目的探讨瘦素在2型糖尿病(T2DM)患者下肢动脉钙化中的作用及可能机制。
我们招募了59名男性T2DM患者和39名非糖尿病男性参与者。所有参与者都接受了下肢动脉的计算机断层扫描扫描。采用标准化软件对钙化评分(CSs)进行分析。通过放射免疫分析试剂盒测定血浆瘦素水平。通过β-甘油磷酸和氯化钙诱导建立人血管平滑肌细胞(VSMCs)钙化模型。钙沉积和矿化通过邻甲酚酞络合物方法和茜素红染色测量。骨形态发生蛋白2(BMP2)的mRNA表达,runt相关转录因子2(Runx2),通过定量RT-PCR测定骨钙蛋白(OCN)和骨桥蛋白(OPN)。通过蛋白质印迹分析确定BMP2,Runx2,α-平滑肌肌动蛋白(α-SMA)和(p)-Akt的蛋白质水平,α-SMA也通过免疫荧光分析测量。
与对照组相比,T2DM患者下肢动脉钙化评分中位数较高[286.50(IQR83.41,1082.00)vs68.66(3.41,141.30),p<0.01]。钙化评分≥300的患者血浆瘦素水平高于≥100(252.67±98.57vs189.38±44.19pg/ml,p<0.05)。与钙化介质相比,瘦素(200、400和800ng/ml)联合钙化培养基[11.99±3.63、15.18±4.55和24.14±5.85mg/ml,分别,vs7.27±1.54毫克/毫升,所有p<0.01]。与钙化介质相比,茜素红染色显示钙分布更明显,BMP2、Runx2和OCN的mRNA水平显著升高,免疫荧光和Westernblot分析表明,瘦素(400ng/ml)联合钙化培养基处理的VSMCs中α-SMA的表达下调,分别。与钙化介质相比,在瘦素(400ng/ml)联合钙化培养基处理的VSMC中,BMP2和Runx2的蛋白水平上调。此外,阻断PI3K/Akt信号通路可降低Leptin(400ng/ml)联合钙化介质处理的VSMCs中BMP2和Runx2蛋白的表达。
瘦素通过PI3K/Akt信号通路上调BMP2和Runx2的表达,调节VSMCs的表型转换,促进T2DM下肢动脉钙化。
We aimed to explore the role and possible mechanism of leptin in lower-extremity artery calcification in patients with type 2 diabetes mellitus (T2DM).
We recruited 59 male patients with T2DM and 39 non-diabetic male participants. All participants underwent computed tomography scan of lower-extremity arteries. The calcification scores (CSs) were analyzed by standardized software. Plasma leptin level was determined by radioimmunoassay kits. Human vascular smooth muscle cells (VSMCs) calcification model was established by beta-glycerophosphate and calcium chlorideinduction. Calcium deposition and mineralization were measured by the o-cresolphthalein complexone method and Alizarin Red staining. The mRNA expression of bone morphogenic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) was determined by quantitative RT-PCR. The protein levels of BMP2, Runx2, α-smooth muscle actin (α-SMA) and (p)-Akt was determined by Western-blot analysis, and α-SMA was also measured by immunofluorescence analysis.
Compared with controls, patients with T2DM showed higher median calcification score in lower-extremity artery [286.50 (IQR 83.41, 1082.00) vs 68.66 (3.41, 141.30), p<0.01]. Plasma leptin level was higher in patients with calcification score ≥300 than ≥100 (252.67 ± 98.57 vs 189.38 ± 44.19 pg/ml, p<0.05). Compared with calcification medium, intracellular calcium content was significantly increased in VSMCs treated by leptin (200, 400 and 800 ng/ml) combined with calcification medium [11.99 ± 3.63, 15.18 ± 4.55, and 24.14 ± 5.85 mg/ml, respectively, vs 7.27 ± 1.54 mg/ml, all p<0.01]. Compared with calcification medium, Alizarin Red staining showed calcium disposition was more obvious, and the mRNA level of BMP2, Runx2 and OCN was significantly increased, and immunofluorescence and Western blot analysis showed that the expression of α-SMA was downregulated in VSMCs treated by leptin (400 ng/ml) combined with calcification medium, respectively. Compared with calcification medium, the protein level of BMP2 and Runx2 was upregulated in VSMCs treated by leptin (400 ng/ml) combined with calcification medium. Moreover, blocking PI3K/Akt signaling pathway can decrease the protein expression of BMP2 and Runx2 in VSMCs treated by leptin (400 ng/ml) combined with calcification medium.
Leptin promoted lower-extremity artery calcification of T2DM by upregulating the expression of BMP2 and Runx2, and regulating phenotypic switch of VSMCs via PI3K/Akt signaling pathway.