目的：观察四妙勇安汤药物血清对高胰岛素/高糖诱导的兔血管平滑肌细胞（VSMC）细胞外基质（ECM）分泌的影响及其可能的作用机制。方法采用血清药理学方法制备药物血清，高胰岛素/高糖诱导兔VSMC增殖。实验分为空白组〔10%胎牛血清（FBS）的正常DMEM〕、正常组（10%FBS的正常DMEM+正常血清）、模型组（10%FBS的高糖DMEM+100 U/L胰岛素+正常血清）、西药组（10%FBS的高糖DMEM+100 U/L胰岛素+辛伐他汀血清）、中药组（10%FBS的高糖DMEM+100 U/L胰岛素+四妙勇安汤血清）。用消化法检测细胞培养上清液中羟脯氨酸（Hyp）含量；逆转录-聚合酶链反应（RT-PCR）检测含药血清对兔VSMC的四型胶原蛋白（COL-Ⅳ）、基质金属蛋白酶-2（MMP-2）及其组织抑制因子（TIMP-1）的mRNA表达和对MMP-2/TIMP-1比值的影响。结果与模型组比较，中药组、西药组细胞培养上清液Hyp含量明显降低（均P＜0.05），且中药组明显低于西药组（mg/L：234.19±26.43比266.73±30.00，P＜0.05）。与正常组比较，模型组COL-Ⅳ、MMP-2的mRNA呈显著高表达（COL-ⅣmRNA：0.78±0.03比0.41±0.02，MMP-2 mRNA：0.80±0.12比0.41±0.02，均P＜0.05），TIMP-1 mRNA表达水平降低（0.35±0.04比0.79±0.07，P＜0.05），MMP-2/TIMP-1比值增加（2.30±0.35比0.52±0.05，P＜0.05）。与模型组比较，中药组、西药组COL-Ⅳ、MMP-2 mRNA表达水平均降低，TIMP-1 mRNA水平提高，MMP-2/TIMP-1比值有所下降（COL-ⅣmRNA：0.55±0.04、0.58±0.03比0.78±0.03，MMP-2 mRNA：0.62±0.05、0.67±0.08比0.80±0.12，TIMP-1 mRNA：0.56±0.02、0.60±0.01比0.35±0.04，MMP-2/TIMP-1：1.11±0.06、1.16±0.15比2.30±0.35，均P＜0.05），但中药组、西药组比较差异无统计学意义（均P＞0.05）。结论四妙勇安汤能够减少高胰岛素/高糖诱导的VSMC分泌ECM，其可能机制与减少Hyp合成，影响COL-Ⅳ、MMP-2、TIMP-1 mRNA表达水平，调整MMP-2/TIMP-1的平衡有关。%Objective To observe the effects of serum containing Simiao Yongan decoction (SYD) on secretion of extracellular matrix (ECM) of rabbit vascular smooth muscle cells (VSMC) induced by high insulin/high glucose and its mechanism. Methods Serum containing SYD was prepared by serum pharmacological method, and the proliferation of rabbit VSMC was induced by high concentrations of glucose and insulin. The experiments were divided into following groups: blank group [normal glucose DMEM containing 10% fetal bovine serum (FBS)], normal group (normal glucose DMEM containing 10%FBS+normal serum), model group (high glucose DMEM containing 10%FBS+100 U/L insulin+normal serum), western medicine group (high glucose DMEM containing 10%FBS+100 U/L insulin+serum containing simvastatin), traditional Chinese medicine group (high glucose DMEM containing 10% FBS +100 U/L insulin+serum containing SYD). The content of hydroxy proline (Hyp) in cell culture supernatant was detected by digestion method. The reverse transcription-polymerase chain reaction (RT-PCR) was used to assay the effects of serum containing drug on the mRNA expressions of collagen-Ⅳ(COL-Ⅳ), matrix metalloproteinase-2 (MMP-2), tissue inhibitors metalloproteinase-1 (TIMP-1) and the MMP-2/TIMP-1 ratio of rabbit VSMC. Results Compared with the model group, the contents of Hyp in cell culture supernatant in traditional Chinese medicine group and western medicine group were significantly reduced (both P<0.05), and the content in traditional Chinese medicine group was much lower than that in western medicine group (mg/L:234.19±26.43 vs. 266.73±30.00, P<0.05). Compared with normal group, the mRNA expressions of COL-Ⅳand MMP-2 were significantly increased (COL-ⅣmRNA:0.78±0.03 vs. 0.41±0.02, MMP-2 mRNA: 0.80±0.12 vs. 0.41±0.02, both P < 0.05), the expression of TIMP-1 mRNA was decreased remarkably (0.35±0.04 vs. 0.79±0.07, P<0.05), and the ratio of MMP-2/TIMP-1 was increased obviously (2.30±0.35 vs. 0.52±0.05, P < 0.05) in model group. Compared with the model group, the mRNA expressions of COL-Ⅳand MMP-2 in traditional Chinese medicine group and western medicine group were decreased significantly, the expression of TIMP-1 mRNA was increased obviously, and the ratio of MMP-2/TIMP-1 was decreased (COL-ⅣmRNA: 0.55±0.04, 0.58±0.03 vs. 0.78±0.03, MMP-2 mRNA: 0.62±0.05, 0.67±0.08 vs. 0.80±0.12, TIMP-1 mRNA:0.56±0.02, 0.60±0.01 vs. 0.35±0.04, MMP-2/TIMP-1:1.11±0.06, 1.16±0.15 vs. 2.30±0.35, all P<0.05), but no statistically significant difference was found between traditional Chinese medicine group and western medicine group (all P > 0.05). Conclusion Serum containing SYD can reduce the secretion of ECM of the rabbit VSMC during the high state of glucose and insulin, and the mechanisms are possibly related to reducing the synthesis of Hyp, influencing the mRNA expression levels of COL-Ⅳ, MMP-2, TIMP-1, and adjusting the balance of MMP-2/TIMP-1.
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