成纤维细胞生长因子21对大鼠血管平滑肌钙化的影响及机制

Effects and mechanism of fibroblast growth factor 21 on rat vascular smooth muscle cells calcification

目的 通过建立离体血管平滑肌细胞钙化模型,观察成纤维细胞生长因子21 （FGF21）对血管钙化的影响,并探讨其机制.方法 使用氯化钙和β磷酸甘油诱导离体大鼠主动脉血管平滑肌细胞钙化.将细胞分为对照组（使用常规培养基）、钙化组（使用钙化培养基）、FGF21组（使用钙化培养基和FGF21）、PD166866组[使用钙化培养基、FGF21和成纤维细胞生长因子受体1（FGFR1）抑制剂PD166866]和GW9962组（使用钙化培养基、FGF21和过氧化物酶体增殖因子受体γ抑制剂GW9662）.通过细胞内钙含量、碱性磷酸酶活性和茜素红染色检测血管平滑肌细胞钙化情况.FGFR1、β-Klotho、骨钙素和平滑肌22α的蛋白含量和mRNA表达水平分别用Western blot和Real-timePCR检测.结果 （1）与对照组比较,钙化组的FGFR1和β-Klotho蛋白水平（P＜0.05）和mRNA水平（P＜0.01）均较低.（2）与钙化组比较,FGF21组钙含量、碱性磷酸酶活性和骨钙素的表达量较少（P ＜0.05或0.01）,同时平滑肌22α的蛋白和mRNA水平较高（P均＜0.05）.茜素红染色显示,与钙化组比较,FGF21组的红色钙化结节较少.（3）PD166866组与钙化组之间的钙含量、碱性磷酸酶活性和茜素红染色结果差异均无统计学意义（P均＞0.05）.（4） GW9662组与钙化组之间的钙含量、碱性磷酸酶活性和茜素红染色结果差异均无统计学意义（P均＞0.05）.结论 在大鼠血管平滑肌钙化模型中,FGFR1和β-Klotho的蛋白及mRNA水平下调,降低了FGF21抑制血管平滑肌细胞钙化的作用;FGF21通过过氧化物酶体增殖因子受体γ通路减轻血管钙化.

Objective To observe the effect and mechanism of fibroblast growth factor 21 （FGF21） on rat vascular smooth muscle cells （VSMCs） calcification in vitro.Methods VSMCs was treated with calcification medium containing calcium chloride and β-glycerophosphate to induce rat VSMCs calcification in vitro.VSMCs were divided into 5 groups： the control group （cultured in normal medium）, the calcification group （incubated in calcified medium）, the FGF21 group （cultured in calcified medium and FGF21）, the PD166866 group （cultured in calcified medium and FGF21 and PD166866, inhibitor of fibroblast growth factor receptor-1 （FGFR1））, the GW9662 group （cultured in calcified medium and FGF21 and GW9662, inhibitor of peroxisome proliferators activated receptor-γ （PPAR-γ））.The calcification of VSMCs was detected by calcium content, alkaline phosphatase activity and alizarin red staining.The protein and mRNA expression of FGFR1, β-Klotho, osteocalcin and smooth muscle 22α （SM22α） were determined by western blot analysis and realtime-PCR, respectively.Results （1） The mRNA （P 〈 0.01） and protein expressions of β-Klotho and FGFR1 were significantly downregulated in calcification group compared with control group （P 〈 0.05 or 0.01）.（2）The protein levels and mRNA expression of calcium content, alkaline phosphatase activity and osteocalcin were significantly downregulated, while the protein levels and mRNA of SM22α were significantly increased in FGF21 group compared with calcification group （all P 〈 0.05）.Moreover, alizarin red staining verified positive red nodules on calcified VSMCs was significantly reduced in FGF21 group than in calcification group.（3）Calcium content, alkaline phosphatase activity and alizarin red staining were similar between PD166866 group and calcification group （all P 〉 0.05）.（4） Calcium content, alkaline phosphatase activity and alizarin red staining were similar between GW9662 group and calcification group （all P 〉 0.05）.Conclusion The inhibition of VSMCs calcification by FGF21 is mediated by further downregulating FGFR1 and β-Klotho while activating PPAR-γpathways.