化橘红水提物对酒精诱导的急性肝损伤保护作用

Protective Effect of Water Extract of Citri Grandis Exocarpium on Alcohol-induced Acute Liver Injury

目的:探究化橘红水提物对酒精诱导急性肝损伤小鼠的药效学作用,为化橘红解酒保肝作用的开发应用提供数据支撑。方法:利用高效液相色谱法(HPLC)确定化橘红水提物的主要成分。根据体质量将60只Balb/c小鼠随机分为6个组:正常组、模型组、化橘红提取物低、中、高剂量组(0.5、1.0、2.0 g·kg^(-1))及海王金樽片组(2.0 g·kg^(-1))。正常组及模型组给予等体积0.5%羧甲基纤维素钠溶液灌胃。每天灌胃给药1次,连续给药14 d。给药结束前1 d,小鼠禁食不禁水12 h,除正常组外其他各组按体质量灌胃56°白酒(13 mL·kg^(-1)),2 h后摘眼球取血。解剖获得肝脏,称其湿重。全自动生化仪检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)及乙醇脱氢酶(ADH)的表达水平;苏木素-伊红(HE)染色检测小鼠肝脏组织病理变化;原位末端标记法(TUNEL)/DAB双染法观察阳性细胞数的比例判断细胞凋亡情况;蛋白免疫印迹法(Western blot)检测凋亡相关蛋白表达情况。结果:化橘红水提物HPLC特征图谱显示主要成分是野漆树苷和柚皮苷。与正常组比较,模型组小鼠肝体比显著增加(P<0.01),肝脏损伤酶ALT和AST的表达明显增加(P<0.05,P<0.01),乙醇脱氢酶(ADH)表达水平明显降低(P<0.05),肝小叶结构显模糊,肝组织呈现不同程度的病理改变,细胞胞浆显著疏松、水肿,脂肪变性较严重,TUNEL阳性率显著增加(P<0.01),B细胞淋巴瘤-2(Bcl-2)表达显著降低(P<0.01),Bcl-2相关X蛋白(Bax)和胱天蛋白酶-3(Caspase-3)显著升高(P<0.01)。与模型组比较,化橘红水提物中剂量组能明显降低肝体比(P<0.05),化橘红水提物各剂量组能明显降低小鼠血清中ALT、AST活性(P<0.05,P<0.01),且能明显上调ADH的表达水平(P<0.05),能够明显改善由酒精引起的细胞胞浆疏松、水肿、脂肪变性的病理特征,明显降低肝细胞TUNEL阳性率(P<0.05,P<0.01),提升抗凋亡蛋白Bcl-2的表达(P<0.05),降低促凋亡蛋白Bax和Caspase-3的表达水平(P<0.01)。结论:化橘红水提物通过调节ALT、AST、ADH的表达水平、改善肝脏脂肪病变和肝细胞凋亡,保护急性肝损伤。

Objective:To explore the pharmacodynamic effect of the water extract of Citri Grandis exocarpium(WEC)on mice with alcohol-induced acute liver injury and provide data support for the development of this medicinal for anti-alcoholism and liver protection.Method:The main components of WEC were determined by high performance liquid chromatography(HPLC).Sixty Balb/c mice were randomized into 6 groups:control group(equal volume of 0.5%carboxymethyl cellulose sodium solution),model group(equal volume of 0.5%carboxymethyl cellulose sodium solution),low-,medium-,and high-dose WEC groups(0.5,1.0,2.0 g·kg^(-1)),and Haiwang Jinzun tablet positive control group(2.0 g·kg^(-1)).The administration lasted 14 days.One day before the end of the administration,mice were fasted for 12 h with free access to water.The mice,except the control group,were given 56°Chinese liquor(13 mL·kg^(-1)).After 2 h,blood was taken from eyeballs and the liver was dissected and weighed.Automatic biochemical analyzer was employed to detect the expression of alanine aminotransferase(ALT),aspartate aminotransferase(AST),and alcohol dehydrogenase(ADH).The pathological changes of liver tissues were observed based on hematoxylin-eosin(HE)staining,and apoptosis of hepatocytes based on TUNEL/DAB staining.The expression of proteins related to apoptosis was detected by Western blot.Result:According to the HPLC fingerprint,the main components of WEC were rhoifolin and naringin.Compared with the control group,the model group showed increase in liver/body weight ratio(P<0.01)and the expression of ALT and AST(P<0.05,P<0.01),decrease in the expression of ADH(P<0.05),blurred structure of hepatic lobules,pathological changes of liver tissue,loose cytoplasm with edema,severe steatosis,rise of the TUNEL-positive rate(P<0.01),reduction in expression of Bcl-2(P<0.01),and increase in Bax and Caspase-3(P<0.01).Compared with the model group,medium-dose WEC lowered liver/body weight ratio(P<0.05).All doses of WEC depressed the activity of ALT and AST(P<0.05,P<0.01),upregulated the expression of ADH(P<0.05),significantly improved the pathological features of alcohol-induced cytoplasmic porosity,edema,and steatosis,down-regulated the TUNEL-positive rate(P<0.05,P<0.01),enhanced the expression of Bcl-2(P<0.05),and decreased Bax and Caspase-3(P<0.01).Conclusion:WEC regulates the expression of ALT,AST,and ADH and improves hepatic steatosis and hepatocyte apoptosis to fight against acute liver injury.