硫化氢对巨噬细胞源性泡沫细胞形成和动脉粥样硬化斑块脂质蓄积的影响

发布时间：2018-01-16 19:09

本文关键词：硫化氢对巨噬细胞源性泡沫细胞形成和动脉粥样硬化斑块脂质蓄积的影响　出处：《南华大学》2011年博士论文　论文类型：学位论文

【摘要】：研究背景：内源性硫化氢(hydrogen sulfide, H2S)可在许多哺乳动物组织、细胞中产生，其合成途径主要为胱硫醚γ-裂解酶(cystathionine-γ-lyase,CSE)和胱硫醚-β合酶(cystathionine-synthase,CBS)催化L-半胱氨酸而生成。H2S具有广泛的作用，，被认为是第三个气体信号分子。冠心病人冠状动脉动脉粥样硬化(atherosclerosis,As)病变程度和病变血管数量与血浆H2S水平呈负相关，但机制有待阐明。单核源性巨噬细胞是促进As发生发展的一类重要的炎症细胞。氧化低密度脂蛋白(oxidized low-densitylipoprotein, oxLDL)是As的独立危险因素，可激活巨噬细胞并调节巨噬细胞功能。然而，人单核源性巨噬细胞是否存在H2S生成体系，oxLDL是否调节巨噬细胞H2S的生成，目前尚无报道。目的：研究人单核源性巨噬细胞是否存在H2S生成体系以及oxLDL对巨噬细胞生成H2S的调节作用。方法：采用亚甲基蓝法结合分光光度仪测H2S的生成，RT-PCR和Western blot检测CSE和CBS的mRNA和蛋白水平。结果：人单核源性巨噬细胞有丰富的CSE和少量的CBSmRNA表达，并内源性产生H2S。L-半胱氨酸（L-cysteine, CSE和CBS的作用底物，2mmol/L）增加巨噬细胞内H2S产生和释放，较对照组增加约28.1%(P 0.05)；而炔丙基甘氨酸(DL-propargylglycine，PPG： CSE抑制剂，3mmol/L）和羟胺（hydroxylamine， HA：CBS抑制剂，100μmol/L）抑制巨噬细胞内H2S的产生和释放，分别较对照组减少约26.8%和27.6%(P 0.05)。OxLDL（100μg/ml）降低巨噬细胞H2S合成酶活性约23.3%，呈时间和剂量依赖性减少细胞上清中H2S水平，并解除L-半胱氨酸对H2S生成的促进作用，但与PPG和HA协同抑制H2S产生。进一步研究发现，OxLDL明显下调巨噬细胞CSE mRNA和蛋白水平，轻微下调CBS mRNA水平。结论：1.人单核源性巨噬细胞内存在H2S生成系统，且以H2S/CSE为主，能生成内源性H2S。 2. OxLDL抑制巨噬细胞内源性H2S生成。研究背景：研究显示H2S对As发生的一些环节具有拮抗效应，如抑制大鼠主动脉平滑肌细胞(smooth muscle cells,SMC)增殖，抑制动脉内皮细胞粘附分子表达等。由于As形成是一个极其复杂的过程，因此有必要进一步探索H2S的潜在抗As效应。巨噬细胞源性泡沫细胞在As发生发展中起至关重要的作用。H2S是否影响泡沫细胞形成，尚未有报道，值得探索。目的：探索硫化氢对巨噬细胞源性泡沫细胞形成的影响及其机制。方法：采用佛玻酯诱导单核细胞（人外周血原始单核细胞或THP-1细胞）分化为巨噬细胞。用油红O染色和高效液相色谱分析巨噬细胞内脂质及总胆固醇(total cholesterol，TC)、胆固醇酯(cholesterol ester，CE)含量，荧光显微镜分析oxLDL结合和摄取；以Western blot检测清道夫受体A (scavenger receptor A，SR-A)， CD36和酰基辅酶A:胆固醇酰基转移酶-1(acyl-coenzyme A:cholesterol acyltransferase-1，ACAT-1)表达，ELISA法测细胞上清TNF-α水平。结果：油红O染色和高效液相色谱结果显示，巨噬细胞与oxLDL单独孵育可明显增加细胞内中性脂质及细胞内TC、CE含量。硫氢化钠(sodium hydrosulfide，NaHS：H2S供体)显著减轻oxLDL诱导的细胞内脂质蓄积，减少TC、CE含量和CE/TC比值。而PPG（H2S生成酶CSE抑制剂）促进细胞内脂质蓄积，增加TC、CE含量和CE/TC比值。巨噬细胞孵育DiI-oxLDL后大量结合并摄取DiI-oxLDL，NaHS抑制巨噬细胞结合和摄取DiI-oxLDL， PPG促进巨噬细胞结合和摄取DiI-oxLDL。进一步研究发现，oxLDL可显著诱导巨噬细胞CD36、SR-A和ACAT-1表达，而NaHS明显抑制oxLDL诱导的CD36、SR-A和ACAT-1表达。KATP通道阻滞剂格列苯脲阻止、ERK1/2抑制剂PD98059促进NaHS的上述效应。NaHS也可减少TNF-α的产生和分泌，并部分抑制TNF-α诱导的CD36表达。结论： H2S可能部分经KATP/ERK1/2环节或部分经抑制TNF-α分泌下调巨噬细胞CD36或SR-A、ACAT-1表达，减少oxLDL结合和摄取，抑制巨噬细胞源性泡沫细胞形成。研究背景：文献报道哺乳动物的动脉组织(主动脉、冠状动脉、小动脉)都含有H2S生成酶CSE，能合成和产生H2S，提示H2S可能参与血管功能的调节。本课题的前期研究显示H2S可抑制巨噬细胞内脂质蓄积，提示这一气体信号分子可能减少As斑块脂质含量，抑制粥样斑块形成。目的：研究H2S对As斑块脂质蓄积的影响。方法：8周龄雄性apoE-/-小鼠，饲喂高脂高胆固醇饮食，分别腹腔注射溶媒（生理盐水）、NaHS、PPG。8周龄雄性C57BL/6J小鼠用作空白对照。20周后，取材分析。采用油红O和苏丹IV染色检测主动脉根部病变及主动脉内膜面脂质含量。采用HE、Movat5’套染检测斑块泡沫细胞。以免疫组织化学法或免疫荧光法测CSE、SR-A、CD36和ACAT-1抗原水平及巨噬细胞含量，Real-time PCR测SR-A、CD36及ACAT-1mRNA水平。用ELISA法测血清TNF-α水平。亚甲基蓝法结合分光光度仪测血浆H2S浓度和主动脉H2S产率。以酶法检测血脂水平。结果：各组小鼠的血脂水平和体重无明显不同。ApoE-/-主动脉根部病变有CSE表达。与C57BL/6小鼠比较，apoE-/-生理盐水组小鼠的血浆H2S水平减少。而与生理盐水组比较，NaHS提高apoE-/-小鼠血浆H2S水平约21.4%（P㩳0.05），PPG不仅使主动脉H2S合成活性降低约35.6%（P㩳0.05），还使血浆H2S水平降低约22.4%（P㩳0.05）。油红O和苏丹IV染色结果显示，NaHS减少主动脉根部病变脂质阳性区百分比和主动脉内膜面脂质阳性区面积，分别较生理盐水组减少23.2%(P㩳0.05)和49.0%(P㩳0.05)。而与生理盐水组比较，PPG组主动脉根部病变脂质阳性区百分比增加31.3%(P㩳0.01)，主动脉内膜面脂质阳性区面积扩大75.4%(P㩳0.01)。与脂质阳性区面积变化一致的是，NaHS减少主动脉根部病变总面积(P㩳0.05)，而PPG增加主动脉根部病变总面积(P㩳0.05)。Movat’5和HE染色结果显示，NaHS较生理盐水显著减少主动脉根部泡沫细胞累积和泡沫细胞面积；而PPG明显增加泡沫细胞累积和泡沫细胞面积。与Movat’5结果相关的是，NaHS抑制主动脉根部病变巨噬细胞聚积、减少SR-A、CD36和ACAT-1表达；而PPG促进主动脉病变巨噬细胞蓄积和增加SR-A、CD36、ACAT-1表达。NaHS也明显降低血清TNF-α水平，而PPG增加血清TNF-α水平。结论：H2S可抑制apoE-/-小鼠As斑块脂质蓄积，其机制可能与减少斑块内巨噬细胞含量、下调SR-A、CD36、ACAT-1表达、降低血清TNF-α水平有关。
[Abstract]:Background: endogenous hydrogen sulfide (hydrogen sulfide, H2S) in many mammalian tissues, cells, the synthesis pathway for cystathionine gamma lyase (cystathionine- R -lyase, CSE) and cystathionine beta synthase (cystathionine-synthase, CBS) L- generated.H2S catalytic cysteine has many functions. Third is considered to be a gaseous signal molecule. Coronary heart disease coronary artery atherosclerosis (atherosclerosis, As) and the severity of lesion number was negatively correlated with the level of H2S, but the mechanism remains to be elucidated. Monocyte derived macrophages are an important class of inflammatory cells and promote the occurrence and development of As. The oxidation of low density lipoprotein (oxidized Low-densitylipoprotein, oxLDL) is an independent risk factor of As, can activate macrophages and macrophage function. However, whether the human monocyte derived macrophages H2S generation system o It is not yet reported whether xLDL regulates the formation of macrophage H2S.
Objective: To investigate whether there is a H2S generation system in human mononuclear macrophages and the regulation of oxLDL on the formation of H2S in macrophages.
Methods: the formation of H2S was measured by methylene blue and spectrophotometer, and the levels of mRNA and protein of CSE and CBS were detected by RT-PCR and Western blot.
Results: human monocyte derived macrophages have a rich CSE and a small amount of expression of CBSmRNA and H2S.L- cysteine (L-cysteine, endogenous substrates, CSE and CBS 2mmol/L) increased macrophage H2S production and release, compared with the control group increased by about 28.1% (P 0.05); and propargylglycine (DL-propargylglycine, PPG:CSE inhibitor. 3mmol/L (hydroxylamine, HA:CBS) and hydroxylamine inhibitor, 100 mol/L) inhibited the production and release of H2S in macrophages, about 26.8% and 27.6% respectively compared with the control group decreased (P 0.05).OxLDL (100 g/ml) reduced H2S synthase activity and low macrophage of about 23.3%, in a time and dose-dependent decrease of H2S level in the culture supernatant. And the release of L- cysteine on the formation of H2S role, but with the PPG and HA synergistic inhibition of H2S. Further studies showed that OxLDL significantly reduced macrophage CSE and protein levels of mRNA, CBS slightly reduced MRNA level. Conclusion: there is a H2S generation system in 1. human mononuclear macrophages, which is based on H2S/CSE and can produce endogenous H2S..
2. OxLDL inhibited the endogenous H2S formation of macrophages.
Background: studies show that H2S has an antagonistic effect on some aspects of As, such as inhibition of rat aortic smooth muscle cells (smooth muscle cells, SMC) proliferation, inhibit the expression of adhesion molecules in endothelial cells. The As formation is a very complicated process, so there is potential for further exploration of the anti As effect of H2S. Macrophage derived foam cells in play crucial role in the.H2S affect the formation of foam cells in the occurrence and development of As have not been reported, is worth exploring.
Objective: To explore the effect of hydrogen sulfide on the formation of macrophage derived foam cells and its mechanism.
Methods: the Buddha ester induced monocyte (glassy original human peripheral blood mononuclear cells or THP-1 cells) differentiated into macrophages. Macrophages and lipid analysis of total cholesterol in oil red O staining and high performance liquid chromatography (total cholesterol, TC (cholesterol), cholesterol ester ester, CE) content, oxLDL binding and analysis the uptake of fluorescence microscope; Western blot A (scavenger receptor detection of scavenger receptor A, CD36 and SR-A), acyl coenzyme A: cholesterol acyltransferase (acyl-coenzyme -1 A:cholesterol Acyltransferase-1, ACAT-1) expression measured cell supernatant levels of TNF- alpha ELISA method.
Results: the results of oil red O staining and HPLC showed that macrophages incubated with oxLDL alone significantly increased the neutral lipid and intracellular TC, CE. The content of sodium hydrosulfide (sodium hydrosulfide, NaHS:H2S donor) significantly reduced the oxLDL induced lipid accumulation in cells, decrease TC, CE content and CE/TC ratio. PPG (H2S CSE synthase inhibitor) to promote lipid accumulation, increase TC, CE content and CE/TC ratio. Macrophages after DiI-oxLDL incubation and combining with a large number of intake of DiI-oxLDL, NaHS inhibited macrophage binding and uptake of DiI-oxLDL, PPG promote macrophage binding and uptake DiI-oxLDL. further study found that oxLDL can significantly induce the expression of SR-A and CD36 in macrophages. ACAT-1 and NaHS significantly inhibited oxLDL induced CD36, SR-A and ACAT-1 expression of.KATP channel blocker glibenclamide block, ERK1/2 inhibitor PD98059 on.N the effect of NaHS AHS also reduces the production and secretion of TNF- alpha, and partially inhibits the expression of CD36 induced by TNF- alpha.
Conclusion: H2S may partly inhibit TNF- alpha secretion by KATP/ERK1/2 or part, down regulate macrophage CD36 or SR-A and ACAT-1 expression, reduce oxLDL binding and uptake, and inhibit macrophage derived foam cell formation.
Background: arterial tissues reported in mammals (aorta, coronary artery, arteriole) contains H2S synthase CSE, synthesis and production of H2S, suggesting that H2S may participate in the regulation of vascular function. Our previous study showed that H2S can inhibit lipid accumulation in macrophages, suggesting a gaseous signal molecule may reduce As lipid plaque the content of inhibition of plaque formation.
Objective: To study the effect of H2S on the accumulation of lipid in As plaque.
Methods: 8 week old male apoE-/- mice were fed with high fat and high cholesterol diet were injected solvent (saline), NaHS, PPG.8 week old male C57BL/6J mice were used as blank control. After.20 weeks, specimens were harvested and analyzed by oil red O and Sultan IV staining of aortic root lesions and aortic intima lipid content by HE. Movat5 ', or detect plaque foam cells by immunohistochemical method and immunofluorescence method to measure CSE, SR-A, CD36 and ACAT-1 antigen levels and macrophage content, Real-time PCR SR-A, CD36 and ACAT-1mRNA level. Serum TNF- levels were measured by ELISA method. The methylene blue method combined with the measurement of plasma H2S concentration and aortic H2S yield points light photometer. To detect the level of lipids enzyme method.
Results: the mice serum lipid levels and body weight not significantly different.ApoE-/- aortic root disease has the expression of CSE. Compared with C57BL/6 mice, the levels of plasma H2S apoE-/- normal saline group were reduced. Compared with the normal saline group, NaHS increased the levels of H2S apoE-/- in plasma of about 21.4% (P? 0.05), PPG not only makes the aortic H2S synthesis activity was decreased by 35.6% (P? 0.05), the plasma level of H2S decreased by about 22.4% (P? 0.05). Oil red O and Sultan IV staining showed that NaHS positive area decreased aortic root disease and aortic intimal lipid percentage of positive area lipid area, respectively, compared with the saline group decreased 23.2% (P? 0.05) and 49% (P? 0.05). Compared with the normal saline group, PPG group of aortic root lesions was 31.3% percentage increase in lipid (P? 0.01), aortic intimal area was enlarged by 75.4% surface lipid (P? 0.01). The area change and positive area of lipid The consensus is that NaHS reduced the total area of the aortic root lesions (P? 0.05), but PPG increased aortic root disease total area (P? 0.05).Movat 5 and HE staining showed that NaHS was significantly reduced compared with the saline area of the aortic root cells and foam foam cell accumulation; while PPG significantly increased the area of cells and foam cell accumulation. And Movat '5 results are related to the inhibition of NaHS, aortic root disease reduced SR-A, macrophage accumulation, the expression of CD36 and ACAT-1; and PPG promote macrophage accumulation and increased aortic SR-A, CD36, ACAT-1 expression of.NaHS also decreased serum TNF- levels, while PPG increased serum TNF- levels.
Conclusion: H2S can inhibit the accumulation of As plaques in apoE-/- mice, and its mechanism may be related to the reduction of macrophage content in plaques, down regulation of SR-A, CD36, ACAT-1 expression and decrease of serum TNF- alpha level.

【学位授予单位】：南华大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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