摘要
目的探讨腓骨蛋白-1(Fibulin-1)在高磷诱导的大鼠平滑肌细胞衰老相关钙化过程中的作用及其机制。方法2020年9月至2021年9月自10只6~8周的雄性SD大鼠胸主动脉和腹主动脉提取大鼠原代血管平滑肌细胞。采用磷酸盐(2.5 mmol/L Pi)刺激大鼠平滑肌细胞钙化作为应激性衰老相关钙化模型。通过β-半乳糖苷酶染色评估细胞衰老。通过茜素红染色和细胞钙含量测定判断细胞钙化程度。蛋白免疫印迹法(Western Blot)检测平滑肌细胞在衰老相关钙化过程中发生表型转化的指标和Fibulin-1表达水平。使用siRNA敲低原代大鼠平滑肌细胞中Fibulin-1表达,免疫荧光检测Psmad3的表达,Western Blot检测Fibulin-1对平滑肌细胞表型转化指标的影响。用重组Fibulin-1培养,并同时加入转化生长因子(TGF)-β抑制剂A83-01和Psmad3抑制剂SIS3,通过Western Blot检测平滑肌细胞衰老和钙化指标。结果在磷酸盐刺激大鼠平滑肌细胞钙化的应激性衰老模型中,Fibulin-1的表达上调(t=11.20,P<0.01),平滑肌细胞收缩表型标志物MHC、SM22α表达下调(t值分别为7.97、10.27,均P<0.01),成骨表型标志物OPN、Bmp2和衰老标志物P53的表达上调(t值分别为4.80、9.56、14.07,均P<0.01)。Fibulin-1敲低后改善了细胞衰老程度和钙沉积(t=12.90,P<0.05)、降低了平滑肌细胞表型转化蛋白OPN、Bmp2和衰老蛋白P53的表达(t值分别为5.92、10.15、8.28,P<0.05或P<0.01),同时TGF-β/信号转导蛋白(Smad3)及Psmad3表达被抑制(t值分别为12.90、7.46,均P<0.01)。加入TGF-β/Smad3通路抑制剂后,重组Fibulin-1对平滑肌细胞表型转化蛋白OPN、SM22α和衰老标志蛋白表达P53的促进作用减弱(t值分别为4.52、9.82、3.85,P<0.01或P<0.05)。结论Fibulin-1可通过TGF-β/smad3信号通路促进血管平滑肌细胞发生衰老相关钙化。
Objective To investigate the role and mechanisms of fibulin-1 in senescence-related calcification of rat vascular smooth muscle cells induced by high-concentrationphosphate treatment.Methods From September 2020 to September 2021,rat primary vascular smooth muscle cells were extracted from the thoracic aorta and abdominal aorta of 10 male SD rats aged 6 to 8 weeks.Phosphate(2.5 mmol/L Pi)was used to stimulate the calcification of vascular smooth muscle cells(VSMCs)in a model of stress-induced senescence-related calcification.Cellular senescence was assessed by SA-β-gal staining.Cellular calcification was determined by alizarin red staining and quantification of calcium deposition.Phenotypic transformation indexes and the expression of fibulin-1 during the process of calcification were detected by Western blot.The expression of fibulin-1 in primary rat vascular smooth muscle cells was knocked down by siRNA,the expression of pSmad3 was detected by immunofluorescence,and the effects of fibulin-1 on phenotypic transformation indexes of smooth muscle cells were detected by Western blot.The cells were cultured with recombinant fibulin-1 while transforming growth factor beta(TGF-β)inhibitor A83-01 and pSmad3 inhibitor SIS3 were also added.The senescence and calcification indexes of smooth muscle cells were detected by Western blot.Results In the stress-induced aging model with phosphate stimulation of calcification in rat VSMCs,the expression of fibulin-1 was up-regulated(t=11.20,P<0.01),the expressions of MHC and SM22αwas down-regulated(t=7.97,P<0.01;t=10.27,P<0.01),and the expression of osteoblastic phenotype markers OPN and Bmp2 and senescence marker P53 was up-regulated(t=4.79,P<0.01;t=9.56,P<0.01;t=14.07,P<0.01).Knockdown of fibulin-1 attenuated the degree of senescence and calcium deposition in VSMCs(t=12.90,P<0.05)and decreased the expression of OPN,Bmp2 and P53(t=5.92,P<0.05;t=10.15,P<0.01;t=8.28,P<0.01),at the same time,and TGF-βand pSmad3 expression was inhibited(t=12.90,P<0.01;t=7.46,P<0.01).After the addition of TGF-β/smad3 pathway inhibitors,the stimulatory effect of recombinant fibulin-1 on phenotypic transformation and senescence protein expression inVSMCs was significantly reduced(t=4.52,P<0.01;t=9.82,P<0.01;t=3.85,P<0.05).Conclusions Fibulin-1 can promote aging-related calcification of vascular smooth muscle cells through the TGF-β/smad3 signaling pathway.
作者
梁小璐
晏丹
罗曼蒂
阮磊
张存泰
Liang Xiaolu;Yan Dan;Luo Mandi;Ruan Lei;Zhang Cuntai(Department of Geriatrics,Tongji Hospital of Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430000,China)
出处
《中华老年医学杂志》
CAS
CSCD
北大核心
2022年第5期580-585,共6页
Chinese Journal of Geriatrics
基金
湖北省技术创新专项重大项目(2019ACA141)。
关键词
腓骨蛋白-1
肌细胞
平滑肌
血管钙化
转化生长因子Β
Fibulin-1
Myocytes,smooth muscle
Vascular calcification
Transforming growth factor beta
