基于Keap1/ARE通路探究氧化苦参碱对肝癌大鼠肝损伤的改善作用

Inhibitory effect of oxymatrine on liver cancer in rats based on Keap1/ARE pathway and its effect on liver injury

目的:分析氧化苦参碱(OMT)对肝癌大鼠肝损伤的改善作用,并探讨Kelch样环氧氯丙烷相关蛋白-1(Keap1)/抗氧化反应元件(ARE)通路在该过程中发挥的作用。方法:大鼠随机分为空白组、模型组、OMT组、Brusatol组、OMT+Bru组。除空白组外所有大鼠腹腔注射二乙基亚硝胺50 mg/kg建立肝癌大鼠模型。各组用不同方法干预20 d。干预结束,检测血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、总胆红素(TBil)、总胆汁酸(TBA)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)水平;HE染色观察肝组织病理学变化;TUNEL染色检测大鼠肝癌细胞凋亡情况;Western Blot法检测肝组织核因子E2相关因子2(Nrf2)、Keap1、ARE蛋白相对表达水平。结果:与模型组比较,OMT组、OMT+Bru组大鼠ALT、AST、TBil、TBA、IL-1β、TNF-α水平降低,Brusatol组大鼠血清ALT、AST、TBil、TBA、IL-1β、TNF-α水平升高,且OMT组血清ALT、AST、TBil、TBA、IL-1β、TNF-α水平低于OMT+Bru组(P<0.05)。HE染色结果显示:空白组肝细胞形态正常,排列整齐。模型组肝细胞排列紊乱,明显肿胀,大量炎细胞浸润。OMT组肝细胞损伤程度明显改善。Brusatol组肝细胞肿胀、坏死、排列紊乱,炎细胞浸润。OMT+Bru组肝细胞损伤有所改善。与模型组比较,OMT组、OMT+Bru组肝癌细胞凋亡率、Nrf2、ARE蛋白相对表达水平升高,Keap1蛋白相对表达水平降低,Brusatol组肝癌细胞凋亡率、Nrf2、ARE蛋白相对表达水平降低,Keap1蛋白相对表达水平升高(P<0.05);OMT组肝癌细胞凋亡率,Nrf2、ARE蛋白相对表达水平高于OMT+Bru组,Keap1蛋白相对表达水平低于OMT+Bru组(P<0.05)。结论:OMT对大鼠肝癌具有抑制作用,能够改善肝损伤,可能是通过激活Keap1/ARE通路实现的。

Objective:To analyze the inhibitory effect of oxymatrine(OMT)on liver cancer in rats and its effect on liver injury,and to explore the role of Kelch-like epichlorohydrin-related protein-1(Keap1)/antioxidant response element(ARE)pathway.Methods:Rats were randomly divided into control group,model group,OMT group,Brusatol group and OMT+Bru group.All rats except the control group were intraperitoneally injected with diethylnitrosamine 50 mg/kg to establish a rat model of liver cancer.Each group used different drugs for intervention.At the end of the intervention,the serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),total bilirubin(TBil),total bile acid(TBA),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)level.HE staining to observe the pathological changes of liver tissue;TUNEL staining to detect the apoptosis of rat liver cancer cells;Western Bloting method to detect the relative expression levels of nuclear factor E2 related factor 2(Nrf2),Keap1 and ARE protein in liver tissue.Results:Compared with the model group,the levels of ALT,AST,TBil,TBA,IL-1β,and TNF-αin the OMT group and OMT+Bru group were decreased,and the serum ALT,AST,TB,TBA,IL-1β,TNF-αlevel in the Brusatol group were increased,and serum ALT,AST,TB,TBA,IL-1βand TNF-αlevels in OMT group were lower than OMT+Bru group(P<0.05).HE staining results showed that the liver cells in the control group were normal in shape and arranged neatly.The liver cells in the model group were arranged disorderly,swollen,and a large number of inflammatory cells infiltrated.The degree of liver cell damage in the OMT group was significantly improved.In Brusatol group,hepatocytes were swollen,necrotic,arranged disorderly,and inflammatory cells infiltrated.The liver cell damage in the OMT+Bru group was improved.Compared with the model group,the apoptotic rate of liver cancer cells,the relative expression levels of Nrf2 and ARE protein in the OMT group and OMT+Bru group were increased,the relative expression levels of Keap1 protein was decreased,the apoptotic rate of liver cancer cells,the relative expression levels of Nrf2,ARE protein in the Brusatol group were decrease,the relative expression level of Keap1 protein was increased(P<0.05),The apoptotic rate of liver cancer cells,the relative expression levels of Nrf2 and ARE protein in the OMT group were higher than that in the OMT+Bru group,and the relative expression level of Keap1 protein was lower than that in the OMT+Bru group(P<0.05).Conclusion:OMT has an inhibitory effect on rat liver cancer and can improve liver injury,which may be achieved by activating the Keap1/ARE pathway.