Zonulin在动脉粥样硬化发病机制中的作用研究与甘氨鹅脱氧胆酸细胞毒性的体外实验研究
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摘要
研究背景：
本论文包括两个部分的内容。
第一部分：zonulin在动脉粥样硬化发病机制中的作用研究
动脉粥样硬化的形成机制一直是心血管疾病研究的焦点之一。除了内皮损伤学说，泡沫细胞形成机制与平滑肌细胞增生理论等经典观点以外，越来越多的证据显示机体持续慢性系统炎症状态与动脉粥样硬化的形成密切相关，最近针对肠道菌群与冠心病发病之间联系的报道也为粥样硬化的形成机制提供了新的观点。肠道菌群通过何种机制参与动脉粥样硬化形成，其促进动脉粥样硬化形成依赖于何种条件，以及在与此相关的发病机制中是否存在可供干预的靶点成为最近粥样硬化机制研究的热点问题。
研究报道在粥样硬化斑块中检测到细菌DNA，其中部分为肠源性细菌产物。针对慢性牙周炎患者患冠心病的机制研究也提示反复菌血症能显著增加冠心病发病率。如能明确细菌是否参与无明显反复感染患者的动脉粥样硬化发病机制，并进一步了解细菌如何进入人体并参与动脉动脉粥样硬化发病机制，可为粥样硬化形成机制的细菌学说提供更多的证据。在此基础上寻找可干预细菌进入人体的关键环节，试通过对关键靶点进行干预减少细菌进入人体的机会，将为预防和治疗动脉粥样硬化提供新的思路。
Zonulin是肠道上皮细胞合成的一种功能蛋白，能够通过调节肠上皮细胞间紧密连接蛋白的结构而改变肠上皮间隙的通透性。研究发现克隆病患者中存在肠道zonulin水平升高与患冠心病几率增加的并存现象，如果zonulin水平升高可增加患冠心病的机会，那么zonulin很可能通过调节肠道上皮屏障的通透性而增加细菌进入人体的机会。为此，本研究用Western Blot方法研究动脉粥样硬化患者血清中Zonulin的表达水平改变，用基于细菌16s rDNA的高通量测序方法寻找无反复感染动脉粥样硬化患者血液中的细菌DNA产物，并试图在体外建立的肠上皮细胞屏障模型中研究部分细菌通过肠上皮屏障的机制。通过上述研究寻找肠道细菌与动脉粥样硬化形成的具体联系机制，以期为动脉粥样硬化的诊治提供新的思路。
第二部分：甘氨鹅脱氧胆酸细胞毒性的体外实验研究
妊娠肝内胆汁淤积综合征（intrahepatic cholestasis of pregnancy ICP）是一组妊娠合并症，发病率因地而异，在长江流域地区可高达4-6%，是该地区危害母婴健康的重要妊娠期疾病。胆酸对于母亲与胎儿的损伤成为ICP母婴损害的主要原因。机体内生理浓度范围内的胆酸对细胞及组织并无毒性作用，但是胆酸升高到一定浓度时，可以产生明显的细胞毒性。胆酸对于细胞的损伤主要以诱导细胞死亡为主，其中最常见的是细胞凋亡。近期研究发现胆酸还可以诱导细胞发生坏死，甚至可能是最近才报道的程序性坏死。研究胆酸对于循环及呼吸系统主要功能细胞的细胞毒性，有助于从细胞水平阐明胆酸对于循环及呼吸系统的功能损害及其分子机制，为揭示ICP相关的子代心肺损伤提供理论基础。
ICP患者子代并发症中，以早产、胎儿宫内窘迫以及新生儿肺损伤最为常见，危害也最大。早产是一系列新生儿并发症的重要原因，胎儿宫内窘迫则是造成子代神经功能损伤的主要原因之一。胆酸对于胎儿血管结构及功能的影响可能是ICP相关早产与宫内窘迫的发病机制。在胎儿血管结构中，与母儿循环中胆酸最直接接触的是血管内皮细胞。而胆酸对于人脐静脉内皮细胞（HUVEC）是否具有损伤作用目前尚未见报道。胆酸诱导的肺泡上皮细胞损伤主要与ICP相关的新生儿肺损伤有关：当母亲患有ICP时，孕妇血中胆酸水平明显增高，羊水及胎儿血中胆酸亦相应升高，胆酸长时间浸润并损伤肺组织，是ICP致新生儿肺损伤的关键因素。基于AECⅡ在肺部的重要作用，研究胆酸对AECⅡ的细胞毒性作用将可能为ICP新生儿肺损伤作出解释。
甘氨鹅脱氧胆酸（Glycochenodeoxycholate，GCDC）是胆酸的一种类型，被发现有强烈的诱导细胞死亡作用，可诱导肝细胞等一系列细胞死亡。但目前尚未见GCDC诱导HUVEC与AECⅡ死亡的报道。
研究目的：
1.检测动脉粥样硬化患者血清Zonulin的表达水平改变。
2.在动脉粥样硬化患者循环中寻找肠道细菌DNA产物并鉴定。
3.建立肠上皮细胞屏障体外模型并以此为基础研究细菌通过肠上皮屏障的机制。
4.明确GCDC能否诱导HUVEC与AECⅡ细胞死亡，及其诱导死亡的具体机制。
5.研究GCDC对HUVEC与AECⅡ结构与功能的影响。
材料方法：
1.采用Western Blot方法检测动脉粥样硬化患者血清中的Zonulin表达变化。
2.用基于ROCHE454平台的细菌16s rDNA的高通量测序方法寻找无反复感染动脉粥样硬化患者血液中的细菌DNA产物。
3.以Caco-2细胞培养结合Transwell方法建立肠上皮细胞屏障体外模型，用电镜及激光共聚焦等方法观察细菌通过该屏障的具体机制。
4.采用DAPI染色，流式细胞学annexin V/PI染色分析，活性caspase3酶免疫荧光染色等方法检测GCDC诱导HUVEC与AECⅡ细胞凋亡与坏死情况。
5.用Real-time PCR等法观察GCDC对HUVEC与AECⅡ结构与功能的影响。
结果：
1.动脉粥样硬化患者血清中存在Zonulin，且其表达水平显著高于对照组。Westernblot结果显示：zonulin为分子量约47KD的蛋白分子，血清中检测到与阳性对照肠上皮细胞中位置相同的条带，且分子量符合zonulin特征。动脉粥样硬化患者与正常对照相比较，血清zonulin的表达增高（n=8，P<0.05）。
2.无感染表现的动脉粥样硬化患者血液中检测到细菌16s rDNA产物。高通量测序结果显示多种肠道来源的细菌16s rDNA在无感染表现的动脉粥样硬化患者血液中检测出，其中以拉恩氏菌属、沙雷氏菌属与荧光假单胞菌属含量最高。
3.体外成功建立了肠上皮细胞屏障模型，微小肠道细菌可能主要从细胞间隙通过肠道上皮屏障。Caco-2细胞经培养21d后分化成熟，细胞间紧密连接形成，跨上皮细胞电阻值显著升高，形成细胞屏障。粘质沙雷氏菌可在不损害细胞的条件下通过肠上皮细胞屏障。
4.采用细胞原代培养方法，体外成功培养出HUVEC与新生大鼠AECⅡ细胞。并对所培养细胞进行了特异性鉴定。GCDC在体外能够诱导原代培养的新生鼠肺泡Ⅱ型上皮细胞（AECII）死亡，包括凋亡和坏死。GCDC诱导的AECII死亡具有浓度依赖性，线粒体形态与功能变化参与细胞死亡过程。
5. GCDC处理的HUVEC出现ET-1的水平变化，3d内以降低ET-1为主，长时间处理后ET-1的变化反而升高。GCDC可有效抑制中AECⅡ肺表面活性物质的分泌。GCDC处理后的AECⅡ出现肺表面活性物质含量的降低。
结论：
1.肠道部分微小体积菌群可能参与动脉粥样硬化的发病机制，其可能经肠上皮细胞间隙通过肠上皮细胞屏障。
2. Zonulin在动脉粥样硬化患者血清中表达升高，可能与其具有调节肠上皮细胞间紧密连接通透性功能有关。
3. ICP相关新生儿肺损伤的发病机制包括GCDC诱导AECⅡ发生凋亡与坏死，以及GCDC抑制AECⅡ肺表面活性物质分泌。
4. ICP相关的早产与胎儿宫内窘迫与GCDC调节HUVEC分泌ET-1相关。
Background:
Part1. Role of zonulin in the pathogenesis of atherosclerosis
Pathogenesis of atherosclerosis is a hot topic of cardiovascular disease. Chronicsystemic inflammation was regarded as an important cause of atherosclerosis. Recent studydiscover the relationship between gut microbiota and atherosclerosis and present new ideafor the pathogenesis of atherosclerosis. However, the following problems are waiting to besolved for further study on atherosclerosis formation: How does gut microbiota induceatherosclerosis? What are the necessary conditions for its effect? Is there any key moleculein the process which can be regulated.
Bacteria DNA was found in the plaque of athesclerosis, some of them are from the gut.Study on chronic periodontitis induced coronary artery disease (CAD) suggested recurrentbacteremia could increase the incidence of CAD. To investigate the role of bacteria inpathigenesis of atherosclerosis in non-infection patients, and understand how does gutmicrobiota enter human circulation, could present more evidence for the theory of bacteriainduced atherosclerosis. To find out the key step of gut microbiota translocation, and stopthe key point of bacteria entrance could be a possible way to prevent and treatatherosclerosis.
Zonulin was a protein produced and secreted by gut epithelium. Zonulin could regulategut permeability via change the structure of tight junction(TJ) between the gut epithelium.Zonulin was reported as an elevated protein with increased incidence of CAD in Crohnsdisease. If elevated zonulin can increase the incidence of CAD, the possible explainationwill be the function of increase the entrance of gut microbiota to the circulation.
So, western blot was used to detect the expression of serum zonulin in atherosclerosispatients. High-throughput sequencing with bacteria16s rDNA detection was applied to findevidence of bacteria in non-infected atherosclerosis patients. After that, gut epithelium barrier model was construced with Caco-2cell culture in vitro to study the mechanism ofbacteris entrance through gutepithelium. These studies focus on the role of zonulin andcould construct the relationship between gut microbiota and atherosclerosis.
Part2. Cell toxicity study of Glycochenodeoxycholate in vitro
Intrahepatic cholestasis of pregnancy is a constant pregnant complication in great areasincluding Yangzi river and around, with an incidence of4-6%. ICP is dangerous to mothersand babies. The pathogenesis of ICP is unclear, but the mothers all had risk a significanthigher serum bile acid level. The elevated bile acid could cause mother and infant injury.Since physiological concentration bile acid is nontoxical to human body, abnormal highlevel bile acid could induce cell toxicity. Bile acid could induce cell death and cause injuryto human body. Most common discovered cell death is cell apoptosis. However, recentstudy showed bile acid also could induce cell necrosis, or programmed cell necrosis, whichis named as necroptosis. To study the cell toxicity of bile acid to cells from vascule and thelung will be much helpful to discover the molecular mechanism of bile acid inducedvascular and lung injury, and give theoretical basis for ICP relevant fetal and newborninjury.
Bile acid induced cell death was confirmed in hepatocyte, myocardium, pancreatic celland tumor cells. The results were considered as an important pathogenesis of cholestasiscomplications. Preterm birth, fetal distress and newborn lung injury are the most constantcomplications of ICP, also the most dangerous types. Preterm birth could bring a lot ofsevere complications, fetal distress could cause future neurological development disable.There may be started from bile acid induced vascular injury. Vascular endothelium was thefirst barrier of the body from circulatory bile acid. However, there is no report about thepossible effect of bile acid to HUVEC. Bile acid induced alveolar epithelium death couldlead to newborn lung injury: when mothers got ICP, bile acid level were elevated both inmaternal serum and amniotic fluid. Fetal lung was infiltrated by amniotic fluid directly.Because AECⅡ is a multifunctional cell in the alveoli, the effect of bile acid toAECⅡ could possiblyexplain bile acid induced newborn lung injury.
The cell toxicity of elevated bile acid level in ICP mother could be the cause of fetaland newborn conlication. Discovering the mechanism of bile acid induced HUVEC andAECⅡ injury could be a complementationof bile acid cell toxicity, also be much helpful for theory base of ICP relevated fetal and newborncomplications. New diagnosis andtherapy strategy could be made with these results.
Glycochenodeoxycholate (GCDC) was a type of bile acid, with strong cell toxicity tohepatocyte and some other types of cells. However, the cell toxicity of GCDC to HUVECand AECⅡ was not reported.
Objects:
1. To discover the relationship between zonulin and atherosclerosis.
2. Find evidence of gut microbiota in the circulation of atherosclerosis patients.
3. Constrct epithelium barrier model in vitro to study the mechanism of bacteriatranslocation through gut gepithelium.
4. To detect GCDC toxicity newborn rat AECⅡ and the mechanism of GCDC inducedHUVEC and AECⅡ deathin vitro.
5. To study the following structure and function injury of GCDC to HUVEC.
Material and Methods:
1. Western blot was applied to detect the expression of zonulin in the serum ofatherosclerosis patients.
2. High-throughput sequencing with bacteria16s rDNA detection was applied to findevidence of bacteria in non-infected atherosclerosis patients.
3. Gut epithelium barrier model was construced with Caco-2cell culture in vitro tostudy the mechanism of bacteris entrance through gutepithelium.
4. DAPI staining, flow cytometry, caspase3activity detection, mitochondrialmorphology analysis were applied to detect cell apptosis and necrosis.
5. Real-time PCR was applied to detect endothelium-1level in HUVEC andpulmonary surfactant assay were applied to GCDC treated AECⅡ.
Results:
1. Zonulin was detected in atherosclerosis patients, it was higher expressed thancontrol. Western blot results showed: zonulin was a47KD protein, it was detected in theserum, which showed a band in the same position with positive contrl, gutepithelium.Serum zonulin in atherosclerosis patients was higher expressed than control.(n=8, P<0.05)
2. Bacteria16s rDNA wasdetected from non-infected athroslerosis blood.High-throughput sequencing for bacteria16s rDNA detection showed the category of bacteria was most from the gut: Rahnella, Serratia and Pseudomonas ranked the top3in allbacterias.
3. Gut epithelium barrier model was successfully construced with21d of Caco-2cellculture in vitro. Caco-2cell differentiated to mature gut epithelium with tight junction.TEER value increased fignificantly. study the mechanism of bacteris entrance throughgutepithelium. Serratia pass through gut epithelial barrier without cell injury.
4. HUVEC and rat AECII were isolated, primary culture and identified. A majority ofapoptotic cells (annexin V-positive cells) was arise in AECII cells treated with GCDC.GCDC produced a dose-dependent increase in cell cytotoxicity measured as ATP releaseand LDH release. Mitochondrial dysfunction and oxidative stress in rat AEC II treated withGCDC.
5. GCDC could regulate ET-1secretion: ET-1in GCDC treated HUVEC was lower in1d and3d, but elevated in5d. GCDC inhibit surfactant secretion from isolated AEC II.
Conclusion：
1. Gut bacteria in short diameter may be related to atherosclerosis formation, theypossibly pass through gut epithelial barrier from the space between epithelium.
2. Zonulin was elevated in serum of atherosclerosis patients, possibly because it couldregulate the permeability of tight junction in gut epithelium barrier.
3. GCDC induced AECⅡ death and inhibition of pulmonary surfactant secretioncontributed to ICP related newborn lung injury.
4. GCDC regulated HUVEC ET-1expression might contribute ICP related pretermbirth and intrauterine distress.
本论文包括两个部分的内容。
第一部分：zonulin在动脉粥样硬化发病机制中的作用研究
动脉粥样硬化的形成机制一直是心血管疾病研究的焦点之一。除了内皮损伤学说，泡沫细胞形成机制与平滑肌细胞增生理论等经典观点以外，越来越多的证据显示机体持续慢性系统炎症状态与动脉粥样硬化的形成密切相关，最近针对肠道菌群与冠心病发病之间联系的报道也为粥样硬化的形成机制提供了新的观点。肠道菌群通过何种机制参与动脉粥样硬化形成，其促进动脉粥样硬化形成依赖于何种条件，以及在与此相关的发病机制中是否存在可供干预的靶点成为最近粥样硬化机制研究的热点问题。
研究报道在粥样硬化斑块中检测到细菌DNA，其中部分为肠源性细菌产物。针对慢性牙周炎患者患冠心病的机制研究也提示反复菌血症能显著增加冠心病发病率。如能明确细菌是否参与无明显反复感染患者的动脉粥样硬化发病机制，并进一步了解细菌如何进入人体并参与动脉动脉粥样硬化发病机制，可为粥样硬化形成机制的细菌学说提供更多的证据。在此基础上寻找可干预细菌进入人体的关键环节，试通过对关键靶点进行干预减少细菌进入人体的机会，将为预防和治疗动脉粥样硬化提供新的思路。
Zonulin是肠道上皮细胞合成的一种功能蛋白，能够通过调节肠上皮细胞间紧密连接蛋白的结构而改变肠上皮间隙的通透性。研究发现克隆病患者中存在肠道zonulin水平升高与患冠心病几率增加的并存现象，如果zonulin水平升高可增加患冠心病的机会，那么zonulin很可能通过调节肠道上皮屏障的通透性而增加细菌进入人体的机会。为此，本研究用Western Blot方法研究动脉粥样硬化患者血清中Zonulin的表达水平改变，用基于细菌16s rDNA的高通量测序方法寻找无反复感染动脉粥样硬化患者血液中的细菌DNA产物，并试图在体外建立的肠上皮细胞屏障模型中研究部分细菌通过肠上皮屏障的机制。通过上述研究寻找肠道细菌与动脉粥样硬化形成的具体联系机制，以期为动脉粥样硬化的诊治提供新的思路。
第二部分：甘氨鹅脱氧胆酸细胞毒性的体外实验研究
妊娠肝内胆汁淤积综合征（intrahepatic cholestasis of pregnancy ICP）是一组妊娠合并症，发病率因地而异，在长江流域地区可高达4-6%，是该地区危害母婴健康的重要妊娠期疾病。胆酸对于母亲与胎儿的损伤成为ICP母婴损害的主要原因。机体内生理浓度范围内的胆酸对细胞及组织并无毒性作用，但是胆酸升高到一定浓度时，可以产生明显的细胞毒性。胆酸对于细胞的损伤主要以诱导细胞死亡为主，其中最常见的是细胞凋亡。近期研究发现胆酸还可以诱导细胞发生坏死，甚至可能是最近才报道的程序性坏死。研究胆酸对于循环及呼吸系统主要功能细胞的细胞毒性，有助于从细胞水平阐明胆酸对于循环及呼吸系统的功能损害及其分子机制，为揭示ICP相关的子代心肺损伤提供理论基础。
ICP患者子代并发症中，以早产、胎儿宫内窘迫以及新生儿肺损伤最为常见，危害也最大。早产是一系列新生儿并发症的重要原因，胎儿宫内窘迫则是造成子代神经功能损伤的主要原因之一。胆酸对于胎儿血管结构及功能的影响可能是ICP相关早产与宫内窘迫的发病机制。在胎儿血管结构中，与母儿循环中胆酸最直接接触的是血管内皮细胞。而胆酸对于人脐静脉内皮细胞（HUVEC）是否具有损伤作用目前尚未见报道。胆酸诱导的肺泡上皮细胞损伤主要与ICP相关的新生儿肺损伤有关：当母亲患有ICP时，孕妇血中胆酸水平明显增高，羊水及胎儿血中胆酸亦相应升高，胆酸长时间浸润并损伤肺组织，是ICP致新生儿肺损伤的关键因素。基于AECⅡ在肺部的重要作用，研究胆酸对AECⅡ的细胞毒性作用将可能为ICP新生儿肺损伤作出解释。
甘氨鹅脱氧胆酸（Glycochenodeoxycholate，GCDC）是胆酸的一种类型，被发现有强烈的诱导细胞死亡作用，可诱导肝细胞等一系列细胞死亡。但目前尚未见GCDC诱导HUVEC与AECⅡ死亡的报道。
研究目的：
1.检测动脉粥样硬化患者血清Zonulin的表达水平改变。
2.在动脉粥样硬化患者循环中寻找肠道细菌DNA产物并鉴定。
3.建立肠上皮细胞屏障体外模型并以此为基础研究细菌通过肠上皮屏障的机制。
4.明确GCDC能否诱导HUVEC与AECⅡ细胞死亡，及其诱导死亡的具体机制。
5.研究GCDC对HUVEC与AECⅡ结构与功能的影响。
材料方法：
1.采用Western Blot方法检测动脉粥样硬化患者血清中的Zonulin表达变化。
2.用基于ROCHE454平台的细菌16s rDNA的高通量测序方法寻找无反复感染动脉粥样硬化患者血液中的细菌DNA产物。
3.以Caco-2细胞培养结合Transwell方法建立肠上皮细胞屏障体外模型，用电镜及激光共聚焦等方法观察细菌通过该屏障的具体机制。
4.采用DAPI染色，流式细胞学annexin V/PI染色分析，活性caspase3酶免疫荧光染色等方法检测GCDC诱导HUVEC与AECⅡ细胞凋亡与坏死情况。
5.用Real-time PCR等法观察GCDC对HUVEC与AECⅡ结构与功能的影响。
结果：
1.动脉粥样硬化患者血清中存在Zonulin，且其表达水平显著高于对照组。Westernblot结果显示：zonulin为分子量约47KD的蛋白分子，血清中检测到与阳性对照肠上皮细胞中位置相同的条带，且分子量符合zonulin特征。动脉粥样硬化患者与正常对照相比较，血清zonulin的表达增高（n=8，P<0.05）。
2.无感染表现的动脉粥样硬化患者血液中检测到细菌16s rDNA产物。高通量测序结果显示多种肠道来源的细菌16s rDNA在无感染表现的动脉粥样硬化患者血液中检测出，其中以拉恩氏菌属、沙雷氏菌属与荧光假单胞菌属含量最高。
3.体外成功建立了肠上皮细胞屏障模型，微小肠道细菌可能主要从细胞间隙通过肠道上皮屏障。Caco-2细胞经培养21d后分化成熟，细胞间紧密连接形成，跨上皮细胞电阻值显著升高，形成细胞屏障。粘质沙雷氏菌可在不损害细胞的条件下通过肠上皮细胞屏障。
4.采用细胞原代培养方法，体外成功培养出HUVEC与新生大鼠AECⅡ细胞。并对所培养细胞进行了特异性鉴定。GCDC在体外能够诱导原代培养的新生鼠肺泡Ⅱ型上皮细胞（AECII）死亡，包括凋亡和坏死。GCDC诱导的AECII死亡具有浓度依赖性，线粒体形态与功能变化参与细胞死亡过程。
5. GCDC处理的HUVEC出现ET-1的水平变化，3d内以降低ET-1为主，长时间处理后ET-1的变化反而升高。GCDC可有效抑制中AECⅡ肺表面活性物质的分泌。GCDC处理后的AECⅡ出现肺表面活性物质含量的降低。
结论：
1.肠道部分微小体积菌群可能参与动脉粥样硬化的发病机制，其可能经肠上皮细胞间隙通过肠上皮细胞屏障。
2. Zonulin在动脉粥样硬化患者血清中表达升高，可能与其具有调节肠上皮细胞间紧密连接通透性功能有关。
3. ICP相关新生儿肺损伤的发病机制包括GCDC诱导AECⅡ发生凋亡与坏死，以及GCDC抑制AECⅡ肺表面活性物质分泌。
4. ICP相关的早产与胎儿宫内窘迫与GCDC调节HUVEC分泌ET-1相关。
Background:
Part1. Role of zonulin in the pathogenesis of atherosclerosis
Pathogenesis of atherosclerosis is a hot topic of cardiovascular disease. Chronicsystemic inflammation was regarded as an important cause of atherosclerosis. Recent studydiscover the relationship between gut microbiota and atherosclerosis and present new ideafor the pathogenesis of atherosclerosis. However, the following problems are waiting to besolved for further study on atherosclerosis formation: How does gut microbiota induceatherosclerosis? What are the necessary conditions for its effect? Is there any key moleculein the process which can be regulated.
Bacteria DNA was found in the plaque of athesclerosis, some of them are from the gut.Study on chronic periodontitis induced coronary artery disease (CAD) suggested recurrentbacteremia could increase the incidence of CAD. To investigate the role of bacteria inpathigenesis of atherosclerosis in non-infection patients, and understand how does gutmicrobiota enter human circulation, could present more evidence for the theory of bacteriainduced atherosclerosis. To find out the key step of gut microbiota translocation, and stopthe key point of bacteria entrance could be a possible way to prevent and treatatherosclerosis.
Zonulin was a protein produced and secreted by gut epithelium. Zonulin could regulategut permeability via change the structure of tight junction(TJ) between the gut epithelium.Zonulin was reported as an elevated protein with increased incidence of CAD in Crohnsdisease. If elevated zonulin can increase the incidence of CAD, the possible explainationwill be the function of increase the entrance of gut microbiota to the circulation.
So, western blot was used to detect the expression of serum zonulin in atherosclerosispatients. High-throughput sequencing with bacteria16s rDNA detection was applied to findevidence of bacteria in non-infected atherosclerosis patients. After that, gut epithelium barrier model was construced with Caco-2cell culture in vitro to study the mechanism ofbacteris entrance through gutepithelium. These studies focus on the role of zonulin andcould construct the relationship between gut microbiota and atherosclerosis.
Part2. Cell toxicity study of Glycochenodeoxycholate in vitro
Intrahepatic cholestasis of pregnancy is a constant pregnant complication in great areasincluding Yangzi river and around, with an incidence of4-6%. ICP is dangerous to mothersand babies. The pathogenesis of ICP is unclear, but the mothers all had risk a significanthigher serum bile acid level. The elevated bile acid could cause mother and infant injury.Since physiological concentration bile acid is nontoxical to human body, abnormal highlevel bile acid could induce cell toxicity. Bile acid could induce cell death and cause injuryto human body. Most common discovered cell death is cell apoptosis. However, recentstudy showed bile acid also could induce cell necrosis, or programmed cell necrosis, whichis named as necroptosis. To study the cell toxicity of bile acid to cells from vascule and thelung will be much helpful to discover the molecular mechanism of bile acid inducedvascular and lung injury, and give theoretical basis for ICP relevant fetal and newborninjury.
Bile acid induced cell death was confirmed in hepatocyte, myocardium, pancreatic celland tumor cells. The results were considered as an important pathogenesis of cholestasiscomplications. Preterm birth, fetal distress and newborn lung injury are the most constantcomplications of ICP, also the most dangerous types. Preterm birth could bring a lot ofsevere complications, fetal distress could cause future neurological development disable.There may be started from bile acid induced vascular injury. Vascular endothelium was thefirst barrier of the body from circulatory bile acid. However, there is no report about thepossible effect of bile acid to HUVEC. Bile acid induced alveolar epithelium death couldlead to newborn lung injury: when mothers got ICP, bile acid level were elevated both inmaternal serum and amniotic fluid. Fetal lung was infiltrated by amniotic fluid directly.Because AECⅡ is a multifunctional cell in the alveoli, the effect of bile acid toAECⅡ could possiblyexplain bile acid induced newborn lung injury.
The cell toxicity of elevated bile acid level in ICP mother could be the cause of fetaland newborn conlication. Discovering the mechanism of bile acid induced HUVEC andAECⅡ injury could be a complementationof bile acid cell toxicity, also be much helpful for theory base of ICP relevated fetal and newborncomplications. New diagnosis andtherapy strategy could be made with these results.
Glycochenodeoxycholate (GCDC) was a type of bile acid, with strong cell toxicity tohepatocyte and some other types of cells. However, the cell toxicity of GCDC to HUVECand AECⅡ was not reported.
Objects:
1. To discover the relationship between zonulin and atherosclerosis.
2. Find evidence of gut microbiota in the circulation of atherosclerosis patients.
3. Constrct epithelium barrier model in vitro to study the mechanism of bacteriatranslocation through gut gepithelium.
4. To detect GCDC toxicity newborn rat AECⅡ and the mechanism of GCDC inducedHUVEC and AECⅡ deathin vitro.
5. To study the following structure and function injury of GCDC to HUVEC.
Material and Methods:
1. Western blot was applied to detect the expression of zonulin in the serum ofatherosclerosis patients.
2. High-throughput sequencing with bacteria16s rDNA detection was applied to findevidence of bacteria in non-infected atherosclerosis patients.
3. Gut epithelium barrier model was construced with Caco-2cell culture in vitro tostudy the mechanism of bacteris entrance through gutepithelium.
4. DAPI staining, flow cytometry, caspase3activity detection, mitochondrialmorphology analysis were applied to detect cell apptosis and necrosis.
5. Real-time PCR was applied to detect endothelium-1level in HUVEC andpulmonary surfactant assay were applied to GCDC treated AECⅡ.
Results:
1. Zonulin was detected in atherosclerosis patients, it was higher expressed thancontrol. Western blot results showed: zonulin was a47KD protein, it was detected in theserum, which showed a band in the same position with positive contrl, gutepithelium.Serum zonulin in atherosclerosis patients was higher expressed than control.(n=8, P<0.05)
2. Bacteria16s rDNA wasdetected from non-infected athroslerosis blood.High-throughput sequencing for bacteria16s rDNA detection showed the category of bacteria was most from the gut: Rahnella, Serratia and Pseudomonas ranked the top3in allbacterias.
3. Gut epithelium barrier model was successfully construced with21d of Caco-2cellculture in vitro. Caco-2cell differentiated to mature gut epithelium with tight junction.TEER value increased fignificantly. study the mechanism of bacteris entrance throughgutepithelium. Serratia pass through gut epithelial barrier without cell injury.
4. HUVEC and rat AECII were isolated, primary culture and identified. A majority ofapoptotic cells (annexin V-positive cells) was arise in AECII cells treated with GCDC.GCDC produced a dose-dependent increase in cell cytotoxicity measured as ATP releaseand LDH release. Mitochondrial dysfunction and oxidative stress in rat AEC II treated withGCDC.
5. GCDC could regulate ET-1secretion: ET-1in GCDC treated HUVEC was lower in1d and3d, but elevated in5d. GCDC inhibit surfactant secretion from isolated AEC II.
Conclusion：
1. Gut bacteria in short diameter may be related to atherosclerosis formation, theypossibly pass through gut epithelial barrier from the space between epithelium.
2. Zonulin was elevated in serum of atherosclerosis patients, possibly because it couldregulate the permeability of tight junction in gut epithelium barrier.
3. GCDC induced AECⅡ death and inhibition of pulmonary surfactant secretioncontributed to ICP related newborn lung injury.
4. GCDC regulated HUVEC ET-1expression might contribute ICP related pretermbirth and intrauterine distress.
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