Yuan Q X, Nagao Y, Gaal K, Hu B, French S W
Harbor-UCLA Medical Center, Torrance, California 90509, USA.
Exp Mol Pathol. 1998 Oct;65(2):87-103. doi: 10.1006/exmp.1998.2231.
Drug-primed mice form Mallory bodies in their liver after various types of liver injury such as heat shock, drug refeeding, or ethanol ingestion. However, the mechanisms involved that lead to Mallory body formation after these different treatments are unknown. There may be a common pathway of Mallory body formation that is initiated by these different types of injuries. Recently it was shown that the phosphatase 1/2A inhibitor okadaic acid induced Mallory body formation, suggesting that the mechanism of formation involves hyperphosphorylation or oxidative stress-induced NFkappaB activation. To test this hypothesis we exposed drug-primed mice to okadaic acid and measured phosphorylation of Mallory body proteins immunohistochemically and by immunoblot chemiluminescence using an antibody specific for phosphothreonine. NFkappaB activation was measured by a gel shift retardation assay of nuclear lysates. Beginning 15 min after okadaic acid injection, complex changes were progressively seen in the liver cells focally including aggregation of cytokeratins 8 and 18 in hepatocytes which otherwise failed to stain normally with cytokeratin antibody. The aggregates stained positive with ubiquitin and phosphothreonine antibodies. Immunoblots showed a progressive increase in positive staining of the Mallory body band with the antibody to phosphothreonine. NFkappaB activation was progressive up to 2 h after okadaic acid treatment but was downregulated 7 days later. In summary we show for the first time the effect of okadaic acid on the liver cytokeratins in vivo. We conclude that hyperphosphorylation and NFkappaB activation may play a role in the early phases of Mallory body formation.
药物预处理的小鼠在经历各种类型的肝损伤（如热休克、重新给予药物或摄入乙醇）后，肝脏中会形成马洛里小体。然而，这些不同处理后导致马洛里小体形成的相关机制尚不清楚。可能存在一条由这些不同类型损伤引发的马洛里小体形成的共同途径。最近有研究表明，磷酸酶1/2A抑制剂冈田酸可诱导马洛里小体形成，这表明其形成机制涉及过度磷酸化或氧化应激诱导的核因子κB激活。为了验证这一假设，我们将药物预处理的小鼠暴露于冈田酸中，并通过免疫组织化学以及使用针对磷酸苏氨酸的特异性抗体进行免疫印迹化学发光法来检测马洛里小体蛋白的磷酸化情况。通过对核裂解物进行凝胶迁移阻滞试验来检测核因子κB的激活情况。在注射冈田酸后15分钟开始，在肝细胞中逐渐观察到复杂的变化，包括细胞角蛋白8和18在肝细胞中的聚集，而这些肝细胞原本不能被细胞角蛋白抗体正常染色。这些聚集体用泛素和磷酸苏氨酸抗体染色呈阳性。免疫印迹显示，马洛里小体条带与磷酸苏氨酸抗体的阳性染色呈逐渐增加趋势。在冈田酸处理后2小时内，核因子κB的激活呈逐渐上升趋势，但在7天后下调。总之，我们首次展示了冈田酸在体内对肝脏细胞角蛋白的影响。我们得出结论，过度磷酸化和核因子κB激活可能在马洛里小体形成的早期阶段起作用。
