| 1. Background:Oxidative stress, which is defined as an imbalance between prooxidant and antioxidant systems in tissue or intracellular, refers to the body after suffering a variety of harmful stimuli, all kinds of free radicals, such as reactive oxygen species (ROS) or reativenitrogen species (RNS) can not be cleared in a timely manner, and in vivo or in cell accumulation induced series of pathophysiological reaction, leading to the process of tissue damage. The body continuous with oxidative stress, vascular tissue formation process becomes uncontrollable and loss the balance between the needs of the organization, while intimal serious injury induced by a variety of vascular diseases, such as:atherosclerosis. Its occurrence and development with aging is closely related to obesity, diabetes, chronic renal failure, cancer and genetic diseases. The prevalence of patients with these diseases in vivo is redox imbalance characterized by "oxidative stress."Advanced oxidation protein products (AOPPs) was initially found to be elevated as an important macromolecule uremic toxin in the plasma of CRF patients by Witko-Sarsat in 1996, which are the dityrosine-containing and cross-linking protein products formed during oxidative stress by reaction of plasma protein with chlorinated oxidants through neutrophil myeloperoxidase-catalyzing reaction. High levels of plasma AOPPs can not also increase oxidative stress is a potential virulence factor, but also promote the development of inflammation, playe the role of inflammatory mediators. Currently considered, AOPPs participates obesity, diabetes, coronary heart disease and kidney fibrosis, the disease course, related diseases such as cataract also play an important role in the aging and elderly. Numerous experiments show that AOPPs closely associated with atherosclerosis, but the specific mechanisms of AOPPs to promote atherosclerosis is not clear.Recent studies show, AOPPs through binding to RAGE, activating of NADPH oxidase, and then promouting a large amount of ROS, and Nox2, Nox4 subunit are the main function of NADPH oxidase to produce ROS in the endothelial cells. The balance between cytosolic Ca2+ level, ROS and ATP content will be broken by a large number of intracellular ROS in endothelial cell. A large number of high levels of ROS and Ca2+ can cause mitochondrial membrane potential decreased, so that ATP synthesis was inhibited, whats more, when a mitochondrial calcium overload, the mitochondrial permeability became shift, swelling, rupture, and cause cell death. Another study found that the AOPPs stimulate could significantly increased hypoxia inducible factor (HIF-1α) and the vascular endothelial growth factor (VEGF) levels in blood. And cardiovascular disease is associated with abnormally elevated HIF-1, and the VEGF levels in blood. For example, studies have shown that atherosclerotic plaques (atherosclerosis, AS) prone to instability, loss, resulting in blood vessel blockage and cause serious consequences, and in the early AS plaque that is the formation of new blood vessels, and plaque neovascularization AS the late stage is a significant feature mouth, resulting in plaque instability and disease progression. HIF-1 a is considered to play a specific transcription factor activity in the absence of oxygen, in AS plaques to promote angiogenesis, leading to plaque instability plays an important role, in addition to the downstream signal of HIF-la, closely related to the formation of VEGF stimulated by HIF-la can induce migration of smooth muscle cells, and vascular smooth muscle migration and plaque. The evidence shows that: AOPPs is an important risk factor for cardiovascular disease.Paeoniflorin is one of the main active ingredients of peony, and it is a terpene glycoside compound alone. Domestic and foreign scholars unfold a more in-depth research for Paeoniflorin, and found that it has a variety of biological effects, and lesser side effects. It has been reported that Paeoniflorin has a pharmacological activity of antioxidation, and could inhibited intracellular calcium overload. In cytology and animal experiments, Paeoniflorin suppresses mitochondria-mediated apoptosis of PC 12 cells induced by 6-OHDA, and anti-apoptotic effects of Paeoniflorin on PC 12 cells might mainly result from its antioxidant capability by increasing glutathione (GSH). Some scholars have found another discovery, Paeoniflorin can dramatically attenuate the 6-OHDA-induced nuclear factor κB (NF-κB) translocation without affecting phosphorylation of Akt, JNK, p38, and ERK1/2. There is also evidence Showed, Paeoniflorin could attenuate ROS production induced by Ab25-35 in SH-SY5Y cells. In addition, Paeoniflorin strikingly inhibited Ab25-35-induced mitochondrial dysfunction. Studies have also shown that Paeoniflorin is beneficial to the prevention of arteriosclerosis through inhibiting oxidized low density lipoprotein (ox-LDL)-induced proliferation of vascular smooth muscle cells (VSMCs) What’s more, it can protect endothelial cells from hypoxic damage through protecting the function of endothelial cells, enhancing the production of nitric oxide (NO) and decreasing the expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1).However, whether Paeoniflorin can eliminate the destructive effects of AOPPs in vascular endothelial cells? Whether it can inhibit ROS formation by regulating the function subunit of NADPH:Nox2, Nox4, and then reduce the oxidative damage? Whether through intervention AOPPs stimulation RAGE-NADPH (Nox2/Nox4)-ROS-NF-KB-HIF-1α/VEGF signal transduction pathways, to achieve its course of intervention for AS? These assumptions require us to further test proved.2. Methods:2.1 Preparation of AOPPsAOPPs-bovine serum albumin (AOPPs-BSA) was prepared as described previously. We use UV spectrophotometry of Witko-Sarsat to detect the AOPPs relative concentration of chloramine-T (μM); BCA assay to detect the total protein content of AOPPs and unmodified BSA samples (mg/ml); Endotoxin levels in the preparation were determined with the amebocyte lysate assay kit (Sigma).2.2 MTT assay to detect the effects of AOPPs or Paeoniflorin on HUVECs activityDetermine the effect of different concentrations of AOPPs and 200 μg/ml BSA and a various concentrations of Paeoniflorin on cell survival in this experiment by using the MTT assay. In addition, we determine the effect of different concentrations of Paeoniflorin pretreated on AOPPs induced HUVECs cell survival.2.3 DCFH-DA fluorescent probe to measure the effect of AOPPs on ROS level in HUVECs as well as the intervention effect of PaeoniflorinThe intracellular ROS production of different concentrations of AOPPs on HUVECs was measured via Fluorescence with a 2’, 7’-dichlorofluorescein-diacetate (DCFH-DA) staining. In order to verify the source of ROS, we observe the blocking effects of different concentrations of Paeoniflorin and different inhibitors:FPS-ZM1 (230 nM), Apocynin (100 μM), NAC (5 mM) and (200 μM) Paeoniflorin for AOPPs on ROS generation in HUVECs.2.4 Fluo-3 AM to measure the effect of AOPPs on intracellular free Ca2+ level in HUVECs as well as the intervention effect of PaeoniflorinFluo-3 AM was used to measure the mitochondrial Ca2+ fluctuations of different concentrations of AOPPs and BSA stimulation HUVECs. In addition, we also measure the effect of different inhibitors FPS-ZM1, Apocynin, NAC and 200 μM Paeoniflorin on AOPPs stimulate mitochondrial Ca2+ fluctuations in HUVECs.2.5 FCM and confocal microscopy to evaluate the effect of AOPPs on mitochondrial membrane potential in HUVECs as well as the intervention effect of PaeoniflorinWe use MitoTracker(?) Deep Red and MitoTracker(?) Green stained HUVECs to measure the effects of different concentrations of AOPPs or 200 μg/ml BSA on the mitochondrial membrane potential in HUVECs by using flow cytometry. Furthermore we also measure the restor effects of different inhibitors:Apocynin, within the FPS-ZM1, NAC and Paeoniflorin on mitochondrial membrane potential dysfunction caused by AOPPs.In addition, we also use confocal microscopy to observe the mitochondrial membrane potential of AOPPs and BSA on the mitochondrial membrane potential in HUVECs.2.6 ATP Determination Kit to measure the effect of AOPPs on ATP level in HUVECs as well as the intervention effect of PaeoniflorinThe effect of different concentrations of AOPPs and BSA on the ATP level in the HUVECs cells was measured using an ATP Determination Kit according to the manufacturer’s protocol. In addition, we also measure restore effect of different inhibitors:Apocynin, FPS-ZM1, NAC and Paeoniflorin on the depletion of ATP on AOPPs induce HUVECs.2.7 Real-Time Quantitative PCR to detect the expression of AOPPs induce RAGE in HUVECs as well as the intervention effect of PaeoniflorinHUVECs were incubated with AOPPs (200 μg/ml) for 15 min,30 min,60 min and 90 min or HUVECs were preincubated with inhibitor (FPS-ZM1) or Paeoniflorin for 30 min prior to incubation with AOPPs (200 μg/ml) for 90 min. After incubation, then the gene expression of RAGE was detected by RT-PCR.2.8 Western blot to detect the expression of AOPPs induce Nox2, Nox4, HIF-la, VEGF and NF-κB p65 in HUVECs as well as the intervention effect of PaeoniflorinIn brief, cells were incubated with AOPPs or preincubation with various inhibitors (FPS-ZM1, Apocynin, NAC and BAY-117 082) and Paeoniflorin for 1 h before exposing to the AOPPs for various time, and then Nox2, Nox4, HIF-la, VEGF and NF-κB p65 were detected by western blot.3. Results3.1 Characterization of AOPPsEndotoxin levels in the preparation were determined with the amebocyte lysate assay kit (Sigma) and were found to be below 0.025 EU/ml. The content of AOPPs was 142.4 ± 9.8 μmol/g protein in prepared AOPPs-BSA and 0.2 ± 0.02 μmol/g protein in native BSA3.2 Effect of AOPPs or Paeoniflorin on Cell ViabilityWe evaluated the cytotoxic effects of Paeoniflorin on HUVECs in the present or absent of AOPPs via the MTT assay. The results show AOPPs exhibited toxity on HUVECs at the concentrations of ≥200 μg/ml(p<0.01), while compared to the unmodified BSA and control group.50-400 μM Paeoniflorin compared with NC group, no significant effect on cell viability. Paeoniflorin pretreatment 1 h, then added AOPPs incubated 24 h, showed 100-400 μM Paeoniflorin group compared with AOPPs, were able to increase cell survival (p> 0.05).3.3 Paeoniflorin attenuates AOPPs-induced ROS production in HUVECsThe DCF-fluorescence intensity increased by AOPPs treatment and peaked at the concentration 200 μg/ml after 90 min (p<0.01). And Paeoniflorin could effectively reduced ROS level in dose-dependent manner (p<0.01). The same result of ROS suppression also happened in the FPS-ZM1-, Apocynin- and NAC-pretreated groups (p<0.01).3.4 Paeoniflorin suppresses abnormal cytoplasmic Ca2+fluxThe effect of AOPPs on Ca2+ flux was in dose-dependent manner (p<0.01). Furthermore Paeoniflorin, together with FPS-ZM1, Apocynin and NAC, obviously suppress the Ca2+ flux (p<0.01).3.5 Paeoniflorin restores mitochondria dysfunction via ROS suppressionThe results indicated the level of MMP decreased in dose-dependent manner in HUVECs treated with AOPPs compared to the unmodified BSA and control groups (p<0.05). The observation results using cofocal microscopy also confirmed the changes on MMP by flow cytometry. The results indicated Paeoniflorin, togther with FPS-ZM1 Apocynin and NAC, successfully decreased the ratio of compromised mitochondria (p<0.01).3.6 Paeoniflorin restores ATP depletionThe results indicated the ATP decreased in dose-dependent manner in HUVECs treated with AOPPs compared to the unmodified BSA and control groups (p<0.05). Furthermore Paeoniflorin, togther with FPS-ZM1 Apocynin and NAC, successfully effectively restore ATP level (p<0.01).3.7 Inhibitory effect of Paeoniflorin on RAGE and Nox2/Nox4 expressionThe results indicated, RAGE was activiated within 15min, maintained until 90 min (p<0.05), and then declined. Nox2/Nox4 was also activiated whith 15 min, maintained until 90 min (p<0.01). Furthermore, both FPS-ZM1 and Paeoniflorin effectively decreased RAGE expression (at the posttranscription level) (p<0.01). Moreover, Paeoniflorin, same as FPS-ZM1, Apocynin and NAC, obviously decreased the expressions of Nox2/Nox4 (at the posttranscription level) (p<0.01).3.8 Inhibitory effect of Paeoniflorin on NF-κBp65 and HIF-1α/VEGF expressionAfter long time ineubation with AOPPs, the protein levels of NF-κB p65 and HIF-1α/VEGF were upregulated in HUVECs. Paeoniflorin, same as FPS-ZM1, Apocynin, NAC and BAY-117 082 can signifieantly reduce the upregulation of NF-κBp65 and HIF-1α/VEGF by AOPPs.4. ConclusionsPaeoniflorin decreased the levels of angiofactors (HIF-1α and VEGF) in HUVECs, mainly through signals mediated by RAGE-NADPH-ROS, which involving in ROS generation, abnormal Ca2+ flux and ATP depletion. Moreover, Paeoniflorin can also suppress the NF-κB actviation, which could indirectly increase the angiofactors (HIF-1α、VEGF) expression. This suggests that Paeoniflorin might be useful as a therapeutic agent for cardiovascular disease contributing to oxidative stress injury. |
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