基于Klotho/FGF23轴探讨益气活血通络方对高磷诱导血管平滑肌细胞钙化的影响  

Effect of Yiqi Huoxue Tongluo Prescription on the Calcification of Vascular Smooth Muscle Cells Induced by High Phosphorus Based on the Klotho/FGF23 Axis

作　　者：李旭峰 冷冬月 LI Xufeng;LENG Dongyue(Shiyan People′s Hospital,Shiyan 442000,Hubei,China)

机构地区：[1]十堰市人民医院,湖北十堰442000 [2]十堰市太和医院,湖北十堰442000

出　　处：《中西医结合心脑血管病杂志》2023年第19期3532-3539,共8页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

摘　　要：目的:基于Klotho/成纤维细胞生长因子23(FGF23)轴探讨益气活血通络方对高磷诱导血管平滑肌细胞钙化的影响。方法:制备益气活血通络方含药大鼠血清,并提取和培养原代大鼠胸主动脉血管平滑肌细胞(VSMCs)。采用β-甘油磷酸(β-GP)诱导大鼠VSMCs钙化模型,加入不同浓度的益气活血通络方含药大鼠血清进行干预,实验设置8组:对照组、模型组、益气活血通络方低剂量组(模型+10%含药血清)、益气活血通络方高剂量组(模型+20%含药血清)、益气活血通络方+sh-Klotho组(模型+20%含药血清+sh-Klotho)、益气活血通络方+阴性对照质粒(sh-NC)组(模型+20%含药血清+sh-NC)、益气活血通络方+成纤维细胞生长因子23基因过表达腺病毒(Ad-FGF23)组(模型+20%含药血清+Ad-FGF23)、益气活血通络方+重组腺病毒空载体(AdLaz)组(模型+20%含药血清+AdLaz)。显微镜下观察VSMCs的形态学变化并采用α-平滑肌肌动蛋白(α-SMA)免疫荧光染色鉴定大鼠VSMCs;细胞计数(CCK-8)法检测细胞增殖情况;茜素红S染色观察钙结节的形成情况;按照钙离子(Ca^(2+))浓度及碱性磷酸酶(ALP)活性检测试剂盒检测细胞内Ca^(2+)浓度、ALP活性;实时荧光定量逆转录聚合酶链式反应(qRT-PCR)和蛋白免疫印迹法(Western Blot)检测细胞Klotho、FGF23、α-SMA、Runt相关转录因子2(RUNX2)、骨形态发生蛋白-2(BMP2)的mRNA和蛋白表达。结果:与对照组比较,模型组可见橘红色钙化结节明显增多,钙化结节面积、Ca^(2+)浓度和ALP活性以及FGF23、RUNX2、BMP2的mRNA和蛋白表达明显增加(P<0.05),Klotho、α-SMA的mRNA和蛋白表达明显降低(P<0.05);与模型组比较,益气活血通络方低、高剂量组钙化结节明显减少,钙化结节面积、Ca^(2+)浓度和ALP活性以及FGF23、RUNX2、BMP2的mRNA和蛋白表达明显降低(P<0.05),Klotho、α-SMA的mRNA和蛋白表达明显升高(P<0.05);而沉默Klotho或上调FGF23的表达能明显逆转益气活血通络�Objective:Based on the Klotho/fibroblast growth factor 23(FGF23)axis to explore the effect of Yiqi Huoxue Tongluo Prescription on the calcification of vascular smooth muscle cells induced by high phosphorus.Methods:Rat serum medicated Yiqi Huoxue Tongluo Prescription was prepared,and primary rat thoracic aortic vascular smooth muscle cells(VSMCs)were extracted and cultured.The calcification model of rat VSMCs was induced byβ-glycerophosphate(β-GP),and added different concentrations of Yiqi Huoxue Tongluo Prescription drug-medicated rat serum for intervention,the experimental rats were divided into eight groups:control group,model group,Yiqi Huoxue Tongluo Prescription low(modeling+10%medicated serum),high(modeling+20%medicated serum)dose groups,Yiqi Huoxue Tongluo Prescription+sh-Klotho group(modeling+20%medicated serum+sh-Klotho),Yiqi Huoxue Tongluo Prescription+sh-NC group(modeling+20%medicated serum+sh-NC),Yiqi Huoxue Tongluo Prescription+Ad-FGF23 group(modeling+20%medicated serum+Ad-FGF23),and Yiqi Huoxue Tongluo Prescription+AdLaz group(modeling+20%medicated serum+AdLaz).The morphological changes of VSMCs were observed under a microscope,and the rat VSMCs were identified byα-smooth muscle actin(α-SMA)immunofluorescence staining;cell proliferation was detected by CCK-8 method.The calcium nodules was observed by Alizarin Red S staining;intracellular calcium ion(Ca^(2+))concentration and alkaline phosphatase(ALP)activity were detected by the kits.The mRNA and protein expression of Klotho,FGF23,α-SMA,Runt-related transcription factor 2(RUNX2),and bone morphogenetic protein-2(BMP2)were detected by real-time fluorescent quantitative PCR(qRT-PCR)and Western Blot.Results:Compared with the control group,the model group showed a significant increase in orange-red calcified nodules,the calcified nodule area,Ca^(2+)concentration and ALP activity,FGF23,RUNX2,and BMP2 mRNA and protein expression significantly increased(P<0.05),Klotho andα-SMA mRNA and protein expression significantly reduced(P<0.05);compared with th

关 键 词：慢性肾病 血管钙化 血管平滑肌细胞 益气活血通络方 Klotho/成纤维细胞生长因子23 实验研究 

分 类 号：R285[医药卫生—中药学]