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

中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (7): 2820-2831.doi: 10.16431/j.cnki.1671-7236.2023.07.022

• 预防兽医 • 上一篇下一篇

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

孙伟翔^1,2,3, 宗莹莹⁶, 刘博¹, 庄远¹, 秦枫^1,2, 陈毓¹, 朱善元^1,2,3, 许惠琴^4,5

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

收稿日期:2022-10-31 发布日期:2023-06-30
通讯作者: 朱善元, 许惠琴 E-mail:jstzmy922@163.com;hqxu309@sina.com
作者简介:孙伟翔,E-mail:407422171@qq.com。
基金资助:
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）

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

SUN Weixiang^1,2,3, ZONG Yingying⁶, LIU Bo¹, ZHUANG Yuan¹, QIN Feng^1,2, CHEN Yu¹, ZHU Shanyuan^1,2,3, XU Huiqin^4,5

1. Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300, China;
2. Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou 225300, China;
3. Engineering Technology Research Center for Modern Animal Science and Novel Veterinary Pharmaceutic Development, Taizhou 225300, China;
4. School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China;
5. Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing 210023, China;
6. Nanjing University of Chinese Medicine Hanlin College, Taizhou 225300, China

Received:2022-10-31 Published:2023-06-30

摘要/Abstract

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、10 μmol/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10 μmol/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（1 μmol/L梓醇）、Cat10（10 μmol/L梓醇）和阳性对照GB1107组（1 μmol/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200 μg/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200 μg/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，10 μmol/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10 μmol/L梓醇孵育48 h为最适试验条件。与AGEs组相比，Cat1和Cat10组改善AGEs导致的细胞损伤，RLSECs上清液LDH活力及MCP-1、ICAM-1的释放量显著降低（P<0.05）。与AGEs组相比，梓醇组抑制巨噬细胞RAW264.7的激活作用，细胞NO的释放量显著降低（P<0.05）。通过慢病毒载体过表达和敲低RLSECs和RAW264.7的Galectin-3，证明了梓醇通过抑制该分子表达可显著改善巨噬细胞的浸润作用（P<0.05）。与AGEs组相比，Cat10组Galectin-3与CD146的结合被显著抑制（P<0.05），且不影响两种分子的各自表达量（P>0.05）。【结论】梓醇通过促进AGEs致Galectin-3和CD146分子复合物的解偶联作用，改善AGEs致肝窦血管内皮细胞的损伤以及促炎因子的释放，降低巨噬细胞的激活和NO的分泌，发挥肝窦血管内皮的保护作用，通过离体试验初步证明了其发挥改善糖尿病AGEs沉积造成的肝损伤的作用机制。

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

Abstract: 【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 10 μmol/L catalpol for 48 h, the effect of cell proliferation was observed by CCK-8 method.Incubating RLSECs with 10 μmol/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 (1 μ mol/L catalpol), Cat10 (10 μ mol/L catalpol) and positive control GB1107 (1 μmol/L GB1107) groups.After Cat1, Cat10 and GB1107 groups were incubated with drugs for 30 min, then all groups were stimulated by 200 μg/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 200 μg/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 from Control, AGEs and Cat10 groups were used to detect the interaction between Galectin-3 and CD146 and their respective expression levels by Western blotting and Co-IP methods.【Result】 Compared with Control group, there was no significant difference in cell proliferation rate of RLSECs in Cat0.1 group (P>0.05), while the cell proliferation rate of RLSECs in Cat1 and Cat10 groups was significantly increased (P<0.05).Compared with the group incubated for 0 h, the cell profiferation rate of RLSECs was significantly increased in the group with 10 μmol/L catalpol incubated for 48 h. Therefore, cells were incubated with 10 μmol/L catatpol for 48 h for subsequent experiments. Compared with AGEs group, in the Cat1 and Cat10 groups the cell damage induced by AGEs was improved, and the LDH activity of RLSECs supernatant, the release of MCP-1 and ICAM-1 were significantly decreased (P<0.05).Compared with AGEs group, in the catalpol groups the activation of macrophage RAW264.7 was inhibited and the release of NO from cells was significantly reduced (P<0.05).By overexpression of lentivirus vector and knockdown of Galectin-3 of RLSECs and RAW264.7, it was proved that catalpol could significantly improve the infiltration of macrophages by inhibiting the expression of this molecule (P<0.05).Compared with AGEs group, in Cat10 group the binding of Galectin-3 and CD146 was significantly inhibited (P<0.05) and the respective expression of the two molecules did not affect (P>0.05).【Conclusion】 Catalpol could promote the uncoupling effect of Galectin-3 and CD146 molecular complexes induced by AGEs, improved the damage of sinusoidal vascular endothelial cells induced by AGEs and the release of proinflammatory factors, reduced the activation of macrophages and the secretion of NO, and played a protective role in sinusoidal vascular endothelial cells.The mechanism of catalpol to improve the liver injury caused by AGEs deposition in diabetes was preliminarily proved through in vitro experiments.

Key words: catalpol; diabetic liver injury; advanced glycation end products(AGEs); Galectin-3; CD146; inflammatory injury

中图分类号:

S852
R285.5

孙伟翔, 宗莹莹, 刘博, 庄远, 秦枫, 陈毓, 朱善元, 许惠琴. 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[J]. 中国畜牧兽医, 2023, 50(7): 2820-2831.

SUN Weixiang, ZONG Yingying, LIU Bo, ZHUANG Yuan, QIN Feng, CHEN Yu, ZHU Shanyuan, XU Huiqin. Catalpol Alleviates the Damaging Effect of AGEs on the Endothelial Cells in the Hepatic Sinus by Disrupting the Interaction Between Galectin-3 and CD146[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2820-2831.

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