MCLR-elicited hepatic fibrosis and carcinogenic gene expression changes persist in rats with diet-induced nonalcoholic steatohepatitis through a 4-week recovery period

Nonalcoholic steatohepatitis (NASH) causes liver extracellular matrix (ECM) remodeling and is a risk factor for fibrosis and hepatocellular carcinoma (HCC). Microcystin-LR (MCLR) is a hepatotoxin produced by fresh-water cyanobacteria that causes a NASH-like phenotype, liver fibrosis, and is also a risk factor for HCC. The focus of the current study was to investigate and compare hepatic recovery after cessation of MCLR exposure in healthy versus NASH animals. Male Sprague-Dawley rats were fed either a control or a high fat/high cholesterol (HFHC) diet for eight weeks. Animals received either vehicle or 30 µg/kg MCLR (i.p: 2 weeks, alternate days). Animals were euthanized at one of three time points: at the completion of the MCLR exposure period and after 2 and 4 weeks of recovery. Histological staining suggested that after four weeks of recovery the MCLR-exposed HFHC group had less steatosis and more fibrosis compared to the vehicle-exposed HFHC group and MCLR-exposed control group. RNA-Seq analysis revealed dysregulation of ECM genes after MCLR exposure in both control and HFHC groups that persisted only in the HFHC groups during recovery. After 4 weeks of recovery, MCLR hepatotoxicity in pre-existing NASH persistently dysregulated genes related to cellular differentiation and HCC. These data demonstrate impaired hepatic recovery and persistent carcinogenic changes after MCLR toxicity in pre-existing NASH.非酒精性脂肪性肝炎（Nonalcoholic steatohepatitis, NASH）可介导肝脏细胞外基质（extracellular matrix, ECM）重塑，同时作为肝纤维化与肝细胞癌（hepatocellular carcinoma, HCC）的危险因素。微囊藻毒素-LR（Microcystin-LR, MCLR）是由淡水蓝藻产生的肝毒素，可诱导出类NASH表型、引发肝纤维化，同时也是HCC的危险因素。本研究旨在探究并对比：停止MCLR暴露后，健康动物与NASH模型动物的肝脏恢复情况。将雄性斯普拉格-道利大鼠（Sprague-Dawley rats）分为两组，分别饲喂对照饮食与高脂高胆固醇（high fat/high cholesterol, HFHC）饮食，持续8周。随后分别给两组大鼠注射赋形剂，或30 μg/kg的MCLR（腹腔注射，每两天一次，持续2周）。实验分别在三个时间点对大鼠实施安乐处死：MCLR暴露期结束时，以及恢复2周、恢复4周时。组织学染色结果显示，恢复4周后，暴露于MCLR的HFHC组大鼠相较于赋形剂暴露的HFHC组与MCLR暴露的对照组，其肝脏脂肪变性程度更轻，但纤维化程度更高。RNA测序（RNA-Seq）分析显示，MCLR暴露后，对照组与HFHC组的ECM相关基因均出现表达失调，但该失调仅在HFHC组的恢复阶段持续存在。恢复4周后，在预先存在NASH的动物中，MCLR的肝毒性会导致与细胞分化及HCC相关的基因出现持续性表达失调。上述数据表明，当机体预先存在NASH时，MCLR中毒后的肝脏恢复能力受损，且会出现持续性致癌相关变化。