摘要/Abstract
摘要： 目的 ·探究 SIRT7（sirtuin 7）对脂多糖（ lipopolysaccharide，LPS）或 D-氨基半乳糖（ D-galactosamine，D-GalN）/LPS诱导的急性肝损伤的作用及机制。方法 ·将 13周龄的 C57BL/6J小鼠随机分为生理盐水组（ n5）、LPS组（n7）和 D-GalN/LPS组（n8），分别腹腔注射生理盐水、 LPS和 D-GalN/LPS。24 h后收集生理盐水组和 LPS组的小鼠血清和肝脏， D-GalN/LPS组于注射后 8 h收集血清和肝脏。利用苏木精 -伊红（ H-E）染色比较 3组小鼠肝脏病理学改变，同时观察血清学生化指标的变化；利用 TUNEL染色和 F4/80染色明确小鼠肝脏内细胞凋亡和炎症细胞浸润程度；利用 realtime-PCR检测各组小鼠肝组织中 SIRT7、白介素 -1β（interleukin 1β，IL-1β）、IL-6、肿瘤坏死因子 α（tumor necrosis factor α，TNF-α）等的 mRNA水平；利用 Western blotting检测各组小鼠肝组织中 SIRT7、激活型胱天蛋白酶 3（cleaved-caspase3）和伴侣葡萄糖调节蛋白（the 78 kDa glucose regulated protein，GRP78）等的蛋白水平。体外实验中，在正常小鼠肝细胞 AML-12细胞中过表达 SIRT7，并给予 LPS刺激，利用 Western blotting明确 SIRT7对 LPS引起的内质网应激信号通路分子的调控。结果 ·注射 LPS或 D-GalN/LPS后，小鼠肝脏出现明显的炎症细胞浸润、充血及肝细胞凋亡，血清中谷丙转氨酶（ glutamic-pyruvic transaminase，GPT）和谷草转氨酶（ glutamic-oxalacetic transaminase，GOT）水平明显升高，肝组织中 SIRT7的 mRNA和蛋白水平均明显降低，内质网应激蛋白 GRP78表达明显上调。在 AML-12细胞系中， SIRT7过表达可明显抑制 LPS引起的 GRP78 蛋白上调。结论 · SIRT7通过抑制内质网应激中的 GRP78减轻 LPS或 D-GalN/LPS诱导的肝细胞凋亡。
关键词: 去乙酰化酶（sirtuin）, 脓毒症, SIRT7, 急性肝损伤, 内质网应激, 细胞凋亡
Abstract:
Objective · To investigate the effects of SIRT7 on acute liver injury inducedlipopolysaccharide (LPS) or D-galactosamine (D-GalN)/LPS and its mechanisms. Methods · Thirteen-week-old C57BL/6J mice were randomly divided into normal saline group (n5), LPS group (n7), and D-GalN/ LPS group (n8), which were respectively intraperitoneally injected with normal saline, LPS or D-GalN/LPS. The serum and livers of normal saline group and LPS group mice were collected 24 hours after the injection, and the samples of D-GalN/LPS group were collected 8 hours after the injection. Liver pathological changes were comparedusing H-E staining, and serological indicators of the mice three groups were also compared. Liver apoptosis and inflammatory cells infiltration were determinedTUNEL staining and F4/80 staining. Meanwhile, the mRNA levels of SIRT7 and inflammatory factors, including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α) in livers were detectedrealtime-PCR. Western blotting was used to detect the protein levels of SIRT7, cleaved-caspase3 and the 78 kDa glucose regulated protein (GRP78) in moliver tissues. AML-12 cell line overexpressing SIRT7 was stimulated with LPS, and Western blotting was used to study the roles of SIRT7 in the endoplasmic reticulum (ER) stress inducedLPS in vitro. Results · LPS orD-GalN/LPSinduced inflammatorycellsinfiltration,hyperemia and hepatocytesapoptosis inlivers.Meanwhile, serum glutamic-pyruvic transaminase (GPT) and glutamic-oxalacetic transaminase (GOT) in the mice treatedLPS or D-GalN/LPS were significantly increased. Moreover, both liver SIRT7 mRNA and protein levels were down-regulated, while GRP78 protein in ER stress pathway was up-regulated. In AML-12 cells, SIRT7 over inhibited LPS-induced up-regulation of GRP78. Conclusion · SIRT7 protects against LPS or D-GalN/LPS-induced hepatocytes apoptosisattenuating ER stress via inactivating GRP78.
Key words: sirtuin, sepsis, SIRT7, acute liver injury, endoplasmic reticulum stress, apoptosis
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