Study On Micro-mechanics Response Of Sinusoidal Endothelial Cell To Astragalus Polysaccharide

Hepatic fibrosis (HF) is a necessary pathological processes of a variety chronic liver disease to cirrhosis. Within the liver fibrosis mechanical environment will change. Sinusoidal endothelial cells (SECS), as liver microvascular endothelial cells, plays an important role in this process. Astragalus, as monarch herb of Qizhu Granules, reinforcing Qi and activating blood and other effects, anti-liver fibrosis. We study its main active ingredient Astragalus polysaccharide (APS) on cultured state SECS impact of micro-mechanics. Use of the laboratory has established joint imaging and measurement system for SECS morphology and micro-mechanics visual observation and measurement, combined with based on blood flow, blood and blood vessels biological force pharmacology theory mechanics perspectives from Astragalus antifibrotic mechanism. Thus Astragalus treatment with SECS injury as a model of liver fibrosis biomechanical pharmacological basis for the study. For further study by changing the structure and mechanical properties of SECS finally able to reverse liver fibrosis in experimental basis. And for famous doctor of Chinese medicine theory connotation of anti-hepatic fibers provide a scientific basis.The study is divided into the following four parts:Part I:SECS’s original culture and identificationObjective:SECS as the research object, first isolated, purified and stable culture SECS, as a prerequisite for further study; while using immunofluorescence staining SECS through the surface markers CD14and factor Ⅷ related antigen antibody, and using environmental scanning electron microscope on the ultrastructure of cells to purified after SECS identification and verification of purity.Methods:Improvement based on the research groupon the pre-methods, the use of the portal vein intubation, the rat liver in situ with D-Hanks solution rinse the blood, and then, the use of type IV collagenase perfusion in situ, IV collagenase, DNA enzyme perfused in vitro enzyme digestion, centrifugal washing out the non-parenchymal liver cells were Percoll density gradient centrifugation of SECS, exclusive selection of endothelial cells in primary culture medium SECS. The SECS by CD14and factor Ⅷ related antigen antibody and an anti-binding, and then with fluorescent secondary antibody, and using DAPI staining nuclei, with fast confocal fluorescence microscope for fluorescence staining for SECS identification. The SECS fixed, dehydrated, dried in low vacuum mode using environmental scanning electron microscope cell ultrastructureResults:Completed SECS isolation and culture, some cells passaged even finished. Observed under an optical microscope, SECS in good condition, in vitro cell growth good condition, morphology typical, showing a typical round after inoculation from the deformation of the spindle, and continued proliferation. Electron microscope, the cells have the typical composition of the sieve fenestrae by a cluster-like structure; also observed that, as the culture time, the aperture will be reduced cytoplasmic and decreases as the cell deformation and spreading outwards, fenestrae also migrate outwards from around the nucleus. Identification of CD14immunofluorescence positive identification rate of approximately99%, Ⅷ factor related antigen-antibody positive identification rate of about2%.Conclusion:In this study, liver collagenase perfusion in situ, in vitro digestion combined percoll density gradient centrifugation successful implementation of the SECS separation and purification, and filter out a suitable SECS endothelial cell-specific medium; cell ultrastructure and by observing CD14and factor Ⅷ related antigen antibody fluorescent staining results confirmed the successful isolation and culture of SECS and SECS purity is high.Part II:Different concentrations of Astragalus polysaccharides on the SECS Young’s modulusObjective:In this study, in vitro SECS as the research object, atomic force microscope (AFM)/fast laser scanning confocal microscope combined imaging and measurement systems, the use of atomic force microscopy force spectroscopy techniques to achieve the effect of different concentrations of APS after24hours of SECS Young modulus measurements to explore SECS APS at different concentrations after intervention micro mechanical response in order to identify different concentrations of astragalus polysaccharides on the mechanical properties of SECSMethods:Study was divided into blank group and experimental group, which numbered blank group Group1:APS0μg/ml, the experimental group according to the concentration of APS divided into five groups:group number2:12.5μg/ml,3groups:25μg/ml,4groups:50μg/ml,5group:100μg/ml,6groups:200μg/ml. According to experimental groups, APS solution was added to24hours after inoculation primary cultured SECS culture medium and placed5%CO2,37℃incubator for24hours. With atomic force microscopy/Fast laser scanning confocal microscope, silica microspheres modified cantilever is biased tool for different concentrations of APS for24hours after the SECS for Young’s modulus measurements, the resulting force curves through Related software to calculate, using the statistical software SAS9.1indent depth of300nm its Young’s modulus.Results: Each experimental group obtained40force curves, a total of240force curves, statistics revealed that the experimental group than the control group, the Young’s modulus of a large group, the difference was statistically significant (P<0.05), APS concentration from12.5μg/ml to200μg/ml of five groups, SECS Young’s modulus fluctuating upward trend, a low concentration of12.5μg/ml group and the group with a high concentration of25μg/ml200μg/ml group difference was statistically significant (P<0.05). While the middle group and the concentration50ug/ml100μg/ml group with low concentration and high concentration differences were not statistically significant (P>0.05).Conclusion:Astragalus polysaccharides action after24hours,the SECS’s Young’s modulus was increases, In other words,astragalus polysaccharide enable the SECS to become "harder" and APS were positively correlated in the experimental concentration range. The SECS becomes "harder" may reduce resistance to blood flow in the hepatic sinusoids, which serve to improve the microcirculation of the liver.Part III:Different concentrations of Astragalus Polysaccharide on SECS spreading and fenestraeObjective:In this study, in vitro SECS as the research object, using environmental scanning electron microscope observation of different concentrations of astragalus polysaccharides on the role after24hours of cell spreading and aperture characteristics SECS effects to clarify the role of different concentrations of APS after24hours for SECS physiological structure.Methods:Same as above, Study was divided into blank group and experimental group, which numbered blank group Group1:APS0μg/ml, the experimental group according to the concentration of APS divided into five groups:group2:12.5μg/ml, groups3:25μg/ml, groups4:50μg/ml, group5:100μg/ml,groups6:200μg/ml.According to experimental groups, APS solution was added to24hours after inoculation primary cultured SECS culture medium and placed5%CO2,37℃incubator for24hours. The SECS fixed with glutaraldehyde, and dehydrated with ethanol concentration, dehydration, critical point drying apparatus and then through the CO2critical point drying method for drying. With environmental scanning electron microscope in the low vacuum mode to observe and capture images. Then each group randomly selected ten cells, the use of image processing software for cell area and the aperture data collection, data input will be collected in an Excel table, and use SAS9.1for statistical analysisResults: APS effect was observed after24hours, SECS spreading area fluctuated upward trend in the experimental group than the control group spreading large cell area, where APS25μg/ml group and200μg/ml group and the control group there was significant difference between (P<0.05). APS role SECS24hours after the experimental group than the control group were fenestrae, and the experimental group with APS concentration increased, the number of fenestrae upward trend, especially in higher concentrations APS group100μg/ml group and200μg/ml group, the number of fenestrae in the control group were more than2times. APS enables cells have a single fenestrae size becomes smaller, and with the increasing concentration of APS, a single fenestrae area there tends to be smaller, APS high concentration group25μg/ml group,50μg/ml group,100μg/ml group,200μg/ml group and the control group there was significant difference between (P<0.05), low concentration group12.5μg/ml group and the control group was no statistical difference between significance (P>0.05). However, the increase in the number of fenestrae on the total fenestrae area of greater than greater than a single fenestrae size decreases, ie, APS acting on SECS24hours after the causes cells to the total fenestrae area increases. Conclusion:APS acting on SECS24hours later facilitate its spread; APS acting on SECS24hours later its fenestrae have a positive impact, and with APS were positively correlated. Indirectly validated state SECS Astragalus increases the total cultivation area of fenestrae, improved sinusoidal capillarization facilitate the SECS communication on both sides of the material, which play a role in liver fibrosis.Part Ⅳ:The impact of Astragalus Polysaccharide on SECS intracellular NO synthesisObjective:Nitric oxide (NO) is an important second messenger in vivo and neurotransmitters, vasodilation of blood vessels. In this study, in vitro SECS as the research object, using fast confocal fluorescence microscopy, NO fluorescent probe, the effects of different concentrations of astragalus polysaccharides on SECS effects of NO synthesis in order to identify different concentrations of astragalus polysaccharides on the physiological function of SECS.Method:First, the state of the SECS culture of NO probe DAF-FM DA installation. With PBS solution as the blank control group, the experimental group divided according to the concentration of APS APS25μg/ml group and APS50μg/ml group, with fast confocal fluorescence microscopy/combined total internal reflection fluorescence microscopy imaging system, the installation of NO of DAF-FM DA probe for real-time fluorescence monitoring of SECS, at10sec/frames pictures, and500seconds in PBS was added to the solution or astragalus polysaccharide solution, observed experimental mean fluorescence intensity region, and using software to time as abscissa, mean fluorescence intensity of the vertical axis, the experimental area, the average fluorescence intensity graphed, astragalus polysaccharides on SECS to judge the impact of NO synthesis.Results:As can be seen, the control group from the experimental1500s beginning to end of the experiment, the experimental area decreased the fluorescence intensity, a decrease of5%, the first500s PBS solution was added did not affect the change trend. APS25ug/ml groups from the start of the experiment to the500S, the experimental area, the average fluorescence intensity decreased, before joining the APS, the mean fluorescence intensity increased rapidly rise by about15%, and the mean fluorescence with time intensity gradually decreased, the end of the experiment the1500s, the fluorescence intensity recovered to the level before dosing. APS50ug/ml Similarly to500S from the beginning of the experiment, the experimental area, the average fluorescence intensity decreased, after the addition of APS, mean fluorescence intensity increased rapidly rise about11%, and the mean fluorescence with time intensity gradually decreased to the1500s when the end of the experiment, the fluorescence intensity recovered to the level before dosing.Conclusion:NO probe DAF-FM DA under the fluorescent light may have some quenched; APS makes SECS vitro state of NO synthesis increased rapidly. SECS increased NO synthesis, can play the intrahepatic vascular dilation, which play a role in improving the hepatic circulation.