Mechanisms Of Salidroside To Improve Endothelial Function And Alleviate Atherosclerosis

Part Ⅰ Salidroside Improves Endothelial Function and Alleviates AtherosclerosisObjective:Salidroside (SAL) is an active component of Rhodiola Rosea with anti-atherosclerosis properties. It has been reported that SAL can efficiently decrease the atherosclerotic plaque formation, but the mechanism is not clear. Endothelial dysfunction characterized by endothelial nitric oxide synthase (eNOS) dysfunction play an important role in the development of atherosclerosis. This study is to elucidate whether SAL can stimulate eNOS activation to alleviate atherosclerosis. We also explore the effects of adenosine5’-monophosphate (AMP)-activated protein kinase (AMPK), Phosphatidylinositol3-kinase (PI3K/Akt) on the activation of eNOS.Methods:(1) After adaptive feeding for a week, the apoK-/-mice were assigned into two groups:1. A Normal-diet group (ND, n=12):received a standard diet; and2. A high-fat-diet group (HFD, n=38):received a high fat diet (15%fat,0.15%cholesterol in mouse food, HFK) for8weeks. The HFD groups were then further randomly assigned into three groups:HFD, SAL25mg/kg/day group and SAL50mg/kg/day group, SAL was given by gavage. In ND and HFD groups, normal saline was given by gavage. At an8-week administration, serum was collected for testing biochemical parameters. Lipid deposition in the whole aorta and aortic sinus were detected by Oil-Red O. The vasodilatation of thoracic aortas from apoE-/-mice was measured using isolated perfusion method. Western blot was used to study the effects of SAL on the phosphorylations and expressions of eNOS, AMP-activated protein kinase (AMPK), and Akt in aortas.(2) The vasodilatation of thoracic aortas from Wistar rats was measured using isolated perfusion method.(3) Western blot was used to study the effects of SAL on the phosphorylations and expressions of eNOS, AMP-activated protein kinase (AMPK), and Akt in HUVECs (human umbilical vein endothelial cells). AMPK inhibitor Compound C, AMPKal/2siRNA and Akt inhibitor LY294002were used to evaluate the cause-and-effect relationship between AMPK or Akt phosphorylation and eNOS activation. HPLC analysis was used to evaluate the effects of SAL on the content of ATP and AMP in HUVECs. Specific fluorescent dyes3-Amino,4-aminomethyl-2’,7’-difluorescein diacetate (DAF-FM-DA),(5,5’,6,6’-tetrachloro-1,1’,3,3’-, tetraethyl-benzimidazolyl-carbocyanine iodid, JC-1) and dihydroethidium(DHE) were used to measured the NO production, mitochondrial membrane potential (△ψm) and superoxide (O2-) production respectively, the fluorescence density was detected with fluorescence microplate reader to evaluate the effects of SAL.Results:(1) Compared to high fat diet (HFD) group, the relative surface area of the atherosclerotic lesion was significantly smaller in the SAL (50mg/kg) group. SAL (50mg/kg) treatment significantly improved the endothelium-dependent relaxation in response to ACh compared with the HFD group. Meanwhile, SAL significantly increased eNOS expression and activity in the mice aortas, as well as the AMPK and Akt phosphorylation. SAL reduced the serum concentrations of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-c), the mechanism of these lipid modulating effects may be due to the activation of AMPK in the livers.(2) SAL induced endothelium-dependent vasodilatation in thoracic aorta of rat. The endothelium-dependent vasodilation was blocked by endothelium removal and the nitric oxide synthase (NOS) inhibitor NG-Nitro-L-arginine Methyl Ester (L-NAME).(3) In HUVECs, SAL increased eNOS ser1177phosphorylation and concomitantly decreased eNOS thr495phosphorylation in time and concentration dependent manner, a pattern indicative of increasing eNOS activity. Meanwhile, DAF-FM-DA fluorescence intensity was also increased in time and concentration dependent manner. After SAL administration, AMPK phosphorylation was enhanced within30min, and then recovered in6h. But, intriguingly, SAL stimulated Akt-Ser473phosphorylation in a different time course, which began to increase after30min and persist for over24h. Treatment cells with AMPK inhibitor or siRNA blocked the effects of SAL on phosphorylation of eNOS ser1177and Akt ser473, suggesting that AMPK is responsible for eNOS and Akt phosphorylation. The effects of SAL on eNOS phosphorylation and expression were almost completely inhibited by pretreatment with PI3K inhibitor LY294002in a long-trem. SAL was found to decrease the△ψm, accordingly decreased cellular ATP content which caused the increasing of AMP/ATP ratio to induce AMPK activation.Conclusions:The action of SAL to reduce atherosclerosis lesion formation may partly attribute to its effect to improve endothelial function by promoting eNOS activation and nitric oxide (NO) production, which is associated with mitochondria depolarization and subsequent activation of AMPK/PI3K/Akt/eNOS pathway. Part ⅡSalidroside Protects against H2O2-induced Endothelial DysfunctionObjective:Overproduced ROS are known to harm the normal vascular function and lead to endothelial dysfunction. As the main sourec of ROS, there are increasing evidences have indicated the importance of mitochondrial dysfunction in atherosclerosis. The purpose of this study was to explore the mechanism of the protective effect of SAL on hydrogen peroxide (H2O2)-induced endothelial dysfunction. The effects of SAL on mitochondrial biogenesis and function, and NF-κB activation were investigate to illustrate its mechanism of improving endothelial function.Methods:The oxidative stress model was established with H2O2as the damage factor. Effects of SAL on cell viability were detected by CCK-8kit, and the activity of transcription factor NF-kappa B (NF-κB) was detected by an ELISA-based transcription activity assay. The vasodilatation of thoracic aortas from Wistar rats were measured using isolated perfusion method. Western blot was used to study the effects of SAL on the activation of eNOS, AMPK, Akt induced by H2O2in HUVECs. The proteins, PGC-la (peroxisome proliferators-activated receptor-γ coactivator-la) and TEAM (mitochondrial transcription factor A), related to mitochondrial biogenesis were also detected by western blot. The mitochondria mass was detected by Mito Tracker green. Specific fluorescent dyes DAF-FM-DA, JC-1and DHE were used to measured the NO production,△ψm and O2-production, respectively. Cellular ATP content was examined using HPLC.Results:Pretreatment HUVECs with SAL significantly reduced the cytotoxicity induced by H2O2. Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation impaired by H2O2. Meanwhile, SAL pretreatment inhibited H2O2-induced O2·-and NO production. The underlying mechanisms involved the inhibition of over-activation of eNOS, AMPK, and Akt induced by H2O2. SAL also inhibited the acitivty of transcription factor NF-κB activated by H2O2(0.1μM). H2O2at the concentration of100μM slightly reduced rather than increased the NF-κB activity. Meanwhile, pretreatment with SAL further reduced this activity. SAL also increased mitochondrial mass, simultaneously up-regulated the mitochondrial biogenesis factors, PGC-1α and TFAM expression in the endothelial cells. eNOS inhibitor L-NAME blocked the upregulation of TFAM and PGC-1α induced by SAL. H2O2-induced mitochondrial dysfunction, as demonstrated by the reduction of△Ψm and ATP production was rescued by SAL pre-treatment.Conclusion:SAL protects endothelium against H2O2-induced injury via promoting mitochondrial biogenesis and recovering mitochondrial function, thus preventing the over-activation of oxidative stress-related downstream signaling pathways.