Astragaloside Ⅳ Protects SD Rat Aortic Smooth Muscle Cells Against Angiotensin Ⅱ Induced Mitochondrial Dysfunction

BackgroundAstragaloside Ⅳ (As-Ⅳ) is one of the main active components derived from a traditional Chinese herbal medicine, Radix Astragalus. Chemically, As-Ⅳ is a cycloartane triterpene saponin with a clear formula and definite molecular weight and it has multiple pharmacological functions including anti-inflammation, antivirus, anti-apoptosis and so on. As-Ⅳ can protect heart against ischemia and reperfusion injury via energy regulation mechanism, and prevents acute kidney injury by inhibiting oxidative stress. It can also protect against oxidative stress induced injury in mesangial cell and cardiomyocytes. These evidences suggest a possible anti-oxidant role of As-Ⅳ, however, the underlying mechanism remains to be fully elucidated.Mitochondria are the main sites of ATP production as the source of energy for a large number of cellular processes, as well as the main organelles of reactive oxygen species (ROS) generation, which physiologically plays an important role in regulating varied cellular processes including differentiation, autophagy, metabolic adaptation, and immune cell activation. Indeed, mitochondria are now considered as organelles that receive, integrate and transmit signals, thus playing a critical role in cellular responses to a variety of stimuli. Therefore, mitochondrial damage may lead to the impairment of various aspects of tissue functioning. Angiotensin Ⅱ (Ang Ⅱ), a main active substance of renin-angiotensin system, involved in cardiovascular diseases, can induce mitochondrial dysfunction. This mitochondrial dysfunction participated in the pathophysiology of diseases such as hypertension, atherosclerosis and restenosis after vascular intervention, all of which are related to vascular dysfunction. In the stimulation of Ang Ⅱ, vascular smooth muscle cells (VSMCs) showed growth, hypertrophy, migration, apoptosis and secretion disorders, events that contribute to abnormal vascular function, and to disease progression. The prior research in our laboratory showed that As-Ⅳ improved the vascular wall compliance of renal hypertensive rats and lowered the blood pressure. However, the protective effect of As-Ⅳ on mitochondrial dysfunction of AoSMCs injured by Ang Ⅱ has yet to be determined.ObjectiveBased on the anti-oxidant property of As-Ⅳ, the present study was designed to investigate whether and how As-Ⅳ can attenuate Ang Ⅱ-induced mitochondrial injury of AoSMCs.Materials and Methods1. Cell Culture. Aortic smooth muscle cells (AoSMCs) were grown from explants of thoracic aorta from male Sprague-Dawley (SD) rats (180-220 g). Identification of AoSMCs was conducted by morphology examination as well as by immunocytochemistry with anti-α-smooth muscle actin antibody. All the cells were used within 4 to 6 passage.2. Astragaloside Ⅳ material. Astragaloside Ⅳ was dissolved in hydroxypropyl-beta-cyclodextrin (HPBCD) completely to prepare stock solution and the working solution was diluted with DMEM culture medium. The cell toxicity of HPBCD or As-Ⅳ on rat AoSMCs was determined by the measurement of LDH in supernatants and MTT assay, respectively.3. Cell viability assay. Ang Ⅱ diluted with culture medium at graded concentrations from 0.01 to 10μmol/L, was applied to the AoSMCs for optimal Ang Ⅱ density. Cell viability was determined by tetrazolium dye (MTT) assay. Ang Ⅱ at the concentration of 1μM was chosen for the rest of experiments. The effect of As-Ⅳ and losartan (10μM), an antagonist of Ang Ⅱ receptor, on AoSMC viability was carried out as mentioned above.4. Assessment of the As-Ⅳ’s protection on mitochondrial dysfunction induced by Ang Ⅱ.1) Mitochondria morphology changes were observed by transmission electron microscopy;2) Fluorescent probe JC-1 was used to assess the changes of mitochondrial membrane potential (△Ψm) and mitochondrial ATP production was measured with a luciferase-based luminescence assay kit according to manufacturer’s instructions;3) The intracellular ROS generation was determined by the fluorescent probe of 6-carboxy-2,7-dichlorodihydrofluorescein diacetate (H2-DCFDA) according to manufacturer’s instructions. The fluorescence of H2-DCFDA was detected with FACS (excitation,488 nm; emission,530 nm);4) Quantitative Real-time PCR was used to detect mitochondrial DNA (mtDNA) copy number.5. To study the underling mechanism of how As-Ⅳ can attenuate Ang Ⅱ-induced mitochondrial injury of AoSMCs.1) The main enzyme activities of ROS generation resources, NADPH oxidase and xanthine oxidase, were assessed by commercial kits according to manufacturer’s instructions, respectively.2) The main ROS scavenging enzyme activities, the activities of total SOD and Mn-SOD activity, were measured with CuZn/Mn-SOD Assay Kit (Beyotime, Nantong, China) according to the manufacturer’s protocol.3) The mRNA and protein changes of mitochondria biosynthesis PGC-la-NRFl-Tfam pathway genes were detected by qRT-PCR and western blotting, respectively.Results1. Cultured aortic AoSMCs from rats treated with Ang II resulted in swollen, hollow and with reduced or missing cristae of mitochondria. The treatment also decreased mitochondrial membrane potential, ATP production and mitochondrial biogenesis as well as increased intracellular ROS production of the cells.2. As-Ⅳ, co-treatment with Ang II showed significantly beneficial effect on mitochondrial morphologic changes and reversed mitochondrial dysfunction.3. The co-treatment with As-Ⅳ significantly reversed Ang Ⅱ induced increase of NADPH oxidase and xanthine oxidase activity and decrease of total SOD and Mn-SOD activity of AoSMCs.4. In addition, As-Ⅳ also normalized Ang Ⅱ induced expression change of peroxisome proliferator-activated receptor y coactivator-la (PGC-1a), nuclear respiratory factor1 (NRF1), mitochondrial transcription factor A (Tfam) and mitochondrial DNA (mtDNA)Conclusions1. As-Ⅳ, co-treatment with Ang Ⅱ showed beneficial effect on mitochondrial morphologic changes and reversed mitochondrial dysfunction of AoSMCs induced by Ang Ⅱ.2. As-Ⅳ has a beneficial effect by rescuing the mitochondrial dysfunction, possibly through the inhibition of NADPH oxidase and xanthine oxidase activities, the enhancement of total SOD and Mn-SOD activities and the activation of PGC-1α-NRF-1-Tfam pathway.