血管生成抑制剂ZM 306416对高脂饮食诱导的非酒精性脂肪性肝病小鼠的保护作用

Protective effects of angiogenesis inhibitor ZM 306416 on non-alcoholic fatty liver disease induced by high-fat diet in mice

目的探讨血管生成抑制剂ZM 306316调节内脏脂肪的功能,分析其对高脂饮食诱导的非酒精性脂肪性肝病(non-alcohol fatty liver disease,NAFLD)小鼠的保护作用及可能机制。方法将32只C57BL/6J小鼠按照随机数字表法分为3组:高脂饮食组(含130 kcal l%的脂肪饲料,11只,HFD组)、低脂饮食组(含45 kcal l%的脂肪饲料,11只、LFD组)和高脂饮食-ZM组(添加0.8%ZM 306416,10只,HFD-ZM组),饲养15周,定时检测小鼠体质量。处死小鼠后,检测小鼠血清生物化学指标,包括丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、总胆固醇(total cholesterol,TC)、甘油三酯(triglyceride,TG)和游离脂肪酸(free fatty acids,FFA)水平。采用免疫组织化学分析附睾脂肪组织中血管性假血友病因子(von Willebrand factor,vWF)阳性细胞数;采用实时荧光定量聚合酶链反应检测各组织中相关基因的表达。结果与HFD组相比,HFD-ZM组小鼠体质量[(37.53±3.64)g vs(45.17±3.22)g]、VAT质量[(2.25±0.85)g vs(3.34±0.54)g]、内脏脂肪细胞大小[(7854.9±60.4)mm2 vs(10800.6±52.7)mm2]、vWF阳性细胞[(47.32±6.2)个vs(31.2±5.4)个]、血管密度[(320.47±10.66)%vs(184.26±14.94)%]及附睾组织中血管生成因子VEGF-A mRNA相对表达量(0.54±0.16 vs 0.24±0.12)均显著降低(P均<0.05),而抗血管生成因子TSP-1的mRNA相对表达量(0.32±0.07 vs 0.42±0.14)显著升高(P<0.05);HFD-ZM组小鼠ALT[(122.47±4.11)U/L vs(95.28±2.21)U/L]、AST[(172.17±5.20)U/L vs(124.39±2.54)U/L]、TG[(4.91±1.13)mmol/L vs(3.56±1.07)mmol/L]、FFA[(1640.55±22.51)μEq/L vs(1131.62±18.33)uEq/L]和TC[(8.10±2.11)mmol/L vs(6.11±2.19)mmol/L]水平均显著降低,差异均有统计学意义(P均<0.05),小鼠肝小叶及门管区可见少量胶原纤维增生,主要位于小叶中央静脉周围,α-SMA阳性细胞数目显著减少。与HFD组相比,HFD-ZM组脂肪酸氧化基因mRNA相对表达量(CPT-1:1.64±0.17 vs 0.97±0.24;MCAD:1.23±0.18 vs 0.91±0.16)显著升高(P均<0.05)、脂肪形成相关基因mRNA相对表达量(PPARγ:0.63±0.24 vs 1.03±0.27)显著降低(P<0.05)、胆固醇生成相关基因mRNA相对表达量(FXR:0.76±0.03 vs 0.65±0.10;HMGCR:1.04±0.14 vs 0.89±0.17)显著升高(P均<0.05),炎症相关基因(TNF-α:0.57±0.13 vs 1.01±0.23;CD68:1.02±0.19 vs 1.62±0.09;MCP-1:0.59±0.11 vs 1.01±0.09;MARCO:0.54±0.13 vs 1.02±0.12;ICAM-1:0.94±0.20 vs 1.56±0.13;VCAM-1:1.12±0.23 vs 1.89±0.16)和肝纤维化相关基因(TGFβ:0.64±0.16 vs 1.02±0.19;α-SMA:0.66±0.11 vs 1.03±0.06)mRNA相对表达量显著降低(P均<0.05),抗氧化基因(SOD2:0.83±0.12 vs 0.71±0.13)和凋亡基因(Bcl-2:0.78±0.12 vs 0.58±0.03)mRNA相对表达量显著升高(P均<0.05)。结论血管生成抑制剂ZM 306416可通过调控小鼠内脏脂肪生成、调节肝脏脂肪变性来改善内脏脂肪组织功能和脂质代谢,抑制由高脂饮食诱导的小鼠NAFLD。

Objective To investigate the angiogenesis inhibitor ZM 306316 in regulating visceral fat function and analyze the protective effect on non-alcoholic fatty liver disease(NAFLD)induced by high-fat diet in mice and its possible mechanism.Methods Total of thirty-two C57BL/6J mice were randomly divided into high fat diet group(130 kcal l%fat diet,11 cases,HFD group),low fat diet group(45 kcal l%fat diet,11 cases,LFD group)and high fat diet-ZM group(0.8%ZM 306416 was added,10 cases,HFD-ZM group),the mice were fed on different diets for 15 weeks.The body weight of the mice were measured regularly.The serum biochemistry index of the mice were detected,including alanine aminotransferase(ALT),aspartate aminotransferase(AST),total cholesterol(TC),triglyceride(TG)and free fatty acids(FFA).The number of vascular pseudophlebrand factor(von Willebrand factor,vWF)positive cells in epididymal adipose tissue was analyzed by immunohistochemistry.Real-time fluorescent quantitative polymerase chain reaction(FQ-PCR)was used to detect the expression of relevant genes in each tissue.Results The body mass[(37.53±3.64)g vs(45.17±3.22)g],VAT mass[(2.25±0.85)g vs(3.34±0.54)g],visceral adipocyte size[(7854.9±60.4)mm2 vs(10800.6±52.7)mm2],vWF positive cells(47.32±6.2 vs 31.2±5.4),vascular density[(320.47±10.66)%vs(184.26±14.94)%]and angiogenic factor VEGF-A mRNA relative expression(0.54±0.16 vs 0.24±0.12)of mice in HFD-ZM group were significantly lower than those in HFD group,and the relative mRNA expression of the anti-angiogenic factor TSP-1 was significantly higher(0.32±0.07 vs 0.42±0.14),the differences were statistically significant(all P<0.05).The level of ALT[(122.47±4.11)U/L vs(95.28±2.21)U/L],AST[(172.17±5.20)U/L vs(124.39±2.54)U/L],TG[(4.91±1.13)mmol/L vs(3.56±1.07)mmol/L],FFA[(1640.55±22.51)μEq/L vs(1131.62±18.33)μEq/L]and TC[(8.10±2.11)mmol/L vs(6.11±2.19)mmol/L]of mice in HFD-ZM group were significantly lower than those in HFP group,the differences were statistically significant(all P<0.05).A small amount of collagen fibroplasia was seen in the liver lobe and portal canal,mainly around the central vein,with a significant decrease in the number ofα-SMA positive cells.Compared with those in HFD group,the relative mRNA expression of fatty acid oxidation genes(CPT-1:1.64±0.17 vs 0.97±0.24;MCAD:1.23±0.18 vs 0.91±0.16)of mice in HFD-ZM group increased significantly(all P<0.05),the relative mRNA expression of adipogenesis gene(PPARγ:0.63±0.24 vs 1.03±0.27)decreased significantly(P<0.05),the relative mRNA expression of cholesterol generation related genes(FXR:0.76±0.03 vs 0.65±0.10;HMGCR:1.04±0.14 vs 0.89±0.17)increased significantly(all P<0.05),the relative mRNA expression of inflammation-related genes(TNF-α:0.57±0.13 vs 1.01±0.23;CD68:1.02±0.19 vs 1.62±0.09;MCP-1:0.59±0.11 vs 1.01±0.09;MARCO:0.54±0.13 vs 1.02±0.12;ICAM-1:0.94±0.20 vs 1.56±0.13;VCAM-1:1.12±0.23 vs 1.89±0.16)and liver fibrosis-related genes(TGFβ:0.64±0.16 vs 1.02±0.19;α-SMA:0.66±0.11 vs 1.03±0.06)decreased significantly(all P<0.05),the relative mRNA expression of antioxidant genes(SOD2:0.83±0.12 vs 0.71±0.13)and apoptotic genes(Bcl-2:0.78±0.12 vs 0.58±0.03)increased significantly(all P<0.05).Conclusions ZM 306416,an angiogenesis inhibitor,can improve visceral adipose tissue function and lipid metabolism by regulating adipogenesis and liver steatosis,and can inhibit NAFLD induced by high-fat diet in mice.