Table_1_Characterization of the CDAA Diet-Induced Non-alcoholic Steatohepatitis Model: Sex-Specific Differences in Inflammation, Fibrosis, and Cholesterol Metabolism in Middle-Aged Mice.docx

BackgroundThe prevalence of non-alcoholic steatohepatitis (NASH) rapidly increases with associated metabolic disorders such as dyslipidemia; therefore, NASH is now considered an independent risk factor of cardiovascular diseases. NASH displays sex-linked epidemiological, phenotypical, and molecular differences; however, little is known about the background of these sex-specific differences on the molecular level.
ObjectivesWe aimed to assess sex-specific differences in the expression of inflammatory and fibrotic genes, as well as in cholesterol metabolism, focusing on the expression of Pcsk9 in several tissues in a mouse model of NASH that shows the typical features of the human condition.
Methods and ResultsWe fed 10-months-old male and female C57Bl/6J mice with a NASH-inducing CDAA or corresponding control diet for 8 weeks. We found that, compared to the control male mice baseline, hepatic Pcsk9 expression as well as serum PCSK9 level was significantly higher in females, and both circulating PCSK9 level and the hepatic Pcsk9 gene were markedly decreased in female mice during NASH development. Histological analysis revealed that male and female mice develop a similar degree of steatosis; however, fibrosis was more pronounced in males upon CDAA diet feeding. Strikingly, female mice have higher hepatic expression of the pro-inflammatory cytokines (Il1b, Ifng), and increased IL-1β cleavage by the NLRP3 inflammasome, and a decrease in Clec4f+ resident Kupffer cell population in comparison to males in the CDAA-fed groups.
ConclusionThis is the first demonstration that there are critical sex-specific differences during NASH development in middle-aged mice regarding inflammation, fibrosis, and cholesterol metabolism and that changes in PCSK9 and IL-1β are likely important contributors to sex-specific changes during the transition to NASH.

研究背景：非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）的患病率随血脂异常等伴随代谢紊乱的进展而快速升高，目前已被认定为心血管疾病的独立危险因素。NASH存在性别相关的流行病学、表型及分子水平差异，但目前对这类性别特异性差异的分子机制尚不清楚。
研究目的：本研究旨在采用具备人类疾病典型特征的NASH小鼠模型，评估炎症与纤维化基因表达、胆固醇代谢的性别特异性差异，重点关注前蛋白转化酶枯草溶菌素9（Pcsk9）在多种组织中的表达水平。
方法与结果：我们将10月龄的雄性与雌性C57BL/6J小鼠分为两组，分别用诱导NASH的CDAA饲料及对应对照饲料喂养8周。结果显示，与对照组雄性小鼠的基线水平相比，雌性小鼠的肝脏Pcsk9表达及血清PCSK9水平显著更高；而在NASH进展过程中，雌性小鼠的循环PCSK9水平及肝脏Pcsk9基因表达均显著降低。组织学分析显示，雌雄小鼠的脂肪变性程度相似，但CDAA饲料喂养后雄性小鼠的纤维化程度更为显著。值得注意的是，与雄性小鼠相比，CDAA饲料喂养的雌性小鼠肝脏促炎细胞因子白细胞介素1β（Il1b）、干扰素γ（Ifng）表达水平更高，NLRP3炎症小体（NLRP3 inflammasome）介导的IL-1β剪切活性增强，且Clec4f阳性的常驻库普弗细胞（Kupffer cell）群数量减少。
研究结论：本研究首次证实，中年小鼠NASH进展过程中存在与炎症、纤维化及胆固醇代谢相关的关键性别特异性差异，且PCSK9与IL-1β的变化可能是向NASH转化阶段性别特异性改变的重要介导因素。