人参-三七-川芎提取物延缓高糖诱导的小鼠血管钙化的机制

Mechanism of Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma Extracts in Delaying High Glucose-induced Vascular Calcification in Mice

目的:从小鼠颈总动脉、胸主动脉中骨桥蛋白(OPN)和平滑肌22α蛋白(SM22α)表达及血管钙盐沉积程度,探讨人参-三七-川芎提取物对高糖诱导的小鼠血管钙化的保护作用。方法:130只雄性C57BL/6小鼠先随机分为正常组和高糖组。高糖组腹腔注射链脲佐菌素(STZ)后,高脂饮食连续喂养7个月,之后再次随机分为模型组、人参-三七-川芎提取物低、高剂量组(0.819,1.638 g·kg^-1)及二甲双胍组(150 mg·kg^-1)。每组灌胃给药,每天1次,连续9周,并监测血糖变化。给药结束前7 d,购进一批4周龄雄性C57BL/6小鼠,正常喂养1周,作为青年组。全部给药结束后,收集小鼠颈总动脉和胸主动脉组织。分别采用冯库萨(Von Kossa)染色判断小鼠颈总动脉、胸主动脉钙盐沉积程度,免疫组化检测小鼠颈总动脉、胸主动脉中OPN与SM22α蛋白表达,蛋白免疫印迹法(Western blot)测定小鼠颈总动脉中OPN与SM22α蛋白表达情况。结果:与青年组比较,正常组小鼠血糖升高无统计学差异,颈总动脉、胸主动脉结构染色均匀,未见黑色颗粒状沉淀;与正常组比较,模型组血糖显著升高(P<0.01),血管中内膜弹性纤维内有大量棕黑色颗粒沉积,钙盐沉积程度明显;与模型组比较,各给药组血糖明显下降(P<0.05,P<0.01),血管钙盐沉积程度明显减轻。与青年组比较,正常组小鼠颈总动脉、胸主动脉中OPN蛋白和SM22α蛋白表达无显著改变;与正常组比较,模型组颈总动脉、胸主动脉中内膜OPN蛋白表达呈阳性,SM22α蛋白表达呈弱阳性,颈总动脉中OPN蛋白表达灰度值显著升高(P<0.01),SM22α蛋白表达灰度值显著降低(P<0.01);与模型组比较,各给药组颈总动脉、胸主动脉中内膜OPN蛋白和SM22α蛋白表达显著改善,颈总动脉中OPN蛋白表达灰度值明显降低(P<0.05,P<0.01),SM22α蛋白表达显著升高(P<0.01)。结论:高糖能够诱导小鼠颈总动脉、胸主动脉钙化,加速血管老化,这一形成过程可能与OPN与SM22α表达有关。人参-三七-川芎提取物可通过调节OPN与SM22α表达,减轻血管钙化,延缓血管老化。

Objective:To investigate the protective effect of Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts on vascular calcification induced by high glucose in mice by observing the expression of osteopontin(OPN)and smooth muscle 22α(SM22α)as well as vascular calcium deposition in the common carotid artery and thoracic aorta of mice.Method:Totally 130 male C57 BL/6 mice were randomly divided into normal control group and high glucose group.The mice in high glucose group were intraperitoneally injected with streptozotocin(STZ),and fed on a high-fat diet for 7 months.Then,the mice were randomly divided into model group,low-dose and high-dose Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts groups(0.819,1.638 g·kg^-1),and metformin group(150 mg·kg^-1).Each group was intragastrically administered once a day for 9 weeks.The changes in blood glucose were measured.Seven days before the end of the administration,a group of 4-week old male C57 BL/6 mice were purchased and fed normally for one week as a youth group.At the end of the administration,the common carotid artery and thoracic aorta tissues of the mice were collected.Von Kossa staining was used to determine the degree of calcium deposition in the common carotid artery and thoracic aorta.The expression levels of OPN and SM22αprotein in the common carotid artery and thoracic aorta were detected by immunohistochemistry.The expression of OPN and SM22αprotein in the common carotid artery of mice was determined by Western blot.Result:As compared with the young group,the blood glucose of the normal control group was slightly increased without statistical difference,the common carotid artery and thoracic aorta were uniformly stained,and no black granular precipitate was observed.As compared with the normal control group,the blood glucose of the model group was increased(P<0.01),with a large amount of brown-black particles deposited in the intimal elastic fibers,showing obvious calcium salt deposition.As compared with the model group,blood glucose was significantly decreased in each administration group(P<0.05,P<0.01),and the degree of vascular calcium salt deposition was significantly reduced.There were no significant changes in expression levels of OPN protein and SM22αprotein in the common carotid artery and thoracic aorta between the youth group and normal control group.As compared with the normal control group,the expression of intimal OPN protein in the common carotid artery and thoracic aorta of the model group was positive,SM22αprotein expression was weakly positive,and the gray value of OPN protein expression in the common carotid artery was significantly increased(P<0.01),while the gray value of SM22αprotein was decreased significantly(P<0.01).As compared with the model group,the expression levels of intimal OPN protein and SM22αprotein in the common carotid artery and thoracic aorta of each administration group were significantly improved,and the gray value of OPN protein expression in the common carotid artery was reduced(P<0.05,P<0.01),while SM22αprotein expression was significantly increased(P<0.01).Conclusion:High glucose can induce calcification of common carotid artery and thoracic aorta in mice and accelerate vascular aging.This formation process may be related to the expression of OPN and SM22α.Ginseng Radix et Rhizoma,Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts can reduce vascular calcification and delay vascular aging by regulating the expression of OPN and SM22α.