论文标题:普伐他汀干预p38 MAPK、IFN-gamma/STAT1、IL-6/STAT3信号传导通路预防动脉粥样硬化的实验研究
Pravastatin Preventing for Atherosclerosis in apoE~(-/-) Mice via Effects on p38 MAPK, IFN-gamma/STAT1 and IL-6/STAT3 Signal Pathway
论文作者
论文导师 孙宝贵,论文学位 博士,论文专业 内科学
论文单位 上海交通大学,点击次数 398,论文页数 111页File Size1273K
论文网 http://www.lw23.com/lunwen_994942/
pravastatin;; atherosclerosis;; p38 MAPK;; IFN-gamma;; STAT1;; IL-6;; STAT3
【目的和意义】 本研究旨在观察普伐他汀在预防动脉粥样硬化(atherosclerosis, AS)形成过程中对p38 MAPK、IFN-gamma(γ)/STAT1及IL-6/STAT3信号传导通路的影响,研究结果将为普伐他汀干预动脉粥样硬化提供新的途径,为防治动脉粥样硬化提供更有临床价值的信息。 【实验方法】 1.采用含1.25%(wt/wt)胆固醇的饲料喂养8周龄apoE~(-/-)小鼠诱导AS模型,同时普伐他汀灌胃(80mg kg-1)干预,8周后,处死小鼠,收集血清,分离主动脉,根部用于组织形态学分析,胸腹主动脉用于mRNA和蛋白分析。 2、采用生化法测定血清内TC和LDL-C变化。 3、主动脉根用做石蜡切片,HE染色后,检测AS的面积和炎症反应程度。 4、real-time PCR法检测胸腹主动脉内IFN-γ和TNF-α的mRNA水平。 5、采用酶联免疫吸附实验(ELISA)法检测血清内IL-6、IFN-γ、TNF-α和ox-LDL含量,以及胸腹主动脉内IL-6、IFN-γ、TNF-α的含量。 6、检测胸腹主动脉内MDA和GSH的含量,检测胸腹主动脉内SOD、GSH-Px、CAT、GST的活性。 7、采用Western-Blot法检测胸腹主动脉内3-nitrotyrosine、iNOS、p38 MAPK、p-p38-MAPK、p53、STAT1、pSTAT1、STAT3、pSTAT3、IRF-1、SOCS1蛋白表达的水平。 【结果】 1、动脉粥样硬化小鼠AS病变的相对面积(30.84﹪)显著高于空白对照组(4.62﹪)(P <0.05),普伐他汀干预小鼠AS病变的相对面积(7.91﹪)显著低于动脉粥样硬化小鼠(P <0.05)。 2、动脉粥样硬化小鼠血清内TC(26.59±7.25 mmol/L)和LDL-C(21.76±6.29 mmol/L)的含量显著升高(P<0.05),普伐他汀干预后血清内TC(21.62±12.78 mmol/L)和LDL-C(17.45±11.08 mmol/L)的含量与动脉粥样硬化小鼠比较差异无统计学意义。 3、动脉粥样硬化小鼠血清内氧化产物ox-LDL和炎症因子IL-6、IFN-γ、TNF-α的含量显著升高(P <0.01),普伐他汀可显著逐渐减少小鼠血清内ox-LDL(P <0.05)和IL-6(P <0.01)、IFN-γ(P <0.01)、TNF-α(P <0.01)的含量。 4、动脉粥样硬化小鼠主动脉AS病变内IFN-γ和TNF-α的mRNA水平显著升高(P <0.05),普伐他汀干预后可显著降低主动脉内IFN-γ和TNF-α的mRNA水平(P <0.05)。 5、动脉粥样硬化小鼠主动脉内IL-6、IFN-γ、TNF-α的含量显著升高(P <0.01),普伐他汀可显著降低主动脉内IL-6 (P <0.01)、IFN-γ(P <0.01)、TNF-α(P <0.05)的含量。 6、动脉粥样硬化小鼠主动脉内MDA的含量显著增加(P <0.01),GSH含量和SOD、CAT、GST、GSH-Px的活性显著降低(P <0.01),普伐他汀干预后可显著降低主动脉内MDA的含量(P <0.01),显著增加GSH含量(P <0.01)和SOD(P <0.01)、CAT(P<0.01)、GST(P <0.01)、GSH-Px(P <0.05)的活性。 7、动脉粥样硬化小鼠主动脉内3-nitrotyrosine、iNOS的表达显著增加,普伐他汀干预后可显著减少主动脉内3-nitrotyrosine、iNOS的表达。 8、动脉粥样硬化小鼠氧化应激诱导的p38 MAPK信号通路的活性因子p-p38 MAPK及该通路的下游因子p53的表达显著增加,普伐他汀干预后可显著降低主动脉内p-p38 MAPK及p53的表达。 9、动脉粥样硬化小鼠主动脉内IFN-γ/STAT1和IL-6/STAT3信号传导通路的活性因子pSTAT1和pSTAT3的表达增加,普伐他汀干预后显著降低主动脉内pSTAT1和pSTAT3的表达。 10、动脉粥样硬化小鼠主动脉内IFN-γ/STAT1和IL-6/STAT3信号传导通路的抑制因子SOCS1的表达显著降低,普伐他汀干预后显著增强主动脉SOCS1的表达。 【结论】 1、普伐他汀可预防高胆固醇饮食诱导的apoE~(-/-)小鼠AS。 2、AS时血清胆固醇含量增高,普伐他汀没有降低apoE~(-/-)小鼠血清胆固醇的作用,普伐他汀预防apoE~(-/-)小鼠AS的效应不依赖其降胆固醇作用。 3、AS时氧化还原系统失平衡和炎症反应增强,普伐他汀通过减轻氧化应激、硝基化应激、炎症反应预防AS。 4、AS时p38 MAPK信号传导通路可被激活,普伐他汀有通过调节p38 MAPK信号传导通路预防AS的作用。 5、AS时IFN-γ/STAT1信号传导通路可被激活,普伐他汀有调节IFN-γ/STAT1信号传导通路预防AS的作用。 6、AS时IL-6/STAT3信号传导通路可被激活,普伐他汀有调节IL-6/STAT3信号传导通路预防AS的作用。
Object To study the effects of pravastatin on p38 MAPK, IFN-γ/STAT1 and IL-6/STAT3 signal pathway during preventing for atheroscleorisis and probe into anti-oxidative and anti-inflammatory mechanisms of pravastatin preventing for atherosclerosis, which may provide new therapeutic approaches for pravastatin preventing for atherosclerosis and clinical evidences for controlling atherosclerosis. Methods 1) Male apoE~(-/-) mice fed a chow diet containing 1.25% cholesterol (wt/wt) were divided into pravastatin group provided with pravastatin (80mg/kg per day) and atherosclerosis group provided without pravastatin. Eight weeks later, mice were anesthetized. Serum was saved. The excised aortic roots were examined for detailed histological analysis. The excised thoracoabdominal aortas were used for mRNA and protein analysis. 2) Concentrations of total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C) in serum were measured by Biochemistry Analyzer. 3) Paraffin sections from the aortic root were stained with Hematoxylin and Eosin. The size of atherosclerotic lesion in each section was evaluated. 4) Levels of IFN-γand TNF-αmRNA in thoracoabdominal aortas were examined by real-time PCR. 5) Concentrations of IL-6, IFN-γand TNF-αin serum and the thoracoabdominal aorta and concentration of ox-LDL in serum were measured by ELISA. 6) Activities of SOD, GSH-Px, CAT and GST and Concentrations of MDA and GSH in thoracoabdominal aorta were measured. 7) Expressions of 3-nitrotyrosine, iNOS, p38 MAPK, p-p38 MAPK, p53, STAT1, pSTAT1, STAT3, pSTAT3, IRF-1 and SOCS1 in thoracoabdominal aorta were examined by Western Blot. Results 1) Pravastatin may prevent for atherosclerosis induced by high-cholesterol diet in apoE~(-/-) mice (P <0.05). 2) Concetrations of LDL-c and TC in pravastatin group were not statistic differences compared with atherosclerosis group. 3) Concentrations of IL-6 (P <0.01), IFN-γ(P <0.01) and TNF-α(P <0.01) and ox-LDL (P <0.05) in serum of apoE~(-/-) mice were significantly decreased in pravastatin group. 4) Levels of IFN-γand TNF-αmRNA in the thoracoabdominal aortas of apoE~(-/-) mice were significantly reduced by pravastatin (P <0.05). 5) Concentrations of IL-6 (P <0.01), IFN-γ(P <0.01) and TNF-α(P <0.05) in the thoracoabdominal aorta of apoE~(-/-) mice were significantly decreased by pravastatin. 6)The concentration of MDA was decreased (P <0.01) and the concentration of GSH was increased (P <0.01) in the thoracoabdominal aortas of apoE~(-/-) mice by pravastatin respectively. 7) Activities of SOD (P <0.01), GSH-Px (P <0.05), CAT (P <0.01) and GST (P <0.01) in the thoracoabdominal aortas of apoE~(-/-) mice were enhanced by pravastatin. 8) Expressions of 3-nitrotyrosine and iNOS in the thoracoabdominal aortas of apoE~(-/-) mice were down-regulated by pravastatin. 9) Expressions of p-p38 MAPK and p53, as the active form and downstream factor of p38 MAPK signal pathway respectively, in the thoracoabdominal aortas of apoE~(-/-) mice were down-regulated by pravastatin. 10) Expressions of pSTAT1 and pSTAT3, as active forms of IFN-γ/STAT1 and IL-6/STAT3 signal pathway respectively, and expression of IRF-1 as a downstream factor of IFN-γ/STAT1 pathway in the thoracoabdominal aortas of apoE~(-/-) mice were down-regulated by pravastatin. 11) The expression of SOCS1 as inhibitory factors of IFN-γ/STAT1 and IL-6/STAT3 signal pathway in the thoracoabdominal aortas of apoE~(-/-) mice were down-regulated by pravastatin. Conclusions 1、Pravastatin may prevent for atherosclerosis induced by high-cholesterol diet in apoE~(-/-) mice. 2、Concentrations of TC and LDL-c were increased in atherosclerosis, and pravavstatin could not decrease that concentrations. The effect of pravastatin preventing for atherosclerosis in apoE~(-/-) mice may not depend on properties of lowering cholesterol. 3、The balance between oxidant and antioxidant system was disturbed and inflammation was enhanced in atherosclerosis. Pravastatin may prevent for atherosclerosis by attenuating oxidative stress, nitrosative stress and inflammation. 4、P38 MAPK signal pathway was activated in atherosclerosis. Pravastatin maybe modulate p38 MAPK signal pathway to prevent afor therosclerosis. 5、IFN-γ/STAT1 signal pathway was activated in atherosclerosis. Pravastatin maybe modulate IFN-γ/STAT1 signal pathway to prevent for atherosclerosis. 6、IL-6/STAT3 signal pathway was activated in atherosclerosis. Pravastatin maybe modulate IL-6/STAT3 signal pathway to prevent for atherosclerosis.
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