白桦脂酸通过RRAGD/mTOR/ULK1促进自噬改善血管平滑肌细胞钙化  

Betulinic Acid Promotes Autophagy and Improves Vascular Smooth Muscle Cell Calcification through RRAGD/mTOR/ULK1

作　　者：侯炽均[1] 陈鑫 曹艳红[2,3,4,5] 张钰冰 张靖悦 王陵军 王婷[1] 叶小汉 郭依宁[1] HOU Chijun;CHEN Xin;CAO Yanhong;ZHANG Yubing;ZHANG Jingyue;WANG Lingjun;WANG Ting;YE Xiaohan;GUO Yining(Dongguan TCM Hospital of Guangzhou University of Chinese Medicine,Dongguan 523127,Guangdong,China;The First Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong,China;First School of Clinic Medicine,Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong,China;Lingnan Medical Research Center,Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong,China;Guangdong Provincial Universities Key Laboratory of Chinese Medicine Prevention and Treatment of Chronic Heart Failure,Guangzhou 510405,Guangdong,China)

机构地区：[1]广州中医药大学东莞医院,广东东莞523127 [2]广州中医药大学第一附属医院,广东广州510405 [3]广州中医药大学第一临床医学院,广东广州510405 [4]广州中医药大学岭南医学研究中心,广东广州510405 [5]广东省普通高校慢性心力衰竭中医药防治重点实验室,广东广州510405

出　　处：《中华中医药学刊》2024年第3期146-151,I0015,共7页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(82274381);广东省基础与应用基础研究基金项目(2020A1515110132);叶小汉东莞市名中医药专家传承工作室项目(粤中医办函[2018]5号)。

摘　　要：目的 探讨白桦脂酸(Betulinic acid, BA)对高磷诱导血管平滑肌细胞钙化的作用机制。方法 采用2.6μmol·g^(-1)高磷诱导小鼠主动脉平滑肌细胞体外钙化模型,CCK8检测白桦脂酸对细胞活力的影响;加入白桦脂酸低、中、高剂量(5μmol·L^(-1)、10μmol·L^(-1)、20μmol·L^(-1))及自噬抑制剂3-甲基腺嘌呤(3-MA,5μmol·g^(-1))。慢病毒过表达Ras相关GTP结合蛋白D(Ras-related GTP-binding Protein D,RRAGD)转染血管平滑肌细胞,通过荧光显微镜、RT-PCR验证转染效率。采用茜素红染色、细胞内钙含量、碱性磷酸酶活性检测细胞钙化程度;Western blot检测血管平滑肌细胞钙化指标RUNT相关转录因子2(Runt-related transcription factor 2,RUNX2),骨形态发生蛋白2(Bone morphogenetic protein, BMP2),平滑肌蛋白22α(Smooth muscle protein 22α,SM22α)和平滑肌肌动蛋白(α-smooth muscle actin, α-SMA);UNC-51样激酶1(UNC-51-like kinase 1,ULK1),自噬蛋白微管相关蛋白1轻链3(LC3Ⅱ/LC3Ⅰ),泛素结合蛋白P62(P62),哺乳动物雷帕霉素靶蛋白(Mammalian target of rapamycin, mTOR)以及RRAGD的表达。结果 根据CCK8细胞活力结果选择白桦脂酸5、10、20μmol·L^(-1)剂量进行后续实验;钙水平、碱性磷酸酶活性及茜素红染色实验结果显示白桦脂酸改善高磷诱导血管平滑肌细胞钙化;Western blot结果显示白桦脂酸下调RUNX2、BMP2、RRAGD、p-mTOR、P62(P<0.05),上调SM22α、α-SMA、LC3Ⅱ/Ⅰ、p-ULK1的蛋白表达(P<0.05)。白桦脂酸减轻高磷诱导血管平滑肌细胞钙化的作用被自噬抑制剂3-MA和过表达RRAGD阻断。结论 白桦脂酸可能通过RRAGD/mTOR/ULK1信号通路促进自噬抑制血管平滑肌细胞钙化。Objective To discuss the mechanism of betulinic acid(BA)on calcification of vascular smooth muscle cells(VSMCs)induced by high phosphorus.Methods In vitro model of high phosphorus induced VSMCs calcification model.CCK8 detected the cell viability on betulinic acid(BA).BA with low,medium and high dose(5μmol·L^(-1),10μmol·L^(-1),20μmol·L^(-1))and 3-MA(5μmol·g^(-1))were added into VSMCs.Slow virus overexpression of Ras related GTP binding protein D(RRAGD)was transfected into VSMCs,and the transfection efficiency was verified by fluorescence microscopy and RT-PCR.Alizarin red staining,intracellular calcium content and alkaline phosphatase activity were used to detect the degree of cell calcification.Western blot was used to detect the expressions of VSMCs calcification indicators RUNT related transcription factor 2(RUNX2)bone morphogenetic protein 2(BMP2)and smooth muscle protein 22α(SM22),α-smooth muscle action(α-SMA),the expressions of UNC-51 like kinase 1(ULK1),autophagy protein microtubule associated protein 1 light chain 3(LC3II/LC3I),ubiquitin binding protein P62(P62),mammalian target of rapamycin(mTOR)and RRAGD.Results According to the results of CCK8,BA doses of 5,10 and 20μmol·L^(-1) were selected for subsequent experiments.Calcium level,ALP activity and alizarin red staining showed that BA improved high phosphorus induced VSMCs calcification.The protein expression levels of RUNX2,BMP2,RRAGD,p-mTOR and P62 were significantly reduced in BA groups while the protein levels of SM22α,α-SMA,LC3Ⅱ/Ⅰand p-ULK1 were increased significantly(P<0.05).The effect of BA on hyperphosphorus-induced VSMCs calcification was blocked by the autophagy inhibitor 3-MA and overexpression lentivirus of RRAGD.Conclusion BA may activate autophagy and inhibit vascular smooth muscle cell calcification through RRAGD/mTOR/ULK1 signaling pathway.

关 键 词：血管平滑肌细胞 细胞钙化 白桦脂酸 自噬 

分 类 号：R282.71[医药卫生—中药学]