Impact of cyclooxygenase 2-dependent generated prostaglandin E2 and other prostanoids in the development of diet-induced non-alcoholic steatohepatitis (NASH)

肥胖以及代谢综合征和2型糖尿病等随之而来的疾病在全球范围内的流行正在迅速增加。非酒精性脂肪性肝病(NAFLD)是以肝脂过度堆积为特征的代谢综合征的肝脏表现，是功能性肝病的主要原因。一些患者发展为严重的非酒精性脂肪性肝炎(NASH)，脂肪变性伴随炎症和纤维化，从而增加了患肝硬变、肝癌和器官衰竭的风险。除细胞因子外，前列腺素E2(PGE2)等前列腺素在炎症的调节中发挥重要作用。前列腺素是一种生物活性脂类，在炎症过程中依赖环氧合酶2(COX2)合成，可以放大或减轻炎症。在HE-7032/1-1项目的范围内，建立了一种NASH诱导饮食，该饮食诱导了野生型小鼠肝脏脂肪变性、炎症和纤维化以及肥胖和胰岛素抵抗，从而显示了代谢综合征中人类NASH的所有临床参数。在这些小鼠以及NASH患者中，肝脏中COX2和PGE2合成的下游酶mPGES1的表达增强，表明PGE2的形成局部增加。前列腺素E_2可抑制促炎细胞因子肿瘤坏死因子α的表达。在喂食NASH诱导饮食的mPGES1缺陷小鼠的肝脏中，与野生型小鼠相比，肿瘤坏死因子α的诱导增加，导致更明显的肝细胞凋亡。与预期相反，mPGES1的缺失只是降低了肝脏PGE2水平，但不足以完全消除饮食诱导的肝脏PGE2生成。因此，不同基因型之间肝脏PGE2水平的微小差异并不会导致更严重的饮食诱导的肝脏炎症。然而，在与COX2抑制剂吲哚美辛孵育的原代巨噬细胞中，炎症介导的PGE2产生的增加完全被取消。后续项目将集中在COX2依赖产生的前列腺素类化合物(如PGE2)在饮食诱导的NASH发展中的影响。在巨噬细胞、肝细胞或这两种细胞类型中COX2特定缺失的小鼠以及它们各自的对照组将接受标准和NASH诱导饮食，并将被描述为NASH的发生和发展。对巨噬细胞群体和不同基因类型的肝细胞进行平行的体外实验，将有助于在细胞水平上确定COX2的促炎或抗炎作用。此外，结果将在人类肝脏探针中得到验证。该项目的第二部分将调查饮食诱导的NASH小鼠肝脏中不同巨噬细胞群的异质性。这些不同的巨噬细胞应该被分离出来，并根据它们对前列腺素E_2等前列腺素的敏感性进行鉴定。

The worldwide prevalence of obesity and the ensuing diseases like the metabolic syndrome and type2 diabetes is increasing rapidly. Non-alcoholic fatty liver disease (NAFLD), which is characterized by an excessive hepatic lipid accumulation, is defined as the hepatic manifestation of the metabolic syndrome and the main reason for functional liver diseases. Some patients develop the severe form of non-alcoholic steatohepatitis (NASH), where steatosis isaccompanied by inflammation and fibrosis, which results in an increased risk for cirrhosis, liver cancer and organ failure. Beside cytokines, prostaglandins like prostaglandin E2 (PGE2) play a Major role in the regulation of the inflammation. Prostaglandins are bioactive lipids that are synthesized cyclooxygenase 2 (Cox2)-dependently during inflammatory processes and can act to amplify or to attenuate inflammation. Within the scope of the project HE-7032/1-1 a NASHinducing diet was established, which induced hepatic steatosis with inflammation and fibrosis as well as obesity and insulin resistance in wildtype mice and thus exhibited all clinical parameters of human NASH in the metabolic syndrome. In these mice, as well as in patients with NASH, the hepatic expression of Cox2 and mPGES1, the Cox2- downstream enzyme in PGE2-synthesis, was enhanced, indicating a local increase in PGE2 formation. PGE2 can inhibit the expression of the pro-inflammatory cytokine TNFα. In livers of mPGES1-deficient mice fed a NASH-inducing diet, the induction of TNFα was increased compared to wild type mice, resulting in more pronounced hepatocyte apoptosis. Against the expectations, a deficiency in mPGES1 only lowered the hepatic PGE2 levels but was not sufficient to completely eliminate the diet-induced hepatic PGE2 generation. Consequently, the minor differences in hepatic PGE2 levels between the genotypes did not result in a more severe diet-induced hepatic inflammation. However, in primary macrophages incubated with the Cox2-inhibitor indomethacin the inflammation-mediated increase in PGE2 production was completely abrogated. The follow-up project will focus on theimpact of the Cox2-dependently generated prostanoids like PGE2 in the development of diet-induced NASH. Mice with a specific deletion of Cox2 in macrophages, hepatocytes or both cell types as well as their respective controls will receive standard and NASH-inducing diets and will be characterized regarding the development and progression of NASH. Parallel in vitro experiments with the macrophage populations and hepatocytes from the individual genotypes will help to determine the pro- or anti-inflammatory role of Cox2 on a cellular level. Additionally, the results will be validated in human liver probes. A second part of the project will investigate the heterogeneity of different macrophage populations in mice livers with diet-induced NASH. These distinct macrophages should be isolated and characterized regarding their sensitivity to prostanoids like PGE2.