补阳还五汤延缓血管平滑肌细胞衰老的机制研究

The Mechanism of Buyang Huanwu Decoction in Preventing the Aging of Vascular Smooth Muscle Cells

目的探讨补阳还五汤抑制血管平滑肌细胞衰老的作用及机制。方法 SD大鼠通过灌胃补阳还五汤汤剂和蒸馏水制备补阳还五汤含药血清和对照血清。用血管紧张素II(AngⅡ)诱导人主动脉血管平滑肌细胞(HA-VSMCs)衰老为模型,分别予金属基质蛋白酶-2(MMP-2)抑制剂ARP101、补阳还五汤含药血清和对照血清干预,并设立年轻对照组。衰老相关β半乳糖苷酶(SA-β-gal)染色测定SA-β-gal的活性;免疫印迹法和明胶酶谱法测定金属基质蛋白酶-2(MMP-2)表达及分泌水平。结果与对照组相比,Ang II诱导的衰老组HA-VSMC中SA-β-gal阳性细胞百分率显著升高[(74.10±4.64)%vs.(10.30±1.92)%,p<0.001];同时MMP-2蛋白表达及分泌水平均显著增加。补阳还五汤含药血清干预的衰老HA-VSMC中SA-β-gal阳性细胞百分率较未干预的衰老细胞显著降低[(14.08±5.15)vs.(74.10±4.64)%,p<0.001],同时MMP-2蛋白表达及分泌水平均显著减少[(0.39±0.16)vs.(0.93±0.20)和(0.67±0.13)vs.(1.81±0.26),均p<0.05];而类似的结果未在对照血清干预的衰老HA-VSMC中观察到。ARP101干预也可降低衰老HAVSMC中SA-β-gal阳性细胞百分率,而补阳还五汤含药血清对衰老细胞SA-β-gal阳性细胞百分率的抑制作用可被ARP101干预减弱。结论补阳还五汤治疗可能通过抑制VSMCs中的MMP-2来延缓血管衰老。

Objective To study the effects and mechanism of serum containing Buyang Huanwu Decoction(BYHWD) on vascular aging in human aorta vascular smooth muscle cells(HA-VAMCs). Methods Serum containing BYHWD and control was prepared through gavaging to SD rats correspondingly. HA-VSMCs aging was induced by angiotensin(Ang II). Aging cells were incubated with ARP101, a selective matrix metalloprotease type 2(MMP-2) inhibitor, serum containing BYHWD and young control group. Senescence-associated β-galactosidase(SA β-gal) Assay was used to measure SA β-gal activity. Western blotting analysis and Gelatin zymography were used to measure MMP-2 production and secretion in HA-VSMC. Results Compared to control group, the percentage of SA β-gal-positive cells was significantly increased in Ang II-induced aging group [(74.10 ± 4.64)% vs.(10.30 ± 1.92)%, p< 0.001]. The same trends were also found about MMP-2 production and secretion. The percentage of SA β-gal-positive cells and MMP-2 production and secretion in serum containing BYHWD group were much lower than those found in Ang II-induced aging group [(14.08 ± 5.15) vs.(74.10 ± 4.64)%,(0.39 ± 0.16) vs.(0.93 ± 0.20) and(0.67 ± 0.13) vs.(1.81 ± 0.26), all p<0.05]. However, similar inhibition was not observed in serum containing control. Similarly, ARP101 incubation in aging HA-VSMCs significantly reduced the percentage of SA β-gal-positive cells, and weakened partially the protective effects of serum containing BYHWD on this role.Conclusion BYHWD may delay vascular aging by inhibiting MMP-2 in HA-VSMCs.