【作者】 贾乙；

【导师】 李晓辉；

【作者基本信息】 第三军医大学 ， 药理学， 2008， 博士

【摘要】 脂代谢的任一环节出现异常均可出现脂代谢紊乱,表现为血脂各成分水平的异常改变和组织中脂质的异常沉积。大量流行病学调查显示,脂代谢紊乱与多种严重威胁人类健康的疾病密切相关,包括脑卒中、动脉粥样硬化和糖尿病等。既往观点认为,遗传性因素是脂代谢紊乱最重要的发病原因,其次是饮食习惯等后天因素。本室研究结果提示,持续性炎症可导致脂代谢异常,全基因组基因芯片检测结果也进一步印证了这一观点。同时,近年来国外大规模人群流行病学调查结果显示系统性红斑狼疮和风湿性关节炎等慢性炎症疾病患者出现了明显的血脂代谢紊乱,进一步说明持续性炎症刺激极可能是发生脂代谢紊乱的新的重要因素。目前国内外在此方面的研究也鲜有报道,特别是基础实验研究尚未见报道。另外,脂质的异常沉积,尤其是脂质在动脉壁下的沉积,即动脉粥样硬化的形成,对健康的危害巨大。而巨噬细胞源性泡沫细胞形成是动脉壁下的脂质沉积最重要的启动因素,为此,本研究采用体外培养泡沫细胞为模型,观察炎症刺激对泡沫细胞形成的影响。三七总皂苷(PNS)是三七中主要的活性成分,既往研究表明,PNS具有保护心肌、降低动脉血压、改善脂代谢紊乱及显著的抗炎能力。IH901是PNS在利用模拟生物体肠道微生态环境中反应得到的活性代谢单体。本研究通过在体实验观察了PNS作用,并在离体实验中观察了IH901的作用。方法为观察持续性炎症刺激对高脂饮食大鼠血脂代谢情况的影响,将动物分为高脂饮食、叠加炎症、PDTC和PNS组。所有动物均给予高脂饮食,叠加炎症组动物腹腔注射酵母多糖-石蜡混悬液(20mg/kg, i.p.,5天1次),PDTC和PNS组动物除按叠加炎症组处理外,分别每天给予PDTC(100mg/kg, i.p.)和PNS(120mg/kg, i.g.)。为探讨持续性炎症刺激对大鼠血脂代谢影响的原因,将动物分为正常对照、单纯炎症、PDTC治疗和PNS治疗组。所有动物均给予正常饮食,单纯炎症组动物腹腔注射酵母多糖-石蜡混悬液(20mg/kg, i.p.,5天1次),PDTC治疗和PNS治疗组动物除按单纯炎症组处理外,分别每天给予PDTC(100mg/kg, i.p.)和PNS(120mg/kg, i.g.)。为研究炎症刺激对泡沫细胞内脂质沉积的影响,将培养的泡沫细胞分为泡沫细胞、炎症刺激、PDTC和IH901组。炎症刺激组给予LPS(20mg/L)进行刺激,在炎症刺激的基础上,PDTC和IH901组分别给予相应的试剂(PDTC 25μM; IH901 25μM)。TNF-α采用ELISA法测定;TG、LDL、HDL、TC、CE、LPL活性使用相应的试剂盒测量;LPL、HMG-CoA还原酶、PPARα、PPARγ、CD36、ABCA1、Perilipin和LXR mRNA水平用RT-PCR法检测,LPL、perilipin和NF-κB蛋白表达水平用western blotting法测量,泡沫细胞染色采用油红O。结果1.持续性炎症能够明显加重高脂饮食动物的血脂代谢紊乱情况。叠加炎症组动物血清中TC、TG和LDL较高脂饮食组有明显升高,而HDL水平明显下降,其变化与动物体内炎症反应强度(TNF-α水平)呈平行性变化。持续性炎症能诱发正常饮食动物血脂紊乱,使TC、TG、LDL升高,HDL降低,与动物体内炎症反应强度变化平行。2.持续性炎症刺激能减少LPL的表达(mRNA和蛋白水平)、降低了LPL的活性,同时增加了HMG-CoA还原酶mRNA的表达,并降低了PPARα/γ的表达。3.同给予持续性炎症刺激动物相比,PDTC能够显著降低动物血清中的TNF-α水平,提高LPL的活性,增高LPL的表达(mRNA和蛋白水平),降低HMG-CoA还原酶mRNA的表达,增高PPARα/γ的表达。4. PNS能够降低TNF-α、TC、TG、LDL水平,升高HDL水平,增加LPL的表达、提高LPL的活性,同时降低HMG-CoA还原酶的表达,并增高PPARα/γ的表达。5.炎症刺激使泡沫细胞内的脂质沉积显著升高(TC、CE和CE/TC水平均升高),与炎症反应强度(TNF-α水平)变化平行。6.炎症刺激导致perilipin表达升高(mRNA和蛋白水平)及CD36、ABCA1、PPARγ和LXR mRNA表达降低。7. PDTC能够降低炎症刺激导致的脂质沉积增加,使CD36、ABCA1、PPARγ和LXR mRNA表达增高,perilipin表达降低(mRNA和蛋白水平)。8. IH 901能够显著降低炎症刺激导致的泡沫细胞内的脂质沉积增加,使ABCA1、PPARγ和LXR mRNA表达增高,CD36 mRNA、perilipin(mRNA和蛋白)表达降低。结论1.持续性炎症刺激可以通过降低LPL和增高HMG-CoA还原酶表达以诱发/加重大鼠的脂代谢紊乱,其机制与持续性炎症刺激对PPARα/γ的影响相关。2.炎症刺激可以引起体外培养的泡沫细胞内脂质沉积增加,其机制与炎症刺激导致perilipin表达增高和ABCA1表达降低有关。3.三七总皂苷口服能够明显减轻炎症刺激诱发/加重的大鼠脂代谢紊乱,机制与增高LPL和降低HMG-CoA还原酶表达有关。4. PNS活性代谢单体IH901能够降低炎症刺激导致的泡沫细胞内脂质沉积增多,机制与其降低perilipin表达,增高ABCA1表达和减少CD36表达有关。更多还原

【Abstract】 Lipid metabolism is one of the most important metabolic systems in organism. The disorder of lipid metabolism included the abnormal serum lipid level and the abnormal aggregation of lipid in tissues. Epidemiological studies have shown the close correlation between the disorder of lipid metabolism and many severe diseases, including stroke, atherosclerosis, diabetes and so on. In traditional views, the disorder of lipid metabolism was mostly considered as a genetic disease, and was affected by living habits partially. However, it was found that persistent inflammation caused by Zymosan could induce the aggravation of serum lipid disorder in our recently studies, which implied that persistent inflammation might play an important role in lipid metabolism process. Meanwhile, the disorder of serum lipid level occurred in patients with chronic inflammation disease, such as rheumatic arthritis and lupus erythematosus. Besides the serum lipid level, the abnormal aggregation of lipid in tissues was a health care problem yet. Among that, the lipid aggregation under endarterium was the most danger one, because it would induce atherosclerosis. And the key point of atherosclerosis progression was the formation of foam cell. Therefore, this study was designed to explore the action of persistent inflammation in lipid metabolism in vivo and in vitro.Panax notoginseng saponins (PNS) are the principal ingredient extracted from the traditional Chinese herb Panax notoginseng and have extent effects on lipid metabolism and cardiovascular diseases. IH 901 is the metabolized product of the PNS by intestinal bacteria in human and rat,which was regarded as the main effective monomer of metabolized Ginsenosides in vivo. Effects of PNS and IH901 were also observed in this study.Methods1. To observe the action of persistent inflammation on lipid metabolism in hyper-fat diet rats, animals were divided into four groups including hyper-fat diet group (high fat diet), inflammation group (high fat diet + zymosan, 20 mg/kg, i.p., once every 5 days), PDTC group (high fat diet +zymosan, 20 mg/kg, i.p., once every 5 days + PDTC, 100 mg/kg, i.p., daily) and PNS group (high fat diet + zymosan, 20 mg/kg, i.p., once every 5 days + PNS 120 mg/kg, i.g., daily).2. To study the effect of persistent inflammation on lipid metabolism in normal rats, animals were divided into four groups including control group (normal diet), simple-inflammation group (normal diet + zymosan, 20 mg/kg, i.p., once every 5 days), PDTC group (normal diet + zymosan, 20 mg/kg, i.p., once every 5 days + PDTC, 100 mg/kg, i.p., daily) and PNS group (normal diet + zymosan, 20 mg/kg, i.p., once every 5 days + PNS 120 mg/kg, i.g., daily).3. To explore the action of inflammation on the formation of foam cell, cells were divided into four groups as control, inflammation group, PDTC group and IH901 group. Rat’s peritoneal macrophages were incubated with ox-LDL for 48h as foam cell group. LPS (20mg/L) was added into the medium as inflammation stimuli in inflammation group. Besides LPS, PDTC (25μM) and IH901 (25μM) were given in PDTC and IH901 groups respectively.4. TNF-αlevel was measured with ELISA technique.5. The foam cells were stained by oil red-O after immobilization.6. The level of TG, LDL, HDL, TC, CE and LPL activity were measured using commercial kits.7. The expression of HMG-CoA reductase, PPARα/γ, CD36, ABCA1, PPARγand LXR mRNA were measured by RT-PCR.8. The protein level of LPL, perilipin and NF-κB were measured with western blotting.Results1. Persistent inflammation could induce and aggravate the lipid metabolism disorder in normal rats and hyper-fat diet rats respectively. Compared with control group and hyper-fat diet group, the serum TNF-α, TC, TG and LDL levels increased significantly, and the level of HDL decreased noticeably.2. Persistent inflammation reduced the LPL activity, the expression of LPL and its mRNA, PPARα/γmRNA and up-regulated the expression of HMG-CoA reductase significantly.3. In PDTC and PDTC therapy group, the levels of TNF-α, TC, TG, LDL, the expression of HMG-CoA reductase were decreased and the HDL level, the expression of PPARα/γmRNA, LPL (mRNA and protein), the activity of LPL were increased by comparison of that in inflammation group and simple-inflammation group respectively.4. The treatment of PNS (i.g.) could improve the lipid metabolism disorder caused by inflammation. In PNS and PNS therapy group, the levels of TNF-α, TC, TG, LDL, the expression of HMG-CoA reductase were decreased and the HDL level, the expression of PPARα/γmRNA, LPL (mRNA and protein), the activity of LPL were increased by comparison of that in inflammation group and simple-inflammation group respectively.5. The inflammation could increase the lipid aggregation in foam cells. The levels of TC, CE, CE/TC ratio and TNF-αincreased significantly compared with foam cell group.6. Inflammation reduced the expression of CD36, ABCA1, PPARγand LXR mRNA and increased the expression of perilipin (mRNA and protein) significantly.7. The treatment of PDTC could up-regulate the expression of CD36, ABCA1, PPARγand LXR mRNA and down-regulate the expression of perilipin (mRNA and protein) significantly.8. The treatment of IH901 could up-regulate the expression of ABCA1, PPARγand LXR mRNA and down-regulate the expression of CD36 mRNA, perilipin (mRNA and protein) noticeably. The levels of TC, CE, CE/TC ratio and TNF-αdecreased significantly compared withinflammation group.Conclusion1. Persistent inflammation could induce and aggravate serum lipid metabolism disorder in rats through down-regulating the expression of LPL and up-regulated the expression of HMG-CoA reductase. PPARα/γwere involved in the mechanism of that.2. Inflammation could increase lipid aggregation in foam cells in vitro, which might be related with the increased expression of perilipin and the decreased expression of ABCA1.3. The treatment of PNS could ameliorate the lipid metabolism disorder induced by persistent inflammation via up-regulating the expression of LPL and down-regulated the expression of HMG-CoA reductase. 4. The metabolite monomer of PNS, IH901 could reduce the excessive lipid aggregation in foam cells caused by inflammation stimulation. The mechanism might be related with the decreased expression of perilipin, the increased expression of ABCA1 and the decreased expression of CD36.更多还原