以草鱼为研究对象，系统研究了溶血对肝脏的损伤机制，以揭示血红蛋白对机体的损伤作用。首先在体内注射血红蛋白，通过苏木精-伊红染色(H.E)发现注射血红蛋白组的肝脏组织中坏死细胞明显增多，普鲁士蓝染色揭示肝脏中存在大量的铁沉积，进一步的实时定量PCR (qRT-PCR)检测结果显示，注射的血红蛋白激活了铁代谢相关基因的表达。为进一步探究体内出血对肝脏的影响，实验通过体内注射苯肼模拟体内出血，H.E和普鲁士蓝染色结果表明，大量的溶血导致肝脏细胞坏死和铁的沉积增加，通过检测肝脏中血红蛋白和铁含量发现，苯肼组中，肝脏组织血红蛋白和铁含量随着时间延长而显著增加，而铁含量的增加同时激活了肝脏组织中铁代谢相关基因的表达。其次，实验检测了注射苯肼后肝脏组织中炎症因子的表达情况，qRT-PCR结果显示，高剂量的血红蛋白激活了多种细胞因子的基因表达，如促炎因子TNF-α、IL-1β和IL-6，抑炎因子IL-10以及趋化因子IL-4和IL-8等。为进一步探究高氧化活性的血红蛋白对肝脏组织的氧化损伤作用，实验检测了肝脏组织中丙二醛(MDA)、脂质过氧化物(LPO)以及β-半乳糖苷酶的含量，检测结果表明，鱼体内的出血显著增加了肝脏氧化损伤作用，同时，qRT-PCR和酶活性检测结果揭示，血红蛋白的氧化损伤作用促进了肝脏组织细胞凋亡的发生。最后，实验检测了肝脏组织中抗氧化酶的表达情况，结果显示，体内的出血显著激活了肝脏组织中的抗氧化系统。研究表明，鱼体内大量出血释放的高氧化活性的血红蛋白显著激活了肝脏组织炎症和氧化损伤的发生，促进了肝脏组织中的细胞凋亡，同时也激活了机体内的抗氧化系统。

A variety of bacterial or viral diseases usually cause massive hemolysis in fish, and release a large amount of cytotoxic hemoglobin into the tissues. However, the damage mechanism of hemolysis to fish tissues is still not well known. Therefore, to explore the damage mechanism of hemolysis in fish to the tissues, Ctenopharyngodon idella was used to systematically study the damage mechanism of hemolysis to the liver. Firstly, the hemoglobin was injected into the C. idella, hematoxylin eosin (H.E) staining assay results showed that injection of hemoglobin caused the dead cells to increase obviously in liver, and Prussian blue staining revealed that more iron deposited in liver. Quantitative real-time PCR (qRT-PCR) detection results showed that the injection of hemoglobin activated the expression of iron metabolism related genes. In order to further explore the damage of hemolysis to the liver, we injected phenylhydrazine into C. idella, H.E and Prussian blue staining results showed that the hemolysis caused the liver cell necrosis and iron deposits to increase, and the content of hemoglobin and iron in the liver all significantly increased with time. Furthermore, the increase of iron also activated the expression of iron metabolism related genes. Then, we detected the expression of inflammatory cytokines in liver after the injection of phenylhydrazine, and qRT-PCR results showed that high dose of hemoglobin activated the expression of various cytokines, such as pro-inflammatory cytokines TNF-α, IL-1β and IL-6, anti-inflammatory cytokines IL-10, and chemokines IL-4 and IL-8. In order to further explore the oxidation damage of hemoglobin in liver, we tested the content of malondialdehyde (MDA) and lipid peroxide (LPO) and β - galactose glucoside enzyme in liver, and the results showed that the hemolysis significantly increased liver oxidative damage effect, at the same time, the qRT-PCR and enzyme activity test results revealed that the oxidative damage of hemolysis induced the occurrence of liver cell apoptosis. Finally, we also examined the expression of antioxidant enzymes in liver, and the results showed that in vivo hemolysis significantly activated the antioxidant system in liver. In conclusion, the present study revealed that massive hemorrhage in fish released the high oxidative activity of hemoglobin which activated the occurrence of inflammation, oxidative damage and apoptosis in liver, and simultaneously up-regulated the expression of antioxidant system. The results of this study will enrich the blood immunology of fish, and also provide theoretical reference for the healthy culture of C. idella.

S941.4

国家自然科学基金(31902409, 31872606, 31572657, 31602190, U1701233)