能酶标仪(美国BIO-RAD公司);UVP-GDS-8000凝胶成像分析系统(美国BIO-RAD公司)。

1.3 实验动物与分组

SPF级雄性Wistar大鼠50只,体质量(190±10) g,购自山东鲁抗医药股份有限公司,动物生产许可证号:SCXK(鲁)2014-0001。大鼠于室温(23±2) ℃、相对湿度50%~60%、12 h光照循环的条件下进行饲养。

将适应性饲养7 d后的大鼠根据随机数字表进行完全随机分组,分为正常对照组、模型对照组和熊果酸小、中、大剂量组,共5组,每组10只。给药方式参考马浩然等[2]报道的方法,即模型对照组参考给予50%(V/V)乙醇8 mL·kg-1·d-1×2周+12 mL·kg-1·d-1×6周;正常对照组给予等体积的0.9%氯化钠溶液;熊果酸小、中、大剂量组分别给予熊果酸50,100,150 mg·kg-1·d-1,1 h后给予和模型对照组等体积乙醇。持续灌胃8周,最后一次灌胃后禁食不禁水12 h,将大鼠称重后用3%戊巴比妥钠30 mg·kg-1麻醉。腹主动脉取血,将血清以3000 r·min-1离心15 min(r=15 cm);留取肝组织,将部分肝组织进行固定,其余组织均置于-80 ℃冰箱中冻存。

1.4 指标检测

1.4.1 苏木精-伊红(HE)染色法观察每组大鼠肝组织病理情况 将0.9 cm×0.9 cm×0.5 cm新鲜肝大叶组织放置于10%甲醛中固定,石蜡包埋切片,HE染色,中性树胶封片,光学显微镜下观察各组大鼠肝组织形态结构的改变。

1.4.2 沙棘熊果酸对各组大鼠血清转氨酶及肝组织TG含量的影响 各组大鼠ALT和AST水平检测严格按照ALT/AST测定试剂盒说明书进行,用酶标仪测量505 nm处吸光度(A值)并计算ALT和AST的含量。

取新鲜肝组织1 g,经过漂洗,拭干,剪碎后,加入适量磁珠及0.9%氯化钠溶液900 μL匀浆5 min,经3000 r·min-1离心15 min(r=15 cm)后取上清液,检测方法严格按照TG说明书进行,用酶标仪测量500 nm处A值,样本含量与A值成正比,以mmol·g-1表示。

1.4.3 TUNEL染色法检测各组大鼠肝细胞的凋亡情况 取新鲜肝脏组织做石蜡切片,严格按照TUNEL细胞凋亡试剂盒进行操作,光镜下(×200)观察,以凋亡细胞核内为黄色或棕黄色颗粒沉淀物为判定标准,计算肝细胞总数、凋亡肝细胞数、肝细胞凋亡率,分析大鼠肝细胞凋亡情况。

1.4.4 免疫印迹法(Western blotting)测定每组大鼠肝组织Bcl-2、Bax及cleaved caspase-3的蛋白表达量 裂解大鼠肝组织提取总蛋白,12 000 ×g离心20 min,并收集上清液,采用BCA法测定蛋白浓度。按5份样品体积与1份loading buffer混匀,100 ℃孵育3 min,上样体积为10~20 μL,经5%~12% SDS-PAGE分离样品后将蛋白转至硝酸纤维素膜上,以5%脱脂奶粉封闭2 h,按操作加入相应的一抗(GAPDH抗体1:800、Bcl-2抗体1:1000、Bax抗体1:1000、cleaved caspase-3抗体1:1000)及适当稀释倍数的二抗。凝胶成像系统摄相分析。以GAPDH作为内参,用靶蛋白积分吸光度(IA)值/GAPDH(IA)值显示靶蛋白相对水平。

1.5 统计学方法

应用SPSS17.0版软件整理和分析实验数据,计量资料数据以均数±标准差($\overline{x}$±s)表示,采用单因素方差分析(one-way ANOVA)和LSD-t检验,以 P<0.05为差异有统计学意义。

2 结果
2.1 沙棘熊果酸对酒精诱导肝损伤大鼠肝组织病理改变的影响

HE染色结果显示,正常对照组肝细胞轮廓完整且结构紧密、以中央静脉为中心向四周呈放射状分布;模型对照组肝细胞排列紊乱,且细胞质内散在空泡,肝组织内炎症细胞聚集,肝细胞呈现片状坏死;而熊果酸小、中、大剂量组肝细胞索排列均有不同程度的改善,炎症细胞浸润程度及坏死区域降低明显(图1)。

图1 肝脏的组织病理学情况(×400)
A.正常对照组;B.模型对照组;C.熊果酸大剂量组;D.熊果酸中剂量组;E.熊果酸小剂量组。

Fig.1 Histopathological structure of the livers(×400)
A.normal control group;B.model control group;C.high-dose ursolic acid group;D.medium-dose ursolic acid group;E.low-dose ursolic acid group.

2.2 沙棘熊果酸对酒精所致肝损伤大鼠血清ALT、AST及肝脏TG含量的影响

表1。模型对照组大鼠血清ALT、AST及肝脏TG含量均明显高于正常对照组(P<0.05);而熊果酸中、大剂量组大鼠血清ALT、AST的活力及肝脏TG含量均明显降低(P<0.05),且逐渐接近于正常对照组,熊果酸小剂量组大鼠血清ALT、AST的活力及肝脏TG含量与模型对照组差异无统计学意义(P>0.05)。

表1 5组大鼠血清ALT、AST及肝脏TG含量比较
Tab.1 Comparison of the serum levels of ALT,AST and hepatic TG contents among five groups of rats $\overline{x}$±s,n=10
组别 ALT AST TG/
(mmol·g-1)
(U·L-1)
正常对照组 46.333±3.570 80.571±12.699 0.316±0.025
模型对照组 74.444±7.011 141.000±14.004 1.069±0.069
熊果酸
大剂量组 60.000±9.433②③ 88.142±8.971②③ 0.453±0.028
中剂量组 62.750±11.646②③ 88.666±11.129②③ 0.393±0.024②③
小剂量组 75.777±13.863 119.140±20.540 0.862±0.064

①与正常对照组比较,t=-8.752~-3.912,P<0.05;②与模型对照组比较,t=2.107~7.785,P<0.05;③与熊果酸小剂量组比较,t=1.762~3.388,P<0.05。

①Compared with normal control group,t=-8.752--3.912,P<0.05;②Compared with model control group,t=2.107-7.785,P<0.05;③Compared with low-dose ursolic acid group,t=1.762-3.388,P<0.05.

表1 5组大鼠血清ALT、AST及肝脏TG含量比较

Tab.1 Comparison of the serum levels of ALT,AST and hepatic TG contents among five groups of rats $\overline{x}$±s,n=10

2.3 沙棘熊果酸对酒精诱导肝损伤大鼠肝细胞凋亡的影响

TUNEL结果显示,正常对照组染色较淡,偶见凋亡细胞;模型对照组染色不均匀,可见大量棕褐色固缩或呈碎片状的细胞核,且染色的阳性细胞多集中在肝小叶、中央静脉肝附近,肝细胞凋亡率高达77%,与正常对照组比较,明显增高(P<0.05);与模型对照组比较,熊果酸小、中、大3个剂量组染色阳性细胞逐渐减少,染色变浅,核碎裂现象明显降低,凋亡率分别为63%,47%和30%,且熊果酸小剂量组与熊果酸大剂量组比较,肝细胞凋亡率明显升高(P<0.05)(图2和图3)。

图2 5组大鼠肝细胞凋亡情况(TUNEL法,×200)
A.正常对照组;B.模型对照组;C.熊果酸大剂量组;D.熊果酸中剂量组;E.熊果酸小剂量组。

Fig.2 Hepatocyte apoptosis in five groups of rats(TUNEL,×200)
A.normal control group;B.model control group;C.high-dose ursolic acid group;D.medium-dose ursolic acid group;E.low-dose ursolic acid group.

图3 5组大鼠肝细胞凋亡率比较($\overline{x}$±s,n=10)
A.正常对照组;B.模型对照组;C.熊果酸大剂量组;D.熊果酸中剂量组;E.熊果酸小剂量组。 ①与正常对照组比较,t=-69.823~-9.457,P<0.05;②与模型对照组比较,t=3.561~17.611,P<0.05;③与熊果酸小剂量组比较,t=-6.767,P<0.05。

Fig.3 Comparison of hepatocyte apoptosis rate among five groups of rats($\overline{x}$±s,n=10)
A.normal control group;B.model control group;C.high-dose ursolic acid group;D.medium-dose ursolic acid group;E.low-dose ursolic acid group.①Compared with normal control group,t=-69.823--9.457,P<0.05;②Compared with model control group,t=3.561-17.611,P<0.05;③Compared with low-dose ursolic acid group,t=-6.767,P<0.05.

2.4 沙棘熊果酸对ALD大鼠肝组织Bcl-2、Bax、cleaved caspase-3蛋白表达的影响

与正常对照组比较,模型对照组大鼠肝组织中Bcl-2蛋白表达量降低明显(P<0.05),Bax及cleaved caspase-3蛋白表达量均明显增高(P<0.05);熊果酸中、大剂量干预的大鼠相较模型对照组而言,肝组织Bcl-2蛋白表达量明显增高(P<0.05),而熊果酸小剂量组大鼠肝组织Bcl-2蛋白表达量无明显差异,熊果酸大剂量组大鼠肝组织Bax蛋白表达量明显减少(P<0.05),熊果酸小剂量组、中剂量组大鼠肝组织Bax蛋白表达量与模型对照组差异无统计学意义(P>0.05),熊果酸中、大剂量组大鼠肝组织cleaved caspase-3蛋白表达量较模型对照组明显降低(P<0.05),但熊果酸小剂量组差异无统计学意义(P>0.05)。见图4。

图4 5组大鼠肝组织中Bcl-2、Bax、cleaved caspase-3蛋白表达量的比较($\overline{x}$±s,n=10)
A.正常对照组;B.模型对照组;C.熊果酸大剂量组;D.熊果酸中剂量组;E.熊果酸小剂量组;①与正常对照组比较,t=-19.682~12.778,P<0.05;②与模型对照组比较,t=-14.409~20.195,P<0.05;③与熊果酸小剂量组比较,t=-11.288~15.734,P<0.05。

Fig.4 Comparison of Bcl-2,Bax and cleaved caspase-3 protein expression in liver tissue among five groups of rats($\overline{x}$±s,n=10)
A.normal control group;B.model control group;C.high-dose ursolic acid group;D.medium-dose ursolic acid group;E.low-dose ursolic acid group.①Compared with normal control group,t=-19.682-12.778,P<0.05;②Compared with model control group,t=-14.409-20.195,P<0.05;③Compared with low-dose ursolic acid group,t=-11.288-15.734,P<0.05.

3 讨论

目前关于大鼠酒精致肝损伤的造模方式有4种:自由饮用模型、胃内喂养模型、腹腔注射模型及酒精灌胃模型[8],但由于饮用量无法控制、摄入途径不符合等不可控因素,本研究选用酒精灌胃方式,与酒精诱导肝损伤的生理生化和病理特征及人的饮酒习惯相结合,给予阶梯式灌胃,成功建立酒精致肝损伤大鼠模型[9]。HE染色观察肝组织变化是临床上反映肝脏损伤程度的金标准[10],ALT和AST是检测肝脏损伤的敏感指标[11],其活性水平的高低可在一定程度上体现生物体内肝细胞损伤的情况[12]。实验表明,慢性酒精中毒可引起肝细胞中TG不能正常输出,导致肝脏中TG的堆积[13]。本研究表明,模型对照组血清ALT、AST及肝组织TG含量均明显升高,肝细胞部分呈现片状坏死,肝细胞索排列不规则,且存在大量脂肪空泡,这与肝损伤导致TG堆积互相印证。而经熊果酸干预8周后,大鼠肝细胞坏死程度明显降低,肝细胞索排列较为整齐,血清ALT、AST及肝组织TG含量均有不同程度下降。

长期摄入酒精可致生物体内肝细胞异常凋亡,其凋亡程度与酒精致肝损伤程度呈正相关[14],故如何减轻肝细胞凋亡程度对ALD的预防与治疗具有重要意义。参与肝细胞凋亡发生发展的基因有许多,与其关系最密切的调控基因为Bcl-2家族的Bcl-2及Bax基因[15]。Bcl-2基因具有抗肝细胞凋亡的作用,而Bax基因可促进肝细胞凋亡,这对基因分别以同源及异源二聚体存在[16]。当Bcl-2过表达时形成异源二聚体Bcl-2-Bax,可有效减轻多因素诱导的肝细胞凋亡;当Bax过表达时形成Bax同源二聚体,可直接诱导肝细胞发生凋亡[17]。因此,Bcl-2与Bax的表达在体内常呈现一种平衡状态。一旦失衡,将对肝细胞的存活或凋亡产生影响。通过增加Bcl-2的表达可抑制caspase-3的激活而减缓肝细胞凋亡的进程[18]。细胞凋亡的程序启动大多由天冬氨酸的特异水解酶(caspase)引发,caspase-3是caspase家族中细胞凋亡促进过程中最主要的执行者[19]。caspase-3在一般情况下以无活性的酶原(Pro-caspase)形式存在,当肝细胞凋亡程序被激活时,Pro-caspase将转变为有活性的裂解caspase-3,其可促进肝细胞的凋亡过程[20]。所以,cleaved caspase-3常被作为判断肝细胞凋亡情况的重要指标之一。caspase-3激活将使Bcl-2的功能由抑制凋亡转变为触发凋亡,而阻止caspase-3的激活将抑制Bcl-2的降解[21]。本研究结果显示,熊果酸组大鼠肝组织中Bax蛋白表达明显受到抑制,而Bcl-2蛋白的表达明显增加,阻止caspase-3的活化。提示熊果酸可能通过调节肝组织中Bcl-2、Bax、cleaved caspase-3蛋白的表达起到抗凋亡作用,进而发挥防治ALD的作用。

综上所述,沙棘熊果酸能够明显改善ALD,其机制可能为调节抑凋亡蛋白Bcl-2、促凋亡蛋白Bax和执行凋亡蛋白cleaved caspase-3的表达,进而降低肝细胞异常凋亡。因此,沙棘熊果酸可作为一种具有潜在保肝作用及药用价值的天然植物化合物,值得进一步开发和研究。

参考文献

[1] PEACOCK A,LEUNG J,LARNEY S.Global statistics on alcohol,tobacco and illicit drug use:2017 status report[J].Addiction,2018,113(10):1905-1926.
AIMS: This review provides an up-to-date curated source of information on alcohol, tobacco and illicit drug use and their associated mortality and burden of disease. Limitations in the data are also discussed, including how these can be addressed in the future. METHODS: Online data sources were identified through expert review. Data were obtained mainly from the World Health Organization, United Nations Office on Drugs and Crime and Institute for Health Metrics and Evaluation. RESULTS: In 2015, the estimated prevalence among the adult population was 18.4% for heavy episodic alcohol use (in the past 30 days); 15.2% for daily tobacco smoking; and 3.8, 0.77, 0.37 and 0.35% for past-year cannabis, amphetamine, opioid and cocaine use, respectively. European regions had the highest prevalence of heavy episodic alcohol use and daily tobacco use. The age-standardized prevalence of alcohol dependence was 843.2 per 100 000 people; for cannabis, opioids, amphetamines and cocaine dependence it was 259.3, 220.4, 86.0 and 52.5 per 100 000 people, respectively. High-income North America region had among the highest rates of cannabis, opioid and cocaine dependence. Attributable disability-adjusted life-years (DALYs) were highest for tobacco smoking (170.9 million DALYs), followed by alcohol (85.0 million) and illicit drugs (27.8 million). Substance-attributable mortality rates were highest for tobacco smoking (110.7 deaths per 100 000 people), followed by alcohol and illicit drugs (33.0 and 6.9 deaths per 100 000 people, respectively). Attributable age-standardized mortality rates and DALYs for alcohol and illicit drugs were highest in eastern Europe; attributable age-standardized tobacco mortality rates and DALYs were highest in Oceania. CONCLUSIONS: In 2015 alcohol use and tobacco smoking use between them cost the human population more than a quarter of a billion disability-adjusted life years, with illicit drugs costing further tens of millions. Europeans suffered proportionately more, but in absolute terms the mortality rate was greatest in low- and middle-income countries with large populations and where the quality of data was more limited. Better standardized and rigorous methods for data collection, collation and reporting are needed to assess more accurately the geographical and temporal trends in substance use and its disease burden.
DOI:10.1111/add.14234      PMID:29749059      URL    
[本文引用:1]
[2] 马浩然,戈娜,赵应杰,.酒精性肝损伤的啮齿类动物模型[J].包头医学院学报,2015,31(11):146-148.
[本文引用:2]
[3] XIAO J,WANG F,WONG N K,et al.Global liver disease burdens and research trends:analysis from a China perspective[J].J Hepatol,2019,71(1):212-221.
Liver diseases affect millions of people worldwide. In most developed countries, the incidence of viral hepatitis is waning as a result of modern advances in disease prevention, diagnosis, and therapies. Expanded programmes for systematic immunisation against hepatitis B virus have also significantly brought down the number of new cases in many countries, including China. In contrast, with the improvement in living standards, the prevalence of metabolic liver diseases including non-alcoholic fatty liver disease and alcohol-related liver disease is set to rise, ultimately leading to more cases of end-stage liver diseases (liver failure, cirrhosis, and liver cancer). Over the past 30years, visionary governments of major nations have provided strong incentives for basic/clinical research, vaccination programmes, and drug discovery and development in the field of hepatology. To get rid of her unflattering title as the
DOI:10.1016/j.jhep.2019.03.004      PMID:30871980      URL    
[本文引用:1]
[4] 张程慧,祁玉霞,程康蓉,.沙棘的综合价值研究进展[J].食品工业科技,2017,38(22):331-335.
[本文引用:1]
[5] OLAS B,KONTEK B,SZCZESNA M,et al.Inhibition of blood platelet adhesion by phenolics' rich fraction of Hippophae rhamnoides L.fruits[J].J Physiol Pharmacol,2017,68:223-229.
Beneficial influence of fruits on human health may be their ability to prevent the hyperactivation of blood platelets and cardiovascular disorders. Effects of the phenolic fraction from Hippophae rhamnoides fruit on different stages of blood platelet activation (platelet adhesion and aggregation) were studied in vitro. We also examined effects of the H. rhamnoides fraction on metabolism of thiol groups, which plays an important role in platelet functions. The effects of the H. rhamnoides fraction on adhesion of blood platelets to collagen and fibrinogen were determined with Tuszynski's and Murphy's method. The platelet aggregation was determined with turbidimetry. The action of the H. rhamnoides fraction on the level of thiol groups in platelet proteins and a level of glutathione (GSH) in platelets was estimated with 5,5'-dithio-bis(2-nitro-benzoic acid). The tested fraction of H. rhamnoides (0.5 - 50 mug/ml; 30 min of the incubation time 30 min) inhibited blood platelets adhesion to collagen and fibrinogen. The effect of the tested fraction on blood platelet adhesion depended on concentration of fraction. In presence of the highest tested concentration which was 50 mug/ml, inhibition of platelet adhesion for thrombin-activated platelets was about 55%. On the other hand, tested plant fraction had no anti-aggregatory properties. Our results showed anti-adhesive properties of phenolic fraction from H. rhamnoides fruit and we suggest that it may be beneficial for prevention of cardiovascular diseases.
PMID:28614772      URL    
[本文引用:1]
[6] SEO D Y,LEE S R,HEO J W,et al.Ursolic acid in health and disease[J].Korean J Physiol Pharmacol,2018,22(3):235-248.
Ursolic acid (UA) is a natural triterpene compound found in various fruits and vegetables. There is a growing interest in UA because of its beneficial effects, which include anti-inflammatory, anti-oxidant, anti-apoptotic, and anti-carcinogenic effects. It exerts these effects in various tissues and organs: by suppressing nuclear factor-kappa B signaling in cancer cells, improving insulin signaling in adipose tissues, reducing the expression of markers of cardiac damage in the heart, decreasing inflammation and increasing the level of anti-oxidants in the brain, reducing apoptotic signaling and the level of oxidants in the liver, and reducing atrophy and increasing the expression levels of adenosine monophosphate-activated protein kinase and irisin in skeletal muscles. Moreover, UA can be used as an alternative medicine for the treatment and prevention of cancer, obesity/diabetes, cardiovascular disease, brain disease, liver disease, and muscle wasting (sarcopenia). In this review, we have summarized recent data on the beneficial effects and possible uses of UA in health and disease managements.
DOI:10.4196/kjpp.2018.22.3.235      PMID:29719446      URL    
[本文引用:1]
[7] 张男男,侯瑞丽,李可欣,.沙棘熊果酸对H22荷瘤小鼠抑瘤活性及其机制的探讨[J].食品研究与开发,2019,40(10):6-12.
[本文引用:1]
[8] OSNA N A,DONOHUE T M J R,KHARBANDA K K.Alcoholic liver disease:pathogenesis and current management[J].Alcohol Res,2017,38(2):147-161.
Excessive alcohol consumption is a global healthcare problem. The liver sustains the greatest degree of tissue injury by heavy drinking because it is the primary site of ethanol metabolism. Chronic and excessive alcohol consumption produces a wide spectrum of hepatic lesions, the most characteristic of which are steatosis, hepatitis, and fibrosis/cirrhosis. Steatosis is the earliest response to heavy drinking and is characterized by the deposition of fat in hepatocytes. Steatosis can progress to steatohepatitis, which is a more severe, inflammatory type of liver injury. This stage of liver disease can lead to the development of fibrosis, during which there is excessive deposition of extracellular matrix proteins. The fibrotic response begins with active pericellular fibrosis, which may progress to cirrhosis, characterized by excessive liver scarring, vascular alterations, and eventual liver failure. Among problem drinkers, about 35 percent develop advanced liver disease because a number of disease modifiers exacerbate, slow, or prevent alcoholic liver disease progression. There are still no FDA-approved pharmacological or nutritional therapies for treating patients with alcoholic liver disease. Cessation of drinking (i.e., abstinence) is an integral part of therapy. Liver transplantation remains the life-saving strategy for patients with end-stage alcoholic liver disease.
PMID:28988570      URL    
[本文引用:1]
[9] GE N,LIANG H,ZHAO Y Y,et al.Aplysin protects against alcohol-induced liver injury via alleviating oxidative damage and modulating endogenous apoptosis-related genes expression in rats[J].J Food Sci,2018,83(10):2612-2621.
We investigated the protective effects and possible mechanisms of Aplysin against alcohol-induced liver injury. Rats were given daily either alcohol only (alcohol model group; 8 to 12 mL/kg body weight), one of three doses of Aplysin (50, 100, or 150 mg/kg Aplysin) plus alcohol, or volume-matched saline. After 6 weeks, the effects of Aplysin were assessed in terms of changes in histology, biochemical indices, and DNA oxidative damage. Potential mechanisms were analyzed through measurements of lipid peroxidation, antioxidant defense systems, expression of cytochrome P450 2E1, and expression of apoptosis-related genes. We found that Aplysin significantly protected the liver against alcohol-induced oxidative injury, evidenced by improved hepatic histological structure, inhibited alcohol-induced elevation of serum biochemical indices, attenuated extents of hepatocellular DNA damage. At a mechanistic level, Aplysin alleviated alcohol-induced oxidative stress as illustrated by the revivification of erythrocyte membrane fluidity, the attenuation of glutathione depletion, the restoration of antioxidase activities, and reduced malondialdehyde overproduction. Furthermore, the mRNA levels of Bax, cytochrome c, and cytochrome P450 2E1 were significantly down-regulated, whereas those of Bcl-2 and caspase-9 and caspase-3 were markedly up-regulated. These findings suggest that Aplysin provides significant protection against alcohol-induced liver injury, possibly through alleviating oxidative damage and modulating endogenous apoptosis-related genes expression. PRACTICAL APPLICATION: Many natural components derived from alga have been used in the food, cosmetics, and biomedicine industries. Aplysin, a marine bromosesquiterpene, was extracted from the red alga Laurencia tristicha, which could effectively protect against alcohol-induced liver injury, might be a potential natural sources for preventing alcoholic liver damage.
DOI:10.1111/1750-3841.14320      PMID:30192013      URL    
[本文引用:1]
[10] SZABO G,PETRASEK J.Gut-liver axis and sterile signals in the development of alcoholic liver disease[J].Alcohol Alcohol,2017,52(4):414-424.
Background: Innate immunity plays a critical role in the development of alcohol-induced liver inflammation. Understanding the inter-relationship of signals from within and outside of the liver that trigger liver inflammation is pivotal for development of novel therapeutic targets of alcoholic liver disease (ALD). Aim: The aim of this paper is to review recent advances in the field of alcohol-induced liver inflammation. Methods: A detailed literature review was performed using the PubMed database published between January 1980 and December 2016. Results: We provide an update on the role of intestinal microbiome, metabolome and the gut-liver axis in ALD, discuss the growing body of evidence on the diversity of liver macrophages and their differential contribution to alcohol-induced liver inflammation, and highlight the crucial role of inflammasomes in integration of inflammatory signals in ALD. Studies to date have identified a multitude of new therapeutic targets, some of which are currently being tested in patients with severe alcoholic hepatitis. These treatments aim to strengthen the intestinal barrier, ameliorate liver inflammation and augment hepatocyte regeneration. Conclusion: Given the complexity of inflammation in ALD, multiple pathobiological mechanisms may need to be targeted at the same time as it seems unlikely that there is a single dominant pathogenic pathway in ALD that would be easily targeted using a single target drug approach. Short summary: Here, we focus on recent advances in immunopathogenesis of alcoholic liver disease (ALD), including gut-liver axis, hepatic macrophage activation, sterile inflammation and synergy between bacterial and sterile signals. We propose a multiple parallel hit model of inflammation in ALD and discuss its implications for clinical trials in alcoholic hepatitis.
DOI:10.1093/alcalc/agx025      PMID:28482064      URL    
[本文引用:1]
[11] 郑铁生,鄢盛恺.临床生物化学检验[M].2版.北京:中国医药科技出版社,2010:273.
[本文引用:1]
[12] CARTER A R,BORGES M C,BENN M,et al.Combined association of body mass index and alcohol consumption with biomarkers for liver injury and incidence of liver disease:a mendelian randomization study[J].JAMA Netw Open,2019,2(3):e190305.
Importance: Individually, higher body mass index (BMI) and alcohol consumption increase the risk of liver disease. Evidence of a joint association is mixed; however, previous studies have not used causal inference methods robust to confounding and reverse causation. Understanding any true effect is key to developing effective interventions to reduce liver disease. Objective: To investigate the joint association of BMI and alcohol consumption with liver injury biomarkers and incident liver disease using factorial mendelian randomization (MR). Design, Setting, and Participants: A population-based cohort study (Copenhagen General Population Study) recruited a random sample of Copenhagen, Denmark, residents aged 20 years or older of white, Danish descent (N = 98 643) between November 25, 2003, and July 1, 2014. Data were also obtained from ongoing links to national registers, and then analyzed from September 30, 2016, to April 23, 2018. Exposures: High and low BMI and alcohol consumption categories from baseline-measured or self-reported observational data and genetic variants predicting BMI and alcohol consumption. Main Outcomes and Measures: Plasma biomarkers of liver injury (alanine aminotransferase [ALT] and gamma-glutamyltransferase [GGT]) and incident cases of liver disease from hospital records were the outcomes. Results: Of the 98643 individuals recruited, 91552 (54 299 [45.2%] women; mean [SD] age, 58 [13.05] years) with no baseline liver disease were included in main analyses. Individuals had a mean (SD) BMI of 26.2 (4.3) and consumed a mean (SD) of 10.6 (10.2) U/wk of alcohol. In factorial MR analyses, considering the high BMI/high alcohol group as the reference, mean circulating ALT and GGT levels were lowest in the low BMI/low alcohol group (ALT: -2.32%; 95% CI, -4.29% to -0.35%, and GGT: -3.56%; 95% CI, -5.88% to -1.24%). Individuals with low BMI/high alcohol use and high BMI/low alcohol use also had lower mean circulating ALT levels (low BMI/high alcohol use: -1.31%; 95% CI, -1.88% to -0.73%, and high BMI/low alcohol use: -0.81%; 95% CI, -2.86% to 1.22%) and GGT levels (low BMI/high alcohol use: -0.91%; 95% CI, -1.60% to -0.22%, and high BMI/low alcohol use: -1.13%; 95% CI, -3.55% to 1.30%) compared with the high BMI/high alcohol use reference group. These patterns were similar in multivariable factorial analyses. For incident liver disease (N = 580), factorial MR results were less conclusive (odds ratio of liver disease vs high BMI/high alcohol group: 1.01; 95% CI, 0.84 to 1.18, for the low BMI/high alcohol group, 1.22; 95% CI, 0.56 to 1.88 for the high BMI/low alcohol group, and 0.99 (95% CI, 0.41 to 1.56 for the low BMI/low alcohol group). Conclusions and Relevance: Interventions to reduce both BMI and alcohol consumption might reduce population levels of biomarkers of liver injury more than interventions aimed at either BMI or alcohol use alone. However, it is not clear whether this intervention will directly translate to a reduced risk of liver disease.
DOI:10.1001/jamanetworkopen.2019.0305      PMID:30848805      URL    
[本文引用:1]
[13] SINGH S,OSNA N A,KHARBANDA K K.Treatment options for alcoholic and non-alcoholic fatty liver disease:A review[J].World J Gastroenterol,2017,23(36):6549-6570.
Alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are serious health problems worldwide. These two diseases have similar pathological spectra, ranging from simple steatosis to hepatitis to cirrhosis and hepatocellular carcinoma. Although most people with excessive alcohol or calorie intake display abnormal fat accumulation in the liver (simple steatosis), a small percentage develops progressive liver disease. Despite extensive research on understanding the pathophysiology of both these diseases there are still no targeted therapies available. The treatment for ALD remains as it was 50 years ago: abstinence, nutritional support and corticosteroids (or pentoxifylline as an alternative if steroids are contraindicated). As for NAFLD, the treatment modality is mainly directed toward weight loss and co-morbidity management. Therefore, new pathophysiology directed therapies are urgently needed. However, the involvement of several inter-related pathways in the pathogenesis of these diseases suggests that a single therapeutic agent is unlikely to be an effective treatment strategy. Hence, a combination therapy towards multiple targets would eventually be required. In this review, we delineate the treatment options in ALD and NAFLD, including various new targeted therapies that are currently under investigation. We hope that soon we will be having an effective multi-therapeutic regimen for each disease.
DOI:10.3748/wjg.v23.i36.6549      PMID:29085205      URL    
[本文引用:1]
[14] ZWOLAK A,SURDACKA A,DANILUK J.Bcl-2 and Fas expression in peripheral blood leukocytes of patients with alcoholic and autoimmune liver disorders[J].Human Exp Toxicol,2016,35(8):799-807.
DOI:10.1177/0960327115607078      URL    
[本文引用:1]
[15] 章雅丹. 新型芹菜素纳米脂质体对糖尿病心肌病大鼠心肌细胞凋亡的影响[J].医药导报,2019,38(5):555-559.
[本文引用:1]
[16] KNIGHT T,LUEDTKE D,EDWARDS H,et al.A delicate balance-the Bcl-2 family and its role in apoptosis,oncogenesis,and cancer therapeutics[J].Biochem Pharmaco,2019,162:250-261.
DOI:10.1016/j.bcp.2019.01.015      URL    
[本文引用:1]
[17] LIAO W,LIU J,LIU B,et al.JIB04 induces cell apoptosis via activation of the p53/Bcl2/caspase pathway in MHCC97H and HepG2 cells[J].Oncol Rep,2018,40(6):3812-3820.
JIB04 is a structurally unique small molecule, known to exhibit anticancer activity and to inhibit the growth of human lung cancer and prostate cancer cell lines. However, the anticancer effect of JIB04 against human hepatic carcinoma, and its underlying mechanisms, are still unclear. In the present study, MHCC97H and HepG2 cells were employed to investigate the anticancer effects of JIB04 on cell viability and apoptosis. Annexin V/PI staining, a CCK8 assay and western blot analysis demonstrated that JIB04 induced apoptosis in MHCC97H and HepG2 cells, which was evidenced by the expression of proapoptotic and apoptotic proteins including p53, Bak, Bax, caspase3 and caspase9. Subsequently, the expression trends of Bcl2 and p53 were reversed after cotreatment with pifithrinalpha (PFTalpha, a p53 inhibitor). The results revealed that JIB04 suppressed the cell viability of MHCC97H and HepG2 cells in a concentrationdependent manner. Meanwhile, it was also demonstrated that JIB04 effectively triggered MHCC97H and HepG2 cell apoptosis by downregulating Bcl2/Bax expression, and upregulating proapoptotic and apoptotic protein expression via the p53/Bcl2/caspase signaling pathway. JIB04 had effects on the inhibition of cell viability and the induction of apoptosis in MHCC97H and HepG2 cells. The underlying mechanism of action of JIB04 was associated with the p53/Bcl2/caspase signaling pathway. Our findings provide a foundation for understanding the anticancer effect of JIB04 on MHCC97H and HepG2 cells, and suggested that JIB04 may be a promising therapeutic agent in human liver cancer.
DOI:10.3892/or.2018.6737      PMID:30272369      URL    
[本文引用:1]
[18] KALE J,OSTERLUND E J,ANDREWS D W.Bcl-2 family proteins:changing partners in the dance towards death[J].Cell Death Differ,2018,25(1):65-80.
The BCL-2 family of proteins controls cell death primarily by direct binding interactions that regulate mitochondrial outer membrane permeabilization (MOMP) leading to the irreversible release of intermembrane space proteins, subsequent caspase activation and apoptosis. The affinities and relative abundance of the BCL-2 family proteins dictate the predominate interactions between anti-apoptotic and pro-apoptotic BCL-2 family proteins that regulate MOMP. We highlight the core mechanisms of BCL-2 family regulation of MOMP with an emphasis on how the interactions between the BCL-2 family proteins govern cell fate. We address the critical importance of both the concentration and affinities of BCL-2 family proteins and show how differences in either can greatly change the outcome. Further, we explain the importance of using full-length BCL-2 family proteins (versus truncated versions or peptides) to parse out the core mechanisms of MOMP regulation by the BCL-2 family. Finally, we discuss how post-translational modifications and differing intracellular localizations alter the mechanisms of apoptosis regulation by BCL-2 family proteins. Successful therapeutic intervention of MOMP regulation in human disease requires an understanding of the factors that mediate the major binding interactions between BCL-2 family proteins in cells.
DOI:10.1038/cdd.2017.186      PMID:29149100      URL    
[本文引用:1]
[19] WANG X,CHEN J,WANG H,et al.Memantine can reduce ethanol-induced caspase-3 activity and apoptosis in H4 cells by decreasing intracellular calcium[J].J Mol Neurosci,2017,62(3/4):402-411.
DOI:10.1007/s12031-017-0948-3      URL    
[本文引用:1]
[20] ZHANG Y,YANG X,GE X H,et al.Puerarin attenuates neurological deficits via Bcl-2/Bax/cleaved caspase-3 and Sirt3/SOD2 apoptotic pathways in subarachnoid hemorrhage mice[J].Biomed Pharmacother,2019,109:726-733.
BACKGROUND: Subarachnoid hemorrhage (SAH) results in many brain dysfunctions and the neuroprotective function of puerarin after brain damage has been demonstrated in several studies. But whether puerarin can reduce brain nerve damage after SAH is not clear.In this study, we hypothesized that puerarin had the neuroprotective effect after SAH, and this protection could be mediated by bothBcl-2/Bax/Cleaved caspase-3 and SIRT3/SOD2 apoptotic signaling pathways. METHODS: First, we used neurological score, brain water content and so on to detect the neurological deficits after SAH. Then apoptosis neuron rate was detected by TUNEL staining. Western blot was carried out to explore the alteration of Blc-2, Bax, cleaved caspase-3 and Sirt3.Also, ROS acitivity and As-lysine level of SOD2 should be detected with assays. RESULTS: We demonstrate that puerarin attenuated the neurological deficits, effectively relieves cerebral edema, and reduce BBB disruption in SAH mice.And we revealed that a reduced rate of apoptosis neuron has been found out in puerarin treatment after SAH. In addition, obviously higher ratio of Blc-2/Bax and decreased expression of cleaved caspase-3 in puerarin-treated SAH micecomparing with vehicle-treated SAH animals had been found. Furthermore, puerarin effectively reversed these alterations in expression and inhibits ROSproduction induced by SAH. Also, puerarin can increase SOD activation after SAH and protect the expression of Sirt3 after SAH. CONCLUSIONS: In coclusion, puerarin can provide potential neuroprotection from the SAH damages, and can be act as a novel therapy for SAH.
DOI:10.1016/j.biopha.2018.10.161      PMID:30551525      URL    
[本文引用:1]
[21] WANG Y,SONG Q,HUANG X,et al.Long noncoding RNA GAS5 promotes apoptosis in primary nucleus pulposus cells derived from the human intervertebral disc via Bcl-2 downregulation and caspase-3 upregulation[J].Mol Med Rep,2019,19(3):2164-2172.
Nucleus pulposus cell (NPC) apoptosis serves an important role in intervertebral disc degeneration (IDD); however, the roles of long noncoding RNAs (lncRNAs) in this process remain unknown. The present study aimed to determine the effects of the lncRNA growth arrestspecific transcript 5 (GAS5) on the apoptosis of primary human NPCs derived from the intervertebral disc, and to investigate the underlying mechanisms. TargetScan was used to predict the lncRNAs targeted by microRNA155 (miR155). Then, NPCs were subjected to lentivirusmediated transduction of miR155 or GAS5. A human lncRNA and mRNA array was used to screen differentially expressed lncRNAs following miR155 overexpression. GAS5 and miR155 expression levels were determined by reverse transcriptionquantitative polymerase chain reaction. After GAS5 overexpression, apoptosis was assessed by flow cytometry via Annexin V/propidium iodide staining. Western blotting was employed to determine the expression of apoptosisassociated proteins, including caspase3 and B cell lymphoma 2 (Bcl2). TargetScan indicated GAS5 had one binding site for miR155. Following exogenous transfection of miR155 mimics, GAS5 expression levels in NPCs were significantly decreased (P<0.05). Interestingly, miR155 overexpression in NPCs resulted in 721 differentially expressed lncRNAs compared with the negative control group (P<0.05), including 492 and 229 upregulated and downregulated lncRNAs respectively. In addition, 18 transcripts of GAS5 exhibited a downregulated expression profile. GAS5 overexpression in NPCs resulted in enhanced caspase3 decreased Bcl2 expression levels; the apoptosis of NPCs was significantly increased (P<0.05). The results of the present study revealed that overexpression of lncRNA GAS5 may promotes NPC apoptosis via Bcl2 downregulation and caspase3 upregulation, which may be associated with miR155. The results of the present study suggest that lncRNA GAS5silenced NPCs, or lentivirusmediated lncRNA GAS5 knockdown may be precise and effective therapeutic strategies in the treatment of IDD.
DOI:10.3892/mmr.2019.9883      PMID:30747227      URL    
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关键词(key words)
熊果酸
沙棘
肝病
酒精性
细胞凋亡

Ursolic acid
L.
Liver disease
alcohol
Cell apoptosis

作者
李可欣
张男男
侯瑞丽
张文龙
高龙
苗晓涵
戈娜

LI Kexin
ZHANG Nannan
HOU Ruili
ZHANG Wenlong
GAO Long
MIAO Xiaohan
GE Na