Increased hemoglobin expression in NASH livers

BACKGROUND & AIMS: Recent studies revealed that hemoglobin is expressed in some non-erythrocytes and it suppresses oxidative stress when overexpressed. Oxidative stress plays a critical role in the pathogenesis of non-alcoholic steatohepatitis (NASH). This study was to investigate whether hemoglobin is expressed in hepatocytes and how it is related to oxidative stress in NASH patients. METHODS: Microarray was performed to identify differentially expressed genes in NASH. Quantitative real time PCR (qRT-PCR) was used to examine gene expression levels. Western blotting and immunofluorescence staining were employed to examine hemoglobin proteins. Flow cytometry was used to measure intracellular oxidative stress. RESULTS: Analysis of microarray gene expression data has revealed a significant increase in the expression of hemoglobin alpha (HBA1) and beta (HBB) in liver biopspies from NASH patients. Increased hemoglobin expression in NASH was validated by qRT-PCR. However, the expression of erythrocyte specific marker genes such as SPTA, SPTB, GYPA, GATA1, and ALAS2 did not change, indicating that increased hemoglobin expression in NASH was not from erythropoiesis, but could result from increased expression in hepatocytes. Immunofluorescence staining demonstrated positive HBA1 and HBB expression in the hepatocytes of NASH livers. Hemoglobin expression was also observed in human hepatocellular carcinoma HepG2 cell line. Furthermore, treatment with hydrogen peroxide, a known oxidative stress inducer, induced a dose dependent increase in HBA1 expression in HepG2 cells. Intriguingly, forced hemoglobin expression suppressed oxidative stress. CONCLUSIONS: Oxidative stress upregulates hemoglobin expression in hepatocytes. Suppression of oxidative stress by hemoglobin could be a mechanism to protect hepatocytes from oxidative damage. These findings suggest that hemoglobin is an inducible antioxidant in hepatocytes in response to increased oxidative stress as found in NASH livers. Twelve biopsy diagnosed NASH patients were included in this study. For control groups, total RNA from 5 different subjects were purchased from ADMET. These subjects are free from liver disease.

研究背景与目的：近期研究表明，血红蛋白（hemoglobin）可在部分非红细胞中表达，且过表达时可抑制氧化应激（oxidative stress）。氧化应激在非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）的发病机制中发挥关键作用。本研究旨在探讨血红蛋白是否在肝细胞中表达，以及其与NASH患者体内氧化应激的关联。

研究方法：采用微阵列芯片（microarray）技术筛选NASH患者组织中的差异表达基因；运用定量实时聚合酶链反应（quantitative real time PCR, qRT-PCR）检测基因表达水平；通过蛋白质印迹法（Western blotting）与免疫荧光染色法（immunofluorescence staining）检测血红蛋白蛋白的表达；利用流式细胞术（flow cytometry）检测细胞内氧化应激水平。

研究结果：对微阵列基因表达数据的分析显示，NASH患者肝活检组织中，血红蛋白α链（HBA1）与β链（HBB）的表达水平显著升高。qRT-PCR验证了NASH患者中血红蛋白表达的上调。然而，红细胞特异性标志物基因（如SPTA、SPTB、GYPA、GATA1及ALAS2）的表达并未发生改变，这表明NASH患者中血红蛋白表达的上调并非源于红细胞生成（erythropoiesis），而是可能由肝细胞内血红蛋白表达增加所致。免疫荧光染色结果显示，NASH患者肝脏的肝细胞中可见HBA1与HBB的阳性表达。在人肝细胞癌HepG2细胞系中同样检测到血红蛋白的表达。此外，用已知的氧化应激诱导剂过氧化氢（hydrogen peroxide）处理HepG2细胞，可呈剂量依赖性上调HBA1的表达。值得注意的是，强制过表达血红蛋白可抑制氧化应激。

研究结论：氧化应激可上调肝细胞内血红蛋白的表达。血红蛋白通过抑制氧化应激，可能是肝细胞免受氧化损伤的一种保护机制。上述研究结果表明，血红蛋白可作为一种可诱导的抗氧化剂，在肝细胞中应对NASH肝脏中出现的氧化应激升高。本研究共纳入12例经活检确诊的NASH患者。对照组的总RNA购自ADMET公司，来源于5名无肝脏疾病的健康受试者。