Role Of Zonulin In The Pathogenesis Of Atherosclerosis&Cell Toxicity Study Of Glycochenodeoxycholate In Vitro

Background:Part1. Role of zonulin in the pathogenesis of atherosclerosisPathogenesis 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 vitroIntrahepatic 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.