自然衰老大鼠血管老化中细胞骨架的改变及人参三七川芎提取物的干预作用

Changes on Cytoskeleton in the Process of Vascular Aging of Natural Aging Rats and Intervention Effects of Extracts from Radix Ginseng,Radix Notoginseng,and Rhizoma Chuanxiong

目的观察人参三七川芎提取物对自然衰老大鼠胸主动脉及骨架蛋白的影响。方法 50只SD雄性大鼠(8~9月龄)按体重随机分为模型组、中药低、中、高剂量组及白藜芦醇组,每组各10只,模型组大鼠正常饲养至21月龄;各给药组大鼠分别从18月龄开始药物干预,中药低、中、高剂量组给药剂量分别为576、1 152、2 305 mg/kg,白藜芦醇组给药剂量为50 mg/kg,模型组按体重给予等体积的纯净水,每日1次,连续灌胃3个月;同时以10只SD雄性大鼠(3月龄)作为青年对照组(青年组)。应用HE染色观察胸主动脉血管形态的改变,β-半乳糖苷酶(SA-β-gal)特异染色法计算蓝染组织比率,免疫荧光染色观察骨架蛋白F-actin形态的改变,Western blot法检测骨架蛋白F-actin和SM22α表达情况。结果与青年组比较,模型组血管内膜损伤且不光滑,中膜明显增生且排列紊乱,弹力板扭曲、断裂、结构不完整,外膜增生明显,SA-β-gal染色组织蓝染比率明显增加(P<0.01),F-actin荧光强度及蛋白表达明显增多(P<0.01),而SM22α蛋白表达明显减少(P<0.01)。药物干预后,各给药组血管壁内皮轻度受损,弹力膜结构较完整,中膜及外膜厚度明显降低(P<0.01);SA-β-gal染色中药中剂量及白藜芦醇组蓝染比率减少(P<0.05),而中药低、高剂量蓝染比率改变无统计学意义(P>0.05);各给药组F-actin荧光强度明显降低(P<0.01);蛋白表达方面,中药中、高剂量组及白藜芦醇组F-actin蛋白表达减少(P<0.01),而中药低剂量组改变则无统计学意义(P>0.05),各给药组SM22α蛋白表达增加(P<0.01)。结论细胞骨架微丝蛋白F-actin形态及蛋白表达在衰老过程中改变明显,其相关蛋白SM22α蛋白表达亦改变明显,可能共同参与了血管老化的进程;人参三七川芎提取物可以一定程度上延缓血管老化,且对于血管F-actin和SM22α蛋白有明显干预作用,可能间接通过此作用延缓了血管老化的进程。

Objective To observe the effect of extracts fromRadix Ginseng,Radix Notoginseng,andRhizoma Chuanxiong（ RGRNRCX） on the thoracic aorta and skeleton protein of the natural aging rats. Methods Totally 50 SD male rats（ 8-9 months old） were randomly divided into model group,low dose,middle dose and high dose RGRNRCX treated group（ 576,1 152,2 305 mg/kg）,and resveratrol group（ 50 mg/kg） according to body weight,10 in each group. Rats in the model group were fed till 21 months old. Rats in each interventional group were intervened from month 18. Equal volume of pure water was given to rats in the model group according to body weight. All medication was administered once per day by gastrogavage for 3 consecutive months. Meanwhile,10 male 3-month-old SD rats were recruited as a young control group. HE staining was used to observe the changes of vascular morphology of the thoracic aorta. SA-β-gal was used to calculate blue dye cell ratio. The configuration changes of F-actin were observed by immunofluorescent staining. The expressions of skeleton pro-tein F-actin and SM22α were detected by Western blot. Results Compared with the young control group,the intima was injured and not smooth in the model group,the tunica media was hyperplasia obviously and disarranged. The elastic plate was twisted and broken,with incomplete structure. The tunica externa was obviously proliferated. The blue dye cell ratio of SA-β-gal staining increased significantly（P〈0. 01）. The fluorescence intensity and protein expression of F-actin increased obviously（P〈0. 01）,while the protein expression of SM22α obviously decreased（P〈0. 01）. The damage of intima was not so severe with quite complete elastic membrane in each drug intervention groups. The thickness of tunica media and tunica externa obviously decreased（P〈0. 01）. The blue dye cell ratio of SA-β-gal staining was reduced in middle dose RGRNRCX treated group and the resveratrol group（P〈0. 05）. Changed blue dye cell ratio of SA-β-gal staining was not statistically different in low and high dose RGRNRCX treated groups（P〈0. 05）. F-actin fluorescence intensity decreased obviously in each intervention group（P〈0. 01）. F-actin protein expression decreased in each intervention group（P〈0. 01）. F-actin protein expression decreased in high and middle dose RGRNRCX treated groups and the resveratrol group（P〈0. 01）. Changed F-actin protein expression was not statistically different in low dose RGRNRCX treated group（P〈0. 05）. SM22α protein expression increased in each intervention group（P〈0. 01）. Conclusions The configuration and protein expression of F-actin changed obviously in the process of cell aging. SM22α expression changed obviously. They might be involved in vascular aging. The extracts from RGRNRCX could delay vascular aging to some extent and have obvious intervention effect on F-actin and SM22α. It might delay vascular aging indirectly.