NLRP6过表达通过AMPK/CPT1A/PGC1A通路促进肝细胞脂肪氧化分解改善非酒精性脂肪肝

doi:10.12122/j.issn.1673-4254.2025.01.15

摘要/Abstract

摘要：

目的研究核苷酸结合寡聚化结构域样受体含pyrin结构域蛋白6（NLRP6）在肝脏脂代谢及非酒精性脂肪性肝病（NAFLD）发展过程中的作用及机制。方法构建高脂饲料喂养（HFD）模型小鼠，分为对照组（正常喂养）、模型组［高脂（脂质含量60%饮食）喂养］，6只/组，持续喂养16周。构建蛋氨酸胆碱缺乏饮食（MCD）小鼠模型，分为对照组［喂养蛋氨酸-胆碱充足（MCS）饮食］和模型组（喂养蛋氨酸-MCD饮食），6只/组，持续喂养8周。使用HE染色及油红O染色鉴定模型是否造模成功，免疫组化染色（IHC）检测两种小鼠模型肝脏中NLRP6的表达。在正常人肝细胞系LO2细胞中使用腺病毒过表达 NLRP6，或 siRNA 敲低 NLRP6，使用棕榈酸（PA）构建NAFLD细胞模型，NLRP6过表达处理分组Ad-Vec组、Ad-NLRP6组、Ad-Vec+PA组、Ad-NLRP6+PA组；NLRP6敲低处理分组阴性对照（NC）组、sh-NLRP6组、NC+PA组、sh-NLRP6+PA组。采用甘油三酯（TG）试剂盒、油红染色、qPCR、Western blotting、ATP 试剂盒和 β-羟基丁酸试剂盒检测NLRP6对脂质代谢的影响及NLRP6对AMPK通路及使用AMPK抑制剂Compound C后AMPK通路的变化。结果在HFD、MCD饮食诱导NAFLD小鼠模型小鼠肝脏中NLRP6表达降低（P<0.001）。在细胞实验中LO2细胞过表达NLRP6后，与Ad-Vec+PA组相比，Ad-NLRP6+PA组细胞内TG含量降低（P<0.0001），且脂质沉积减少；敲低NLRP6，与NC+PA组相比，sh-NLRP6+PA组细胞内TG含量增加（P<0.05），并且脂质沉积加重；与Ad-Vec+PA组相比，Ad-NLRP6+PA组PGC1A、CPT1A mRNA及蛋白表达水平增加（P<0.01）。脂质氧化分解代谢产物ATP、β-羟丁酸含量升高（P<0.05）。与Ad-Vec+PA组相比，Ad-NLRP6+PA组肝细胞中AMPK通路磷酸化水平升高（P<0.05）；使用AMPK抑制剂后，Ad-NLRP6引起的脂质氧化分解作用被抑制关。结论 NLRP6通过AMPK/CPT1A/PGC1A促进肝细胞脂肪氧化分解，改善脂质沉积。

关键词: 非酒精性脂肪肝, NLRP6, AMPK, 脂肪酸氧化

Abstract:

Objective To investigate the regulatory role of nucleotide-bound oligomerized domain-like receptor containing pyrin-domain protein 6 (NLRP6) in liver lipid metabolism and non-alcoholic fatty liver disease (NAFLD). Methods Mouse models with high-fat diet (HFD) feeding for 16 weeks (n=6) or with methionine choline-deficient diet (MCD) feeding for 8 weeks (n=6) were examined for the development of NAFLD using HE and oil red O staining, and hepatic expressions of NLRP6 were detected with RT-qPCR, Western blotting, and immunohistochemical staining. Cultured human hepatocytes (LO2 cells) with adenovirus-mediated NLRP6 overexpression or knock-down were treated with palmitic acid (PA) in the presence or absence of compound C (an AMPK inhibitor), and the changes in cellular lipid metabolism were examined by measuring triglyceride, ATP and β-hydroxybutyrate levels and using oil red staining, RT-qPCR, and Western blotting. Results HFD and MCD feeding both resulted in the development of NAFLD in mice, which showed significantly decreased NLRP6 expression in the liver. In PA-treated LO2 cells, NLRP6 overexpression significantly decreased cellular TG content and lipid deposition, while NLRP6 knockdown caused the opposite effects. NLRP6 overexpression in PA-treated LO2 cells also increased mRNA and protein expressions of PGC1A and CPT1A, levels of ATP and β-hydroxybutyrate, and the phosphorylation level of AMPK pathway; the oxidative decomposition of lipids induced by Ad-NLRP6 was inhibited by the use of AMPK inhibitors. Conclusion NLRP6 overexpression promotes lipid oxidation and decomposition through AMPK/CPT1A/PGC1A to alleviate lipid deposition in hepatocytes.

Key words: non-alcoholic fatty liver disease, NLRP6, AMPK, fatty acid oxidation

石情, 冉苏叶, 宋铃榆, 杨红, 王文娟, 刘晗琳, 刘琦. NLRP6过表达通过AMPK/CPT1A/PGC1A通路促进肝细胞脂肪氧化分解改善非酒精性脂肪肝[J]. 南方医科大学学报, 2025, 45(1): 118-125.

Qing SHI, Suye RAN, Lingyu SONG, Hong YANG, Wenjuan WANG, Hanlin LIU, Qi LIU. NLRP6 overexpression improves nonalcoholic fatty liver disease by promoting lipid oxidation and decomposition in hepatocytes through the AMPK/CPT1A/PGC1A pathway[J]. Journal of Southern Medical University, 2025, 45(1): 118-125.

图/表 5

表1 用于qPCR实验的引物序列

Tab.1 Primers for RT-qPCR

Gene	Forward primer	Reverse primer
Homo CD36	ACGCTGAGGACAACACAGTCT	GCCACAGCCAGATTGAGAAC
Homo FATP1	ACGCGATATACCAGGAGCTG	ATCTTGAAGGTGCCTGTGGT
Homo PPARG	CATAAAGTCCTTCCCGCTGA	TCTGTGATCTCCTGCACAGC
Homo SREBF1	GCTGTTGGTGCTCGTCTCCTTG	GCTTGCGATGCCTCCAGAAGTAC
Homo FASN	GTCCACCAGCAACATCAGC	GTTCTCCAGCAAGCCATCTC
Homo CPT1A	GCACATCGTCGTGTACCATC	AATAGGCCTGACGACACCTG
Homo PGC1A	GAAGCAGACAAGACCGGTGA	AATAGGCCTGACGACACCTG
Homo MTTP	CTTCCTGGCCTTCATTCAGC	AGCAGAGGTGACAGCATCCA
Homo GAPDH	CTCCTCCTGTTCGACAGTCAGC	CCCAATACGACCAAATCCGTT
Homo NLRP6	GGAGTTGCTGTACTGCCTGT	CCTCAGAAAGGTCTCGGCAG
Mus NLRP6	CGGGACGAGAGGAAGGCAGAG	CACACGATCCAGCACACGAAGG
Mus β-actin	TCATCACTATTGGCAACGAGC	AACAGTCCGCCTAGAAGCAC

图1 NLRP6在NAFLD小鼠模型肝脏中表达降低

Fig. 1 NLRP6 expression is decreased in the liver of mouse models of NAFLD. A, B: HE and oil red O staining for detecting lipid deposition in the liver. C, D: NLRP6 immunohistochemical staining of the liver of NAFLD mice (**P<0.01 vs Control, ## #P<0.001 vs control MCS). E: NLRP6 mRNA expression level in the liver of NAFLD mice (**P<0.01 vs Control, ##P<0.01 vs MCS).

图2 肝细胞NLRP6对肝细胞脂质沉积的影响

Fig.2 Effect of NLRP6 on lipid deposition in cultured hepatocytes A: Relative NLRP6 mRNA levels in NLRP6-overexpressing hepatocytes (****P<0.0001). B: Relative NLRP6 mRNA levels in hepatocytes with NLRP6 knockdown (***P<0.001). C, D: Relative NLRP6 protein levels in NLRP6-overexpressing hepatocytes (***P<0.001). E, F: Relative NLRP6 protein levels in hepatocytes with NLRP6 knockdown (***P<0.001). G: oil red O staining of hepatocytes with NLRP6 overexpression or knockdown. H: TG content in NLRP6-overexpressing hepatocytes (****P<0.0001 vs Ad-Vec+PA group ). I: TG content in hepatocytes with NLRP6 knockdown (*P<0.05 vs NC+PA group).

图3 肝细胞NLRP6对脂质氧化分解的影响

Fig.3 Effect of NLRP6 on lipid oxidation and decomposition in hepatocytes A: mRNA expression levels of lipid metabolism-related genes in PA-treated liver cells overexpressing NLRP6 (***P<0.001 vs Ad-Vec+PA group). B, C: Western blotting of CPT1A and PGC1A in NLRP6-overexpressing hepatocytes (**P<0.01, ***P<0.001 vs Ad-Vec+PA group). D: ATP content in NLRP6-overexpressing hepatocytes (*P<0.05 vs Ad-Vec+PA group). E: β-hydroxybutyrate content in NLRP6-overexpressing hepatocytes (*P<0.05 vs Ad-Vec+PA group).

图4 肝细胞NLRP6对AMPK通路的影响

Fig.4 Effect of NLRP6 on AMPK pathway in hepatocytes. A, B: Western blotting for p-AMPK in NLRP6-overexpressing hepatocytes (*P<0.05 vs Ad-Vec+PA ). C, D: Western blotting for p-AMPK in NLRP6-overexpressing hepatocytes treated with AMPK inhibitor ( ###P<0.001 vs Ad-NLRP6+PA). E, F: Western blotting for CPT1A and PGC1A in NLRP6-overexpressing hepatocytes treated with AMPK inhibitor (###P<0.001, ####P<0.0001 vs Ad-NLRP6+PA). G: ATP content in NLRP6-overexpressing hepatocytes treated with AMPK inhibitor (###P<0.001 vs Ad-NLRP6+PA). H: β‑hydroxybutyrate content in NLRP6-overexpressing hepatocytes treated with AMPK inhibitor (###P<0.001 vs Ad-NLRP6+PA) .

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