6-O-脱硫酸化肝素通过Syndecan-1/c-Met/Akt途径延缓博来霉素诱导的肺损伤  

6-desulfated heparin inhibits heparan sulfate shedding and epithelial cell damage during bleomycin induced pulmonary injury

作　　者：杨晶 刘晓妮 吴青青 翟艳铎 杜珊珊 季洋 邢新会[2] 陈敬华 闫昳姝 YANG Jing;LIU Xiao-ni;WU Qing-qing;ZHAI Yan-duo;DU Shan-shan;JI Yang;XING Xin-hui;CHEN Jing-hua;YAN Yi-shu(Dept of Pharmaceutics,School of Life sciences and Health Engineering,Jiangnan University,Wuxi Jiangsu 214122,China;MOE Key Laboratory of Industrial Biocatalysis,Institute of Biochemical Engineering,Dept of Chemical Engineering,Tsinghua University,Beijing 100084,China)

机构地区：[1]江南大学生命科学与健康工程学院,江苏无锡214122 [2]清华大学化学工程系生化工程研究所工业生物催化教育部重点实验室,北京100084

出　　处：《中国药理学通报》2022年第8期1147-1155,共9页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No.21978114,81900560);江苏省自然科学基金(No.BK20180294);中国博士后基金(No.2020M671348)。

摘　　要：目的探究不同硫酸化方式的肝素类药物对博来霉素(bleomycin,BLM)诱导肺损伤保护作用的分子机制。方法首先,合成不同硫酸化方式的肝素衍生物——6-脱硫酸肝素(6-DeH)和N-乙酰化肝素(N-AcH);其次,通过乳酸脱氢酶活性测定、MTT实验、Annexin V/PI染色和Hoechst 33258染色等系列实验评价各化合物对BLM诱导支气管上皮细胞(BEARS-2B)损伤的保护作用;接着,通过免疫荧光染色、蛋白免疫印迹等方法考察了6-DeH对Syndecan-1脱落的抑制作用,以及对下游c-Met/Akt信号通路的影响;最后,构建了BLM诱导的肺损伤小鼠模型,通过HE染色、Syndecan-1免疫染色、体质量、生存率等参数测定,进一步验证6-DeH对BLM诱导肺损伤的作用。结果通过构建BLM诱导BEARS-2B细胞损伤模型,发现6-DeH可与细胞表面硫酸乙酰肝素竞争性结合BLM,减少BLM对细胞的损伤,减少细胞表面Syndecan-1脱落,并激活c-Met/Akt信号通路,从而提高细胞存活率;通过BLM诱导的肺损伤小鼠模型,进一步验证了6-DeH能够在肺损伤的早期减少Syndecan-1的脱落,并延缓肺损伤、纤维化进程。结论6-DeH具有保护BLM诱导肺损伤的作用,其机制与抑制Syndecan-1的脱落和激活c-Met/Akt信号通路有关。Aim To study the effect of different heparin sulfation patterns on bleomycin induced lung injury.Methods First,heparin derivatives with different sulfation patterns,6-desulfated heparin(6-DeH)and N-acetylated heparin(N-AcH),were synthesized.Secondly,the effect of these compounds on BLM-induced bronchial epithelial cell(BEARS-2B)injury was evaluated via lactate dehydrogenase activity,MTT experiment,Annexin V/PI staining and Hoechst 33258 staining.Then,immunofluorescence staining and Western blotting were used to investigate the shedding of Syndecan-1 and the activation of c-Met by 6-DeH/Akt signaling pathway.Finally,a BLM-induced lung injury mouse model was used to further verify the protective effect of 6-DeH by HE staining,Syndecan-1 immunostaining,body weight change,and survival rate.Results In the BLM-induced BEARS-2B injury model,6-DeH was selected as the best candidate,which exerted their effect by competitively binding to BLM,thereby reducing the damage of heparan sulfate barrier(Syndecan-1)on cell surface,and improving cell survival by activating the downstream c-Met/Akt pathway.In the BLM-induced lung injury mouse model,it was further confirmed that 6-DeH reduced the shedding of Syndecan-1 in the early stage,and delayed the lung injury and fibrosis process.Conclusions 6-DeH protects the bronchial epithelial cells against BLM-induced lung injury through inhibiting the shedding of Syndecan-1 and activating the c-Met/Akt signaling pathway.

关 键 词：肺损伤 上皮细胞通透性 肝素 选择性脱硫 SYNDECAN-1 c-Met/Akt信号通路 

分 类 号：R-332[医药卫生] R322.35R329.25R563R973.2