天麻钩藤饮调控MFN2表达干预自发性高血压病模型大鼠血管衰老的作用机制

Tianma Gouteng Decoction regulates MFN2 expression to delay vascular aging in spontaneously hypertensive rats

研究天麻钩藤饮对自发性高血压病模型大鼠血管衰老的作用,探讨其是否通过调控线粒体融合蛋白2(mitofusin 2,MFN2)表达改善血管衰老的分子机制。将20只64周龄自发性高血压病模型大鼠(spontaneously hypertensive rats, SHR)随机分为衰老组、治疗组(天麻钩藤饮5.48 mg·kg),64周龄Wistar-Kyoto (WKY)大鼠为正常组,14周龄自发性高血压病模型大鼠为青年组;进行天麻钩藤饮干预12周。通过HE染色、Masson染色和电子显微镜观察胸主动脉形态学变化;检测丙二醛(malondialdehyde, MDA)含量、总超氧化物歧化酶(superoxide dismutase, SOD)活性、呼吸链复合物Ⅲ水平和硫氧还蛋白过氧化物酶(thioredoxin peroxidase, TPX)活性,β-半乳糖苷酶(senescence-associated beta-galactosidase, SA-β-Gal)检测血管衰老。免疫组织化学/荧光法、qRT-PCR和Western blot法检测胸主动脉组织MFN2及衰老标志蛋白沉默信息调节因子1(silent information regulator 1, SIRT1)、Klotho、p21和p53的表达。结果显示,与青年组和正常组比较,衰老组中血压明显升高,血管内膜明显增厚导致管腔狭窄,抗氧化指标SOD和TPX显著下降,而促氧化指标MDA和线粒体呼吸链复合物Ⅲ升高,引起血管衰老明显增多;分子机制研究表明MFN2表达下降,衰老相关分子SIRT1和Klotho表达下降,但p21和p53表达增强(P<0.01或P<0.05)。通过天麻钩藤饮干预,能够显著降低血压,血管内膜增厚减轻,抗氧化指标SOD和TPX显著升高,而促氧化指标MDA和线粒体呼吸链复合物Ⅲ下降,引起血管衰老明显减弱;同时MFN2表达增强,衰老相关分子SIRT1和Klotho表达增强,但p21和p53表达下降(P<0.01或P<0.05)。以上结果表明天麻钩藤饮能够显著延缓高血压病血管衰老作用,其机制可能通过增强MFN2表达,调控衰老相关蛋白表达,改善氧化应激。

We studied the effect of Tianma Gouteng Decoction on vascular aging in spontaneously hypertensive rats(SHRs) to explore the molecular mechanism of the decoction in improving arterial vascular aging by regulating the expression of mitofusin 2(MFN2).Twenty 64-weeks-old SHRs were randomly assigned into the aging group and the treatment group(Tianma Gouteng Decoction 5.48 mg·kg).Wistar-Kyoto(WKY) rats of 64 weeks old were taken as the normal group and SHR rats of 14 weeks old as the young group.The intervention with Tianma Gouteng Decoction lasted for 12 weeks.We then employed HE staining and Masson staining to observe the morphology of thoracic aorta under an electron microscope and measured the malondialdehyde(MDA) content, superoxide dismutase(SOD) activity, respiratory chain complex Ⅲ level, and thioredoxin peroxidase(TPX) activity.The vascular aging was detected via the biomarker senescence-associated beta-galactosidase(SA-β-Gal).The expression levels of MFN2 and aging marker proteins silent information regulator 1(SIRT1), Klotho, p21, and p53 in thoracic aorta were detected by immunohistochemistry/fluorescence, qRT-PCR, and Western blot.Compared with the young group and the normal group, the aging group had risen blood pressure, lumen stenosis caused by thickened intima of blood vessels, decreased SOD and TPX activities, increased MDA and mitochondrial respiratory chain complex Ⅲ levels, down-regulated expression of MFN2, SIRT1, and Klotho, and up-regulated expression of p21 and p53(P<0.01 or P<0.05).The treatment with Tianma Gouteng Decoction significantly lowered blood pressure, mitigated vascular intimal thickening, increased SOD and TPX activities, and decreased MDA and mitochondrial respiratory chain complex Ⅲ levels, thus alleviating vascular aging.At the same time, the decoction up-regulated the expression of MFN2, SIRT1, and Klotho, while down-regulated that of p21 and p53(P<0.01 or P<0.05).In summary, Tianma Gouteng Decoction can significantly delay the vascular aging in hypertension.Specifically, it may up-regulate the expression of MFN2 and regulate the expression of aging-related proteins to alleviate oxidative stress.