斑蟊素致肝脏慢性损伤的研究

Preliminary research on chronic liver injury induced by cantharidin

目的:探讨斑蟊素致肝脏慢性损伤的可能机制。方法:用不同剂量斑蟊素持续对昆明小鼠灌胃14天,检测其肝功能、肝脏指数和肝脏病理变化,检测ATF-6、XBP1、GRP78、ATF-4、CHOP、BAX、Bcl-2、caspase3、caspase8和caspase9蛋白的表达。用不同浓度斑蟊素连续培养人肝细胞LO2系,显微观察生长,CCK-8检测活性,检测GRP78和CHOP的mRNA的表达,检测GRP78、ATF-4、CHOP、BAX、Bcl-2、caspase3、caspase8和caspase9蛋白的表达。结果:斑蟊素(≥1.0 mg/kg)能增高小鼠转氨酶和肝脏指数,肝细胞坏死及凋亡,且程度与其剂量正相关;ATF-6、XBP1、GRP78、ATF-4、CHOP、BAX、caspase3、caspase8和caspase9蛋白的表达上调,Bcl-2蛋白的表达下调。斑蟊素(≥0.392μg/ml)抑制LO2系细胞活性,增高GRP78和CHOP基因mRNA的表达,GRP78、ATF-4、CHOP、BAX、caspase3、caspase8和caspase9蛋白的表达上调,Bcl-2蛋白的表达下调。结论:斑蟊素可能主要通过内质网应激途径诱导肝细胞凋亡从而导致肝脏的慢性损伤。

Objective：To investigate the possible mechanism of chronic injury induced by cantharidin in mice liver and human hepatocytes. Methods： The mice were orally administered eantharidin for 14 days. The hepatic function, liver index and morphological examination were measured. Western blot was used to identify the expression of ATF-6, XBP1, GRP78, ATF-4, CHOP, BAX, Bel-2, caspase3, caspase8 and easpase9 proteins. Human LO2 hepatoeytes were incubated with different concentrations of eantharidin. Microscope was used to observe the cell growth. CCK-8 assay was used to measure the proliferation. Real-time PCR was performed to quantify the mRNA expression of GRP78 and CHOP. The expressions of GRP78, ATF-4, CHOP, BAX, caspase3,caspase8,easpase9 and Bcl-2 protein were examined by Western blot. Results： Cantharidin （ 1〉 1.0mg/kg） eoald increase transaminases and liver index. Liver cell necrosis and apoptosis were enhanced. It was positively correlated with the dose. The expressions of ATF-6, XBP1, GRP78, ATF-4, CHOP, BAX, caspase3, caspase8 and caspase9 protein were increased and the expression of Bcl- 2 protein decreased （ P 〈 0.05 ）. Proliferation of LO2 hepatoeytes was inhibited by Cantharidin （ ≥ 0. 392 μg/ml） . The expressions of GRP78 and CHOP mRNA increased. The expression of GRP78, ATF-4, CHOP, BAX, caspase3, easpase8 and caspase9 were up-regulated and the expression of Bcl-2 protein was down-regulated （ P 〈 0.05 ） ; Conclusion ： Cantharidin can induce liver cell apoptosis by endoplasmie reticulum stress and damaging hepatoeytes.