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Effects of Astragalus Polysaccharide on Mechanical Characterization of liver sinusoidal endothelial cells by Atomic Force Microscopy at Nanoscale

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Chinese Journal of Integrative Medicine 2018年第6期24卷 455-459页

作者： LU Wen-liang LI Juan-mei YANG Jiao XU Chen-guang ZHANG Sha-sha YAN Jie ZHANG Ting-ting ZHAO Hui-hui Department of liver Diseases Guang'anmen Hospital China Academy of Chinese Medical Sciences Beijing 100053 China Department of Traditional Chinese Medicine The First People's Hospital of Huzhou Huzhou Zhejiang Province 313000 China Department of Traditional Chinese Medicine The First People's Hospital of Yuhang District Hangzhou 311100 China Department of Traditional Chinese Medicine Ningxia People's Hospital Yinchuan 750021 China

Objective： To study the effects of Astragalus polysaccharide （APS）, the primary effective component of the Chinese herb medicine Astragalus membranaceus （frequently used for its anti-hepatic fibrosis effects）, on nanoscale mechanical properties of liver sinusoidal endothelial cells （SECs）. Methods： Using endothelial cell medium as the control, 5 experimental groups were established utilizing different concentrations of APS, i.e. 12.5, 25, 50, 100, and 200μg/mL. By using atomic force microscopy along with a microcantilever modified with a silicon dioxide microsphere as powerful tools, the value of Young＇s modulus in each group was calculated. SAS 9.1 software was applied to analyze the values of Young＇s modulus at the pressed depth of 300 nm. Environmental scanning electron microscopy was performed to observe the surface microtopography of the SECs. Results： The value of Young＇s modulus in each APS experimental group was significantly greater than that of the control group： as APS concentration increased, the value of Young＇s modulus presented as an increasing trend. The difference between the low-concentration （12.5 and 25 μg/mL） and high-concentration （200μg/mL） groups was statistically significant （P〈0.05）, but no significant differences were observed between moderate-concentration （50 and 100μg/mL） groups versus low- or high-concentration groups （P〉0.05）. Surface topography demonstrated that APS was capable of increasing the total area of fenestrae. Conclusions： The values of Young＇s modulus increased along with increasing concentrations of APS, suggesting that the stiffness of SECs increases gradually as a function of APS concentration. The observed changes in SEC mechanical properties may provide a new avenue for mechanistic rasearch of anti-hepatic fibrosis treatments in Chinese medicine.

关键词： liver sinusoidal endothelial cell atomic force microscope Astragalus polysaccharide Young's modulus nanoscale Chinese medicine

Effects of ethanol on liver sinusoidal endothelial cells-fenestrae of rats

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Hepatobiliary & Pancreatic Diseases International 2005年第3期4卷 422-426页

作者： Bing-Yuan Wang, Xiao-Hua Ju, Bao-Yu Fu, Jian Zhang and Yan-Xue Cao Department of Gastroenterology, First Hospital of China Medical University, Shenyang 110001 , China Department of Gastroenterology First Hospital of China Medical University Shenyang 110001 China. wangby@***

Important advances have been made in research into the mechanism of alcoholic liver disease (ALD) over the past few years,but the role of liver sinusoidal endothelial cell (LSEC) in ALD has not been elucidated adequately. This study was undertaken to investigate the effect of ethanol on fenestrae of LSECs in rats. METHODS: A rat model of alcoholic liver disease was established by means of direct intragastric instillation of ethanol. Fifty-five rats of experimental (35 rats) and control (20) groups were sacrificed at the end of 4,8,12 weeks respectively, and also at the end of 12-week abstinence. After heart perfusion, the liver tissue was fixed and stained with hematoxylin and eosin for observation of serial changes of LSEC-fenestrae under a transmission electron microscope. RESULTS: Normal LESC was flat with a nucleus and organelles arranged regularly. The distal cytoplasm displayed as a lamina with many fenestrae, lacking the basement membrane(BM) underneath the endothelium. At the end of 4-week alcohol feeding, the number of fenestrae decreased at the distal cytoplasm in some LSECs, without the formation of the BM underneath the endothelium. At the end of 8 weeks, the number of fenestrae decreased significantly or even disappeared. The BM began to develop incompletely underneath the endothelium, while the active fibroblast appeared. At the end of 12 weeks, the number of fenestrae decreased more significantly and the complete BM could even be seen. But the changes were mostly limited in the single or adjoining sinus, and fibrosis was scarcely formed. At the end of 12-week abstinence, defenestration and formation of the endothelial BM lightened significantly. CONCLUSIONS:Defenestration and formation of the BM in LSECs develop gradually with the chronic stimulation of ethanol. Hepatic sinusoidal capillarization and fibrosis will be seen if their state is more serious. These early changes, i. e., limited and regional defenestration and capillarization may be the basi

关键词： alcoholic liver disease hepatic fibrosis liver sinusoidal endothelial cell liver sinusoidal capillarization

Discussion on Serum Technique and Cryopreservation Technique in the Culture of liver sinusoidal endothelial cells

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Agricultural Biotechnology 2021年第1期10卷 129-132,135页

作者： Yue PENG Aolei SU Tiejian ZHAO Peng LIU Qing WANG Zhihao SHANG Guangxi University of Chinese Medicine Nanning 530200China Guangxi Health Technical Vocational College Nanning 530023China

Hepatic sinusoidal endothelial cells are a kind of highly differentiated cells in hepatic sinusoids, which play an important role in the occurrence and development of liver fibrosis. Hepatic sinusoidal endothelial cells are often used in the study of liver fibrosis. In the process of culturing liver sinusoidal endothelial cells, the treatment of serum for culture and cryopreservation of cells are relatively complicated and error-prone. If the technical details of serum treatment and cell cryopreservation are not handled properly, it will have a more obvious effect on the effect of hepatic sinusoidal endothelial cell culture. We have gained experience in the theoretical study of liver fibrosis and the operation of cell culture experiments, and this paper summarized the details of liver sinusoidal endothelial cell culture such as the problems of serum preservation, thawing, precipitates and heat inactivation, as well as methods and precautions for cell cryopreservation.

关键词： liver sinusoidal endothelial cell cell culture Serum cell cryopreservation

Reconstruction of liver organoid using a bioreactor

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World Journal of Gastroenterology 2006年第12期12卷 1881-1888页

作者： Masaya Saito Tomokazu Matsuura Takahiro Masaki Tetsuro Suzuki Haruka Maehashi Keiko Shimizu Yoshiaki Hataba Tohru Iwahori Filip Braet Division of Gastroenterology and Hepatology Department of Internal MedicineThe Jikei University School of MedicineTokyoJapan Department of Laboratory Medicine The Jikei University School of MedicineTokyoJapan Department First Department of Biochemistry The Jikei University School of MedicineTokyoJapan DNA Medical Institute The Jikei University School of MedicineTokyoJapan Fifth Division of Blood Purification Department of SurgeryTokyo Medical UniversityTokyoJapan Australian Key Center for Microscopy & Microanalysis Electron Microscope UnitThe University of SydneyNSW 2006 Australia

AIM： To develop the effective technology for reconstruction of a liver organ in vitro using a bio-artificial liver. METHODS： We previously reported that a radial-flow bioreactor （RFB） could provide a three-dimensional highdensity culture system. We presently reconstructed the liver organoid using a functional human hepatocellular carcinoma cell line （FLC-5） as hepatocytes together with mouse immortalized sinusoidal endothelial cell （SEC） line M1 and mouse immortalized hepatic stellate cell （HSC） line A7 as non parenchymal cells in the RFB. Two × 10^7 FLC-5 cells were incubated in the RFB. After 5 d, 2 × 10^7 A7 cells were added in a similar manner followed by another addition of 10^7 M1 cells 5 d later. After three days of perfusion, some cellulose beads with the adherent cells were harvested. The last incubation period included perfusion with 200 nmol/L swinholide A for 2 h and then the remaining cellulose beads along with adherent cells were harvested from the RFB. The cell morphology was observed by transmission electron microscopy （TEM） and scanning electron microscopy （SEM）. To assess hepatocyte function, we compared mRNA expression for urea cycle enzymes as well as albumin synthesis by FLC-5 in monolayer cultures compared to those of single-type cultures and cocultures in the RFB. RESULTS： By transmission electron microscopy, FLC-5, M1, and A7 were arranged in relation to the perfusion side in a liver-like organization. Structures resembling bile canaliculi were seen between FCL-5 cells. Scanning electron microscopy demonstrated fenestrae on SEC surfaces. The number of vesiculo-vacuolar organelles （WO） and fenestrae increased when we introduced the actin-binding agent swinholide-A in the RFB for 2h. With respect to liver function, urea was found in the medium, and expression of mRNAs encoding arginosuccinate synthetase and arginase increased when the three cell types were cocultured in the RFB. However, albumin synthesis decreased. CONCLUSION： Co-culture

关键词： liver organoid Organ reconstruction Bioartificial liver Coculture liver sinusoidal endothelial cell Hepatocytes Fenestrae Vesiculo vacuolar organelles Radial flow bioreactor

Research Progress and Research Ideas on Anti-hepatic Fibrosis and Promoting Blood Circulation and Removing Blood Stasis of the National Drug Plumbapin Based on W-P Body

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Plant Diseases and Pests 2020年第4期11卷 39-46页

作者： Peng Yue Su Aolei Zhao Tiejian Zheng Yang Shang Zhihao Guangxi University of Chinese Medicine Nanning 530200China Medical Basic Department of Guangxi Medical College Nanning 530021China

Hepatic sinusoidal endothelial cells(HSEC)are the most important cells that constitute the microcirculation barrier of ***,W-P bodies have been detected in the HSEC cells of most patients with liver fibrosis,and these bodies have become the synthesis and storage sites of vW factor,ET-1 and other factors of liver fibrosis;during pathological stimulation,W-P bodies will excrete the above factors into the cytoplasm,which can make the structure and function of HSEC maladjusted,cause the disturbance of liver microcirculation,and aggravate the process of liver ***,previous studies have found that Plumbagin,the active ingredient of Guangxi specialty ethnic medicine,has the definite function of promoting blood circulation and removing blood stasis and anti-liver fibrosis,but its mechanism is not *** this study,the research progress of the above problems was reviewed,and further research ideas were derived from it as follows:the role of Plumbagin in promoting blood circulation and removing blood stasis and anti-liver fibrosis is produced by affecting the formation quantity of W-P bodies,affecting the synthesis and storage of the contents of W-P bodies,and interfering with their exocytosis ability.

关键词： Plumbagin Anti-hepatic fibrosis Promoting circulation and removing stasis liver sinusoidal endothelial cell W-P body Animal moulding

Function and Mechanism Analysis of liver endothelial cells

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Function and Mechanism Analysis of Liver Endothelial Cells

Xiayuxue Decoction(下瘀血汤)Attenuates Hepatic Stellate cell Activation and sinusoidal Endothelium Defenestration in CCI_4-Induced Fibrotic liver of Mice

第十一届全国免疫学学术大会

作者： Yu Qing Luo Jinzhuo Wang Qin Liu Yanan Wang Lu Yang Shangqing Zhu Dan Pan Wen Liu Jia Yang Dongliang Department of Infectious Diseases Institute of Infection and Immunology Union Hospital Tongji Medical College Huazhong University of Science and Technology

Backgrounds&Aims:liver sinusoidal endothelial cells(LSECs)play a crucial role in maintaining the homeostasis of the hepatic microenvironment through induction of Ag-specific T cell ***,certain virus infection can indu... 详细信息

关键词： Hepatitis B virus liver sinusoidal endothelial cell T cell immunity PD-L1

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Chinese Journal of Integrative Medicine 2014年第7期20卷 516-523页

作者：章李军孙明瑜宁冰冰张文萌陈高峰慕永平张华刘佳边艳琴刘平 Institute of liver Diseases Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine E-Institute of Traditional Chinese Internal Medicine Shanghai Municipal Education Commission Key Laboratory of liver and Kidney Diseases Shanghai University of Traditional Chinese Medicine E-Institute of Traditional Chinese Internal Medicine

Objective： To investigate the effects of ancient Chinese medical formula Xiayuxue Decoction （下瘀血汤, XYXD） on activation of hepatic stellate cells （HSCs） and defenestration of sinusoidal endothelial cells （SECs） in CCI4-induced fibrotic liver of mice. Methods： High performance liquid chromatography was used to identify the main components of XYXD and control the quality of extraction. C57BL/6 mice were induced liver fibrosis by CCI4 exposure and administered with XYXD for 6 weeks simultaneously. liver tissue was investigated by hematoxylin-eosin and Sirius-red staining. sinusoidal fenestrations were observed by scanning electronic microscopy and fluorescent immunohistochemistry of PECAM-1 （CD31）. Whole liver lysates were detected of α-smooth muscle actin （α-SMA） and type-I collagen by Western blot. Primary rat HSCs-T6 cells were analyzed by detecting α-SMA, F-actin, DNA fragmentation through confocal microscopy, Western blot, terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） assay and cellomics arrayscan, respectively. Results： Amygdalin and emodin in XYXD were identified. XYXD （993 mg/kg） inhibited Sirius red positive area up to 70.1% （P〈0.01）, as well as protein levels of α-SMA and type- I collagen by 42.0% and 18.5% （P〈0.05） respectively. In vitro, XYXD （12.5 μg/mL, 50 μg/mL） suppressed the activation of HSCs and reversed the myofibroblastic HSCs into quiescent, demonstrated as inhibition of fluorescent F-actin by 32.3% and 46.6% （P〈0.05）. Besides, XYXD induced the apoptosis of HSC-T6 cells by 20.0% （P〈0.05） and 49.5% （P〈0.01）, evidenced by enhanced TUNEL positivity. Moreover, ultrastructural observation suggested XYXD inhibited defenestration of SECs, which was confirmed by 31.1% reduction of protein level of CD31 （P〈0.05）. Conclusions： XYXD inhibited both HSCs activation and SECs defenestration which accompany chronic liver injuries. These data may help to understand the underlying mechanisms of XYXD for prevetion of chronic liver diseases

关键词： liver fibrosis cirrhosis hepatic stellate cell liver sinusoidal endothelial cell Xiayuxue Decoction