瞬时受体电位香草素1型对高脂饮食诱导的小胶质细胞代谢的调控

doi:10.3969/j.issn.1674-8115.2023.12.004

摘要/Abstract

摘要：

目的·利用转录组以及脂质组分析技术研究瞬时受体电位香草素1型（transient receptor potential vanilloid type 1，TRPV1）通道的激活对高脂饮食诱导的小胶质细胞代谢的调控作用。方法·以8周龄C57BL/6J小鼠（WT）和Trpv1^-/-（KO）小鼠为实验动物，高脂饲料（high-fat diet，HFD）分别喂养3d、7d、8周诱导造模（WT和KO组，n=3；WT-HFD和KO-HFD组，n=4）。通过免疫荧光试验测量WT-HFD和KO-HFD组小鼠大脑中TRPV1通道的表达以及细胞定位。通过RNA测序和液相色谱-质谱法确定WT-HFD和KO-HFD组小鼠的大脑表型。结果·与WT组小鼠相比，WT-HFD组小鼠体内小胶质细胞Trpv1 mRNA的表达水平显著增加。与WT-HFD组小鼠相比，KO-HFD组小鼠的脑脂质代谢、线粒体功能、葡萄糖转移以及糖酵解相关基因的表达水平下调。脂质组分析显示，虽然KO-HFD组小鼠的脑组织中脂质积累，但是Trpv1基因敲除减弱了HFD诱导的小胶质细胞活化，此外，TRPV1激动剂辣椒素在体外减弱棕榈酸诱导的线粒体膜电位去极化。结论·TRPV1通过线粒体驱动的燃料可用性机制调节小胶质细胞的脂质和葡萄糖代谢。

关键词: 瞬时受体电位香草素1型, 小胶质细胞, 代谢, 脂质, 线粒体

Abstract:

Objective ·Transcriptomic and lipidomic analysis techniques were used to investigate the role of transient receptor potential vanilloid type 1 (TRPV1) channel activation in the regulation of high-fat diet-induced microglial metabolism. Methods ·Eight-week-old C57BL/6J mice (WT) and Trpv1^-/- (KO) mice were used as experimental animals, and fed high-fat diet (HFD) for 3 days, 7 days, and 8 weeks to induce modelling (WT and KO groups, n = 3; WT-HFD and KO-HFD groups, n = 4). TRPV1 channel expression and cellular localisation were measured by immunofluorescence in the brains of mice in the WT-HFD and KO-HFD group. RNA sequencing and liquid chromatography-mass spectrometry were performed to determine the brain phenotype of mice in the WT-HFD and KO-HFD groups. Results ·The expression level of Trpv1 mRNA in microglia was significantly increased in mice in the WT-HFD group compared to mice in the WT group. The expression levels of genes related to brain lipid metabolism, mitochondrial function, glucose transfer, and glycolysis were down-regulated in the KO-HFD group of mice compared with the WT-HFD group of mice. Lipidomic analysis showed that although lipids accumulated in the brain tissue of mice in the KO-HFD group, Trpv1 knockdown attenuated HFD-induced microglia activation, and in addition the TRPV1 agonist capsaicin attenuated palmitate-induced depolarisation of mitochondrial membrane potential in vitro. Conclusion ·Together, these findings suggest that TRPV1 regulates lipid and glucose metabolism in microglia via fuel availability driven by a mitochondrial mechanism.

Key words: transient receptor potential vanilloid type 1(TRPV1), microglia, metabolism, lipid, mitochondria

中图分类号:

R964

沙旭栋, 王晨飞, 鲁佳, 虞志华. 瞬时受体电位香草素1型对高脂饮食诱导的小胶质细胞代谢的调控[J]. 上海交通大学学报（医学版）, 2023, 43(12): 1493-1506.

SHA Xudong, WANG Chenfei, LU Jia, YU Zhihua. Regulation of high-fat diet-induced microglial metabolism by transient receptor potential vanilloid type 1[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(12): 1493-1506.

图/表 8

表1 用于qRT-PCR的引物序列（5'→3'）

Tab 1 Primer sequences used for qRT-PCR （5'→3'）

Oligonucleotide	SOURCE	IDENTIFIER
mouse trpv1 FWD: TGGCTCATATTTGCCTTCAG mouse trpv1 REV: CAGCCCTAGGAGTTGATFGA	Sango Biotech	N/A
mouse ucp2 FWD: GCTGGTGGTTCGGAGAT mouse ucp2 REV: TGAAGTGGCAAGGGAGG	Sango Biotech	N/A
mouse tnf-α FWD: CAGGAGGGAGAACAGAAACTCCA mouse tnf-α REV: CCTGGTTGGCTGCTT	Sango Biotech	N/A
mouse il-1β FWD: GGAGGTGGTGATAGCCGGTAT mouse il-1β REV: TGGGTAATCCATAGAGCCCAG	Sango Biotech	N/A
mouse gapdh FWD: TGATGGCAACAATCTCCAC mouse gapdh REV: CGTCCCGTAGACAAAATGGT	Sango Biotech	N/A

图1 高脂饮食喂养前后 WT 和 Trpv1 敲除的小鼠的WGCNANote： A. Modules of WT, KO, WT-HFD, and KO-HFD mice (n=3 mice in WT and KO group, n=4 mice in WT-HFD and KO-HFD group). B/E/H. Top 20 pathways of GO and KEGG enrichment analysis of the pink, black and blue module. C/F/I. Network plot of the top 10 genes in pink, black and blue module. D/G/J. Trajectory of the module eigengenes in pink, black and blue module. One-way ANOVA was applied. *P < 0.05, ***P < 0.001, ****P = 0.000.

Fig 1 WGCNA in WT and genetic Trpv1 deletion mice before and after high fat feeding

图2 高脂饮食改变 Trpv1 敲除的小鼠脑内的脂质组成分Note： A. The composition of lipidome profiling of brain cells. B—C. Lipidome profiling of WT-HFD compared to WT and KO-HFD compared to KO mice brain. D. Distribution of PC, PE, and PG chain lengths of WT and KO mice induced by HFD treating. E—G. Distribution of PC, PE, and PG species of WT and KO mice induced by HFD treating. Data present the x±s (WT, WT-HFD, KO, n =3; KO-HFD, n =4). Statistical test: two-sided Student's t-test, two-way ANOVA followed by the Dunnett's post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, **** P = 0.000.

Fig 2 HFD induced liposome switch of brain cells with Trpv1 deficiency

图3 Trpv1 敲除缓解高脂饮食对小鼠大脑转录组的多种影响Note： A. Top lipidome enriched KEGG pathways of WT-HFD compared to WT mice and KO-HFD compared to KO mice. B/C. Gene expression changes, top GO and KEGG pathways enriched pathways of KO compared to WT mice and KO-HFD compared to WT-HFD mice.D—I. Heat maps of KO compared to WT mice and KO-HFD compared to WT-HFD mice.

Fig 3 Genetic Trpv1 deletion reduced HFD-induced multiple effects on brain transcriptome

图4 Trpv1 敲除缓解高脂饮食对大脑代谢途径的多种影响Note： A. The expressions of genes involved in monocarboxylate transporters, gluconeogenesis, glucose transporter, lactate shuttle, glycolysis, TCA cycle, and oxidative phosphorylation of WT and KO mice treated on SCD or 7 d HFD (red, increase; blue, decrease). B—G. The bar graphs show the fold induction of those genes within the class in WT-HFD versus WT mice (blue), and KO-HFD versus KO mice (red). All replicates within a class were averaged to obtain fold induction. Color key on the bottom of the figure indicated the group of samples.

Fig 4 Genetic Trpv1 deletion reduced HFD-induced multiple effects on metabolic pathways of the brain

图5 Trpv1 敲除可改善高脂饮食诱导的小胶质细胞活化Note： A/B. Iba-1+ active microglia and GFAP+ reactive astrocytes of WT or TRPV1 KO mice fed on 3-day HFD. Data represent x±s. ?P < 0.05, **P < 0.01, ***P < 0.001, ****P = 0.000. Scale bar: 50 μm.

Fig 5 Genetic Trpv1 deletion ameliorates HFD-induced microglia activation

图6 高脂饮食的小鼠大脑内的小胶质细胞和星形胶质细胞激活且上调表达TRPV1Note： A—C. Co-staining of TRPV1 and Iba1, TRPV1 and GFAP, TRPV1 and NeuN in SCD and 3-day HFD mice. Nuclei were stained in blue with DAPI. Scale bar: 50 μm; Scale bar: 10 μm.

Fig 6 Up-regulation of TRPV1 in active microglia and reactive astrocytes of HFD mice brains

图7 Trpv1 敲除可减轻高脂饮食诱导的神经炎症和线粒体激活Note： A—D. The mRNA levels of Trpv1, Ucp2, Tnf-α, and Il-1β in isolated cortical microglia (CD11b+ cells) from WT and KO mice mixed gender fed on SCD, 3 d HFD, 7 d HFD, or 8-week HFD (n=3). E/F. Mitochondrial membrane potential was detected by fluorescence microscopy (n=3). G. Indications of quantified lipid classes and acyl chains (circles) and genes (rectangles) of WT and KO mice by HFD. Data represent x±s. ?P < 0.05, ****P = 0.000. Scale bar: 50 μm.

Fig 7 Genetic Trpv1 deletion attenuate HFD-induced neuroinflammation and mitochondrial activation

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