香叶木素对APAP诱导的肝损伤作用及机制研究

Effect and Mechanism of Diosmetin on APAP-Induced Liver Injury

为了探究香叶木素(diosmetin)对对乙酰氨基酚(APAP)诱导的肝损伤作用及机制,将48只昆明小鼠随机分为6组,分别为空白组(NC)、APAP肝损伤模型组(DILI)、香叶木素低、中、高剂量治疗组(XL+DILI、XM+DILI、XH+DILI)、香叶木素高剂量对照组(XHD),每组8只。NC组和XHD组小鼠腹腔注射生理盐水,其余小鼠腹腔注射等体量的APAP注射液(300 mg/kg),每日2次,连续注射4 d。第5天开始NC组和DILI组小鼠灌胃生理盐水,XL+DILI组、XM+DILI组、XH+DILI组和XHD组小鼠分别按照25 mg/kg、50 mg/kg、100 mg/kg、100 mg/kg等体量灌胃香叶木素药液,每日2次,连续3 d。最后一次给药12 h后,检测各组小鼠血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)活性,以及肝组织中谷胱甘肽(GSH)、超氧化物歧化酶(SOD)、过氧化氢(H_(2)O_(2))和丙二醛(MDA)的水平,肝组织切片HE染色观察肝损伤情况,qPCR检测细胞色素P450酶2E1(CYP2E1)mRNA表达水平,Western blot检测CYP2E1、热休克蛋白60(HSP60)和半胱氨酸天冬氨酸蛋白酶3(Caspase 3)在蛋白水平的表达。结果表明:香叶木素可降低ALT和AST活性,提高GSH和SOD水平,显著降低H_(2)O_(2)和MDA水平。香叶木素可显著降低DILI小鼠肝组织中CYP2E1 mRNA和蛋白表达水平,对肝脏组织中HSP60和Caspase 3蛋白的表达具有良好的抑制作用。综上所述,香叶木素具有抗APAP诱导的小鼠肝损伤的作用,其机制可能与改善氧化应激、抑制CYP2E1表达、减缓线粒体应激和细胞凋亡有关。

In order to investigate the effect and mechanism of diosmetin on acetaminophen(APAP)-induced liver injury,forty-eight Kunming mice were randomly divided into six groups with eight mice in each group.The groups included normal control group(NC),APAP liver injury model group(DILI),diosmetin low,medium and high dose treatment groups(XL+DILI,XM+DILI,XH+DILI),diosmetin high dose control group(XHD).The mice in the NC group and XHD group were injected intraperitoneally with saline,while the rest of the mice were injected intraperitoneally with equal amount of APAP injection(300 mg/kg)twice a day for 4 days.On the fifth day,mice in the NC group and DILI group were gavaged with saline,while mice in the XL+DILI,XM+DILI,XH+DILI and XHD groups were gavaged with diosmetin at levels of 25 mg/kg,50 mg/kg,100 mg/kg and 100 mg/kg twice a day for 3 days.Twelve hours after the last gavage,the serum alanine transaminase(ALT),aspartate transferase(AST)activities and the levels of glutathione(GSH),superoxide dismutase(SOD),hydrogen peroxide(H_(2)O_(2))and malondialdehyde(MDA)in liver tissues of the mice were measured,and liver sections were stained with hematoxylin-eosin to observe liver injury.The expression of cytochrome P450 enzyme 2E1(CYP2E1)at the mRNA level was detected by qPCR,and the expression of CYP2E1,heat shock protein 60(HSP60)and cysteine aspartate protease 3(Caspase 3)at the protein level was detected by Western blot.The results showed that diosmetin reduced ALT and AST activity,increased GSH and SOD levels,significantly reduced H_(2)O_(2) and MDA levels.Gene and protein expression of CYP2E1 in liver tissues of DILI mice were significantly reduced,and the expression of HSP60 and Caspase 3 proteins in liver were well inhibited.In summary,diosmetin can protect the liver from APAP induced injury in mice,and the mechanism may be related to the improvement of oxidative stress,inhibition of CYP2E1 expression,moderation of the mitochondrial stress and apoptosis.