健脾祛痰化瘀方调节miR-155/Rheb/mTOR途径介导内皮细胞自噬防治动脉粥样硬化机制研究

Mechanism of Prevention and Treatment of Arteriosclerosis By Jianpi Qutan Huayu Formula(健脾祛痰化瘀方)RegulatingmiR-155/Rheb/mTOR Pathway to Mediate Autophagy of Endothelial Cells

目的探讨健脾祛痰化瘀方调节miR-155/Rheb/mTOR途径介导内皮细胞自噬防治动脉粥样硬化(atherosclerotic,AS)的机制。方法将40只ApoE小鼠随机分为模型组、阿托伐他汀组、中药低剂量组、中药中剂量组、中药高剂量组,8只C57BL/6 Cnc小鼠作为正常组。除正常组给予基础饲料外,其余各组给予高脂饲料。造模12周后,灌胃给药,正常组与模型组给予相应体积的生理盐水,阿托伐他汀组、中药低剂量组、中药中剂量组、中药高剂量组给予相应药物。4周后全自动生物化学分析仪检测血清甘油三酯(triglyceride,TG)、总胆固醇(total cholesterol,TC)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C)和低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)的含量;HE染色观察主动脉病理及斑块变化情况;采用透射电镜观察主动脉自噬体超微结构的变化;实时荧光定量Q-PCR法检测miR-155、LC3、Rheb、GAPDHmRNA的表达;采用Wes全自动蛋白质印迹定量分析系统检测LC3、Rheb、p70s6k、p-p70s6k、mTOR、p-mTOR及β-actin蛋白的表达。结果与正常对照组相比,模型组ApoE小鼠血清中TG、TC、LDL-C水平均显著升高,HDL-C水平显著下降(P<0.01);HE染色结果显示模型组小鼠主动脉管腔中斑块面积范围较大、程度较重;电镜结果显示模型组小鼠主动脉超微结构自噬小体增多;模型组ApoEAS小鼠主动脉LC3-I、LC3-II、miR-155 mRNA表达显著下调(P<0.01),Rheb mRNA表达显著升高(P<0.01);模型组ApoEAS小鼠主动脉LC3-II/LC3-I显著下降(P<0.01),Rheb、p-mTOR/mTOR、p-P70S6K/P70S6K蛋白表达显著升高(P<0.01)。经阿托伐他汀和健脾祛痰化瘀方药低、中、高剂量组治疗后,与模型组相比,阿托伐他汀组以及健脾祛痰化瘀方药低、中、高剂量组血清中TG、TC、LDL-C水平均发生显著性降低(P<0.01),HDL-C水平无明显变化趋势;HE染色结果显示阿托伐他汀和健脾祛痰化瘀方药低、中、高剂量组小鼠主动脉管腔中斑块面积范围明显减小、程度明显减轻;电镜结果显示阿托伐他汀和健脾祛痰化瘀方药低、中、高剂量组小鼠主动脉超微结构显示自噬小体减少;阿托伐他汀组以及健脾祛痰化瘀方药低、中、高剂量组LC3-I、LC3-II、miR-155mRNA表达显著上调(P<0.01或P<0.05),Rheb mRNA表达显著下调(P<0.01或P<0.05);阿托伐他汀组以及健脾祛痰化瘀方药低、中、高剂量组LC3-II/LC3-I表达有所上调(P<0.01或P<0.05),Rheb、p-mTOR/mTOR、p-P70S6K/P70S6K蛋白表达显著下调(P<0.01或P<0.05)。结论健脾祛痰化瘀方可能通过调节miR-155/Rheb/mTOR途径介导内皮细胞自噬从而达到防治AS的作用。

Objective To investigate mechanism of preventing and treating arteriosclerosis(AS)by Jianpi Qutan Huayu Formu-la(健脾祛痰化瘀方)regulaing miR-155/Rheb/mTOR pathway to mediate the autophagy of endohelial ell.Mehods Forty ApoE--mice were randomly divided into model group,atorvastatin group,Chinese medicine low-dose group,Chinese medicine medium-dose group,Chinese medicine high-dose group,and 8 C57BL/6 Cne mice as the normal group.The normal group was given the basic diet.The other groups were given high fat diet.After 12 weeks of modeling,the normal group and model group were given the corresponding volume of normal saline.The atorvastatin group,Chinese medicine low-dose group,Chinese medi-cine medium-dose group and Chinese medicine high-dose group were given the corresponding drugs.Serum levels of triglycer-ide(TG),tolal cholesterol(TC),high-density lipoproteincholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)were detected by automatic biochemical analyzer 4 weeks later.HE staining was used to observe the pathological changes and plaque changes of aorta.The ultrastructure of aortie autophagosome was observed by transmission electron microsco-py.The mRNA expressions of miR-155,LC3,Rheb and glyceraldehyde-3-phosphate dehydrogenase(GAPDH)were detected by real-time fluorescence quantiative Q-PCR.The expressions of LC3,Rheb,P70S6K,P-P70S6K,mTOR,P-mTOR andβ-actin were detected by Wes automated westerm blot quantiative analysis system.Resuls Compared with those of the normal control group,the serum levels of TG,TC and LDL-C of ApoE-/-mice in the model group were signifcanly increased,while the HDL-C level was significantly decreased(P<0.01).HE staining results showed that the area and degree of plaques in the active vascular lumen of mice in the model group were larger and heavier.The results of electron microscope showed that the au-tophagosome of the ulrastructure of aorta in the model group was increased.The mRNA expressions of LC3-I,LC3-II and Mir-155 in aorta of ApoE-/-AS mice in the model group were si gnifcantly down-regulated(P<0.01),the mRNA expression of Rheb was significantly increased(P<0.01).The ratio of LC3-I/LC3-I in aorta of ApoE-/-AS mice in the model group was signifcantly decreased(P<0.01),the protein expressions of Rheb,P-mTOR/mTOR and P-P70S6K/P70S6K were sig-nificantly increased(P<0.01).After the treatment of atorvastatin and the low,medium and high dose of Jianpi Qutan Huayu Formula,compared with those of the model group,the serum levels of TG,TC and LDL-C in the atorvastatin group,Chinese med-medicine low-dose group,Chinese medicine medium-dose group and Chinese medicine high-dose group were significantly decreased(P<0.01).The HDL-C level had no significant change trend.HE staining results showed that the area and degree of active vascular plaques in atorvastatin group,the Chinese medicine low-dose group,Chinese medicine medium-dose group and Chinese medicine high-dose group were significantly reduced.Electron microscope results showed that the aortic ultrastructure of atorvastatin group,the Chinese medicine low-dose group,Chinese medicine medium-dose group and Chinese medicine high-dose group showed decreased autophagosomes.The expressions of LC3-I,LC3-II and miR-155 mRNA in the atorvastatin group,the Chinese medicine low-dose group,Chinese medicine medium-dose group and Chinese medicine high-dose group were significantly up-regulated(P<0.01 or P<0.05)and the mRNA expression of Rheb was significantly down-regulated(P<0.01 or P<0.05).The ratio of LC3-I/LC3-I was up-regulated in the atorvastatin group,the Chinese medicine low-dose group,Chinese medicine medium-dose group and Chinese medicine high-dose group(P<0.01 or P<0.05).The protein expressions of Rheb,p-mTOR/mTOR and p-P70S6K/P70S6K were significantly down-regulated(P<0.01 or P<0.05).Conclusion Jianpi Qutan Huayu Formula may achieve the prevention and treatment of AS by regulating miR-155/Rheb/mTOR pathway to mediate the autophagy of endothelial cells.