淡豆豉异黄酮通过PPARγ/LXRα/ABCA1信号通路改善动脉粥样硬化小鼠脂质代谢的作用

Isoflavones from Sojae Semen Praeparatum Regulate Lipid Metabolism in Atherosclerotic Mice Through PPARγ/LXRα/ABCA1 Signaling Pathway

目的:通过卵巢切除结合高脂饲料喂养制备动脉粥样硬化小鼠模型,观察淡豆豉异黄酮(ISSP)对小鼠脂质代谢的影响,并从调控过氧化物酶体增殖物激活受体γ/肝X受体α/腺苷三磷酸结合盒转运体A1(PPARγ/LXRα/ABCA1)信号通路的角度进一步探讨其作用机制。方法:将50只载脂蛋白E敲除(ApoE^(-/-))小鼠随机分为模型组、西药组(阿托伐他汀钙,3.03 mg·kg^(-1))、中药低、中、高剂量组(淡豆豉异黄酮,2.5、5、10 mg·kg^(-1)),每组10只,以手术切除双侧卵巢同时喂食高脂饲料的方法制备动脉粥样硬化模型小鼠,另取10只ApoE^(-/-)小鼠,以手术切除卵巢旁脂肪同时喂食高脂饲料的方法作为假手术组,其中部分小鼠因术后感染死亡,最终确定每组为6只小鼠,术后1周开始各组灌胃相应药物或等量生理盐水。12周后检测血清及肝脏组织中甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、游离脂肪酸(NEFA)的含量;酶联免疫吸附测定法(ELISA)检测血清中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的含量;苏木素-伊红(HE)及油红O染色观察主动脉斑块形成及肝脏脂质沉积情况;实时荧光定量聚合酶链式反应(Real-time PCR)及蛋白免疫印迹法(Western blot)检测肝脏PPARγ、LXRα、ABCA1、ABCG1 mRNA及蛋白表达。结果:与假手术组比较,模型组小鼠血清TC、TG、LDL-C、TNF-α及IL-6含量显著升高(P<0.01),HDL-C水平显著降低(P<0.01);肝脏指数明显升高(P<0.05),肝脏脂肪空泡明显,脂质沉积显著,肝脏中TC、TG、NEFA含量显著增多(P<0.01);肝脏PPARγ、LXRα、ABCA1 mRNA表达明显降低(P<0.05,P<0.01),ABCG1 mRNA及蛋白表达明显降低(P<0.05,P<0.01);与模型组比较,服用阿托伐他汀钙和淡豆豉异黄酮中、高剂量组可使小鼠血清TC、TG、LDL-C、TNF-α和IL-6含量显著降低(P<0.01),HDL-C水平显著降低(P<0.01);肝脏指数显著降低(P<0.01),肝脏脂肪空泡及脂质沉积显著减少,肝脏中TC、TG、NEFA含量明显增多(P<0.05,P<0.01);肝脏PPARγ、LXRα、ABCA1和ABCG1 mRNA及蛋白表达明显上调(P<0.05,P<0.01)。结论:淡豆豉异黄酮可能通过PPARγ/LXRα/ABCA1信号通路调节脂质代谢,减少血清炎症表达及肝脏脂质沉积从而改善动脉粥样斑块的形成。

Objective:To study the effect of isoflavones from Sojae Semen Praeparatum(ISSP)on lipid metabolism in atherosclerotic mice,and decipher the underlying mechanism via the peroxisome proliferatoractivated receptor gamma/liver X receptor alpha/ATP-binding cassette transporter A1(PPARγ/LXRα/ABCA1)signaling pathway.Method:Fifty ApoE^(-/-)mice were randomly assigned into the model group,western medicine(atorvastatin calcium,3.03 mg·kg^(-1))group,and low-,medium-,and high-dose ISSP(2.5,5,10 mg·kg^(-1),respectively)groups,with 10 rats in each group.Atherosclerosis model mice were established by bilateral ovariectomy and feeding high-fat diet.Another 10 ApoE^(-/-)mice receiving ovariectomy and high-fat diet were taken as the sham group.Some mice died of postoperative infection,and finally 6 mice were included in each group.One week after operation,each group was administrated with corresponding drugs or equivalent amount of normal saline.After 12 weeks,the levels of triglyceride(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),and non-esterified fatty acids(NEFAs)in serum and liver tissue were measured.The levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in serum were detected by enzyme-linked immunosorbent assay(ELISA).Hematoxylineosin(HE)staining and oil red O staining were used for observation of aortic plaque formation and liver lipid deposition.The mRNA and protein levels of PPARγ,LXRα,ABCA1,and ATP-binding cassette transporter G1(ABCG1)in liver were determined by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)and Western blot.Result:Compared with the sham group,the modeling of atherosclerosis increased the aortic plaque area(P<0.01),elevated the serum TC,TG,LDL-C,TNF-α,and IL-6 levels(P<0.01),decreased the level of HDL-C(P<0.01),increased the liver index(P<0.05)and the levels of TC,TG,and NEFAs in liver(P<0.01),and caused obvious hepatic fat vacuoles and lipid deposition.In addition,the modeling down-regulated the mRNA levels of PPARγ,LXRα,ABCA1 in liver(P<0.05,P<0.01),and regulated the mRNA and protein levels of ABCG1(P<0.05,P<0.01).Compared with the model group,atorvastatin calcium and middle-,high-dose ISSP reduced the serum TC,TG,LDL-C,TNF-α,and IL-6 levels(P<0.01),decreased the liver index(P<0.01),alleviated the liver fat vacuoles and lipid deposition,and increased the levels of TC,TG,and NEFAs in the liver(P<0.05,P<0.01).Furthermore,they up-regulated the mRNA and protein levels of PPARγ,LXRα,ABCA1,and ABCG1 in the liver(P<0.05,P<0.01).Conclusion:ISSP may regulate lipid metabolism through PPARγ/LXRα/ABCA1 signaling pathway to downregulate the expression of inflammatory cytokines in serum and alleviate liver lipid deposition,thereby suppressing the formation of atherosclerotic plaque.