Effect of melatonin on GRP-78 and CHOP expression in transplant liver tissues of rats
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摘要:
目的 通过检测褪黑素(MEL)对肝移植大鼠肝脏内质网应激(ERS)通路相关分子葡萄糖调节蛋白-78(GRP-78)及CCAAT增强子结合蛋白同源蛋白(CHOP)表达的影响, 探讨MEL对移植肝的作用。 方法 采用"磁环法"制作大鼠原位肝移植模型, 雄性SD大鼠随机分为3组:假手术组(Sham组)、原位肝移植组(OLT组)、原位肝移植+褪黑素处理组(OLT+MEL组), 每组各8只。术后24 h, 获取血清样本及肝脏标本。检测血清丙氨酸转移酶(ALT)及天冬氨酸转移酶(AST)水平, 苏木素-伊红(HE)染色观察各组肝组织病理学改变, 采用聚合酶链反应和蛋白免疫印迹试验检测GRP-78及CHOP的信使核糖核酸(mRNA)和蛋白表达水平。 结果 与Sham组比较, OLT组血清ALT、AST显著升高(均为P < 0.01), 肝组织损伤严重, GRP-78及CHOP在mRNA和蛋白水平表达明显增加(均为P < 0.01)。与OLT组比较, OLT+MEL组大鼠ALT、AST水平明显降低(均为P < 0.05), 肝组织损伤减轻, GRP-78及CHOP mRNA和蛋白表达水平明显降低(均为P < 0.05)。 结论 褪黑素能降低移植肝ERS相关分子GRP-78及CHOP在mRNA和蛋白水平的表达, 这可能是它减轻移植后肝脏损伤的机制之一。 -
关键词:
- 原位肝移植 /
- 大鼠 /
- 褪黑素 /
- 内质网应激 /
- 葡萄糖调节蛋白-78 /
- CCAAT增强子结合蛋白同源蛋白
Abstract:Objective To investigate the function of melatonin (MEL) on liver transplantation by detecting the effect of MEL on glucose regulated protein-78 (GRP-78) and CCAAT enhancer-binding protein homologous protein (CHOP) involved in the endoplasmic reticulum stress (ERS) pathways in livers of rats with liver transplantation. Methods The rat orthotopic liver transplantation model was established by using'magnetic-loop method'. Male SD rats were randomized into 3 groups: the sham operation group (Sham group), the orthotopic liver transplantation group (OLT group) and the orthotopic liver transplantation+MEL treatment group (OLT+MEL group), 8 rats in each group. Serum sample and liver sample were collected at 24 h after operation. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected. Liver tissues were stained with hematoxylin-eosin (HE) and pathological changes of liver tissues of each group were observed. The messenger ribonucleic acid (mRNA) and protein expression levels of GRP-78 and CHOP were detected by polymerase chain reaction and Western blot. Results Compared with the Sham group, the serum ALT and AST of the OLT group increased significantly (both in P < 0.01), liver tissues injury was serious, and mRNA and protein expression levels of GRP-78 and CHOP increased significantly (all in P < 0.01). Compared with the OLT group, ALT and AST of the OLT+MEL group decreased significantly (both in P < 0.05), liver tissues injury was alleviated, and mRNA and protein expression levels of GRP-78 and CHOP decreased significantly (all in P < 0.05). Conclusions MEL may reduce mRNA and protein expression levels of GRP-78 and CHOP involved in the ERS pathways of transplant liver, which may be one of its mechanisms to alleviate liver injury after liver transplantation. -
图 1 各组大鼠术后24 h肝组织病理学改变(HE，×200)
注：A图为Sham组；B图为OLT组；C图为OLT+MEL组
Figure 1. Changes of liver histology of rats in each group 24 h after surgery
图 2 各组大鼠肝组织GRP-78及CHOP的蛋白表达水平比较
注：OLT组与Sham组比较，aP < 0.01；OLT+MEL组与OLT组比较，bP < 0.05
Figure 2. Comparison of GRP-78 and CHOP protein levels of liver tissue in each group of rats
表 1 本实验所用基因的引物序列
Table 1. Primer sequences of genes in the experiment
基因 引物序列(5' to 3') 引物长度(bp) GRP-78 上游：GAAACTGCCGAGGCGTAT 197 下游：ATGTTCTTCTCTCCCTCTCTCTTA CHOP 上游：CGGAGTGTCCCAGCACCATCA 92 下游：CCCTCTCCTTTGGTCTACCCTCA β-actin 上游：TGTTGCCCTAGACTTCGAGCA 155 下游：CCATACCCAGGAAGGAGG 
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表 2 各组大鼠血清ALT和AST水平的比较
Table 2. Comparison of serum ALT and AST levels in each group of rats (U/L, x±s)
组别 n ALT AST Sham组 8 40±4 132±7 OLT组 8 221±45a 324±49a OLT+MEL组 8 149±35b 255±56b 注：与Sham组比较，aP < 0.01；与OLT组比较，bP < 0.05
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表 3 各组大鼠肝组织GRP-78及CHOP的mRNA表达水平比较
Table 3. Comparison of GRP-78 and CHOP mRNA levels of liver tissue in each group of rats(x±s)
组别 n GRP-78 CHOP Sham组 8 1.01±0.09 1.06±0.09 OLT组 8 7.49±0.39a 9.59±0.67a OLT+MEL组 8 4.69±0.23b 5.31±0.51b 注：与Sham组比较，aP < 0.01；与OLT组比较，bP < 0.05
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