M1型巨噬细胞促进非酒精性脂肪性肝炎

M1 macrophages promote non-alcoholic steatohepatitis

目的探索巨噬细胞在非酒精性脂肪性肝炎(NASH)中的作用、机制和治疗价值。方法通过给予8周龄雄性Alms突变(foz/foz)小鼠高脂饮食6、8和10周,给予8周龄雄性C57BL/6小鼠蛋氨酸胆碱缺乏(MCD)饮食7 d、3周和4周分别构建NASH小鼠模型,对照组小鼠分别给予普通饮食和MCD对照饮食。采用荧光定量聚合酶链反应方法检测NASH模型小鼠和对照组小鼠肝脏的F4/80 mRNA水平,采用免疫荧光染色观察对照组小鼠和NASH模型小鼠肝脏中的巨噬细胞。lysM-Cre/DTR转基因小鼠予MCD饮食5周后,分为转基因实验组(给予白喉毒素清除巨噬细胞)和转基因对照组(给予磷酸盐缓冲液注射)。检测转基因实验组和转基因对照组小鼠肝脏中的甘油三酯水平和脂质过氧化物水平,并对小鼠的肝脏组织进行炎症评分。通过细胞因子分析实验和蛋白质印迹法分析巨噬细胞调节NASH中炎症的作用机制。通过AML-12肝细胞和RAW264.7巨噬细胞条件培养基与细胞共培养观察肝细胞和巨噬细胞的相互作用。通过氯膦酸脂质体清除野生型和NASH模型小鼠中的巨噬细胞,证实其在NASH预防中的价值。统计学方法采用独立样本t检验。结果NASH模型foz/foz小鼠高脂饮食饲养6、8和10周时肝脏中的F4/80 mRNA水平均高于对照组小鼠(1.49±0.19、1.70±0.15、1.93±0.04比1.05±0.22),差异均有统计学意义(t=3.06、4.92、7.92,均P<0.05);NASH模型小鼠MCD饮食饲养7 d和3周时肝脏中的F4/80 mRNA水平均高于对照组小鼠(2.70±0.99和3.08±1.71比1.00±0.83),差异均有统计学意义(t=3.43、3.54,均P<0.01);免疫荧光染色结果显示,与对照组比较,NASH模型小鼠MCD饲养4周时肝脏中F4/80^(+)诱导型一氧化氮合酶(iNOS)^(+)的M1型巨噬细胞增多,F4/80^(+)CD206^(+)的M2型巨噬细胞明显减少。巨噬细胞清除后转基因实验组小鼠的炎症评分、肝脏甘油三酯和脂质过氧化物水平均低于转基因对照组[(0.69±0.32)分比(1.95±0.74)分、(43.97±13.24)g/mg比(63.09±14.85)g/mg、(24.84±6.21)nmol/mg比(37.91±8.91)nmol/mg],差异均有统计学意义(t=3.14、2.72、2.41,均P<0.05)。细胞因子分析实验结果显示,巨噬细胞清除可显著降低血液中白细胞介素(IL)-12和巨噬细胞炎症蛋白-1α的蛋白质水平(差异倍数分别为-3.98、-2.74,均P<0.05)。转基因实验组小鼠细胞核中CCAAT/增强子结合蛋白β蛋白质减少。体外研究发现RAW264.7巨噬细胞条件培养基可促进AML-12肝细胞中脂肪聚集;MCD培养基处理的AML-12肝细胞条件培养基可促进RAW264.7巨噬细胞向M1型极化。经氯膦酸脂质体处理后的野生型小鼠肝脏中的甘油三酯和脂质过氧化物的蛋白质表达水平均低于MCD诱导的NASH模型小鼠[(45.33±14.59)g/mg比(63.10±16.02)g/mg、(2.11±0.48)nmol/mg比(2.73±0.17)nmol/mg],差异均有统计学意义(t=2.84、2.73,均P<0.05)。蛋白质印迹法结果显示,经氯膦酸脂质体处理后的NASH模型小鼠肝脏中的磷酸化蛋白激酶R样内质网激酶、肌醇需求酶-1α、蛋白质二硫键异构酶、葡萄糖调节蛋白78、磷酸化真核起始因子2α的蛋白质相对表达量均低于未经氯膦酸脂质体处理的NASH模型小鼠(1.84±0.36比3.05±0.83、1.50±0.84比6.65±1.47、0.87±0.12比2.28±0.52、1.68±0.43比4.76±1.13、1.42±0.19比2.75±0.79),差异均有统计学意义(t=2.32、5.28、4.56、4.41、2.85,均P<0.05)。结论NASH中巨噬细胞发生M1型极化,并与肝细胞相互作用促进炎症因子分泌、脂肪合成因子上调、氧化应激和内质网应激,引起NASH进展。巨噬细胞清除剂氯膦酸脂质体是预防NASH潜在的新途径。

Objective To investigate the function,mechanism and therapeutic potential of macrophages in non-alcoholic steatohepatitis(NASH).Methods Eight-week-old male foz/foz(Alms mutant)mice were fed with a high fat diet(HFD)for 6,8 and 10 weeks and 8-week-old male C57BL/6 mice were fed with a methionine and choline-deficient(MCD)diet for 7 d,3 weeks and 4 weeks to establish NASH models.The mice of control group were fed with normal diet or MCD control diet.The expression of F4/80 mRNA level in the livers of mice of NASH model group and control group was detected by fluorescence quantitative polymerase chain reaction.Macrophages in the livers of mice of NASH group and control group were determined by immunofluorescence staining.After transgenic lysM-Cre/DTR mice were fed with MCD diet for 5 weeks,they were divided into transgenic experimental group(ablation of macrophages induced by diphtheria-toxin(DTox)injection)and transgenic control group(phosphate buffer saline injection).The levels of triglyceride and lipid peroxide in the livers of transgenic experimental group and transgenic control group were detected,and the inflammation of the livers of the mice was scored.The mechanism of macrophages regulating inflammation in NASH was investigated by cytokine profiliny analysis and Western blotting.The interaction between hepatocytes and macrophages were determined by co-culturing the conditional medium of hepatocytes AML-12 and macrophages RAW264.7.Macrophages of mice of control group and NASH model group were depleted by liposomal clodronate to confirm its value in NASH prevention.Independent sample t-test was used for statistical analysis.Results F4/80 mRNA level in the livers of NASH model foz/foz mice fed with HFD for 6 weeks,8 weeks and 10 weeks was higher than that of control group(1.49±0.19,1.70±0.15 and 1.93±0.04 vs.1.05±0.22),and the differences were statistically significant(t=3.06,4.92 and 7.92,all P<0.05).The expression of F4/80 mRNA level of the livers of NASH model mice fed with MCD for 7 d and 3 weeks was higher than that of control group(2.70±0.99 and 3.08±1.71 vs.1.00±0.83),and the differences were statistically significant(t=3.43 and 3.54,both P<0.01).The results of immunofluorescence demonstrated that compared with that of control group,the number of F4/80^(+)inducible nitric oxide synthase(iNOS)^(+)M1 macrophages were significantly increased,while F4/80^(+)CD206^(+)M2 macrophages were significantly decreased in the livers of NASH model mice fed with MCD for 4 weeks.After macrophages depletion,the inflammation score,the levels of triglyceride and lipid peroxide in the liver of transgenic experimental mice were all lower than those of transgenic control mice(0.69±0.32 vs.1.95±0.74,(43.97±13.24)g/mg vs.(63.09±14.85)g/mg,(24.84±6.21)nmol/mg vs.(37.91±8.91)nmol/mg),and the differences were statistically significant(t=3.14,2.72 and 2.41,all P<0.05).The results of cytokine profiling analysis showed that macrophage depletion could lower the levels of interleukin(IL)-12 and macrophages inflammatory protein-1α(the difference between multiples:-3.98,-2.74,both P<0.05).CCAAT/enhancer binding proteinβwas defected in the nuclear of transgenetic experimental mice.In vitro study showed that RAW264.7 macrophages conditional medium could promote lipid accumulation in AML-12 hepatocytes,while conditional medium from MCD medium-treated AML-12 hepatocytes could promote RAW264.7 macrophages to M1 polarization.After treated with liposomal clodronate,the levels of triglyceride and lipid peroxidation in the liver of control mice were both lower than those of MCD-induced NASH model mice((45.33±14.59)g/mg vs.(63.10±16.02)g/mg,(2.11±0.48)nmol/mg vs.(2.73±0.17)nmol/mg),and the differences were statistically significant(t=2.84 and 2.73,both P<0.05).The results of Western blotting indicated that after treating with liposomal clodronate,the relative content of phosphorylated protein kinase R-like endoplasmic reticulum kinase,inositol requiring enzyme-1α,protein disulfide isomerase,glucose regulatory protein 78,phosphorylated eukaryotic initiation factor 2αin the liver of NASH model mice were all lower than those of NASH model mice without liposomal clodronate treatment(1.84±0.36 vs.3.05±0.83,1.50±0.84 vs.6.65±1.47,0.87±0.12 vs.2.28±0.52,1.68±0.43 vs.4.76±1.13,1.42±0.19 vs.2.75±0.79),and the differences were statistically significant(t=2.32,5.28,4.56,4.41 and 2.85,all P<0.05).Conclusions Macrophages are polarized into M1 phenotype in NASH.M1 macrophages contributed to NASH progression by interacting with hepatocyets to promote the secretion of inflammatory cytokines,up-regulation of lipogenic factors,oxidative stress and endoplasmic reticulum stress,resulting in the progression of NASH.Macrophages depletion by liposomal clodronate is a potential noval approach for NASH prevention.