冠心病血瘀证目标分子的鉴定、验证及功能分析
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摘要
课题组前期应用寡核苷酸基因芯片技术构建了冠心病血瘀证白细胞差异基因表达谱,筛选出FcγRⅢA(免疫球蛋白IgG结晶片段受体Ⅲa)、PKCβ1(蛋白激酶Cpl)、IL-8(白细胞介素-8)、HLA-DQB1(主要组织相容性复合体Ⅱβ1)、FOLR3(叶酸受体γ3)、PTGDS(前列腺素D2合成酶)6个目标基因。运用基因本体论和显著性通路分析方法,从分子水平阐明了冠心病血瘀证与炎症免疫反应的相关性；进一步通过Signal-Net网络构建,使用网络拓扑技术分析发现差异基因FcγRⅢA. PKCβ1和IL-8在冠心病血瘀证疾病网络中处于关键调控环节,并对其中的目标分子IL-8进行了进一步的临床验证和体内、外功能分析。
本课题对另外两个目标分子PKCβ1、FcγRⅢA进行了鉴定、验证,并对FcyRIIIA在冠心病血瘀证形成中的作用进行分析。纳入冠心病血瘀证患者、冠心病非血瘀证患者和健康对照者,抽取外周血,分离白细胞,抽提总RNA,应用实时定量逆转录聚合酶链反应(Real-time reverse transcription polymerase chain reaction, Real-time RT-PCR)技术检测目标分子PKCβ1、FcγRⅢA基因表达水平,结果发现FcγRⅢA mRNA表达水平与前期基因芯片结果一致。我们进一步从临床血清学水平进行了规模验证,并就其细胞来源进行了相关分析；同时根据FcγRⅢA的功能,结合冠心病、血瘀证病理特点,体外通过构建人外周血单核细胞-脐静脉内皮细胞实验体系,观察FcγRⅢA对单核-内皮细胞粘附的影响；体内以ApoE-/-小鼠为研究对象,观察FcγRⅢA高表达及抑制其表达后对小鼠主动脉粥样硬化斑块稳定性及炎症相关因子的影响,以期探索目标分子FcγRⅢA参与冠心病血瘀证疾病过程的可能机制；同时,观察赤芍川芎有效部位对ApoE-/-小鼠FcγRⅢA蛋白水平、主动脉粥样硬化斑块稳定性以及炎症相关因子的影响,明确活血化瘀制剂对冠心病血瘀证靶标分子的干预效应。
本课题研究分为两部分：文献综述和实验研究。
1文献综述：本部分分别从血瘀证与炎症反应、FcγR与心血管疾病研究进展两个方面进行综述。
2实验研究：包括以下六个部分。
研究一冠心病血瘀证目标分子鉴定
目的：应用Real-time RT-PCR技术,对冠心病血瘀证目标分子PKCβ1、FcyRIIIA进行鉴定。
方法：冠心病诊断参照1999年美国心脏病学会(ACC)／美国心脏协会(AHA)／美国医师学会及美国内科学会(ACP-ASIM)联合协定关于《慢性稳定性心绞痛诊疗指南》、《不稳定性心绞痛、无ST段抬高心肌梗死诊疗指南》。同时,参考中国中西医结合学会活血化瘀专业委员会制定的血瘀证诊断标准。筛选符合冠心病、血瘀证、健康人入选标准的冠心病血瘀证组、冠心病非血瘀证组和健康对照组各20例为研究对象。冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血2 mL,分离白细胞,抽提总RNA,应用Real-time RT-PCR技术检测目标分子PKCβ1和FcγRⅢA基因表达水平。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数、病程、合并疾病和用药情况方面无显著性差异,具有可比性。
①PKCβ1 mRNA表达在冠心病血瘀证组(0.74±0.30)和非血瘀证组(0.96±0.31)均较健康对照组(1.03±0.37)降低,且冠心病血瘀证组较非血瘀证组降低显著(P<0.05)。
②FcγRⅢA mRNA表达在冠心病血瘀证组(1.37±0.50)和非血瘀证组(1.37±0.42)均较健康对照组(0.89±0.33)显著升高(P<0.01),且冠心病血瘀证组较非血瘀证有升高趋势。
结论：
①PKCβ1基因参与冠心病血瘀证过程,但与前期研究不一致,尚需进一步扩大样本量进行鉴定。
②FcγRⅢA与冠心病血瘀证具有一定相关性,可能在冠心病血瘀证形成过程中发挥重要作用。
研究二FcγRⅢA血清蛋白水平以及冠心病血瘀证与动脉粥样硬化危险因素的相关性研究
目的：应用酶联免疫吸附(ELISA)法检测FcγRⅢA血清水平(可溶性CD16,sCD16),验证其与冠心病血瘀证的临床相关性,并分析冠心病血瘀证与动脉粥样硬化危险因素如血脂、高敏C反应蛋白(hs-CPR)以及单核细胞参数的相关性。
方法：纳入冠心病血瘀证、冠心病非血瘀证患者各50例和健康对照组40例为研究对象。冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血5 mL,采用双抗体夹心ABC-ELISA法检测血清sCD16浓度,同时对动脉粥样硬化危险因素(血脂、hs-CPR以及单核细胞计数和单核细胞百分比)进行检测。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数、病程和合并疾病方面无显著性差异,具有可比性。
①血清sCD16在冠心病血瘀证组(3.67±0.98 U/mL)和非血瘀证组(2.80±0.80U/mL)均较健康对照组(1.58±0.47 U/mL)显著升高(P<0.01),且冠心病血瘀证组较非血瘀证组升高显著(P<0.05)。
②总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)在冠心病血瘀证组和非血瘀证组均较健康对照组显著升高(P<0.05或0.01),且冠心病血瘀证组较非血瘀证组有升高趋势；而高密度脂蛋白胆固醇(HDL-C)在冠心病血瘀证组和非血瘀证组较健康对照组显著降低(P<0.01),且冠心病血瘀证组较非血瘀证有降低趋势。
③hs-CRP水平在冠心病血瘀证组(2.91±1.98 mg/L)和非血瘀证组(1.43±1.29mg/L)均较健康对照组(0.56±0.09 mg/L)显著升高(P<0.01,0.05),且冠心病血瘀证组较非血瘀证组升高显著(P<0.05)。
④外周血单核细胞计数在冠心病血瘀证组、非血瘀证组和健康对照组之间差异无统计学意义,而单核细胞百分比在冠心病血瘀证组(6.40±1.17%)和非血瘀证组(5.35±1.62%)均较健康对照组(4.99±1.81%)升高,且冠心病血瘀证组较非血瘀证组升高显著(P<0.05)。
结论：
①血清sCD16与冠心病血瘀证具有相关性,从蛋白水平验证了目标分子FcγRⅢA与冠心病血瘀证密切相关。
②冠心病血瘀证患者与动脉粥样硬化危险因素,特别是hs-CRP和单核细胞百分比显著相关。
研究三FcyRIIIA来源分析
目的：应用流式细胞术检测冠心病血瘀证患者外周血单核细胞CD14+CD16+表达以分析目标分子FcyRIIIA来源,并分析细胞间粘附分子(ICAM-1,CD54)在冠心病血瘀证患者单核细胞CD14+CD16+表面的变化,同时检测冠心病血瘀证患者炎症相关因子[可溶性CD14(sCD14)、巨噬细胞集落刺激因子(M-CSF)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1(IL-1)]血清水平。
方法：冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血,采用流式细胞术检测外周血单核细胞CD14+CD16+表达及其表面CD54平均荧光强度；采用双抗体夹心ABC-ELISA法检测sCD14、M-CSF、TNF-α和IL-1血清水平。
结果：
①外周血单核细胞CD14+CD16+表达在冠心病血瘀证组(11.24±7.73%)和非血瘀证组(9.96±3.66%)均较健康对照组(5.64±2.49%)显著升高(P<0.01,0.05),且冠心病血瘀证组较非血瘀证组有升高趋势。
②外周血单核细胞CD14+CD16+表面CD54的平均荧光强度在冠心病血瘀证组(1760.60±457.60)和非血瘀证组(1089.27±426.76)均较健康对照组(849.00±100.91)显著升高,且冠心病血瘀证组较非血瘀证组升高显著(P<0.01)。
③炎症相关因子sCD14、M-CSF、TNF-α和IL-1血清水平在冠心病血瘀证组和非血瘀证组均较健康对照组显著升高(P<0.01),且冠心病血瘀证组较非血瘀证组升高显著(P<0.05)。
结论：
①冠心病血瘀证患者单核细胞处于活化状态。
②冠心病血瘀证患者高水平血清M-CSF能刺激单核细胞CD14+CD16+高表达,进而促进单核细胞CD14+CD16+表面CD54表达升高并产生大量TNF-α和IL-1前炎症因子,从细胞来源方面再次鉴定了目标分子FcyRIIIA与冠心病血瘀证密切相关。
③冠心病血瘀证患者体内炎症水平特别是单核细胞来源的细胞因子处于较高水平。
研究四FcyRIIIA在人外周血单核细胞-脐静脉内皮细胞粘附中的作用分析
目的：研究FcyRIIIA对人单核细胞-脐静脉内皮细胞粘附的影响,对其进行体外功能分析。
方法：①分离、鉴定并培养人外周血单核细胞,实验分为单核细胞(MC)组：单核细胞培养18 h;M-CSF组：重组人M-CSF刺激单核细胞培养18 h;免疫球蛋白IgG (IVIG)组：IVIG提前干预单核细胞30 min后再加重组人M-CSF刺激培养18 h,流式细胞术检测三组单核细胞CD14+CD16+表达情况。②分离、.鉴定并培养人外周血单核细胞、脐静脉内皮细胞,实验分为MC组：单核细胞培养18 h;M-CSF组：重组人M-CSF刺激单核细胞培养18h; IVIG组：IVIG提前干预单核细胞30min后再加重组人M-CSF刺激培养18 h。三组细胞消化后再与脐静脉内皮细胞培养1 h,Bradford法检测三组单核-内皮细胞粘附率。
结果：瑞姬染色显微镜下细胞形态学观察和流式细胞术单核细胞CD14鉴定人外周血单核细胞,倒置相差显微镜细胞形态学观察和Ⅷ因子相关抗原间接免疫荧光方法鉴定人脐静脉内皮细胞。
①经重组人M-CSF刺激培养18 h后单核细胞CD14+CD16+表达较未刺激前显著升高(P<0.01)；提前给予IVIG干预30 min后,单核细胞CD14+CD16+表达较重组人M-CSF刺激培养18 h显著降低(P<0.01)。
②经重组人M-CSF刺激培养18 h后单核-内皮细胞粘附率较单核细胞未刺激前显著升高(P<0.05)；提前给予IVIG干预30 min后,单核-内皮细胞粘附率较重组人M-CSF刺激培养18 h显著降低(P<0.05)。
结论：
①重组人M-CSF在体外能刺激人外周血单核细胞CD14+CD16+表达,IVIG能抑制该作用。
②单核细胞CD14+CD16+能介导人外周血单核-脐静脉内皮细胞粘附。
研究五FcyRIIIA对ApoE-/-小鼠主动脉粥样硬化斑块不稳定性的作用评价
目的：研究目标分子FcyRIIIA高表达和抑制其后对ApoE7-/-小鼠主动脉粥样硬化斑块不稳定性的影响。
方法：66只8周龄雄性ApoE-/-小鼠分为模型组：高脂饲料喂养10 w,ApoE-/-小鼠+腹腔注射免疫球蛋白IgG组(IVIG组)：连续腹腔注射IVIG 5 d+高脂饲料喂养10 w, ApoE-/-小鼠+辛伐他汀组(Sm组)：Sm灌胃每日一次+高脂饲料喂养10 w,每组各22只。与ApoE-/-小鼠具有相同遗传背景的C57BL/6J小鼠22只作为对照组,普通饲料喂养10 w。
①主动脉组织病理学观察：主动脉根部组织石蜡切片H&E染色发现有动脉粥样硬化改变的切片进行Masson染色评价斑块胶原含量,a-actin和CD68免疫组化染色分别评价斑块平滑肌细胞含量和巨噬细胞含量,主动脉根部组织冰冻切片油红O染色评价斑块脂肪组织含量。
②血液学指标检测：生化法检测血脂并计算血浆致动脉粥样硬化指数；流式细胞术检测外周血单核细胞CD14+CD16+表达；双抗体夹心ABC-ELISA法检测血清TNF-α、IL-1和sE-selectin水平。
③MMP-9基因表达和蛋白水平检测：Real-time RT-PCR技术和Western blot法分别检测主动脉MMP-9 mRNA表达和蛋白水平。
④相关性分析：外周血单核细胞CD14+CD16+表达、主动脉MMP-9基因表达和血清TNF-a水平在模型组、IVIG组和Sm组的相关性分析。
结果：
①模型组小鼠主动脉粥样硬化斑块含有大量脂质成分、巨噬细胞和平滑肌细胞,且胶原成分增多。IVIG能通过减少斑块内脂质含量、减少巨噬细胞聚集和平滑肌细胞增殖而发挥改善动脉粥样硬化的作用,该作用与Sm相似。
②模型组小鼠血脂TC、TG、LDL-C水平升高,HDL-C水平降低,外周血单核细胞CD14+CD16+高表达,血清TNF-α、IL-1和sE-selectin水平较对照组显著升高(P<0.01,0.05)；上述指标在IVIG组有不同程度改善且作用与Sm组相似。
③模型组小鼠主动脉MMP-9基因表达和蛋白水平均较对照组显著升高(P<0.01),上述指标在IVIG组有不同程度改善且作用与Sm组相似。
④相关性分析发现,外周血单核细胞CD14+CD16+表达在模型组、IVIG组和Sm组均与主动脉MMP-9基因表达和血清TNF-a水平呈显著正相关。
结论：
①FcyRIIIA介导主动脉粥样硬化模型小鼠炎症相关因子释放增加。
②FcyRIIIA一定程度上影响小鼠主动脉粥样硬化斑块的稳定性。
研究六赤芍川芎有效部位对ApoE-/-小鼠主动脉粥样硬化斑块稳定性的干预效应研究
目的：研究赤芍川芎有效部位(赤芍总苷、川芎总酚)对ApoE-/-小鼠主动脉粥样硬化斑块稳定性的干预效应,并分析其可能的作用靶点。
方法：64只8周龄雄性ApoE-/-小鼠分为模型组：高脂饲料喂养10w, ApoE-/-小鼠+Sm组(Sm组)：Sm灌胃每日一次+高脂饲料喂养10w, ApoE-/-小鼠+芎芍胶囊(XSC)大剂量组(XSCH组)：大剂量XSC灌胃每日一次+高脂饲料喂养10 w和ApoE-/-小鼠+XSC小剂量(XSCL)组：小剂量XSC灌胃每日一次+高脂饲料喂养10 w,每组各16只。各组进行血液学指标、MMP-9基因表达和蛋白水平比较以及相关性分析。
结果：
①XSC对TC、TG、LDL-C水平和AIP均有不同程度降低,同时能降低外周血单核细胞CD14+CD16+表达,不同程度降低血清TNF-α、IL-1和sE-selectin水平,对ApoE-/-小鼠体内炎症水平的改善作用XSCH与Sm相似。
②XSC与Sm类似均能降低ApoE-/-小鼠主动脉MMP-9基因表达和蛋白水平,对动脉粥样硬化斑块具有稳定作用,但对MMP-9基因表达的降低作用XSCH较Sm显著(P<0.05)。
③相关性分析发现,经XSC干预后,主动脉MMP-9基因表达和血清TNF-α水平下降均与外周血单核细胞CD14+CD16+表达降低呈显著正相关。
结论：
①赤芍川芎有效部位能减轻炎症反应、稳定斑块、改善主动脉粥样硬化。
②赤芍川芎有效部位抗主动脉动脉粥样硬化的作用靶点与FcyRⅢA有关。
Coronary heart disease (CHD) is the leading cause of death worldwide. Genetic factors and environmental factors are responsible for the development of CHD. The underlying mechanisms of CHD have not yet been elucidated. Additionally, there is little known about the way of CHD-specific prevention and treatment therapy. Previous studies have investigated the differential gene expression profiles in peripheral leukocytes from CHD patients with blood stasis syndrome (BSS) by oligonucleotide microarray technique. We have shown that the immune inflammatory response is correlated to the development of CHD and Fc receptor III A of immunoglobulin G (FcyRIIIA, also named CD 16), protein kinase C beta 1 (PKCβ1), interleukin-8 (IL-8), HLA-DQB1, FOLR3, PTGDS were involved in the development of CHD with BSS. Of them, FcyRIIIA, PKCβ1 and IL-8 played the key role in the regulation of CHD with BSS. In our previous studies, we have shown that IL-8 was implicated in the development of CHD with BSS and its functional analysis was determined in vitro and vivo.
In the current study, we investigated the mRNA expression of PKCβ1 and FcyRIIIA in the leukocytes in CHD patients with BSS, CHD patients with non-BSS and the healthy control by Real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). There was a significant increase of FcyRIIIA at the mRNA level in leukocytes in CHD patients with BSS, consistent with our previous study. Then, we determined the protein level of both sCD16 in sera and membrane CD 16 on monocytes in CHD patients with BSS. Moreover, we investigated FcyRIIIA-induced molecular events that may be responsible for the development of CHD using human peripheral monocyte-umbilical vein endothelial cells in vitro and aortic atherosclerotic formation in ApoE-/- mouse model. Meanwhile, we determined the intervention effects of active ingredients of Chuanxiong rhizome and red peony root (Chuangxiongol and paeoniflorin) on FcyRIIIA expression, aortic atherosclerotic plaque destabilization and inflammatory factors in ApoE-/- mice.
This study is divided into two parts:literature review and experimental research.
1 Literature review:Including the following two reviews:BSS and inflammation and Investigation of correlation between FcyR and cardiovascular diseases.
2 Experimental research:Including the following six parts. Study I:Identification of related molecules in CHD patients with BSS
Objective:To identify the related molecules PKCβ1 and FcyRIIIA in CHD patients with BSS by Real-time RT-PCR.
Methods:All cases were divided into CHD patients with BSS, CHD patients with non-BSS and the healthy control,20 in each group. CHD patients were diagnosed according to the 1999 ACC/AHA/ACP-ASIM Guidelines for the Management of Patients with Chronic Stable Angina and the 2002 ACC/AHA Guidelines for the Management of Patients with Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. A diameter stenosis of at least 50% was diagnosed by visible estimation in a major coronary artery from standard selective coronary angiography. Patients with BSS were diagnosed according to guidelines for the diagnosis of BSS. mRNA expression of PKCβ1 and FcyRIIIA in the leukocytes was determined in blood samples taken following fasting from CHD patients with BSS, CHD patients with non-BSS and the healthy control by Real-time RT-PCR.
Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels, course of disease, medical history and herbal therapy beween CHD patients with BSS and CHD patients with non-BSS.
①There was a significant decrease of PKCβ1 at the mRNA level in leukocytes in both CHD patients with BSS (0.74±0.30) and CHD patients with non-BSS (0.96±0.31) when compared to the healthy control (1.03±0.37). Additionally, mRNA expression of PKCβ1 in CHD patients with BSS was lower than that in CHD patients with non-BSS (P<0.05)
②There was a significant increase of FcyRIIIA at the mRNA level in leukocytes in both CHD patients with BSS (1.37±0.50) and CHD patients with non-BSS (1.37±0.42) when compared to the healthy control (0.89±0.33, P<0.05). Additionally, mRNA expression of FcyRIIIA in CHD patients with BSS was higher than that in CHD patients with non-BSS without significant differences.
Conclusion:
①PKCβ1 may participate in the development of CHD with BSS. However, results in the current study was in contradiction with our previous study of gene chip. Identification of PKCβ1 in CHD patients with BSS should be analyzed further.
②There is a certain correlation between FcyRIIIA and CHD with BSS. It may be involved in the development of CHD with BSS.
Study II:Investigation of FcyRIIIA at the protein level and the correlation of CHD patients with BSS and risk factors of atherosclerosis
Objective:To determine the protein level of FcyRIIIA (soluble CD 16, sCD16) and analyze the correlation of CHD patients with BSS and risk factors of atherosclerosis such as blood lipid level, high-sensitivity C-reactive protein (hs-CRP) and parameters of monocytes.
Methods:All cases were divided into CHD patients with BSS and CHD patients with non-BSS, fifty in each group and forty healthy individuals were selected as the healthy control. The protein level of sCD16 in sera was determined in blood samples taken following fasting from CHD patients with BSS, CHD patients with non-BSS and the healthy control by Enzyme-linked immunosorbent assay (ELISA). Then, levels of blood lipid and hs-CRP, and parameters of monocytes were analyzed as well.
Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels, course of disease and medical history beween CHD patients with BSS and CHD patients with non-BSS.
①There was a significant increase of the protein level of serum sCD16 in both CHD patients with BSS (3.67±0.98 U/mL) and CHD patients with non-BSS (2.80±0.80 U/mL) when compared to the healthy control (1.58±0.47 U/mL). Additionally, serum level of sCD16 in CHD patients with BSS was higher than that in CHD patients with non-BSS (P<0.05)
②There was a significant increase of blood lipid level such as total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in both CHD patients with BSS and CHD patients with non-BSS when compared to the healthy control (P< 0.01,0.05). However, levels of TC, TG, and LDL-C were increased in CHD patients with BSS, but not significantly, compared with CHD patients with non-BSS. There was a significant decrease of high-density lipoprotein cholesterol (HDL-C) level in both CHD patients with BSS and CHD patients with non-BSS when compared to the healthy control (P<0.01). Additionally, HDL-C level was reduced in CHD patients with BSS, but not significantly, compared with CHD patients with non-BSS.
③There was a significant increase of hs-CRP level in both CHD patients with BSS (2.91±1.98 mg/L) and CHD patients with non-BSS (1.43±1.29 mg/L) when compared to the healthy control (0.56±0.09 mg/L) (P<0.01,0.05). Additionally, hs-CRP level in CHD patients with BSS was higher than that in CHD patients with non-BSS (P< 0.05).④There were no statistically significant differences of monocyte count in peripheral blood among CHD patients with BSS, CHD patients with non-BSS and the healthy control. There was an increase of percentage of monocytes in both CHD patients with BSS (6.40±1.17%) and CHD patients with non-BSS (5.35±1.62%) when compared to the healthy control (4.99±1.81%). Additionally, percentage of monocytes in CHD patients with BSS was higher than that in CHD patients with non-BSS (P<0.05).
Conclusion:
①High level of sCD16 in CHD patients with BSS suggests that there is relevance of FcyRⅢA to CHD patients with BSS at the protein level.
②There was is relevance of risk facotrs of atherosclerosis, especially hs-CPR and percentage of monocytes to CHD patients with BSS.
StudyⅢ:Original analysis of FcγRⅢA:the protein level of membrane CD16 on monocytes
Objective:To investigate the protein level of membrane CD 16 on monocytes in CHD patients with BSS by flow cytometric analysis, and then, expression of intercellular adhesion molecule-1 (ICAM-1, also named CD54) on CD14+CD16+monocytes was determined. Meanwhile, levels of inflammatory factors such as soluble CD 14 (sCD14), macrophage colony stimulating factor (M-CSF), tumor necrosis factor-a (TNF-a) and interleukin-1 (IL-1) in sera were analyzed.
Methods:The protein level of membrane CD 16 on monocytes was determined in blood samples taken following fasting from CHD patients with BSS, CHD patients with non-BSS and the healthy control by flow cytometry. Mean fluorescence intensity (MFI) of ICAM-1 on CD14+CD16+monocytes was analyzed as well. Levels of sCD14, M-CSF, TNF-a and IL-1 in sera were examined by ELISA.
Results:
①There was a significant increase of the protein level of membrane CD 16 on monocytes in both CHD patients with BSS (11.24±7.73%) and CHD patients with non-BSS (9.96±3.66%) when compared to the healthy control (5.64±2.49%) (P< 0.01,0.05). Additionally, this protein level of membrane CD16 on monocytes in CHD patients with BSS was increased, but not significantly, compared with CHD patients with non-BSS.
②There was a significant increase of MFI of CD54 on CD14+CD16+monocytes in both CHD patients with BSS (1760.60±457.60, P<0.01) and CHD patients with non-BSS (1089.27±426.76) when compared to the healthy control (849.00±100.91). Additionally, MFI of CD54 on CD14+CD16+monocytes in CHD patients with BSS was higher than that in CHD patients with non-BSS (P<0.01).
③There was a significant increase of serum levels of inflammatory factors such as sCD14, M-CSF, TNF-a and IL-1 in both CHD patients with BSS and CHD patients with non-BSS when compared to the healthy control. Additionally, levels of all these inflammatory factors in CHD patients with BSS were higher than those in CHD patients with non-BSS (P<0.05).
Conclusion:
①Activated monocytes are found in CHD patients with BSS.
②CD14+CD16+monocytes was stimulated by high serum M-CSF level in CHD patients with BSS, along with increased level of CD54 on CD14+CD16+monocytes and TNF-a and IL-1 in sera. Taken together, these results suggest that FcyRIIIA is correlated to CHD patients with BSS.
③Level of inflammatory factors, especially cytokines from monocytes increase in CHD patients with BSS.
StudyⅣ:Effects of FcyRIIIA on the adherence of monocytes from human peripheral blood to human umbilical vein endothelial cells
Objective:To investigate the effects of FcyRIIIA on the adherence of monocytes from human peripheral blood to human umbilical vein endothelial cells (HUVECs).
Methods:First, we overexpressed and inhibited FcyRIIIA on monocytes by recombinant human M-CSF stimulation for 18 h or IVIG pretreatment for 1 h respectively, according to procedures as follows:monocytes randomly divided into three groups. MC group:monocytes cultured for 18 h; M-CSF group:monocytes stimulated by recombinant human M-CSF (10 ng/mL) for 18 h; IVIG group:monocytes pretreated with IVIG (5 mg/mL) for 30 min, then treated with recombinant human M-CSF (10 ng/mL) for 18 h. Monocytes in these three groups were harvested 18 h later, and the surface expression of CD 14 and CD 16 on monocytes was determined by flow cytometry. Furthermore, the adhesive efficiency of monocytes to HUVECs was determined by Bradford method. Briefly, monocytes were treated with recombinant human M-CSF stimulation for 18 h or with IVIG pretreatment for 1 h and then recombinant human M-CSF stimulation for 18 h or without any intervention. Monocytes were released from the culture dish with 3% EDTA in PBS at a density of 1×109/L. Confluent monolayers of HUVECs were added to monocytes with different treatments and allowed to incubate for 1 h at 37℃in a tissue culture incubator, and the adherent rate was determined.
Results:Monocytes were identified by their characteristic morphology in microscope using Wright-Gimsa staining. The purity of monocyte preparations was about 90% as determined by flow cytometric analysis. HUVECs were identified to be more than 99% endothelial cells by their characteristic cobblestone morphology in an inverted microscope and by immunocytochemical demonstration of factor VIII staining.
①The protein level of membrane CD 16 on monocytes with stimulation by recombinant human M-CSF for 18 h was higher than that without stimulation (P<0.01). After IVIG pretreatment for 30 min, the protein level of membrane CD 16 on monocytes was decreased compared to that on monocytes with M-CSF stimulation for 18 h (P<0.01).
②The adhesive efficiency of monocytes to HUVECs after recombinant human M-CSF stimulation for 18 h was higher than that without M-CSF stimulation (P<0.05). The adhesive efficiency of monocytes to HUVECs after IVIG pretreatment for 30 min decreased obviously compared to that of monocytes with M-CSF stimulation for 18 h (P <0.05).
Conclusion:
①The protein level of membrane CD 16 on monocytes is induced by recombinant human M-CSF in vitro, and IVIG may inhibit this effect.
②The elevated level of FcyRIIIA on monocytes dramatically correlate to the adhesive efficiency to HUVECs in vitro.
Study V:Effects of FcyRIIIA on aortic atherosclerotic plaque destabilization in ApoE-/- mice
Objective:To investigate the effects of FcyRIIIA on aortic atherosclerotic plaque destabilization in ApoE-/- mice.
Methods:Sixty six 8-week-old male ApoE-/- mice were randomly divided into the model group, ApoE-/- mice+intraperitoneal injection immunoglobulin group and ApoE-/- mice+Simvastatin group, twenty two mice in each group. Twenty two 8-week-old male C57BL/6J mice were selected as the control group. Mice in the control group were put on a normal diet, and others fed with a high-fat diet for 10 weeks.
①Deteciton of aortic histopathology. Paraffins slides of aortic root tissue which were found atherosclerosis changes were dyed with Masson to evaluate plaques collagen content and immunohistochemical staining a-actin and CD68 were evaluated contents of plaques vascular smooth muscle cells (VSMCs) and macrophages, respectively. Frozen slides of aortic root tissue was dyed with oil red O to evaluate the content of plaques adipose tissue.
②Deteciton of hematologic indices. Levels of TC, TG, LDL-C and HDL-C were determined using standard enzymatic methods and atherogenic index of plasma (AIP) was calculated. The protein level of membrane CD 16 on monocytes in ApoE-/- mice was analyzed by flow cytometry and levels of TNF-a, IL-1 and sE-selectin in sera were examined by ELISA.
③Detection of mRNA expression and the protein level of MMP-9 in aorta:mRNA expression and the protein level of MMP-9 in aorta was determined by Real-time RT-PCR and Western blot, respectively.
④Correlation analysis:correlation of the protein level of membrane CD16 on monocytes and mRNA expression of MMP-9 in aorta or serum level of TNF-a in ApoE-/- mice with a high-fat diet for 10 w, IVIG pretreatment or Simvastatin treatment was analyzed.
Results:
①Aortic atherosclerotic plaque in ApoE-/- mice contains a lot of lipid, macrophages and VSMCs, whereas collagen composition was reduced. Similar to Simvastatin, IVIG may ameliorate athersoclerosis by decreasing lipid level and macrophages contents and inhibiting VSMCs proliferation.
②High level of TC, TG and LDL-C and increased protein level of membrane CD 16 on monocytes were found in ApoE-/- mice, along with increased levels of TNF-a, IL-1 and sE-selectin in sera. Additionally, similar to Simvastatin, IVIG pretreatment inhibited this response in ApoE-/- mice.
③Increased mRNA expression and the protein level of MMP-9 in aotra were found in ApoE-/- mice compared with C57 mice (P<0.01). Additionally, similar to Simvastatin, IVIG pretreatment inhibited mRNA expression and protein level of MMP-9 in ApoE-/-mice.
④Correlation analyses showed that the protein level of membrane CD 16 on monocytes was positively correlated to mRNA expression of MMP-9 in aotra and serum level of TNF-a among ApoE-/- mice with a high-fat diet for 10 w, IVIG pretreatment, or Simvastatin treatment.
Conclusion:
①cyRIIIA may contribute to the release of inflammatory factors such as TNF-a, IL-1 and sE-selectin in ApoE-/- mice with aortic atherosclerotic plaque.
②FcyRIIIA may be involved in the development of aortic atherosclerotic plaque destabilization.
Study VI:Effects of active ingredients of Chuanxiong rhizome and red peony root on aortic atherosclerotic plaque
Objective:To investigate the effects of active ingredients of Chuanxiong rhizome and red peony root (Chuangxiongol and paeoniflorin) on aortic atherosclerotic plaque and possible mechanisms.
Methods:Sixty four 8-week-old male ApoE-/- mice were randomly divided into the model group, ApoE-/- mice+Simvastatin group, ApoE-/- mice+high dosage of Xiongshao Capsule (XSC) group (XSCH) and ApoE-/- mice+low dosage of XSC group (XSCL), sixteen mice in each group. Methods of detection of hematologic indices, mRNA expression and the protein level of MMP-9 in aorta and correlation analysis in this study were listed in the Study VI.
Results:
①After XSC treatment for 10 weeks, levels of TC, TG, LDL-C and AIP were decreased compared with ApoE-/- mice, along with the decreased levels of both protein level of membrane CD 16 on monocytes and inflammatory factors such as TNF-a, IL-1 and sE-selectin in sera. Similar to Simvastatin, XSCH inhibited this inflammation response in ApoE-/- mice.
②Similar to Simvastatin, XSC inhibited mRNA expression and the protein level of MMP-9 in ApoE-/- mice aotra. However, the stabilized effect of XSCH on atherosclerotic plaques was stronger than that in ApoE-/- mice with Simvastatin treatment (P<0.05).
lation analyses showed that decreased MMP-9 mRNA expression in aotra and serum TNF-a were positively correlated to the low protein level of membrane CD 16 on monocytes in ApoE-/- mice with XSC treatment.
Conclusion:
①Active ingredients of Chuanxiong rhizome and red peony root may alleviate inflammatory reaction, stabilize atherosclerotic plaque and improve atheroslcerotic formation.
②The role of active ingredients of Chuanxiong rhizome and red peony root in preventing atherosclerosis is related to FcyRIIIA.
本课题对另外两个目标分子PKCβ1、FcγRⅢA进行了鉴定、验证,并对FcyRIIIA在冠心病血瘀证形成中的作用进行分析。纳入冠心病血瘀证患者、冠心病非血瘀证患者和健康对照者,抽取外周血,分离白细胞,抽提总RNA,应用实时定量逆转录聚合酶链反应(Real-time reverse transcription polymerase chain reaction, Real-time RT-PCR)技术检测目标分子PKCβ1、FcγRⅢA基因表达水平,结果发现FcγRⅢA mRNA表达水平与前期基因芯片结果一致。我们进一步从临床血清学水平进行了规模验证,并就其细胞来源进行了相关分析；同时根据FcγRⅢA的功能,结合冠心病、血瘀证病理特点,体外通过构建人外周血单核细胞-脐静脉内皮细胞实验体系,观察FcγRⅢA对单核-内皮细胞粘附的影响；体内以ApoE-/-小鼠为研究对象,观察FcγRⅢA高表达及抑制其表达后对小鼠主动脉粥样硬化斑块稳定性及炎症相关因子的影响,以期探索目标分子FcγRⅢA参与冠心病血瘀证疾病过程的可能机制；同时,观察赤芍川芎有效部位对ApoE-/-小鼠FcγRⅢA蛋白水平、主动脉粥样硬化斑块稳定性以及炎症相关因子的影响,明确活血化瘀制剂对冠心病血瘀证靶标分子的干预效应。
本课题研究分为两部分：文献综述和实验研究。
1文献综述：本部分分别从血瘀证与炎症反应、FcγR与心血管疾病研究进展两个方面进行综述。
2实验研究：包括以下六个部分。
研究一冠心病血瘀证目标分子鉴定
目的：应用Real-time RT-PCR技术,对冠心病血瘀证目标分子PKCβ1、FcyRIIIA进行鉴定。
方法：冠心病诊断参照1999年美国心脏病学会(ACC)／美国心脏协会(AHA)／美国医师学会及美国内科学会(ACP-ASIM)联合协定关于《慢性稳定性心绞痛诊疗指南》、《不稳定性心绞痛、无ST段抬高心肌梗死诊疗指南》。同时,参考中国中西医结合学会活血化瘀专业委员会制定的血瘀证诊断标准。筛选符合冠心病、血瘀证、健康人入选标准的冠心病血瘀证组、冠心病非血瘀证组和健康对照组各20例为研究对象。冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血2 mL,分离白细胞,抽提总RNA,应用Real-time RT-PCR技术检测目标分子PKCβ1和FcγRⅢA基因表达水平。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数、病程、合并疾病和用药情况方面无显著性差异,具有可比性。
①PKCβ1 mRNA表达在冠心病血瘀证组(0.74±0.30)和非血瘀证组(0.96±0.31)均较健康对照组(1.03±0.37)降低,且冠心病血瘀证组较非血瘀证组降低显著(P<0.05)。
②FcγRⅢA mRNA表达在冠心病血瘀证组(1.37±0.50)和非血瘀证组(1.37±0.42)均较健康对照组(0.89±0.33)显著升高(P<0.01),且冠心病血瘀证组较非血瘀证有升高趋势。
结论：
①PKCβ1基因参与冠心病血瘀证过程,但与前期研究不一致,尚需进一步扩大样本量进行鉴定。
②FcγRⅢA与冠心病血瘀证具有一定相关性,可能在冠心病血瘀证形成过程中发挥重要作用。
研究二FcγRⅢA血清蛋白水平以及冠心病血瘀证与动脉粥样硬化危险因素的相关性研究
目的：应用酶联免疫吸附(ELISA)法检测FcγRⅢA血清水平(可溶性CD16,sCD16),验证其与冠心病血瘀证的临床相关性,并分析冠心病血瘀证与动脉粥样硬化危险因素如血脂、高敏C反应蛋白(hs-CPR)以及单核细胞参数的相关性。
方法：纳入冠心病血瘀证、冠心病非血瘀证患者各50例和健康对照组40例为研究对象。冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血5 mL,采用双抗体夹心ABC-ELISA法检测血清sCD16浓度,同时对动脉粥样硬化危险因素(血脂、hs-CPR以及单核细胞计数和单核细胞百分比)进行检测。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数、病程和合并疾病方面无显著性差异,具有可比性。
①血清sCD16在冠心病血瘀证组(3.67±0.98 U/mL)和非血瘀证组(2.80±0.80U/mL)均较健康对照组(1.58±0.47 U/mL)显著升高(P<0.01),且冠心病血瘀证组较非血瘀证组升高显著(P<0.05)。
②总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)在冠心病血瘀证组和非血瘀证组均较健康对照组显著升高(P<0.05或0.01),且冠心病血瘀证组较非血瘀证组有升高趋势；而高密度脂蛋白胆固醇(HDL-C)在冠心病血瘀证组和非血瘀证组较健康对照组显著降低(P<0.01),且冠心病血瘀证组较非血瘀证有降低趋势。
③hs-CRP水平在冠心病血瘀证组(2.91±1.98 mg/L)和非血瘀证组(1.43±1.29mg/L)均较健康对照组(0.56±0.09 mg/L)显著升高(P<0.01,0.05),且冠心病血瘀证组较非血瘀证组升高显著(P<0.05)。
④外周血单核细胞计数在冠心病血瘀证组、非血瘀证组和健康对照组之间差异无统计学意义,而单核细胞百分比在冠心病血瘀证组(6.40±1.17%)和非血瘀证组(5.35±1.62%)均较健康对照组(4.99±1.81%)升高,且冠心病血瘀证组较非血瘀证组升高显著(P<0.05)。
结论：
①血清sCD16与冠心病血瘀证具有相关性,从蛋白水平验证了目标分子FcγRⅢA与冠心病血瘀证密切相关。
②冠心病血瘀证患者与动脉粥样硬化危险因素,特别是hs-CRP和单核细胞百分比显著相关。
研究三FcyRIIIA来源分析
目的：应用流式细胞术检测冠心病血瘀证患者外周血单核细胞CD14+CD16+表达以分析目标分子FcyRIIIA来源,并分析细胞间粘附分子(ICAM-1,CD54)在冠心病血瘀证患者单核细胞CD14+CD16+表面的变化,同时检测冠心病血瘀证患者炎症相关因子[可溶性CD14(sCD14)、巨噬细胞集落刺激因子(M-CSF)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1(IL-1)]血清水平。
方法：冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血,采用流式细胞术检测外周血单核细胞CD14+CD16+表达及其表面CD54平均荧光强度；采用双抗体夹心ABC-ELISA法检测sCD14、M-CSF、TNF-α和IL-1血清水平。
结果：
①外周血单核细胞CD14+CD16+表达在冠心病血瘀证组(11.24±7.73%)和非血瘀证组(9.96±3.66%)均较健康对照组(5.64±2.49%)显著升高(P<0.01,0.05),且冠心病血瘀证组较非血瘀证组有升高趋势。
②外周血单核细胞CD14+CD16+表面CD54的平均荧光强度在冠心病血瘀证组(1760.60±457.60)和非血瘀证组(1089.27±426.76)均较健康对照组(849.00±100.91)显著升高,且冠心病血瘀证组较非血瘀证组升高显著(P<0.01)。
③炎症相关因子sCD14、M-CSF、TNF-α和IL-1血清水平在冠心病血瘀证组和非血瘀证组均较健康对照组显著升高(P<0.01),且冠心病血瘀证组较非血瘀证组升高显著(P<0.05)。
结论：
①冠心病血瘀证患者单核细胞处于活化状态。
②冠心病血瘀证患者高水平血清M-CSF能刺激单核细胞CD14+CD16+高表达,进而促进单核细胞CD14+CD16+表面CD54表达升高并产生大量TNF-α和IL-1前炎症因子,从细胞来源方面再次鉴定了目标分子FcyRIIIA与冠心病血瘀证密切相关。
③冠心病血瘀证患者体内炎症水平特别是单核细胞来源的细胞因子处于较高水平。
研究四FcyRIIIA在人外周血单核细胞-脐静脉内皮细胞粘附中的作用分析
目的：研究FcyRIIIA对人单核细胞-脐静脉内皮细胞粘附的影响,对其进行体外功能分析。
方法：①分离、鉴定并培养人外周血单核细胞,实验分为单核细胞(MC)组：单核细胞培养18 h;M-CSF组：重组人M-CSF刺激单核细胞培养18 h;免疫球蛋白IgG (IVIG)组：IVIG提前干预单核细胞30 min后再加重组人M-CSF刺激培养18 h,流式细胞术检测三组单核细胞CD14+CD16+表达情况。②分离、.鉴定并培养人外周血单核细胞、脐静脉内皮细胞,实验分为MC组：单核细胞培养18 h;M-CSF组：重组人M-CSF刺激单核细胞培养18h; IVIG组：IVIG提前干预单核细胞30min后再加重组人M-CSF刺激培养18 h。三组细胞消化后再与脐静脉内皮细胞培养1 h,Bradford法检测三组单核-内皮细胞粘附率。
结果：瑞姬染色显微镜下细胞形态学观察和流式细胞术单核细胞CD14鉴定人外周血单核细胞,倒置相差显微镜细胞形态学观察和Ⅷ因子相关抗原间接免疫荧光方法鉴定人脐静脉内皮细胞。
①经重组人M-CSF刺激培养18 h后单核细胞CD14+CD16+表达较未刺激前显著升高(P<0.01)；提前给予IVIG干预30 min后,单核细胞CD14+CD16+表达较重组人M-CSF刺激培养18 h显著降低(P<0.01)。
②经重组人M-CSF刺激培养18 h后单核-内皮细胞粘附率较单核细胞未刺激前显著升高(P<0.05)；提前给予IVIG干预30 min后,单核-内皮细胞粘附率较重组人M-CSF刺激培养18 h显著降低(P<0.05)。
结论：
①重组人M-CSF在体外能刺激人外周血单核细胞CD14+CD16+表达,IVIG能抑制该作用。
②单核细胞CD14+CD16+能介导人外周血单核-脐静脉内皮细胞粘附。
研究五FcyRIIIA对ApoE-/-小鼠主动脉粥样硬化斑块不稳定性的作用评价
目的：研究目标分子FcyRIIIA高表达和抑制其后对ApoE7-/-小鼠主动脉粥样硬化斑块不稳定性的影响。
方法：66只8周龄雄性ApoE-/-小鼠分为模型组：高脂饲料喂养10 w,ApoE-/-小鼠+腹腔注射免疫球蛋白IgG组(IVIG组)：连续腹腔注射IVIG 5 d+高脂饲料喂养10 w, ApoE-/-小鼠+辛伐他汀组(Sm组)：Sm灌胃每日一次+高脂饲料喂养10 w,每组各22只。与ApoE-/-小鼠具有相同遗传背景的C57BL/6J小鼠22只作为对照组,普通饲料喂养10 w。
①主动脉组织病理学观察：主动脉根部组织石蜡切片H&E染色发现有动脉粥样硬化改变的切片进行Masson染色评价斑块胶原含量,a-actin和CD68免疫组化染色分别评价斑块平滑肌细胞含量和巨噬细胞含量,主动脉根部组织冰冻切片油红O染色评价斑块脂肪组织含量。
②血液学指标检测：生化法检测血脂并计算血浆致动脉粥样硬化指数；流式细胞术检测外周血单核细胞CD14+CD16+表达；双抗体夹心ABC-ELISA法检测血清TNF-α、IL-1和sE-selectin水平。
③MMP-9基因表达和蛋白水平检测：Real-time RT-PCR技术和Western blot法分别检测主动脉MMP-9 mRNA表达和蛋白水平。
④相关性分析：外周血单核细胞CD14+CD16+表达、主动脉MMP-9基因表达和血清TNF-a水平在模型组、IVIG组和Sm组的相关性分析。
结果：
①模型组小鼠主动脉粥样硬化斑块含有大量脂质成分、巨噬细胞和平滑肌细胞,且胶原成分增多。IVIG能通过减少斑块内脂质含量、减少巨噬细胞聚集和平滑肌细胞增殖而发挥改善动脉粥样硬化的作用,该作用与Sm相似。
②模型组小鼠血脂TC、TG、LDL-C水平升高,HDL-C水平降低,外周血单核细胞CD14+CD16+高表达,血清TNF-α、IL-1和sE-selectin水平较对照组显著升高(P<0.01,0.05)；上述指标在IVIG组有不同程度改善且作用与Sm组相似。
③模型组小鼠主动脉MMP-9基因表达和蛋白水平均较对照组显著升高(P<0.01),上述指标在IVIG组有不同程度改善且作用与Sm组相似。
④相关性分析发现,外周血单核细胞CD14+CD16+表达在模型组、IVIG组和Sm组均与主动脉MMP-9基因表达和血清TNF-a水平呈显著正相关。
结论：
①FcyRIIIA介导主动脉粥样硬化模型小鼠炎症相关因子释放增加。
②FcyRIIIA一定程度上影响小鼠主动脉粥样硬化斑块的稳定性。
研究六赤芍川芎有效部位对ApoE-/-小鼠主动脉粥样硬化斑块稳定性的干预效应研究
目的：研究赤芍川芎有效部位(赤芍总苷、川芎总酚)对ApoE-/-小鼠主动脉粥样硬化斑块稳定性的干预效应,并分析其可能的作用靶点。
方法：64只8周龄雄性ApoE-/-小鼠分为模型组：高脂饲料喂养10w, ApoE-/-小鼠+Sm组(Sm组)：Sm灌胃每日一次+高脂饲料喂养10w, ApoE-/-小鼠+芎芍胶囊(XSC)大剂量组(XSCH组)：大剂量XSC灌胃每日一次+高脂饲料喂养10 w和ApoE-/-小鼠+XSC小剂量(XSCL)组：小剂量XSC灌胃每日一次+高脂饲料喂养10 w,每组各16只。各组进行血液学指标、MMP-9基因表达和蛋白水平比较以及相关性分析。
结果：
①XSC对TC、TG、LDL-C水平和AIP均有不同程度降低,同时能降低外周血单核细胞CD14+CD16+表达,不同程度降低血清TNF-α、IL-1和sE-selectin水平,对ApoE-/-小鼠体内炎症水平的改善作用XSCH与Sm相似。
②XSC与Sm类似均能降低ApoE-/-小鼠主动脉MMP-9基因表达和蛋白水平,对动脉粥样硬化斑块具有稳定作用,但对MMP-9基因表达的降低作用XSCH较Sm显著(P<0.05)。
③相关性分析发现,经XSC干预后,主动脉MMP-9基因表达和血清TNF-α水平下降均与外周血单核细胞CD14+CD16+表达降低呈显著正相关。
结论：
①赤芍川芎有效部位能减轻炎症反应、稳定斑块、改善主动脉粥样硬化。
②赤芍川芎有效部位抗主动脉动脉粥样硬化的作用靶点与FcyRⅢA有关。
Coronary heart disease (CHD) is the leading cause of death worldwide. Genetic factors and environmental factors are responsible for the development of CHD. The underlying mechanisms of CHD have not yet been elucidated. Additionally, there is little known about the way of CHD-specific prevention and treatment therapy. Previous studies have investigated the differential gene expression profiles in peripheral leukocytes from CHD patients with blood stasis syndrome (BSS) by oligonucleotide microarray technique. We have shown that the immune inflammatory response is correlated to the development of CHD and Fc receptor III A of immunoglobulin G (FcyRIIIA, also named CD 16), protein kinase C beta 1 (PKCβ1), interleukin-8 (IL-8), HLA-DQB1, FOLR3, PTGDS were involved in the development of CHD with BSS. Of them, FcyRIIIA, PKCβ1 and IL-8 played the key role in the regulation of CHD with BSS. In our previous studies, we have shown that IL-8 was implicated in the development of CHD with BSS and its functional analysis was determined in vitro and vivo.
In the current study, we investigated the mRNA expression of PKCβ1 and FcyRIIIA in the leukocytes in CHD patients with BSS, CHD patients with non-BSS and the healthy control by Real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). There was a significant increase of FcyRIIIA at the mRNA level in leukocytes in CHD patients with BSS, consistent with our previous study. Then, we determined the protein level of both sCD16 in sera and membrane CD 16 on monocytes in CHD patients with BSS. Moreover, we investigated FcyRIIIA-induced molecular events that may be responsible for the development of CHD using human peripheral monocyte-umbilical vein endothelial cells in vitro and aortic atherosclerotic formation in ApoE-/- mouse model. Meanwhile, we determined the intervention effects of active ingredients of Chuanxiong rhizome and red peony root (Chuangxiongol and paeoniflorin) on FcyRIIIA expression, aortic atherosclerotic plaque destabilization and inflammatory factors in ApoE-/- mice.
This study is divided into two parts:literature review and experimental research.
1 Literature review:Including the following two reviews:BSS and inflammation and Investigation of correlation between FcyR and cardiovascular diseases.
2 Experimental research:Including the following six parts. Study I:Identification of related molecules in CHD patients with BSS
Objective:To identify the related molecules PKCβ1 and FcyRIIIA in CHD patients with BSS by Real-time RT-PCR.
Methods:All cases were divided into CHD patients with BSS, CHD patients with non-BSS and the healthy control,20 in each group. CHD patients were diagnosed according to the 1999 ACC/AHA/ACP-ASIM Guidelines for the Management of Patients with Chronic Stable Angina and the 2002 ACC/AHA Guidelines for the Management of Patients with Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. A diameter stenosis of at least 50% was diagnosed by visible estimation in a major coronary artery from standard selective coronary angiography. Patients with BSS were diagnosed according to guidelines for the diagnosis of BSS. mRNA expression of PKCβ1 and FcyRIIIA in the leukocytes was determined in blood samples taken following fasting from CHD patients with BSS, CHD patients with non-BSS and the healthy control by Real-time RT-PCR.
Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels, course of disease, medical history and herbal therapy beween CHD patients with BSS and CHD patients with non-BSS.
①There was a significant decrease of PKCβ1 at the mRNA level in leukocytes in both CHD patients with BSS (0.74±0.30) and CHD patients with non-BSS (0.96±0.31) when compared to the healthy control (1.03±0.37). Additionally, mRNA expression of PKCβ1 in CHD patients with BSS was lower than that in CHD patients with non-BSS (P<0.05)
②There was a significant increase of FcyRIIIA at the mRNA level in leukocytes in both CHD patients with BSS (1.37±0.50) and CHD patients with non-BSS (1.37±0.42) when compared to the healthy control (0.89±0.33, P<0.05). Additionally, mRNA expression of FcyRIIIA in CHD patients with BSS was higher than that in CHD patients with non-BSS without significant differences.
Conclusion:
①PKCβ1 may participate in the development of CHD with BSS. However, results in the current study was in contradiction with our previous study of gene chip. Identification of PKCβ1 in CHD patients with BSS should be analyzed further.
②There is a certain correlation between FcyRIIIA and CHD with BSS. It may be involved in the development of CHD with BSS.
Study II:Investigation of FcyRIIIA at the protein level and the correlation of CHD patients with BSS and risk factors of atherosclerosis
Objective:To determine the protein level of FcyRIIIA (soluble CD 16, sCD16) and analyze the correlation of CHD patients with BSS and risk factors of atherosclerosis such as blood lipid level, high-sensitivity C-reactive protein (hs-CRP) and parameters of monocytes.
Methods:All cases were divided into CHD patients with BSS and CHD patients with non-BSS, fifty in each group and forty healthy individuals were selected as the healthy control. The protein level of sCD16 in sera was determined in blood samples taken following fasting from CHD patients with BSS, CHD patients with non-BSS and the healthy control by Enzyme-linked immunosorbent assay (ELISA). Then, levels of blood lipid and hs-CRP, and parameters of monocytes were analyzed as well.
Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels, course of disease and medical history beween CHD patients with BSS and CHD patients with non-BSS.
①There was a significant increase of the protein level of serum sCD16 in both CHD patients with BSS (3.67±0.98 U/mL) and CHD patients with non-BSS (2.80±0.80 U/mL) when compared to the healthy control (1.58±0.47 U/mL). Additionally, serum level of sCD16 in CHD patients with BSS was higher than that in CHD patients with non-BSS (P<0.05)
②There was a significant increase of blood lipid level such as total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in both CHD patients with BSS and CHD patients with non-BSS when compared to the healthy control (P< 0.01,0.05). However, levels of TC, TG, and LDL-C were increased in CHD patients with BSS, but not significantly, compared with CHD patients with non-BSS. There was a significant decrease of high-density lipoprotein cholesterol (HDL-C) level in both CHD patients with BSS and CHD patients with non-BSS when compared to the healthy control (P<0.01). Additionally, HDL-C level was reduced in CHD patients with BSS, but not significantly, compared with CHD patients with non-BSS.
③There was a significant increase of hs-CRP level in both CHD patients with BSS (2.91±1.98 mg/L) and CHD patients with non-BSS (1.43±1.29 mg/L) when compared to the healthy control (0.56±0.09 mg/L) (P<0.01,0.05). Additionally, hs-CRP level in CHD patients with BSS was higher than that in CHD patients with non-BSS (P< 0.05).④There were no statistically significant differences of monocyte count in peripheral blood among CHD patients with BSS, CHD patients with non-BSS and the healthy control. There was an increase of percentage of monocytes in both CHD patients with BSS (6.40±1.17%) and CHD patients with non-BSS (5.35±1.62%) when compared to the healthy control (4.99±1.81%). Additionally, percentage of monocytes in CHD patients with BSS was higher than that in CHD patients with non-BSS (P<0.05).
Conclusion:
①High level of sCD16 in CHD patients with BSS suggests that there is relevance of FcyRⅢA to CHD patients with BSS at the protein level.
②There was is relevance of risk facotrs of atherosclerosis, especially hs-CPR and percentage of monocytes to CHD patients with BSS.
StudyⅢ:Original analysis of FcγRⅢA:the protein level of membrane CD16 on monocytes
Objective:To investigate the protein level of membrane CD 16 on monocytes in CHD patients with BSS by flow cytometric analysis, and then, expression of intercellular adhesion molecule-1 (ICAM-1, also named CD54) on CD14+CD16+monocytes was determined. Meanwhile, levels of inflammatory factors such as soluble CD 14 (sCD14), macrophage colony stimulating factor (M-CSF), tumor necrosis factor-a (TNF-a) and interleukin-1 (IL-1) in sera were analyzed.
Methods:The protein level of membrane CD 16 on monocytes was determined in blood samples taken following fasting from CHD patients with BSS, CHD patients with non-BSS and the healthy control by flow cytometry. Mean fluorescence intensity (MFI) of ICAM-1 on CD14+CD16+monocytes was analyzed as well. Levels of sCD14, M-CSF, TNF-a and IL-1 in sera were examined by ELISA.
Results:
①There was a significant increase of the protein level of membrane CD 16 on monocytes in both CHD patients with BSS (11.24±7.73%) and CHD patients with non-BSS (9.96±3.66%) when compared to the healthy control (5.64±2.49%) (P< 0.01,0.05). Additionally, this protein level of membrane CD16 on monocytes in CHD patients with BSS was increased, but not significantly, compared with CHD patients with non-BSS.
②There was a significant increase of MFI of CD54 on CD14+CD16+monocytes in both CHD patients with BSS (1760.60±457.60, P<0.01) and CHD patients with non-BSS (1089.27±426.76) when compared to the healthy control (849.00±100.91). Additionally, MFI of CD54 on CD14+CD16+monocytes in CHD patients with BSS was higher than that in CHD patients with non-BSS (P<0.01).
③There was a significant increase of serum levels of inflammatory factors such as sCD14, M-CSF, TNF-a and IL-1 in both CHD patients with BSS and CHD patients with non-BSS when compared to the healthy control. Additionally, levels of all these inflammatory factors in CHD patients with BSS were higher than those in CHD patients with non-BSS (P<0.05).
Conclusion:
①Activated monocytes are found in CHD patients with BSS.
②CD14+CD16+monocytes was stimulated by high serum M-CSF level in CHD patients with BSS, along with increased level of CD54 on CD14+CD16+monocytes and TNF-a and IL-1 in sera. Taken together, these results suggest that FcyRIIIA is correlated to CHD patients with BSS.
③Level of inflammatory factors, especially cytokines from monocytes increase in CHD patients with BSS.
StudyⅣ:Effects of FcyRIIIA on the adherence of monocytes from human peripheral blood to human umbilical vein endothelial cells
Objective:To investigate the effects of FcyRIIIA on the adherence of monocytes from human peripheral blood to human umbilical vein endothelial cells (HUVECs).
Methods:First, we overexpressed and inhibited FcyRIIIA on monocytes by recombinant human M-CSF stimulation for 18 h or IVIG pretreatment for 1 h respectively, according to procedures as follows:monocytes randomly divided into three groups. MC group:monocytes cultured for 18 h; M-CSF group:monocytes stimulated by recombinant human M-CSF (10 ng/mL) for 18 h; IVIG group:monocytes pretreated with IVIG (5 mg/mL) for 30 min, then treated with recombinant human M-CSF (10 ng/mL) for 18 h. Monocytes in these three groups were harvested 18 h later, and the surface expression of CD 14 and CD 16 on monocytes was determined by flow cytometry. Furthermore, the adhesive efficiency of monocytes to HUVECs was determined by Bradford method. Briefly, monocytes were treated with recombinant human M-CSF stimulation for 18 h or with IVIG pretreatment for 1 h and then recombinant human M-CSF stimulation for 18 h or without any intervention. Monocytes were released from the culture dish with 3% EDTA in PBS at a density of 1×109/L. Confluent monolayers of HUVECs were added to monocytes with different treatments and allowed to incubate for 1 h at 37℃in a tissue culture incubator, and the adherent rate was determined.
Results:Monocytes were identified by their characteristic morphology in microscope using Wright-Gimsa staining. The purity of monocyte preparations was about 90% as determined by flow cytometric analysis. HUVECs were identified to be more than 99% endothelial cells by their characteristic cobblestone morphology in an inverted microscope and by immunocytochemical demonstration of factor VIII staining.
①The protein level of membrane CD 16 on monocytes with stimulation by recombinant human M-CSF for 18 h was higher than that without stimulation (P<0.01). After IVIG pretreatment for 30 min, the protein level of membrane CD 16 on monocytes was decreased compared to that on monocytes with M-CSF stimulation for 18 h (P<0.01).
②The adhesive efficiency of monocytes to HUVECs after recombinant human M-CSF stimulation for 18 h was higher than that without M-CSF stimulation (P<0.05). The adhesive efficiency of monocytes to HUVECs after IVIG pretreatment for 30 min decreased obviously compared to that of monocytes with M-CSF stimulation for 18 h (P <0.05).
Conclusion:
①The protein level of membrane CD 16 on monocytes is induced by recombinant human M-CSF in vitro, and IVIG may inhibit this effect.
②The elevated level of FcyRIIIA on monocytes dramatically correlate to the adhesive efficiency to HUVECs in vitro.
Study V:Effects of FcyRIIIA on aortic atherosclerotic plaque destabilization in ApoE-/- mice
Objective:To investigate the effects of FcyRIIIA on aortic atherosclerotic plaque destabilization in ApoE-/- mice.
Methods:Sixty six 8-week-old male ApoE-/- mice were randomly divided into the model group, ApoE-/- mice+intraperitoneal injection immunoglobulin group and ApoE-/- mice+Simvastatin group, twenty two mice in each group. Twenty two 8-week-old male C57BL/6J mice were selected as the control group. Mice in the control group were put on a normal diet, and others fed with a high-fat diet for 10 weeks.
①Deteciton of aortic histopathology. Paraffins slides of aortic root tissue which were found atherosclerosis changes were dyed with Masson to evaluate plaques collagen content and immunohistochemical staining a-actin and CD68 were evaluated contents of plaques vascular smooth muscle cells (VSMCs) and macrophages, respectively. Frozen slides of aortic root tissue was dyed with oil red O to evaluate the content of plaques adipose tissue.
②Deteciton of hematologic indices. Levels of TC, TG, LDL-C and HDL-C were determined using standard enzymatic methods and atherogenic index of plasma (AIP) was calculated. The protein level of membrane CD 16 on monocytes in ApoE-/- mice was analyzed by flow cytometry and levels of TNF-a, IL-1 and sE-selectin in sera were examined by ELISA.
③Detection of mRNA expression and the protein level of MMP-9 in aorta:mRNA expression and the protein level of MMP-9 in aorta was determined by Real-time RT-PCR and Western blot, respectively.
④Correlation analysis:correlation of the protein level of membrane CD16 on monocytes and mRNA expression of MMP-9 in aorta or serum level of TNF-a in ApoE-/- mice with a high-fat diet for 10 w, IVIG pretreatment or Simvastatin treatment was analyzed.
Results:
①Aortic atherosclerotic plaque in ApoE-/- mice contains a lot of lipid, macrophages and VSMCs, whereas collagen composition was reduced. Similar to Simvastatin, IVIG may ameliorate athersoclerosis by decreasing lipid level and macrophages contents and inhibiting VSMCs proliferation.
②High level of TC, TG and LDL-C and increased protein level of membrane CD 16 on monocytes were found in ApoE-/- mice, along with increased levels of TNF-a, IL-1 and sE-selectin in sera. Additionally, similar to Simvastatin, IVIG pretreatment inhibited this response in ApoE-/- mice.
③Increased mRNA expression and the protein level of MMP-9 in aotra were found in ApoE-/- mice compared with C57 mice (P<0.01). Additionally, similar to Simvastatin, IVIG pretreatment inhibited mRNA expression and protein level of MMP-9 in ApoE-/-mice.
④Correlation analyses showed that the protein level of membrane CD 16 on monocytes was positively correlated to mRNA expression of MMP-9 in aotra and serum level of TNF-a among ApoE-/- mice with a high-fat diet for 10 w, IVIG pretreatment, or Simvastatin treatment.
Conclusion:
①cyRIIIA may contribute to the release of inflammatory factors such as TNF-a, IL-1 and sE-selectin in ApoE-/- mice with aortic atherosclerotic plaque.
②FcyRIIIA may be involved in the development of aortic atherosclerotic plaque destabilization.
Study VI:Effects of active ingredients of Chuanxiong rhizome and red peony root on aortic atherosclerotic plaque
Objective:To investigate the effects of active ingredients of Chuanxiong rhizome and red peony root (Chuangxiongol and paeoniflorin) on aortic atherosclerotic plaque and possible mechanisms.
Methods:Sixty four 8-week-old male ApoE-/- mice were randomly divided into the model group, ApoE-/- mice+Simvastatin group, ApoE-/- mice+high dosage of Xiongshao Capsule (XSC) group (XSCH) and ApoE-/- mice+low dosage of XSC group (XSCL), sixteen mice in each group. Methods of detection of hematologic indices, mRNA expression and the protein level of MMP-9 in aorta and correlation analysis in this study were listed in the Study VI.
Results:
①After XSC treatment for 10 weeks, levels of TC, TG, LDL-C and AIP were decreased compared with ApoE-/- mice, along with the decreased levels of both protein level of membrane CD 16 on monocytes and inflammatory factors such as TNF-a, IL-1 and sE-selectin in sera. Similar to Simvastatin, XSCH inhibited this inflammation response in ApoE-/- mice.
②Similar to Simvastatin, XSC inhibited mRNA expression and the protein level of MMP-9 in ApoE-/- mice aotra. However, the stabilized effect of XSCH on atherosclerotic plaques was stronger than that in ApoE-/- mice with Simvastatin treatment (P<0.05).
lation analyses showed that decreased MMP-9 mRNA expression in aotra and serum TNF-a were positively correlated to the low protein level of membrane CD 16 on monocytes in ApoE-/- mice with XSC treatment.
Conclusion:
①Active ingredients of Chuanxiong rhizome and red peony root may alleviate inflammatory reaction, stabilize atherosclerotic plaque and improve atheroslcerotic formation.
②The role of active ingredients of Chuanxiong rhizome and red peony root in preventing atherosclerosis is related to FcyRIIIA.
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