Abstract：
Nonalcoholic fatty liver disease (NAFLD) consists of a broad spectrum of conditions, and nonalcoholic steatohepatitis (NASH) is the advanced form of NAFLD. TAF15 is a DNA and RNA binding protein and is involved in crucial inflammatory signalling pathways. We aimed to investigate the role of TAF15 in the progression of NASH and the underlying molecular mechanism.
We generated mice with hepatocyte-specific knockdown and overexpression of TAF15 using a specific adeno-associated virus (AAV). NASH models were established by feeding mice high-fat and high-cholesterol diets and methionine- and choline-deficient diets. Cleavage under targets and tagmentation and dual-luciferase reporter assays were performed to investigate the effect of TAF15 on FASN transcription. Coimmunoprecipitation and immunofluorescence assays were conducted to explore the interaction of TAF15 and p65. In vitro coculture systems were established to study the interactions of hepatocytes, macrophages and HSCs.
TAF15 was significantly increased in the livers of mouse NASH models and primary hepatocyte NASH model. Knockdown of TAF15 inhibited steatosis, inflammation and fibrosis, while overexpression of TAF15 promoted NASH phenotypes. Mechanistically, TAF15 bound directly to the promoter region of FASN to facilitate its expression, thereby promoting steatosis. Moreover, TAF15 interacted with p65 and activated the NF-κB signalling pathway, increasing the secretion of proinflammatory cytokines and triggering M1 macrophage polarization. Treatment with the FASN inhibitor orlistat partially reversed the phenotypes.
These results suggested that TAF15 exacerbated NASH progression by regulating lipid metabolism and inflammation via transcriptional activation of FASN and interacting with p65 to activate the NF-κB signalling pathway.
We generated mice with hepatocyte-specific knockdown and overexpression of TAF15 using a specific adeno-associated virus (AAV). NASH models were established by feeding mice high-fat and high-cholesterol diets and methionine- and choline-deficient diets. Cleavage under targets and tagmentation and dual-luciferase reporter assays were performed to investigate the effect of TAF15 on FASN transcription. Coimmunoprecipitation and immunofluorescence assays were conducted to explore the interaction of TAF15 and p65. In vitro coculture systems were established to study the interactions of hepatocytes, macrophages and HSCs.
TAF15 was significantly increased in the livers of mouse NASH models and primary hepatocyte NASH model. Knockdown of TAF15 inhibited steatosis, inflammation and fibrosis, while overexpression of TAF15 promoted NASH phenotypes. Mechanistically, TAF15 bound directly to the promoter region of FASN to facilitate its expression, thereby promoting steatosis. Moreover, TAF15 interacted with p65 and activated the NF-κB signalling pathway, increasing the secretion of proinflammatory cytokines and triggering M1 macrophage polarization. Treatment with the FASN inhibitor orlistat partially reversed the phenotypes.
These results suggested that TAF15 exacerbated NASH progression by regulating lipid metabolism and inflammation via transcriptional activation of FASN and interacting with p65 to activate the NF-κB signalling pathway.
摘要：
目的：非酒精性脂肪性肝病（NAFLD）包括广泛的疾病，非酒精性脂肪性肝炎（NASH）是NAFLD的晚期形式。TAF15是一种DNA和RNA结合蛋白，参与关键的炎症信号通路。我们旨在研究TAF15在NASH进展中的作用及其潜在的分子机制。
方法：我们使用特异性腺相关病毒（AAV）产生了肝细胞特异性敲低和TAF15过表达的小鼠。通过喂养小鼠高脂肪和高胆固醇饮食以及蛋氨酸和胆碱缺乏饮食来建立NASH模型。进行靶标下的切割和标签化和双荧光素酶报告基因测定以研究TAF15对FASN转录的影响。进行免疫共沉淀和免疫荧光测定以探索TAF15和p65的相互作用。建立了体外共培养体系来研究肝细胞的相互作用，巨噬细胞和HSC。
结果：TAF15在小鼠NASH模型和原代肝细胞NASH模型的肝脏中显著增加。敲除TAF15抑制脂肪变性，炎症和纤维化，而TAF15的过表达促进NASH表型。机械上,TAF15直接与FASN的启动子区结合以促进其表达，从而促进脂肪变性。此外，TAF15与p65相互作用并激活NF-κB信号通路，增加促炎细胞因子的分泌并触发M1巨噬细胞极化。用FASN抑制剂奥利司他治疗部分逆转了表型。
结论：这些结果表明，TAF15通过FASN的转录激活调节脂质代谢和炎症并与p65相互作用以激活NF-κB信号通路，从而加剧了NASH的进展。
方法：我们使用特异性腺相关病毒（AAV）产生了肝细胞特异性敲低和TAF15过表达的小鼠。通过喂养小鼠高脂肪和高胆固醇饮食以及蛋氨酸和胆碱缺乏饮食来建立NASH模型。进行靶标下的切割和标签化和双荧光素酶报告基因测定以研究TAF15对FASN转录的影响。进行免疫共沉淀和免疫荧光测定以探索TAF15和p65的相互作用。建立了体外共培养体系来研究肝细胞的相互作用，巨噬细胞和HSC。
结果：TAF15在小鼠NASH模型和原代肝细胞NASH模型的肝脏中显著增加。敲除TAF15抑制脂肪变性，炎症和纤维化，而TAF15的过表达促进NASH表型。机械上,TAF15直接与FASN的启动子区结合以促进其表达，从而促进脂肪变性。此外，TAF15与p65相互作用并激活NF-κB信号通路，增加促炎细胞因子的分泌并触发M1巨噬细胞极化。用FASN抑制剂奥利司他治疗部分逆转了表型。
结论：这些结果表明，TAF15通过FASN的转录激活调节脂质代谢和炎症并与p65相互作用以激活NF-κB信号通路，从而加剧了NASH的进展。
