梓醇抑制Galectin-3和CD146互作改善AGEs致肝窦内皮细胞的损伤作用  

Catalpol Alleviates the Damaging Effect of AGEs on the Endothelial Cells in the Hepatic Sinus by Disrupting the Interaction Between Galectin-3 and CD146

作　　者：孙伟翔 宗莹莹 刘博 庄远 秦枫[1,2] 陈毓 朱善元[1,2,3] 许惠琴[4,5] SUN Weixiang;ZONG Yingying;LIU Bo;ZHUANG Yuan;QIN Feng;CHEN Yu;ZHU Shanyuan;XU Huiqin(Jiangsu Agri-animal Husbandry Vocational College,Taizhou 225300,China;Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals,Taizhou 225300,China;Engineering Technology Research Center for Modern Animal Science and Novel Veterinary Pharmaceutic Development,Taizhou 225300,China;School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica,Nanjing 210023,China;Nanjing University of Chinese Medicine Hanlin College,Taizhou 225300,China)

机构地区：[1]江苏农牧科技职业学院,泰州225300 [2]江苏省兽用生物制药高技术研究重点实验室,泰州225300 [3]江苏现代畜牧与新兽药工程技术中心,泰州225300 [4]南京中医药大学药学院,南京210023 [5]江苏省中药药效与安全性评价重点实验室,南京210023 [6]南京中医药大学翰林学院,泰州225300

出　　处：《中国畜牧兽医》2023年第7期2820-2831,共12页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金项目(81874359);江苏省高校自然科学基金面上项目(18KJD360002);江苏农牧科技职业学院科研项目(NSF2021CB04)。

摘　　要：【目的】探讨梓醇通过影响半乳糖凝集素-3(Galectin-3)和CD146的相互作用改善晚期糖基化终末产物(advanced glycation end products,AGEs)导致大鼠肝窦内皮细胞(rat liver sinusoidal endothelial cells,RLSECs)的炎性损伤作用。【方法】用不同浓度(0、0.1、1、10μmol/L)梓醇分别孵育RLSECs 48 h后,采用CCK-8法观察细胞增殖情况。用10μmol/L梓醇孵育RLSECs 0、12、24、48、96 h,同上法观察细胞增殖情况。设Control(空白对照组)、AGEs(AGEs处理)、Cat1(1μmol/L梓醇)、Cat10(10μmol/L梓醇)和阳性对照GB1107组(1μmol/L GB1107),Cat1、Cat10和GB1107组加入相应含量的药物培养基孵育30 min后,除Control组外其他组均在培养基中加入终浓度为200μg/mL的AGEs刺激,观察以上各组的RLSECs形态变化,采用乳酸脱氢酶(lactate hydrogenase,LDH)法检测细胞的损伤程度,采用ELISA法观察各组单核细胞趋化蛋白-1(MCP-1)、细胞间黏附分子-1(ICAM-1)的分泌量。将巨噬细胞RAW264.7种板,同上法分组并给药,48 h后采用Griess法观察各组一氧化氮(nitric oxide,NO)向细胞上清液的释放量。将RAW264.7细胞种于Transwell小室,RLSECs种于孔板底部,设Control、AGEs、Cat10、Cat10+LV-Galectin-3-GFP、Cat10+LV-Galectin-3-shRNA组,后两组先转染慢病毒48 h,再分别给药30 min后,除Control组外在培养基中加入终浓度为200μg/mL的AGEs刺激,48 h后用结晶紫法观察巨噬细胞的透膜细胞数量。将Control、AGEs、Cat10、GB1107组的RLSECs孵育药物48 h后,采用免疫荧光法观察Galectin-3和CD146的共定位情况。将Control、AGEs和Cat10组的蛋白样品通过Western blotting和免疫共沉淀(Co-IP)法检测Galectin-3和CD146的相互作用以及各自的表达量。【结果】与Control组相比,Cat0.1组RLSECs增殖率无显著差异(P>0.05),Cat1和Cat10组RLSECs增殖率显著升高(P<0.05);与孵育0 h组相比,10μmol/L梓醇孵育48 h组RLSECs增殖率显著上升(P<0.05)。因此,后续试验选用10μmol/L梓醇孵育【Objective】The purpose of this study was to investigate the effect of catalpol on the inflammatory injury of rat liver sinusoidal endothelial cells(RLSECs)induced by advanced glycation end products(AGEs)via affecting the interaction between Galectin-3 and CD146.【Method】After incubating RLSECs with 0,0.1,1 and 10μmol/L catalpol for 48 h,the effect of cell proliferation was observed by CCK-8 method.Incubating RLSECs with 10μmol/L catalpol for 0,12,24,48 and 96 h,and the effect of cell proliferation was observed by the same method as above.Set Control(blank control group),AGEs(AGEs treatment),Cat1(1μmol/L catalpol),Cat10(10μmol/L catalpol)and positive control GB1107(1μmol/L GB1107)groups.After Cat1,Cat10 and GB1107 groups were incubated with drugs for 30 min,then all groups were stimulated by 200μg/mL AGEs except for Control group,the morphological changes of RLSECs in the above groups were observed,and the degree of cell damage was detected by lactate dehydrogenase(LDH)method.The secretion of monocyte chemoattractant protein-1(MCP-1)and intercellular adhesion molecule-1(ICAM-1)were measured by ELISA.The macrophages RAW264.7 were divided into groups and administered with the same method as above.After 48 h,the amount of nitric oxide(NO)released to the cell supernatant in each group was observed with Griess method.RAW264.7 cells were seeded in the Transwell chamber and RLSECs were seeded at the bottom of the well plate.Control,AGEs,Cat10,Cat10+LV Galectin-3-GFP and Cat10+LV Galectin-3-shRNA groups were set up.The last two groups were transfected with lentivirus for 48 h,and then administered with drugs for 30 min respectively,AGEs were added to the culture medium with a final concentration of 200μg/mL for stimulation except for Control group,and the number of migrated macrophages was observed by crystal violet method 48 h later.After incubation of RLSECs in Control,AGEs,Cat10 and GB1107 groups for 48 h,the co-localization of Galectin-3 and CD146 was observed by immunofluorescence.The protein samples fr

关 键 词：梓醇 糖尿病肝损伤 晚期糖基化终末产物(AGEs) GALECTIN-3 CD146 炎性损伤 

分 类 号：S852[农业科学—基础兽医学] R285.5[农业科学—兽医学]