Abstract：
BACKGROUND: Metabolic-associated fatty liver disease (MAFLD) is a leading cause of chronic liver disease worldwide. Autophagy plays a pivotal role in lipid metabolism; however, the mechanism underlying the reduced autophagic activity in MAFLD remains elusive.
METHODS: Autophagy was monitored by TUNEL assay and immunofluorescence staining of LC3. The expression of autophagy-related proteins, PPARα, HDAC2, and HRD1 was detected by Western blot. The association between HDAC2 and PPARα promoter was assessed by chromatin immunoprecipitation (ChIP) and dual-luciferase assays, and the HRD1-mediated ubiquitin-proteasomal degradation of HDAC2 was detected by co-immunoprecipitation (co-IP). The in vitro findings were validated in a hypoxia-induced MAFLD mouse model. Histological changes, fibrosis, and apoptosis in liver tissues were detected by hematoxylin and eosin staining, Masson\'s trichrome staining, and TUNEL assay. The immunoreactivities of key molecules were examined by IHC analysis.
RESULTS: Hypoxia-suppressed autophagy in hepatocytes. Hypoxic exposure downregulated HRD1 and PPARα, while upregulating HDAC2 in hepatocytes. Overexpression of PPARα promoted hepatic autophagy, while knocking down HDAC2 or overexpressing HRD1 reduced hypoxia-suppressed autophagy in hepatocytes. Mechanistically, HDAC2 acted as a transcriptional repressor of PPARα, and HRD1 mediated the degradation of HDAC2 through the ubiquitin-proteasome pathway. Functional studies further showed that hypoxia-suppressed hepatic autophagy via the HRD1/HDAC2/PPARα axis in vitro and in vivo.
CONCLUSIONS: HRD1-mediated ubiquitination of HDAC2 regulates PPARα-mediated autophagy and ameliorates hypoxia-induced MAFLD.
METHODS: Autophagy was monitored by TUNEL assay and immunofluorescence staining of LC3. The expression of autophagy-related proteins, PPARα, HDAC2, and HRD1 was detected by Western blot. The association between HDAC2 and PPARα promoter was assessed by chromatin immunoprecipitation (ChIP) and dual-luciferase assays, and the HRD1-mediated ubiquitin-proteasomal degradation of HDAC2 was detected by co-immunoprecipitation (co-IP). The in vitro findings were validated in a hypoxia-induced MAFLD mouse model. Histological changes, fibrosis, and apoptosis in liver tissues were detected by hematoxylin and eosin staining, Masson\'s trichrome staining, and TUNEL assay. The immunoreactivities of key molecules were examined by IHC analysis.
RESULTS: Hypoxia-suppressed autophagy in hepatocytes. Hypoxic exposure downregulated HRD1 and PPARα, while upregulating HDAC2 in hepatocytes. Overexpression of PPARα promoted hepatic autophagy, while knocking down HDAC2 or overexpressing HRD1 reduced hypoxia-suppressed autophagy in hepatocytes. Mechanistically, HDAC2 acted as a transcriptional repressor of PPARα, and HRD1 mediated the degradation of HDAC2 through the ubiquitin-proteasome pathway. Functional studies further showed that hypoxia-suppressed hepatic autophagy via the HRD1/HDAC2/PPARα axis in vitro and in vivo.
CONCLUSIONS: HRD1-mediated ubiquitination of HDAC2 regulates PPARα-mediated autophagy and ameliorates hypoxia-induced MAFLD.
摘要：
背景：代谢相关脂肪性肝病（MAFLD）是全球慢性肝病的主要原因。自噬在脂质代谢中起关键作用;然而,MAFLD中自噬活性降低的潜在机制仍然难以捉摸。
方法：通过TUNEL测定和LC3免疫荧光染色监测自噬。自噬相关蛋白的表达,PPARα,通过Westernblot检测HDAC2和HRD1。通过染色质免疫沉淀（ChIP）和双荧光素酶测定评估HDAC2和PPARα启动子之间的关联，并通过免疫共沉淀（co-IP）检测到HDAC2的HRD1介导的泛素-蛋白酶体降解。在缺氧诱导的MAFLD小鼠模型中验证了体外发现。组织学变化,纤维化，苏木精和伊红染色检测肝组织凋亡，马森三色染色，和TUNEL分析。通过IHC分析检查关键分子的免疫反应性。
结果：缺氧抑制肝细胞自噬。低氧暴露下调HRD1和PPARα，同时上调肝细胞中的HDAC2。PPARα过表达促进肝脏自噬,而敲低HDAC2或过表达HRD1可减少缺氧抑制的肝细胞自噬。机械上,HDAC2作为PPARα的转录抑制因子，HRD1通过泛素-蛋白酶体途径介导HDAC2的降解。功能研究进一步表明，缺氧在体外和体内通过HRD1/HDAC2/PPARα轴抑制肝脏自噬。
结论：HRD1介导的HDAC2泛素化调节PPARα介导的自噬并改善缺氧诱导的MAFLD。
方法：通过TUNEL测定和LC3免疫荧光染色监测自噬。自噬相关蛋白的表达,PPARα,通过Westernblot检测HDAC2和HRD1。通过染色质免疫沉淀（ChIP）和双荧光素酶测定评估HDAC2和PPARα启动子之间的关联，并通过免疫共沉淀（co-IP）检测到HDAC2的HRD1介导的泛素-蛋白酶体降解。在缺氧诱导的MAFLD小鼠模型中验证了体外发现。组织学变化,纤维化，苏木精和伊红染色检测肝组织凋亡，马森三色染色，和TUNEL分析。通过IHC分析检查关键分子的免疫反应性。
结果：缺氧抑制肝细胞自噬。低氧暴露下调HRD1和PPARα，同时上调肝细胞中的HDAC2。PPARα过表达促进肝脏自噬,而敲低HDAC2或过表达HRD1可减少缺氧抑制的肝细胞自噬。机械上,HDAC2作为PPARα的转录抑制因子，HRD1通过泛素-蛋白酶体途径介导HDAC2的降解。功能研究进一步表明，缺氧在体外和体内通过HRD1/HDAC2/PPARα轴抑制肝脏自噬。
结论：HRD1介导的HDAC2泛素化调节PPARα介导的自噬并改善缺氧诱导的MAFLD。
