Flavonoids From Inula Britannica L. Inhibits Injury-induced Neointimal Formation Through Suppressing Oxidative Stress Generation

The use of percutaneous transluminal coronary angioplasty (PTCA) has greatly reduced the number of fatalities in patients who suffer myocardial infarction. Unfortunately, this technique is plagued by a high incidence of vessel renarrowing or restenosis, occurring in 30 to 40% of patients within 6 months of the procedure. Reactive oxygen species (ROS) are thought to play an important role in restenosis. ROS derived mainly from vascular smooth muscle cells (VSMCs) contribute to the proliferation and migration of medial VSMCs leading to neointimal hyperplasia and adverse remodeling and ultimately, vessel restenosis. NAD(P)H oxidases appear to be the major sources of superoxide production in vascular tissues. Association has been made between elevated superoxide levels and neointima formation. This is apparent in models of restenosis and balloon catheter injury. Particularly, the role of NAD(P)H oxidases in neointimal hyperplasia has been demonstrated. Also, sustained downregulation of the expression of extracellular superoxide dismutase (EC-SOD) has been shown to occur after balloon angioplasty, contributing to the imbalance between the production and disposal of ROS and constrictive remodeling.Flavonoids are a large group of polyphenolic compounds abundantly present in the human diet, first identified as plant pigments, but now also recognized as very potent antioxidants and immunomodulators. Large epidemiological studies have shown an inverse association between dietary flavonoid intake and mortality from coronary heart disease. Importantly, therapeutic approaches aiming at augmenting the resistance of tissue to oxidation, such as administration of flavonoids, inhibit vascular remodeling in animal models of hypertension. The beneficial effects of flavonoid consumption on cardiovascular risk are supported by mechanistic and epidemiologic evidence. Inula Britannica L. is a traditional Chinese medicinal herb that has been used to treat bronchitis and inflammation. We have previously demonstrated that 1-O-acetylbritannilactone (ABL) from Inula Britannica L. inhibits NF-κB activation and decreases the level of iNOS and COX-2 gene expression in a dose-dependent manner in RAW 264.7 macrophages stimulated by LPS/IFN-γ, and suppresses neointimal formation induced by balloon injury. Recently, the total flavonoid extracts (TFE) were prepared from Inula Britannica L. Based on a protective effect of antioxidants on neointimal thickening or remodeling after balloon injury, we speculate that the TFE could inhibit the neointimal formation after balloon injury by suppressing oxidative stress generation. In this study, we have investigated whether and how TFE blocks the neointimal hyperplasia in balloon-injured rat carotid arteries.1 TFE suppresses oxidative stress generation and neointimal formation induced by balloon injury in ratTFE was extracted from Inula Britannica L. and identified by HPLC. The yield obtained was 4% relative to the original crude drugs. TFE used in this experiment contained 45.5% quercitrin, 25.85% luteolin, 12.96% 6-methoxyluteolin, 4.33% spinacetin and 1.85% isorhamnetin. The model of rat carotid artery balloon-injury was established as described previously. TFE freshly dissolved in 10% polyglycol-400, was administered orally at doses of 12.5, 25, and 50 mg/kg/d by gastric gavage from 3 days before balloon injury to 2 weeks after the injury.1.1 TFE inhibits injured-induced neointimal hyperplasia of carotid artery in ratAt 14 days after balloon injury, six cross-sections from the middle of each common carotid artery were stained with hematoxylin and eosin (HE). The neointimal and medial areas were calculated using the Image-Pro Plus Analyzer version 5.1 software in a blind manner. Neointimal formation was induced by balloon injury, and increasing I/M ratio of carotid arteries was observed after balloon injury. The injury-induced neoinimal hyperplasia started to increase at day 3 after balloon injury and reached the peak level at day 14. To explore the effect of TFE on neoinimal hyperplasia, the rats were administered orally at doses of TFE 12.5, 25, and 50 mg/kg/d by gastric gavage from 3 days before balloon injury to 2 weeks after the injury, respectively. The animals treated with high dose of TFE (50 mg/kg/d) showed reduction in neointimal hyperplasia, and the ratio of I/M was significantly reduced by over 70% in balloon injured-carotid arteries, compared with injured group. The inhibitory effect of TFE (50 mg/kg/d) on neointimal hyperplasia was almost consistent with atorvastatin that is a positive control. However, the lower doses of TFE (12.5 and 25 mg/kg/d) did not significantly inhibit the injury-induced neointimal formation.1.2 TFE suppresses oxidative stress generation induced by balloon injuryTo characterize O2?- production and localization within the vascular wall, ethidium red fluorescence was analyzed in sections of carotid arteries incubated with DHE that is converted into ethidium by O2?--induced oxidation. Positive red nuclei could be observed in adventitial, medial, and endothelial cells. At 14 days after balloon injury, carotid arteries wall showed an increase in O2?- production that was most evident in the neointimal and medial layer of the vessel. TFE (50 mg/kg/d) significantly prevented injury-induced increase in O2?- production.1.3 Effect of TFE on plasma SOD activity and MDA content after balloon injuryTo test whether the inhibiting neointimal hyperplasia effect of TFE is related to its anti-oxidative properties, the level of MDA and the activity of SOD in plasma were measured before and after balloon injury with or without TFE treatment. The data showed that the plasma MDA content were significantly higher in injured-group than those in sham group. The activity of SOD in plasma was significantly reduced after balloon injury. The animal treated by TFE (50 mg/kg/d) provoked a significant reduction of plasma MDA content, and increment of plasma SOD activity as compared with model group (p<0.01), respectively. Results showed that plasma SOD activity was obviously increased by TFE (50 mg/kg/d) treatment (p<0.01), and plasma MDA production was markedly decreased by TFE (50 mg/kg/d) treatment (p<0.01). Furthermore, the expression and distribution of SOD protein in neointima was detected by Western blot and immunohistochemical analysis, respectively. TFE treatment abolished the decrease in SOD expression induced by balloon injury, and the level of SOD protein approached that of sham group after TFE (50 mg/kg/d) treatment, and increased SOD protein localized in endothelial side of neointima.1.4 TFE inhibits VSMC phenotypic remodeling induced by balloon injuryTo determine whether TFE affects VSMC phenotype, SM22α, a marker of differentiated VSMCs, was detected. The results showed that the expression of SM22αprotein was reduced in neointimal formation induced by balloon injury, indicating that VSMCs change from differentiated state to dedifferentiated state. However, in TFE treatment group, the level of SM22αprotein was almost similar to the level observed in sham group. These results suggest that TFE inhibits VSMC phenotypic remodeling, in consistent with inhibiting neointimal hyperplasia.2 TFE reduces oxidative stress in VSMCs induced by H2O2The present study was designed to investigate crucial mechanisms of quercetin suppressing the oxidative stress in vascular smooth muscle cells (VSMCs).2.1 TFE and quercetin inhibit the formation of O2?- induced by H2O2The production of O2?- in VSMCs was detected by dihydroethidium (DHE) staining. DHE staining showed that the intensity of red fluorescence increased significantly after treatment with H2O2 (200μmol/L) for 12 h in VSMCs. The preincubation with 20 mg/L of TFE and 50μmol/L quercetin resulted in reduction in O2?- production induced by H2O2, respectively. This result suggested that TFE and quercetin can inhibit the formation of O2?- induced by H2O2 in VSMCs.2.2 TFE decreases the generation of MDA induced by H2O2MDA, a production of lipid peroxidation, can be used as a marker of vessel oxidative stress state and oxidative injury. When being treated with 200μmol/L H2O2 for 24 h, the level of MDA in culture medium of VSMCs was increased by 2.78-fold compared with control group (p<0.01). However, the amount of MDA was significantly reduced by 25.33% (p<0.01) in VSMCs pretreated by TFE before H2O2 stimulation compared to H2O2 group, almost similar to that of quercetin that is positive control, suggesting that both TFE and quercetin can inhibit the MDA production induced by H2O2.2.3 TFE enhances SOD activity in VSMCs treated by H2O2SOD is an anti-oxidant enzyme to scavenge oxygen-free radical production. SOD activity in culture medium of VSMCs treated with H2O2 decreased significantly by 42.86%, comparing with control cells (p<0.01). However, the activity of SOD in medium of VSMCs pretreated with TFE increased by 1.46-fold (P<0.01), compared with H2O2 group, suggesting that both TFE and quercetin can restore SOD activity, which is associated with its anti-oxidative activity.3 TFE inhibits p47phox expression and phosphorylationNAD(P)H oxidases are the major sources of superoxide production in vascular tissues. NAD(P)H oxidase system is composed of two cytosolic subunits p47phox and p67phox. The translocation of cytosolic p47phox to the membrane is essential in the assembly process of this complex and plays a major role in NAD(P)H oxidase activity in cardiovascular cells. Other results suggest that decreased NAD(P)H oxidase derived O2?- may be an important mechanism contributing to the prevention of endothelial dysfunction and neointimal hyperplasia by flavonoids.3.1 H2O2 induces the expression of p47phox in VSMCsWestern blot analysis showed that H2O2 triggered p47phox expression. The expression of p47phox increased dramatically in VSMCs treated with H2O2 at different doses (0, 50, 100, 200, 400μmol/L) for 12 h. After treatment by 200μmol/L H2O2 from 0 to 24 h, p47phox protein increased by 1.08-, 1.21-, 1.43-, 1.79- and 1.62-fold compared with control group (P<0.01), respectively. These findings indicated that H2O2 induces the expression of p47phox in a dose- and time-dependent manner. 3.2 TFE and quercetin inhibits the expression of p47phox in vascular tissue induced by balloon injury.Immunohistochemistry assays showed that the increased positive cells for p47phox mainly localized in endothelial side of neointima in model group. However, the number of p47phox positive cells is decreased in TFE group, compare with that in model group. Similarly, Western blot analysis showed that balloon injury induced the expression of p47phox in rat carotid artery, and that the expression of p47phox increased at 3 days after balloon injury and was maximal at 14 days. Administration with TFE (50 mg/kg/d) inhibited the injury-induced increase in p47phox expression in carotid arteries. Immunohistochemical analysis revealed that overexpression of p47phox occurred mainly in the neointima layer and was also inhibited by treatment with TFE (50 mg/kg/d).3.3 TFE and quercetin inhibits the expression of p47phox in VSMCs induced by H2O2In order to confirm the inhibitory effect of TFE on the expression of p47phox, total RNA was extracted from VSMCs by Trizol reagent and then the mRNA of p47phox in VSMCs were detected by RT-PCR. The result showed that treatment with H2O2 resulted in 1.7-fold increase in the level of p47phox, compared with control group. After the cells were pre-incubated with TFE or quercetin, the gene transcriptional activities of p47phox were significantly reduced, and the level of mRNA decreased by 17% or 25%, respectively. However, both agents have no effect on the expression of p47phox in quiescent VSMCs. The data suggested that both TFE and quercetin can inhibit the transcriptional activities of p47phox gene induced by H2O2 in VSMCs.Immunofluorescence analysis confirmed that TFE and quercetin can inhibit the expression of p47phox protein in VSMCs. The p47phox positive cells were increased in H2O2 group. When preincubate with TFE or quercetin, the fluorescence intensities were decreased, indicating that the up-regulation of p47phox expression was inhibited. Western blot analysis showed that treatment with 200μmol/L H2O2 for 12 h resulted in 1.79-fold increase in the level of p47phox protein, compared with control group. After the cells were pre-incubated with TFE or quercetin, p47phox protein was decreased by 38.2% or 39.4% compared with H2O2 group, respectively, coincidence with those in RT-PCR.3.4 TFE and quercetin inhibits the H2O2-induced phosphorylation of p47phox in VSMCsImmunoprecipitation analysis showed that H2O2 triggered p47phox phosphorylation, which reached to peak at 15 min and kept high level up to 30 min and decreased slowly. The pretreating of the cells with TFE resulted in 47.2% decrease in the level of p47phox phosphorylation (P<0.01).4 TFE modulates ERK1/2, Akt and NF-κB signaling pathwaysProtein phosphorylation plays a major role in modulating the activity of transcription factors. The mitogen-activated protein kinase (MAPK) signaling cascades are involved in transducting extracellular signals to nucleus by phosphorylating and activating a variety of transcription factors. ROS has been shown to activation of ERK 1/2. To determine mechanism of TFE inhibiting phosphorylation of p47phox, the role of ERK 1/2, Akt and NF-κB p65 in the signal transduction responses to H2O2 was analyzed by Western blot and immunohistochemistry, respectively.4.1 TFE and quercetin inhibit the phosphorylation of ERK1/2 activated by endothelial injury or H2O2Immunohistochemistry assays and Western blot were used to determine the effect of TFE and quercetin on phosphorylation of ERK1/2 induced by oxidative stress. The results showed that phosphorylated ERK1/2 incresed and localized in neointima 14 days after balloon injury. The phosphorylation of ERK1/2 increased drastically, and reached the peak at 15 min after H2O2 treatment in VSMCs. This data suggest that ERK1/2 pathway can be activated by balloon injury or H2O2. The activation of ERK1/2 signaling pathway can be inhibited significantly by pro-treatment with TFE in vivo and in vitro.4.2 TFE and quercetin inhibit the phosphorylation of Akt activated by H2O2 Western blot analysis showed that total Akt protein among different treatment groups was not different. The level of Akt phosphorylation was increased by 2 folds in VSMCs after H2O2 treatment. However, pre-incubation of TFE or quercetin decreased in H2O2-induced Akt phosphorylation by 33.3% or 37.2%, respectively.4.3 TFE and quercetin inhibit H2O2-induced nuclear translocation of NF-κB p65The nuclear translocation of NF-κB is characterized as the NF-κB activation. We first used Western blot to show the location of NF-κB in nuclei of H2O2-induced VSMCs. The results showed that there was low level of NF-κB p65 in nucleus of resting cells. After induction with H2O2 for different times, p65 was gradually localized to the nuclei of VSMCs, indicating translocation of p65 to the nuclei in VSMCs induced by H2O2. In TFE-preincubated VSMCs, H2O2-induced increase in nuclear p65 was reduced.Conclusions1. TFE inhibits neointimal formation through up-regulating SOD protein expression and activity, and suppressing ROS generation induced by balloon injury. The inhibitory effect of TFE (50 mg) on neointimal hyperplasia is almost consistent with atorvastatin that is a positive control.2. TFE treatment abolishes the decrease in SOD activity, reduces MDA level in plasma and significantly prevents injury-induced increase in O2?- production in rats after balloon injury.3. TFE inhibits the expression and phosphorylation of p47phox induced by ROS in vascular cells, in vivo and in vitro.4. TFE modulate the activation of ERK1/2, Akt and NF-κB signaling pathway in VSMCs treated by H2O2.5. TFE is a very potent antioxidant and inhibits neointimal formation through antioxidant. The decreased NAD(P)H oxidase derived O2?- may be an important mechanism contributing to the prevention of endothelial dysfunction and neointimal hyperplasia by TFE.