目的 探讨维生素D（vitamin D,VD）调控脂肪酸诱导的巨噬细胞M1-M2极化表型转变对肝细胞脂质代谢的直接作用及机制。方法 体外饱和脂肪酸孵育培养小鼠巨噬细胞RAW264.7并予活性VD-1,25(OH)2D3补充干预,制备相应的条件培养液体外培养小鼠肝细胞AML12。Real-time PCR检测巨噬细胞M1/M2极化基因表达水平,Real-time PCR、Western blot分别检测巨噬细胞VD受体（VD receptor, VDR）/过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptor gamma, PPARγ）基因、蛋白表达水平;油红O染色观察肝细胞内脂质沉积表现,Real-time PCR、Western blot分别检测肝细胞脂质合成和分解代谢相关酶类基因、蛋白表达水平。结果 体外饱和脂肪酸作用下,与VD对照组相较,VD补充组巨噬细胞M1极化基因诱生型一氧化氮合酶2（inducible nitric oxide synthase 2, iNOS2）、肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）和白细胞介素-6（interleukin-6, IL-6）及M2极化基因白细胞介素-10（interleukin-10, IL-10）mRNA表达水平均明显降低,巨噬细胞VDR/PPARγ mRNA和蛋白表达水平均明显降低。体外饱和脂肪酸作用的巨噬细胞条件培养液孵育肝细胞,与VD对照组相较,VD补充组肝细胞脂肪变性明显减少,肝细胞脂质合成代谢酶[固醇调节元件结合蛋白1C（sterol-regulatory element binding protein 1C, SREBP1C）、脂肪酸合酶（fatty acid synthase, FASN）] mRNA和蛋白表达水平均明显降低,脂质分解代谢酶肉碱棕榈酰转移酶1A（carnitine palmitoyltransferase 1A, CPT1A）mRNA和蛋白表达水平明显降低。结论 补充VD介导VDR/PPARγ通路逆转脂肪酸诱导的巨噬细胞极化表型直接影响肝细胞脂质代谢水平并改善肝细胞脂肪变性。
Abstract
Objective To investigate the effect of vitamin D (VD) on lipid metabolism by modulating fatty-acid induced macrophage M1-M2 polarization in hepatocytes. Methods Saturated fatty acid treated RAW264.7 macrophages were cultured in DMEM with 1,25(OH)2D3, and the conditioned medium (CM) were prepared. Then, AML12 hepatocytes were cultured in CM for 24 h. The M1/M2 polarization phenotype markers of macrophages were detected by RT- PCR. The mRNA and protein expressions of VDR and PPARγ of macrophages were detected by real-time PCR and Western blot, respectively. Lipid deposition in hepatocytes was examined by oil red O staining. The mRNA and protein expressions of lipometabolic enzymes of hepatocytes were detected by RT- PCR and Western blot, respectively. Results Compared to palmitic acid (PA) treated VD control group, the mRNA expressions of M1 phenotype markers, including iNOS2, TNFα and IL-6, and M2 phenotype marker IL-10, as well as the mRNA and protein expressions of VDR and PPARγ of macrophages in PA plus VD treated group were significantly decreased. Hepatocyte steatosis was obviously alleviated in the CM-PA plus VD treated group compared to the CM-PA plus VD control group. The mRNA and protein expressions of SREBP1C and FASN, as well as CPT1A of hepatocytes were significantly decreased in CM-PA plus VD treated group compared to the CM-PA plus VD control group. Conclusion VD supplementation improves lipid metabolism by reversing fatty-acid induced macrophage polarization through VDR/PPARγ pathway in hepatocytes.
关键词
维生素D /
巨噬细胞 /
极化 /
肝细胞 /
脂质代谢
Key words
vitamin D /
macrophage /
polarization /
hepatocyte /
lipid metabolism
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参考文献
[1] Rinella ME.Nonalcoholic fatty liver disease: a systematic review[J]. JAMA, 2015, 313:2263–2273.
[2] Jablonski KL, Jovanovich A, Holmen J, et al. Low 25-hydroxyvitamin D level is independently associated with non-alcoholic fatty liver disease[J]. Nutr Metab Cardiovasc Dis, 2013, 23:792–798.
[3] Barchetta I, Angelico F, Del Ben M, et al. Strong association between non-alcoholic fatty liver disease (NAFLD) and low 25(OH) vitamin D levels in an adult population with normal serum liver enzymes[J]. BMC Med, 2011, 9:85.
[4] Zhu S, Wang Y, Luo F, et al. The level of vitamin D in children and adolescents with nonalcoholic fatty liver disease: a meta-analysis[J]. Biomed Res Int, 2019, 2019:7643542.
[5] Sakpal M, Satsangi S, Mehta M, et al. Vitamin D supplementation in patients with nonalcoholic fatty liver disease: a randomized controlled trial[J]. JGH Open, 2017, 1:62–67.
[6] Kitson MT, Pham A, Gordon A, et al. High-dose vitamin D supplementation and liver histology in NASH[J]. Gut, 2016, 65:717–718.
[7] Kazankov K, Jørgensen SMD, Thomsen KL, et al. The role of macrophages in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis[J]. Nat Rev Gastroenterol Hepatol, 2019, 16:145–159.
[8] Luo WJ, Xu QY, Wang Q, et al. Effect of modulation of PPAR-γ activity on Kupffer cells M1/M2 polarization in the development of non-alcoholic fatty liver disease[J]. Sci Rep, 2017, 7:44612.
[9] Maina V, Sutti S, Locatelli I, et al. Bias in macrophage activation pattern influences non-alcoholic steatohe- patitis (NASH) in mice[J]. Clin Sci, 2012, 122:545–553.
[10] 罗雯静,董显文,赵巧素,等.维生素D调控Kupffer细胞极化在非酒精性脂肪性肝病防治中的作用[J].浙江医学,2023,45:916–921.
[11] Powell EE, Wong VW, Rinella M.Non-alcoholic fatty liver disease[J]. Lancet, 2021, 397:2212–2224.
[12] Charoenngam N, Holick MF.Immunologic effects of vitamin D on human health and disease[J]. Nutrients, 2020, 12:2097–2124.
[13] Dong B, Zhou Y, Wang W, et al. Vitamin D receptor activation in liver macrophages ameliorates hepatic inflammation, steatosis, and insulin resistance in mice[J]. Hepatology, 2020, 71:1559–1574.
[14] Locati M, Curtale G, Mantovani A.Diversity, mechanisms, and significance of macrophage plasticity[J]. Annu Rev Pathol, 2020, 15:123–147.
[15] Wang C, Ma C, Gong L, et al. Macrophage polarization and its role in liver disease[J]. Front Immunol, 2021, 12:803037.
[16] Bozic M, Guzmán C, Benet M, et al. Hepatocyte vitamin D receptor regulates lipid metabolism and mediates experimental diet-induced steatosis[J]. J Hepatol, 2016, 65:748–757.
[17] Di Rosa M, Malaguarnera G, De Gregorio C, et al. Immuno-modulatory effects of vitamin D3 in human monocyte and macrophages[J]. Cell Immunol, 2012, 280:36–43.
[18] Daniel B, Nagy G, Czimmerer Z, et al. The nuclear receptor PPARγ controls progressive macrophage polarization as a ligand-insensitive epigenomic ratchet of transcriptional memory[J]. Immunity, 2018, 49:615–626.
[19] Alimirah F, Peng X, Yuan L, et al. Crosstalk between the peroxisome proliferator-activated receptor γ(PPARγ) and the vitamin D receptor (VDR) in human breast cancer cells: PPARγ binds to VDR and inhibits 1α,25- dihydroxyvitamin D3 mediated transactivation[J]. Exp Cell Res, 2012, 318:2490–2497.
[20] Ramos LF, Silva CM, Pansa CC, et al. Non-alcoholic fatty liver disease: molecular and cellular interplays of the lipid metabolism in a steatotic liver[J]. Expert Rev Gastroenterol Hepatol, 2021, 15:25–40.
[21] Smith GI, Shankaran M, Yoshino M, et al. Insulin resistance drives hepatic de novo lipogenesis in nonalcoholic fatty liver disease[J]. J Clin Invest, 2020, 130:1453–1460.
[22] Alves-Bezerra M, Cohen DE.Triglyceride metabolism in the liver[J]. Compr Physiol, 2017, 8:1–8.
[23] Friedman SL, Neuschwander-Tetri BA, Rinella M, et al. Mechanisms of NAFLD development and therapeutic strategies[J]. Nat Med, 2018, 24:908–922.
基金
宁波市自然科学基金（No. 202003N4234）; 浙江省医药卫生科技计划（No. 2024KY1477）
